Toolkit: HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis: HIV treatment and care

HIV TREATMENT AND CARE HIV MOLECULAR DIAGNOSTICS TOOLKIT TO IMPROVE ACCESS TO VIRAL LOAD TESTING AND INFANT DIAGNOSIS TOOLKIT JULY 2019 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis ISBN 978-92-4-151621-1 © World Health Organization 2019 Some rights reserved. This work is available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 IGO licence (CC BY-NC-SA 3.0 IGO; https://creativecommons.org/licenses/by-nc-sa/3.0/igo). Under the terms of this licence, you may copy, redistribute and adapt the work for non-commercial purposes, provided the work is appropriately cited, as indicated below. In any use of this work, there should be no suggestion that WHO endorses any specific organization, products or services. The use of the WHO logo is not permitted. If you adapt the work, then you must license your work under the same or equivalent Creative Commons licence. If you create a translation of this work, you should add the following disclaimer along with the suggested citation: “This translation was not created by the World Health Organization (WHO). WHO is not responsible for the content or accuracy of this translation. The original English edition shall be the binding and authentic edition”. Any mediation relating to disputes arising under the licence shall be conducted in accordance with the mediation rules of the World Intellectual Property Organization. Suggested citation. HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis. Geneva: World Health Organization; 2019. Licence: CC BY-NC-SA 3.0 IGO. Cataloguing-in-Publication (CIP) data. CIP data are available at http://apps.who.int/iris. Sales, rights and licensing. To purchase WHO publications, see http://apps.who.int/bookorders. To submit requests for commercial use and queries on rights and licensing, see http://www.who.int/about/licensing. Third-party materials. If you wish to reuse material from this work that is attributed to a third party, such as tables, figures or images, it is your responsibility to determine whether permission is needed for that reuse and to obtain permission from the copyright holder. The risk of claims resulting from infringement of any third-party-owned component in the work rests solely with the user. General disclaimers. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of WHO concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted and dashed lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by WHO in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by WHO to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall WHO be liable for damages arising from its use. Cover photo: © WHO Layout by L’IV Com Sàrl Printed in Switzerland HIV MOLECULAR DIAGNOSTICS TOOLKIT TO IMPROVE ACCESS TO VIRAL LOAD TESTING AND INFANT DIAGNOSIS TOOLKIT – JULY 2019 22 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis CONTENTS Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1. Introduction: molecular diagnostics for HIV viral load testing and infant diagnosis . . . . . . . . . . . . . . . . . . . 4 2. Viral load test result utilization to support clinical management of people living with HIV on antiretroviral therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3. Estimated reach of and access to viral load testing using traditional plasma specimens . . . . . . . . . . . . . . . . 10 4. Specimen stability for HIV viral load testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 5. Technical background: molecular testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 6. Alternative specimen types and technologies for consideration when liquid plasma cannot be used widely for viral load testing due to infrastructure, transport or other constraints. . . . . . . . . . . . . . . . . . . . . 18 6.1. Alternative specimen types and technologies: dried blood spot specimens for HIV viral load testing . . . . . . . . . . 18 6.2. Alternative specimen types and technologies: dried plasma spot specimens for HIV viral load testing . . . . . . . . 20 6.3. Alternative specimen types and technologies: plasma preparation tubes for HIV viral load testing . . . . . . . . . . . 21 6.4. Alternative specimen types and technologies: point-of-care and near-point-of-care tools for HIV viral load testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 7. Operational interventions and considerations in scaling up viral load and infant diagnosis . . . . . . . . . . . . 25 7.1. Specimen transport options for molecular diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 7.2. Infant diagnosis and viral load specimen collection bundles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 7.3. Operational interventions: using viral load as a diagnostic for infants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 7.4. Novel point-of-care tools for early infant diagnosis of HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 7.5. Operational interventions: updated considerations for a comprehensive quality management package for point-of-care testing within national health programmes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Annex 1. Infant diagnosis algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 © W H O 3HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis ACKNOWLEDGEMENTS Several key stakeholders have provided significant input throughout the development process of this publication, including the following. – Robert Luo, Kameko Nichols and Neil Parkin – African Society for Laboratory Medicine: Charles Kiyaga and Anafi Mataka – Clinton Health Access Initiative: Paolo Maggiore, Maria Rosezoil Rioja and Jilian Sacks – Elizabeth Glaser Pediatric AIDS Foundation: Jennifer Cohn – United States Agency for International Development: Dianna Edgil, Matthew Wattleworth and Jason Williams – WHO Regional Office for Africa: Fatim Cham Jallow and Fausta Mosha – WHO Essential Medicines and Health Products Programme: Mercedes Perez and Ute Ströher In particular: – The Clinton Health Access Initiative and the United States Agency for International Development supported the development of Section 3. – Robert Luo supported the development of Section 5 and subsection 6.1. – Kameko Nichols supported the development of subsection 7.1. – The Clinton Health Access Initiative supported the development of subsection 7.2. © W H O 44 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 1. INTRODUCTION: MOLECULAR DIAGNOSTICS FOR HIV VIRAL LOAD TESTING AND INFANT DIAGNOSIS Treatment failure monitoring Monitoring people receiving antiretroviral therapy is important to ensure successful treatment, identify adherence problems and determine whether antiretroviral therapy regimens should be switched in case of treatment failure. In 2013, WHO recommended viral load testing as the preferred monitoring approach to diagnose and confirm antiretroviral therapy failure (1) . Compared with clinical or immunological monitoring, viral load provides an early and more accurate indication of treatment failure. Measuring viral load can help to distinguish between drug resistance and non-adherence when coupled with robust enhanced adherence counselling. Further, viral load can serve as a proxy measure for the risk of transmission and effectiveness of prevention interventions at both the individual and population levels. Updated 2016 WHO guidelines recommend routine viral load monitoring be carried out at 6 months, 12 months after initiation of antiretroviral therapy and then every 12 months thereafter if the person is stable on antiretroviral therapy (2) . If viral load is not routinely available, CD4 count and clinical monitoring should be used to assess treatment failure. Further, dried blood spot specimens using venous or capillary whole blood can be used to determine the HIV viral load. A threshold of 1000 copies/mL should be used to determine treatment failure when using dried blood spot specimens, as similarly defined for testing using plasma. Treatment failure is defined by a persistently detectable viral load exceeding 1000 copies/mL (2) : that is, two consecutive viral load measurements within a three-month interval with adherence support between measurements after at least six months of starting a new antiretroviral therapy regimen (Box 1). In addition, viral load may support differentiated service delivery strategies for people living with HIV, including those who are stable on antiretroviral therapy (2) . Stable individuals are defined as those who have received antiretroviral therapy for at least one year and have no adverse drug reactions that require regular monitoring, no current illnesses or pregnancy, are not currently breastfeeding and have good understanding of lifelong adherence and evidence of treatment success (two consecutive viral load measurements below 1000 copies/mL). The package of care for stable individuals can include less frequent clinic visits and medication pickup, community-based care and cessation of CD4 count monitoring if viral load testing is available. Many national guidelines now recommend and are scaling up access to viral load testing for treatment monitoring (Fig. 1). The proportion of yearly viral load tests performed has increased significantly since 2013 (Fig. 2) (5). About 15 million viral load tests were conducted in 2017, and projections suggest that nearly 29 million tests may be performed in 2022. Despite increasing volumes, the total coverage of the demand of viral load testing remained below 60% in 2017. As national viral load test volumes are large and continue to grow, this will add significantly more costs to national testing budgets. Fortunately, several recent pricing commitments have been negotiated to support viral load testing expansion and access (6–8) . Numerous technologies, both laboratory-based and near- point-of-care assays, currently exist to support the scaling up of viral load testing and infant diagnosis. Several additional technologies are being developed (9,10). Box 1. Assessing advanced HIV disease Since CD4 count is the best predictor of disease status and immediate risk of death, it should be used to identify people with advanced HIV disease. Everyone entering or re-entering care should receive a CD4 test at treatment baseline and as clinically indicated for people who are clinically unstable or have symptoms of advanced HIV disease (2,3). Further, it is strongly recommended that people with advanced HIV disease (CD4 count below 200 cells/mm3 or WHO stage 3 or 4) receive a package of care (4). 5HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 0 1,700 3,400850 Kilometers National policy on routine viral load testing for monitoring ART and level of implementation for adults and adolescents in low- and middle-income countries (situation as of July 2019) Fully implemented Partially implemented Not implemented Targeted viral load testing only No policy on viral load testing Data not reported Fast-Track countries High-income countries Not applicable Source: Global AIDS Monitoring (UNAIDS/WHO/UNICEF) and WHO HIV Country Intelligence Tool, 2019 Fig. 1. National policy on routine viral load testing for monitoring ART and level of implementation for adults and adolescents in low- and middle-income countries (situation s of July 2019) Fig. 2. Estimated viral load forecast in low- and middle-income countries globally Source: 2018 CHAI HIV Market Report. 14.7M 17.3M 20.9M 23.6M 26.4M 28.5M 10.2M 9.6M 7.7M 6.7M 5.M 3.9M 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0 M 5 M 10 M 15 M 20 M 25 M 30 M 35 M 2017 2018 2019 2020 2021 2022 Co ve ra ge VL T es ts Viral Load Demand Forecast Forecasted Demand Unmet Need Coverage 66 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Infant diagnosis Infant diagnosis consists of testing throughout the exposure period of HIV-exposed infants. Depending on the age, this can comprise either nucleic acid–based testing or serological testing. More specifically, early infant diagnosis refers specifically to nucleic acid-based testing of infants within two months of birth. See Annex 1 for the infant diagnosis algorithm. Coverage of early infant diagnosis (testing within two months of birth) has remained stagnant in recent years, with about 51% of HIV-exposed infants receiving a nucleic acid test within the first two months of life in 2018 (11). The proportions of HIV-exposed infants tested at nine months or at the end of the exposure period have been difficult to gather. Current forecasts for nucleic acid testing suggest moderate growth and sustained volumes through 2022 (Fig. 3) (5). About 1.4 million infant nucleic acid tests were performed in 2017, with more than 2 million projected to be needed for 2022. Since 2010, several key recommendations have been made to support access to and expanded scale-up of infant diagnosis (2,12). • An indeterminate range should be used to improve the accuracy of all nucleic acid–based infant diagnosis assays (strong recommendation, moderate-quality evidence). • Among infants with an initial positive nucleic acid test result, it is strongly recommended that antiretroviral therapy be started without delay and, at the same time, a second specimen be collected to confirm the initial positive test (strong recommendation, low-quality evidence). • It is strongly recommended that children (18 months or older) with suspected HIV infection or HIV exposure have HIV serological testing performed according to the standard diagnostic HIV algorithm used for adults to determine final diagnosis (strong recommendation, high-quality evidence). • In generalized epidemic settings, infants and children with unknown HIV status who are admitted for inpatient care or attending malnutrition or TB clinics should be routinely tested for HIV (strong recommendation, low- quality evidence). • In generalized epidemic settings, infants and children with unknown HIV status should be offered HIV testing in outpatient or immunization clinics (conditional recommendation, low-quality evidence). • Nucleic acid–testing technologies that are developed and validated for use at or near to the point of care can be used for infant HIV testing (conditional recommendation, low-quality evidence). • Addition of nucleic acid testing at birth to existing infant diagnosis approaches can be considered to identify HIV infection among HIV-exposed infants (conditional recommendation, low-quality evidence). • Consideration should be given to replace serological testing at nine months of age with nucleic acid–based testing. Fig. 3. Estimated infant diagnosis forecast in low- and middle-income countries globally Source: 2018 CHAI HIV Market Report. 1.4M 1.6M 1.7M 1.9M 2.M 2.2M 922K 921K 912K 875K 750K 541K 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 0.0 M 0.5 M 1.0 M 1.5 M 2.0 M 2.5 M 3.0 M 2017 2018 2019 2020 2021 2022 Co ve ra ge EI D T es ts R un EID Demand Forecast Forecasted Demand Unmet Need Coverage 7HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 2. USING VIRAL LOAD TEST RESULTS TO SUPPORT THE CLINICAL MANAGEMENT OF PEOPLE LIVING WITH HIV RECEIVING ANTIRETROVIRAL THERAPY The 2016 WHO consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection (2) provided a strong recommendation for using viral load testing routinely as the preferred antiretroviral drug monitoring tool. WHO recommends viral load testing at six months after initiating antiretroviral therapy, at 12 months and then annually thereafter to enable early detection of treatment failure, prevent drug resistance, identify people with high viral loads with poor adherence and avoid inappropriate switching of treatment regimens (2). In 2014, UNAIDS launched the 90–90–90 treatment targets to be accomplished by 2020, aimed at helping to end the AIDS epidemic as a public health threat. The third 90 aims to ensure that 90% of the people receiving antiretroviral therapy have suppressed viral loads (13). Antiretroviral therapy and treatment adherence provides remarkable and sustained clinical benefits, even among people with advanced HIV disease. Evidence from national viral load dashboards and the population-based HIV impact assessment (14) results suggests that the suppression rates among people living with HIV on antiretroviral therapy are generally 85–92% (Fig. 4). It is important to understand viral suppression rates at the population level to identify potential hot-spots of Fig. 4. Viral load suppression rates in selected countries Suppressed Non-suppressed Lesotho Malawi Swaziland United Republic of Tanzania Zambia Zimbabwe Kenya Uganda 88.3% 90.8% 91.9% 87.7% 89.2% 86.5% 86.4% 92.2% Population based HIV impact assessments, 2015-2017 National viral load dashboards: 2017 85.0% South Africa Namibia 91.3% Cameroon 80.0% Côte d’Ivoire 75.9% 88 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis transmission, inform national targets and provide targeted programmatic quality improvement efforts, but perhaps more importantly, at the patient level to provide enhanced care and support to people living with HIV. Clinically stable people with undetectable viral loads can be provided with differentiated service delivery options that reduce clinic visits and allow for three- or six-monthly drug prescriptions. Further, viral load testing is critical to ensure that those with detectable viral loads greater than 1000 copies/mL are provided enhanced adherence counselling and more closely monitored to determine whether they need to switch to second-line treatment (Fig. 5). The treatment monitoring algorithm is meant to support clinicians and patients in determining whether elevated viral loads or suspicion of treatment failure is caused by drug resistance or poor adherence. It is undesirable for both people living with HIV and programmes to unnecessarily switch people to more expensive and less-well-tolerated second-line regimens when they are simply non-adherent, primarily because adherence issues will not necessarily improve upon switching. However, continuing on a failing drug regimen when the root cause is drug resistance can lead to further drug resistance, additional immune deterioration and possibly clinical effects. Are viral load test results being used to make clinical decisions? Viral load testing has been significantly scaled up in recent years, from 7 million tests in 2013 to 15 million tests in 2017. However, performing viral load tests should not be the main consideration of viral load programmes. Programmes may want to also focus on how viral load test results are used to inform clinical decision-making. Médecins Sans Frontières carried out an in-depth analysis of the execution of key steps in the viral load treatment monitoring algorithm across six countries and 149 clinical sites supported by their programmes (15) . Among people with an elevated initial viral load (18% mean), an average of 68% received at least one enhanced adherence counselling session, 52% received a second follow-up viral load test, 34% re-suppressed (<1000 copies/mL) and 33% of those eligible switched to second-line treatment. These results are further supported by a preliminary analysis of publicly available data published on national dashboards in three countries in eastern Africa (16). Despite Fig. 5. WHO treatment failure monitoring algorithm 9HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis an increase in viral load testing coverage and encouraging viral suppression proportions, less than 10% of the people with an elevated first viral load went through the viral load algorithm to receive a second follow-up viral load test to determine the need for switching to a second-line regimen (Fig. 6). This trend has remained consistent across years. Key considerations Diagnostic tests are not of significant value unless the test results are used clinically. The laboratory–clinical interface may be the most difficult yet the most critical and rewarding mechanism for improving patient management. To create effective health services that provide optimal care and treatment to people living with HIV, programmes must revitalize and invest in the laboratory–clinical interface and ensure that the right training, tools and environment are available to improve the uptake and use of all diagnostic results in a timely manner. Several tools currently exist to better support the clinical uptake of viral load test results and could be adapted and adopted by national programmes (17–19) . Scaling up successful viral load programmes requires using all test results and integrating them into clinical services to optimize patient care and programmatic success. Fig. 6. Viral load tests conducted in three countries in eastern Africa, 2012–2016 VL VL>1000 2nd viral load 1 500 000 1 000 000 500 000 N um be r o f v ira l l oa d te st s 0 2012 2013 2014 2015 2016 10 10 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 3. ESTIMATED REACH OF AND ACCESS TO VIRAL LOAD TESTING USING TRADITIONAL PLASMA SPECIMENS The 2016 WHO consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection (2) recommend viral load as the preferred monitoring approach to diagnose and confirm treatment failure and plasma specimens as the preferred specimen type for viral load testing. Although significant scale-up has occurred across resource-limited countries with a high burden of HIV infection since the initial recommendation in 2013, several significant barriers have limited full access. In particular, the use of liquid plasma using EDTA tubes (see Section 4) can be limited because of strict specimen storage stability times and temperatures, within which the specimens would need to be transported to the testing laboratory or an intermediary hub for processing. Further, there is limited cold-chain availability between many health-care facilities and testing sites in resource-limited settings. However, traditional EDTA plasma specimens have significant potential for viral load testing. Even within the specimen storage stability times and temperatures, many people still have access to viral load using this type of specimen. An analysis was recently conducted to better understand the radius around testing laboratories or intermediary hubs within which people may access to viral load testing using traditional EDTA plasma specimens. The analysis was conducted across four countries: Eswatini, Nigeria, Rwanda and Zimbabwe. Several assumptions were included, such as: • vehicles travelling at 50 km/h; • straight-line measurements from the health-care facility to the testing laboratory or intermediary hub plus a 17% circuity factor; • testing laboratories or intermediary hubs considered as the final point for separating plasma; • the last specimen collected each day had a maximum wait of two hours at the health-care facility before pick-up and transport to reach the testing laboratory or intermediary hub within the stipulated manufacturer storage stability time; and • this analysis does not incorporate alternative plasma specimen types or consider on-site centrifugation and associated specimen storage and transport. Illustrative plasma radius analysis Across the four countries, just under half of all health- care facilities are near enough to the testing laboratory or intermediary hub to transport traditional EDTA plasma specimens within the time stipulated by the manufacturer. This translates to more than 50% or nearly 1 million people Table 1. Access to viral load testing using traditional EDTA plasma Country Access to viral load testing using traditional EDTA plasma Facilities People Eswatini 260/350 (74%) 250 000/320 000 (78%) Nigeria 750/2 600 (29%) 450 000/1 200 000 (38%) Rwanda 505/550 (92%) 148 000/165 000 (90%) Zimbabwe 700/1 500 (47%) 120 000/190 000 (63%) Total 2 215/5 000 (44%) 968 000/1 875 000 (52%) 11HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis across the four countries analysed having access to viral load testing using traditional EDTA plasma. Even in such a geographically large country as Nigeria, nearly 40% of the people receiving antiretroviral therapy and needing viral load testing would have access using traditional EDTA plasma. Geographically smaller countries, such as Eswatini and Rwanda, may have fewer laboratory facilities but can provide access to viral load testing using traditional EDTA plasma specimens to nearly 80% or more of the people accessing antiretroviral therapy. The high access of viral load testing using traditional EDTA plasma is likely because most viral load testing laboratories are in major urban centres. Likewise, the largest antiretroviral therapy centres where people seek care are often also located in major urban centres. This analysis highlights the link in which more than 50% of the people receiving antiretroviral therapy who need viral load testing are within a specimen transport time of a few hours from the testing laboratory or intermediary hub (Fig. 7). Fig. 7. Illustrative example of plasma access radius around testing laboratories in Zimbabwe Conclusions This illustrative analysis provides a snapshot highlighting the potential access to viral load testing using the preferred plasma specimen. The proportion of people who can access viral load testing using traditional EDTA plasma specimens can vary across settings, depending on several factors including the number of laboratories, road infrastructure and size of the country. Efforts should be made and maximized to ensure access to viral load testing using the preferred plasma specimen. It might be helpful to conduct similar in-depth analyses across countries to determine the facilities and people who may be able to access viral load testing using traditional plasma specimens. Current infrastructure may not always enable the use of traditional EDTA plasma in many settings because of poor roads and infrastructure, large distances, ad hoc specimen transport, etc. Therefore, for the facilities and people without access to viral load testing using traditional EDTA plasma, alternatives could be considered to ensure viral load access, including improved infrastructure, specimen transport networks and alternative specimen types and technologies. This molecular diagnostics toolkit will provide background information and data on several of these alternative strategies to ensure a complementary approach to expanding access. 12 12 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 4. SPECIMEN STABILITY FOR HIV VIRAL LOAD TESTING The 2016 WHO consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection (2) recommend viral load as the preferred monitoring approach to diagnose and confirm treatment failure and plasma specimens as the preferred specimen type for viral load testing. Although significant scale-up has occurred across resource-limited countries with a high burden of HIV infection since the initial recommendation in 2013, several significant barriers have limited full access to viral load testing. In particular, using liquid plasma and using ethylenediaminetetraacetic acid (EDTA) or plasma preparation tubes (see subsection 6.1) can be limited because of strict specimen storage stability times, within which the specimens would need to be transported to the testing facility or an intermediary hub for processing. Highlighted in Table 2, these are the maximum times according to the storage temperature stipulated by the manufacturers from whole-blood specimen collection to plasma separation. Extending storage times before processing beyond these recommendations could affect performance and risks providing incorrect results to clinicians and patients. Table 2. Manufacturer-stated whole-blood stability details Assay Maximum time from whole-blood specimen collection to plasma separation Room temperature (temperature) Refrigeration (temperature) Abbott RealTime HIV-1 (20,21) 24 hours (15–30°C) 48 hours (2–8°C) Abbott m-PIMA HIV-1/2 VL (22,23) 48 hours (18–28°C) NR Biocentric Generic HIV Charge Virale (24) 24 hours (2–25°C) 24 hours (2–25°C) bioMérieux NucliSENS EasyQ® HIV-1 (25,26) NR 24 hours (2–8°C) Cavidi ExaVir Load (27) 4–6 hours (no temperature specified) Cepheid Xpert HIV-1 Viral Load (28,29) 8 hours (15–30°C) 72 hours (2–8°C) Hologic Aptima HIV-1 Quant Dx (30,31) 24 hours (2–30°C) 24 hours (2–30°C) Qiagen artus HI Virus-1 RG (32) 6 hours (no temperature specified) Qiagen artus HI Virus-1 QS-RGQ (33) 6 hours (no temperature specified) Roche COBAS TaqMan HIV-1 (34,35) 24 hours (2–25°C) 24 hours (2–25°C) Roche cobas HIV-1 for cobas 4800 System (36) 24 hours (2–25°C) 24 hours (2–25°C) Roche cobas HIV-1 for cobas 6800/8800 Systems (37) 24 hours (2–25°C) 24 hours (2–25°C) Sacace HIV Real-TM Quant Dx (38) NR 12 hours (2–8°C) Siemens VERSANT HIV-1 RNA 1.5 (39) 6 hours (15–25°C) 24 hours (2–8°C) © W H O NR: not reported. 13HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Once whole-blood specimens are separated into plasma, they can be frozen for long periods of time before testing. However, health-care facilities may lack centrifuges, freezers and/or the associated necessary skills to fully leverage these extended stability times after plasma separation. Whole-blood stability for HIV viral load systematic review A systematic review of nine studies entitled “Expanding access to HIV viral load testing: RNA stability in EDTA tubes and plasma preparation tubes beyond current time and temperature thresholds” was published in 2014 (40) .The systematic review highlighted three key findings. • Whole blood and plasma were stable up to 168 hours after specimen collection when refrigerated. • Whole blood was stable up to 72 hours after specimen collection when stored at 25°C. • Plasma was stable up to 48 hours after specimen collection (plasma preparation tubes) or plasma separation (EDTA) when stored at 25°C. Some important limitations to be considered, however, are that all studies included laboratory analyses rather than active realistic storage and transport times and temperatures and all were conducted in the United States or Europe. Further, only a few relevant studies were available for inclusion, and most had small sample sizes. In addition, few studies included samples that had suppressed viral loads (<1000 copies/mL), making the results difficult to interpret within this range. However, a recently published study observed elevated viral load results of undetectable viral load specimens when plasma was stored beyond 72 hours (41) . Interestingly, 20% of the undetectable viral load results became low-level viraemic at any room temperature or refrigeration. Further, 51% of undetectable specimens became low-level viraemic if the plasma was not centrifuged again before testing after 48 hours of storage. Conclusions Fortunately, since the systematic review was published, several manufacturers have now lengthened their room temperature stability intended claims to allow for 24 hours from whole-blood specimen collection to plasma separation. Although the systematic review suggests that specimens are stable beyond the manufacturer intended claims, countries and laboratories would be responsible for viral load test results under such off-label conditions. Additional research and manufacturer support to extending whole-blood stability intended use claims should be encouraged since broader specimen stability would support the expansion of viral load access using the preferred specimen type: plasma. © W H O 14 14 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 5. TECHNICAL BACKGROUND: NUCLEIC ACID– BASED TESTING What is viral load testing? HIV viral load testing is a way to measure the number of viruses present in a blood sample. Whole blood consists of cellular components (white blood cells, red blood cells and platelets) and cell-free plasma. Nucleic acid–based testing is done using a nucleic acid amplification test, which determines the number of copies of HIV per millilitre of plasma. Nucleic acid amplification tests work by amplifying either HIV genetic material or a probe that binds to HIV (42). The test then uses a chemical reaction to measure the amount of amplification seen during the test, which corresponds to the quantity of HIV present in the sample. The most common type of viral load test is a quantitative polymerase chain reaction (qPCR). Other types of viral load testing include transcription-mediated amplification and branched DNA testing (2). Usefulness of viral load testing • HIV viral load monitoring is important to ensure successful antiretroviral therapy. Viral load monitoring is the preferred approach to diagnose and confirm treatment failure (2) . • Viral load testing provides clients with knowledge, control and motivation to understand their HIV infection and adhere to their treatment (43). • Having low or undetectable viral loads reduce the risk of disease progression and HIV transmission (44,45). © W H O 15HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 3. HIV DNA and RNA in whole blood Blood: cellular portion Blood: plasma portion HIV DNA and RNA White blood cells (such as CD4 cells): HIV DNA is contained inside the cells along with copies of HIV that contain HIV RNA during HIV replication. HIV has also been found to be associated with platelets, most likely on the cell surface, although the virus is not found inside platelets or red blood cells. HIV RNA is found in free virus in plasma. HIV DNA should not be present in any significant proportions, although small amounts of DNA may be found in plasma from cells that have broken open or if cells have carried over into the plasma from insufficient separation of whole blood. Sample type Whole blood contains both the cellular component of blood as well as the plasma. Whole blood contains both HIV DNA, intracellular RNA and cell-free RNA and has been used for early infant diagnosis of HIV and HIV drug resistance testing. Plasma is the preferred sample type for viral load testing, which aims to detect the number of copies of HIV RNA per millilitre of plasma. Plasma can also be used for HIV drug resistance testing if adequate HIV RNA (>400 copies/mL) is present. Testing methods Whole blood is tested either in liquid form or from a dried blood spot. Viral load testing using whole blood may be inaccurate if significant quantities of HIV DNA and/ or intracellular RNA are detected by the assay in addition to the cell-free (plasma) RNA the assay is designed to detect. Plasma is typically tested in liquid form but can also be tested from a dried plasma spot. HIV DNA versus HIV RNA HIV is an RNA virus comprising RNA and proteins. During its replication cycle, the genetic material of HIV exists in both RNA and DNA forms. HIV DNA is the genetic material of HIV that is found inside cells of the body infected by HIV. In whole blood, HIV DNA is mostly found inside white blood cells called CD4 cells, which are an important part of the immune system. HIV integrates its DNA into the DNA of the CD4 cells so it can use the cells to make more copies of itself. In this form, it is known as HIV proviral DNA (46–48). HIV RNA is most commonly found in plasma, which is the part of whole blood after removing all of the cells. Whole blood is typically separated into plasma and its cellular components by centrifuging the blood. HIV exists as an RNA virus in plasma before it infects cells, as intracellular RNA inside cells as copies of the virus are being made and in plasma again once these viral copies are released (6–8). When HIV is suppressed by antiretroviral therapy, HIV DNA remains present inside cells and occasionally as intracellular RNA, but little to no HIV RNA can be detected in plasma since the medicines prevent viral replication. However, when HIV is not suppressed, most of the HIV nucleic acid is typically present as HIV RNA in plasma, with additional intracellular RNA from active viral replication, and a smaller proportion present as HIV DNA inside cells (7). How does viral load testing work? Nucleic acid testing for HIV can detect both HIV DNA and RNA that are present in a sample. Some assays have been designed to preferentially detect DNA or RNA, but since HIV DNA and RNA are copies of the same genetic material, they can also be hard to distinguish. However, viral load testing is designed to measure the amount of HIV RNA in plasma. Plasma is therefore the preferred sample type for viral load testing; however, alternative specimen types and technologies exist to support expanded access to viral load testing, including dried blood spots prepared using whole blood (2,49). Dried blood spot specimens can allow for longer transport and storage times; however, using whole blood results often in detecting HIV proviral DNA, intracellular RNA and cell-free RNA. Together, this can result in excessive quantification of viral load results. Table 3 explains the differences between the two major components of whole blood (DNA and RNA) in HIV viral load testing. © W H O 16 16 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Box 2. Timing versus technologies for diagnosing HIV among infants The nucleic acid–based technique (qPCR) used for viral load testing is very similar, often the same, for infant testing or qualitative assays. HIV “DNA PCR” is a commonly used synonym for HIV infant diagnosis testing. However, it is important to distinguish between the technology used for testing (such as PCR versus an HIV antibody test) and the time frame of testing. Early infant diagnosis specifically refers to nucleic acid–based testing at birth or in the first two months of life, whereas infant diagnosis refers to testing during the exposure period including the nine-month nucleic acid test. Infant diagnosis is often done on whole blood, either in liquid form or on a dried blood spot. These assays can detect HIV DNA, intracellular RNA and cell-free RNA. This is not a problem and will even improve the sensitivity of the assay, since the presence of any HIV genetic material in the blood can indicate HIV infection. Since both HIV DNA and RNA are present, virological testing or HIV nucleic acid amplification testing are more accurate terms for infant PCR testing than HIV DNA PCR. Box 3. Key viral load terms Suppressed viral load: viral load measurements below 1000 copies/mL. An unsuppressed or elevated viral load is a measurement above 1000 copies/mL (2). Undetectable viral load: the absence of any HIV found in a blood specimen by viral load testing. Table 4 shows the detection limits of commercially available viral load assays. © W H O 17HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 4. Summary of HIV viral load assays Manufacturer and test name Sample type Limit of detection (copies/mL) Maximum time from whole-blood specimen collection to plasma separation Regulatory approval Early infant diagnosis testing Abbott: RealTime HIV-1 (20,21)1,2 m-PIMATM HIV-1/2 VL (22,23) Plasma DBS Plasma 40 839 800 24 h at 15–30°C, 48 h at 2–8°C 48 h at 18–28°C CE, FDA, WHO CE, WHO CE, WHO Available, separate test Available, separate test Biocentric GENERIC HIV Charge Virale (24) Plasma 390 24 h at 2–25°C CE Available, separate test bioMérieux NucliSENS EasyQ® HIV-1 v2.0 (25,26) Plasma DBS 25 802 24 h at 2–8°C CE, WHO CE, WHO N/A Cavidi ExaVirTM Load (27) Plasma 200 4–6 h, no temperature specified CE N/A Cepheid Xpert® HIV-1 Viral Load (28,29) Plasma 40 8 h at 15–30°C, 24 h at 15–25°C, 72 h at 2–8°C CE, WHO Available, separate test Hologic Aptima™ HIV-1 Quant Dx (30,31) Plasma 30 24 h at 2–30°C CE, FDA, WHO Same Test Qiagen: artus® HI Virus-1 RG (32) artus® HI Virus-1 QS-RGQ (33) Plasma Plasma 60 45 6 h, no temperature specified 6 h, no temperature specified CE CE N/A N/A Roche: COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0 (34,35) cobas® HIV-1 for cobas® 4800 System (36) cobas® HIV-1 for cobas® 6800/8800 Systems (37) Plasma PSC Plasma PSC Plasma PSC 20 738 20 599 13.2 790 24 h at 2–25°C 24 h at 2–25°C 24h at 2-25°C CE, FDA, WHO CE, WHO CE CE, FDA CE Available, Separate Test Same Test Available, Separate Test Sacace HIV Real-TM Quant DX (38) Plasma 48 IU/mL 12 h at 2–8°C CE N/A Siemens VERSANT® HIV-1 RNA 1.5 (39) Plasma 37 6 h at 15–25°C, 24 h at 2–8°C CE N/A 1 Abbott Laboratories (2014). Abbott RealTime HIV-1 Instructions for Use. 2 WHO Prequalification of Diagnostics Programme (2016). Public Report: Abbott RealTime HIV-1. Available at https://www.who.int/diagnostics_laboratory/evaluations/pq-list/hiv- vrl/180423_amended_final_pqpr_0145_027_00_v11.pdf?ua=1 © W H O h: hours; CE: Conformité Européenne, conforming to European Union regulations; FDA: United States Food and Drug Administration approval; WHO: WHO prequalification of in vitro diagnostics; DBS: dried blood spot; PSC: dried plasma spot from a plasma separation card; N/A: not currently available; IU: international units. 18 18 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 6. ALTERNATIVE SPECIMEN TYPES AND TECHNOLOGIES FOR CONSIDERATION WHEN LIQUID PLASMA CANNOT BE USED WIDELY FOR VIRAL LOAD TESTING BECAUSE OF INFRASTRUCTURE, TRANSPORT OR OTHER CONSTRAINTS 6.1 ALTERNATIVE SPECIMEN TYPES AND TECHNOLOGIES: DRIED BLOOD SPOT SPECIMENS FOR HIV VIRAL LOAD TESTING Although plasma specimens are the standard for viral load testing, their use is restricted by the limited ambient temperature stability of viral biomarkers in whole blood and plasma during storage and transport and the limited cold-chain availability between many health-care facilities in resource-limited settings. Dried blood spot specimens for HIV testing are well established in resource-limited settings and have been routinely used for collecting and shipping infant HIV diagnosis specimens for testing by PCR in centralized laboratories. They are beneficial since they do not require centrifuges, refrigerators or freezers at the specimen collection site, can be stored and transported for weeks at ambient temperature and require a simple finger-prick or heel-stick blood specimen that can be prepared by lower cadres of health-care facility staff. Similar benefits could be achieved by using dried blood spot specimens for viral load testing programmes in resource-limited settings. The required storage and shipping conditions may differ when dried blood spot specimens are used for drug resistance testing. Dried blood spot specimens for viral load testing using nucleic acid–based detection methods use whole blood as the input specimen, which can result in extraction and © W H O 19HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 5. Summarized results from technical evaluation meta-analysis Assay Sample size Sensitivity (95% CI)a Specificity (95% CI)a Abbott RealTime HIV-1, one-spotb 700 88.26% (49.64–98.28) 99.07% (68.38–99.98) Abbott RealTime HIV-1, two-spot 2004 93.13% (83.72–97.27) 91.11% (82.35–95.75) Biocentric Generic HIV Charge Virale 531 94.86% (71.14–99.28) 55.16% (35.01–73.75) bioMérieux NucliSENS EasyQ® HIV-1 1062 82.95% (78.38–86.71) 95.06% (89.29–97.80) Hologic Aptima 382 87.52% (77.93–93.30) 87.18% (59.01–96.98) Roche COBAS TaqMan HIV-1 Free Virus Elution 3076 94.77% (84.59–98.36) 93.93% (71.95–98.94) Roche COBAS TaqMan HIV-1 SPEX 3190 98.23% (95.85–99.26) 48.49% (22.63–75.18) Siemens VERSANT HIV-1 RNA 144 90.97% (69.20–97.83) 87.76% (75.28–94.41) a Sensitivity and specificity using a treatment failure threshold of 1000 copies/mL. b As a change notification, a laboratory evaluation of dried blood spot specimens using the CE-marked protocol was not conducted within WHO prequalification review. detection of proviral DNA and intracellular RNA in addition to the primary biomarker target of free viral RNA circulating in the plasma. Together, this may result in excessive quantification of the viral load result. Limited progress has been made in ensuring the quality of using dried blood spot specimens for HIV viral load testing through international regulatory approval. Dried blood spot specimen regulatory approvals and technical evaluations (countries often consider these approvals when procuring or selecting diagnostic technologies): • CE-IVD (Conformité Européenne in vitro diagnostics): two technologies have received CE- IVD for using dried blood spot specimens for viral load testing: Abbott RealTime HIV-1 and bioMérieux NucliSENS EasyQ® HIV-1; and • WHO prequalification: two technologies have met WHO requirements: bioMérieux NucliSENS EasyQ® HIV-1 in January 2017 and Abbott RealTime HIV-1 (21) on 24 August 2017. The limit of detection of the Abbott RealTime HIV-1 assay using dried blood spot specimens is 839 copies/mL (21) . Independent technical evaluations: the results from 40 technical evaluations of dried blood spot specimens across over 25 countries examining six commercially available viral load testing technologies were included in a comprehensive clinical meta-analysis, which resulted in more than 10 000 paired dried blood spot–plasma data points (Table 5) (50). WHO recommendations The 2016 WHO consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection (2) recommend that dried blood spot specimens using venous or capillary whole blood can be used to determine the HIV viral load. A threshold of 1000 copies/mL should be used to determine treatment failure when using dried blood spot specimens, as defined for testing in plasma. Although plasma specimens are preferred for viral load testing, dried blood spot specimens are recommended for use in settings where logistical, infrastructural or operational barriers prevent routine viral load monitoring using plasma specimens. Current use Dried blood spot specimens provide a way to improve the coverage and reach of viral load testing where the preparation and transport of plasma specimens may be limited by cold-chain requirements or transport challenges. Several countries are currently implementing dried blood spot specimens to support viral load access and scale-up. In 2018, more than 2 million viral load tests were run using dried blood spot specimens across six countries with a high burden of HIV infection. Further, some countries have begun implementing the use of DBS specimens for viral load testing using protocols recommended by manufacturers despite its off-label use. © W H O 20 20 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Conclusions Sufficient evidence has been generated on the performance of dried blood spot specimens for viral load testing to support rapid national regulatory approval and initiation of scale-up. Further technical evaluations of these technologies are unlikely to add value but may instead delay implementation and timely treatment monitoring. However, it is essential that suppliers seek regulatory approval and WHO prequalification of such alternative specimen types to support country scale-up and access to viral load testing. 6.2 ALTERNATIVE SPECIMEN TYPES AND TECHNOLOGIES: DRIED PLASMA SPOT SPECIMENS FOR HIV VIRAL LOAD TESTING An additional alternative to using liquid plasma for viral load testing is dried plasma spot specimens. These specimens use the same or similar filter paper as dried blood spot specimens for viral load or infant diagnosis; however, with the application of plasma instead of whole blood. Plasma separation cards and simple devices are also currently in development or recently available on the market to support the expansion of viral load testing using plasma specimens. Dried plasma spot specimens for HIV testing are an alternative specimen type developed similarly to the well- established dried blood spot specimens (subsection 6.1) that have been routinely used for collecting and shipping infant HIV diagnosis specimens for testing by PCR in centralized laboratories. Although they require centrifugation or collection of plasma for spotting on the card, they can be stored and transported for weeks at ambient temperature. An advantage of dried plasma spot specimens is that plasma separation and use removes the detection and quantification of intracellular RNA and proviral DNA often observed with whole-blood specimens; however, the smaller input specimen volume may limit the perfect comparability with liquid plasma specimens. Table 6. Summarized results from technical evaluation meta-analysis Assay Sample size Sensitivity (95% CI)a Specificity (95% CI)a All technologies 1872 92.54% (87.85–95.52%) 95.15% (87.41–98.23%) Abbott RealTime HIV-1 245 99.39% (95.78–99.91%) 85.37% (75.97–91.50%) Biocentric Generic HIV Charge Virale 148 98.12% (56.78–99.95%) 75.00% (46.90–91.06%) bioMérieux NucliSENS EasyQ® HIV-1 173 77.78% (53.53–91.40%) 99.35% (95.57–99.91%) Roche COBAS TaqMan HIV-1 1077 93.05% (87.75–96.16%) 94.90% (78.59–98.95%) a Sensitivity and specificity using a treatment failure threshold of 1000 copies/mL. Typically, plasma prepared for dried plasma spot specimens or plasma separation cards or devices is derived from whole blood taken in EDTA tubes or plasma preparation tubes (see subsection 6.3). Manufacturers should, therefore, include one or both tubes types in their intended use claims and regulatory approval documentation. Most viral load assays currently on the market include one or both tube types. Independent technical evaluations: the results from 17 technical evaluations across 12 countries and looking at four commercially available technologies were included in a comprehensive meta-analysis, which resulted in nearly 2000 paired dried plasma spot–plasma data points (Table 6) (50). The performance of dried plasma spot specimens across all technologies was comparable to using traditional liquid plasma. As expected, since the input specimen type, plasma, was used, limited upward and downward misclassification was observed. WHO recommendations The 2016 WHO consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection (2) recommend viral load as the preferred monitoring approach to diagnose and confirm treatment failure and prefer plasma specimens for viral load testing. A threshold of 1000 copies/mL can be used to determine treatment failure when using any specimens, including dried plasma spot specimens, as defined for testing in plasma. Current use Dried plasma spot specimens provide a way to improve the coverage and reach of viral load testing, where storage and transport of liquid plasma specimens may be limited by cold-chain requirements or transport challenges. However, preparation of dried plasma spot specimens requires centrifugation to separate plasma from whole blood. This can be done either at the point of specimen collection, if feasible, or within a few hours of specimen collection, depending on manufacturer guidelines, by a hub or regional laboratory. 21HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Conclusions Sufficient evidence has been generated on the performance of dried plasma spot specimens for viral load testing to support the initiation of scale-up, if desired within national operational plans to support country scale-up and access to viral load testing. Further technical evaluations of these technologies are unlikely to add value but may instead delay implementation and timely treatment monitoring. However, information focusing on the feasibility and operational best practices of using dried plasma spot specimens within viral load scale-up plans has been limited. 6.3 ALTERNATIVE SPECIMEN TYPES AND TECHNOLOGIES: PLASMA PREPARATION TUBES FOR HIV VIRAL LOAD TESTING The gold standard plasma specimen for viral load testing is generally collected using whole blood in an EDTA (ethylenediaminetetraacetic acid anti-coagulant) tube (purple or lavender cap). As highlighted in Section 4, whole blood in EDTA tubes must be transported and plasma separated within 6–24 hours, depending on the manufacturer. This can be restrictive for many countries and health-care facilities. However, some alternative plasma specimens can be considered. Plasma preparation tubes as well as plasma collected on cards, such as dried plasma spots (subsection 6.2) and plasma separation cards can also be considered to support scale-up. Unlike standard EDTA blood collection tubes, plasma preparation tubes can facilitate simpler handling and storage of plasma for nucleic acid–based testing. Plasma preparation tubes use the same EDTA anticoagulant but contain a gel that separates the plasma from blood cells after centrifugation. After the blood is collected, the plasma preparation tube is spun in a centrifuge within 24 hours and a gel barrier inside the plasma preparation tube separates the plasma from the rest of the whole blood so the plasma can be used for HIV viral load testing. The same plasma specimen volume is used for the viral load assay; therefore, limits of detection are generally synonymous with EDTA plasma. Plasma preparation tube regulatory approvals: • CE-IVD (Conformité Européenne in vitro diagnostics): seven technologies have received CE-IVD for using plasma preparation tubes for viral load testing: Abbott RealTime HIV-1, Cepheid Xpert® HIV-1 Viral Load, Hologic AptimaTM HIV-1 Quant Dx, Roche COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0, Roche cobas® HIV-1 for cobas® 4800, Roche cobas® HIV-1 for cobas® 6800/8800 and Siemens VERSANT® HIV-1 RNA 1.5. • WHO prequalification: four technologies have met WHO requirements: Abbott RealTime HIV-1, Cepheid Xpert® HIV-1 Viral Load, Hologic AptimaTM HIV-1 Quant Dx and Roche COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0. • FDA (United States Food and Drug Administration): four technologies have received FDA approval for using plasma preparation tubes for viral load testing: Abbott RealTime HIV-1, Hologic AptimaTM HIV-1 Quant Dx, Roche COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0 and Roche cobas® HIV-1 for cobas® 6800/8800. Systematic review and best practices: a systematic review was conducted to examine the accuracy of plasma preparation tubes for HIV viral load testing. The review identified 16 peer-reviewed published studies from 1995 to 2014 that compared plasma preparation tubes to standard EDTA blood collection tubes on HIV viral load assays approved by a stringent regulatory authority. Although the earliest studies demonstrated that plasma preparation tubes could be used with no significant differences in viral load results (51–54) , later studies demonstrated elevated viral loads from plasma preparation tubes, especially at viral loads less than 5000 copies/mL (55–57) . The increase in viral load results likely resulted from the leakage of HIV nucleic acids, such as proviral HIV DNA and intracellular RNA present in the cellular component of whole blood, which moved back through the gel barrier into the plasma. Additional studies found that this issue could be resolved by either aliquoting the plasma into a second tube quickly after the initial centrifugation (58–60) or repeating centrifugation after transport of the plasma preparation tubes to the laboratory before aliquoting and testing (61,62). Four published studies evaluated plasma preparation tubes on currently available viral load assays (Abbott RealTime HIV-1 and Roche COBAS AmpliPREP/COBAS TaqMan HIV- 1 Test, v2.0) (63–66). The three studies using the Abbott viral load assay showed no significant change in viral load results regardless of whether the plasma preparation tubes were frozen and thawed or transported after initial centrifugation and before testing. The three studies using a Roche assay found elevated viral load results if the plasma preparation tubes were frozen or transported without a second centrifugation before testing. These viral load results were found to be between zero and several thousand copies/ mL higher than plasma prepared from a standard EDTA collection tube, with the difference being most noticeable for plasma viral loads less than 1000 copies/mL. Consequently, manufacturer instructions recommend an additional centrifugation step before testing using the Roche assay. For both the Abbott and Roche assays, aliquoting the plasma into a secondary tube after initial centrifugation also ensured accurate viral load results (Table 7). 22 22 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 7. Published handling methods for commercially available plasma preparation tubes and viral load assays Product Published plasma preparation tube handling methods providing accurate viral load results Abbott RealTime HIV- 1 (63–65) • Aliquoting plasma into new tube after initial centrifugation • Freezing plasma preparation tubes at –20°C after initial centrifugation and thawing before testing, without the necessity for another centrifugation step • Transporting plasma preparation tubes after initial centrifugation between sites before testing, without the necessity for another centrifugation step Roche COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0 (64–66) • Aliquoting plasma into new tube after initial centrifugation • Repeat centrifugation after transport or freezing of plasma preparation tubes to ensure complete the separation of the cellular and plasma components of blood before testing Note: In the absence of repeat centrifugation after freezing and thawing of plasma preparation tubes or after transport of plasma preparation tubes, some viral load results were observed to be erroneously high. Repeat centrifugation is not necessary if the plasma has already been aliquoted into a new tube before freezing or transport. BD Vacutainer® PPTTM (67) • Centrifuge for at least 10 minutes at 1100 × g at room temperature, within 6 hours of collecting whole blood to prepare plasma. • Follow assay manufacturer instructions for storage and transport: typically plasma preparation tubes can be stored at ambient temperature for one day or refrigerated at 4°C for up to five days; if longer storage is desired, the plasma should be frozen. Table 8. Advantages and challenges associated with plasma preparation tubes Advantages Challenges • Fewer manual sampling handling steps than standard EDTA tubes • Reduced risk of sample contamination and laboratory errors if plasma is not aliquoted into a new tube • Ability to store plasma for longer periods of time than uncentrifuged whole blood, which can facilitate longer transport times to the laboratory • Higher cost of plasma preparation tubes than standard EDTA tubes • Programmatic complexities involving supply chain logistics, staff training and proper implementation of plasma preparation tubes • Centrifuges are required on-site for immediate plasma separation • Primary tube sampling is not always possible • Inaccurate viral load results may be seen if manufacturer-specific instructions are not followed: for example, a repeat centrifugation step may be necessary before testing • Sample bundling currently unavailable All studies evaluated only BD Vacutainer® PPTTM. Additional plasma preparation tubes exist (also referred to as EDTA with gel separate tubes: Grenier (68) or TUD (69) ); however, no studies have been published. Further, other regulatory-approved and/or WHO-prequalified viral load assays (such as the Cepheid Xpert HIV-1 and Hologic Aptima HIV-1 Quant Dx assay) that include plasma preparation tubes in their instructions for use do not provide any further specific guidance on how they should be used (Table 8). Conclusions Plasma preparation tubes allow plasma to be prepared, stored and transported in the same tube used to collect venous whole blood. Plasma preparation tubes provide equivalent viral load results to plasma from standard EDTA tubes if their proper handling is followed according to manufacturer instructions and guidance from independently published studies. Centrifugation of plasma preparation tubes and/or aliquoting of plasma into a separate tube before viral load testing has been shown to prevent spuriously elevated viral load results. However, not all viral load assays have clear instructions or peer- reviewed evaluations published on their use of plasma preparation tubes, and centrifuges (and the associated skills) are necessary at the point of specimen collection. Plasma preparation tubes may be worth considering in settings in which simpler plasma preparation, reduced cross- contamination risk, and the need for longer sample transport times can facilitate the scaling up of viral load testing. © W H O 23HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 6.4 ALTERNATIVE SPECIMEN TYPES AND TECHNOLOGIES: POINT-OF-CARE AND NEAR-POINT-OF-CARE TOOLS FOR HIV VIRAL LOAD TESTING Technologies developed for use at or near the point of care may also be considered for viral load testing. These technologies can be decentralized and used at the point of care. Point-of-care technologies do not require consistent electricity, temperature-controlled rooms or routine calibration, are relatively easy to use, are automated, have no or minimal third-party commodity requirements and can be operated by non-laboratory professionals. Near-point- of-care technologies are similar but may require the use of consistent electricity and/or temperature-controlled rooms. Further, most technologies currently available require plasma specimens. Significant progress has been made in ensuring the quality of new point-of-care viral load technologies. Point-of-care and near-point-of-care viral load regulatory approvals and technical evaluations (countries often consider these approvals when procuring or selecting diagnostic technologies): • CE-IVD (Conformité Européenne in vitro diagnostics): four technologies have received CE-IVD: Abbott™ m-PIMA HIV-1/2 VL, Cepheid Xpert® HIV-1 Viral Load and Diagnostics for the Real World’s SAMBA I HIV-1 Semi-Quantitative Plasma Test and SAMBA II HIV- 1 Semi-Quantitative Plasma Test; and • WHO prequalification: two technologies have met WHO requirements: Abbott™ m-PIMA HIV-1/2 VL (23) and Cepheid Xpert® HIV-1 Viral Load (29)1 received WHO prequalification on 8 April 2019 and 20 July 2017, respectively. The Abbott™ m-PIMA HIV-1/2 VL assay requires 50 µl of venous EDTA plasma and can detect HIV-1 groups M, N, and O and HIV-2. The limit of detection is 800 copies/mL (23) . The Cepheid Xpert® HIV-1 Viral Load assay requires 1 mL of plasma (can be derived from ACD, EDTA or PPT-EDTA blood specimen tubes) and can detect HIV-1 groups M, N, and O. The limit of detection is 40 copies/mL (29) . Additional specifications of these and products in development are available (10,70). Independent technical evaluations: the results from 13 technical field evaluations of the Cepheid Xpert® HIV-1 Viral Load assay were consolidated across 11 countries into a meta-analysis (Table 9) (71). 1 The Cepheid Xpert® HIV-1 Viral Load assay can be used with a variety of Xpert devices at or near the point of care, from the 1-module EDGE to the 16-module Xpert. © W H O 24 24 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 9. Summarized results from WHO prequalification and independent technical evaluations Assay Evaluator Sample type Sample size Sensitivity (95% CI)a Specificity (95% CI)a Abbott™ m-PIMA HIV- 1/2 VLb WHO prequalification/ United States Centers for Disease Control and Prevention Plasma 421 95.1% (91.7–97.5%) (23) 99.4% (96.8–99.9%) (23) Cepheid Xpert® HIV-1 Viral Load WHO prequalification/ United States Centers for Disease Control and Prevention Plasma 439 94.14% (90.37–96.76%) (29) 98.50% (95.68–99.69%) (29) Meta-analysis Plasma 3790 96.47% (95.10–97.47%) (72) 96.59% (92.90–98.39%) (72) a Sensitivity and specificity using a treatment failure threshold of 1000 copies/mL. b No meta-analysis has yet been prepared because of a lack of published independent technical evaluations. Considerations As of 2019, WHO does not have a recommendation for the consideration of point-of-care viral load technologies; however, this will be reviewed in 2020. Considering some of the challenges in scaling up viral load testing, both clinically and logistically, point-of-care viral load testing may support broader access to viral load, deliver results to clinicians and patients more quickly and accelerate decision-making through same-day testing. Box 4. Setting priorities for viral load testing Several population groups could be considered and given priority for point-of-care viral load testing when overall volumes may overwhelm such technologies. • Pregnant and breastfeeding women, especially around the time of delivery, may benefit from faster result delivery and clinical decision-making to prevent mother-to-child transmission. • Infants and other children living with HIV, who typically are at higher risk of treatment failure and drug resistance because of exposure to maternal antiretroviral therapy and postnatal prophylaxis, may benefit from more rapid delivery of results and more attentive treatment monitoring. • Further, people re-entering care, those who for whom treatment failure is suspected and those with advanced HIV disease may benefit from more rapid delivery of results and clinical decision- making. In addition, several point-of-care technologies are also polyvalent or multi-disease technologies capable of testing different conditions using disease-specific tests on the same platform. Significant existing device footprint may allow for programmatic and diagnostic integration to expand access to viral load testing (73). Conclusions Sufficient evidence has been generated on the performance of some point-of-care viral load assays to support rapid national regulatory approval and the initiation of scale- up. Further technical evaluations of these technologies are unlikely to add value but may instead delay implementation. Studies of the impact on patient management and care, operational feasibility, acceptability and cost–effectiveness are ongoing. However, countries need to individually determine the contextual importance, utility and range of point-of-care viral load assays within their patient care and diagnostic networks. 25HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 7. OPERATIONAL INTERVENTIONS AND CONSIDERATIONS IN SCALING UP VIRAL LOAD TESTING AND INFANT DIAGNOSIS 7.1 OPTIONS FOR TRANSPORTING SPECIMENS FOR NUCLEIC ACID–BASED DIAGNOSTICS Laboratories and testing capacity within a diagnostics network are not present onsite at every health-care facility patients attend. The testing and analysis usually offered at centralized laboratories are critical for managing people living with HIV, such as providing viral load testing and infant diagnosis of HIV, but accessing these services can be a challenge. Testing at or closer to the point of care is one solution to address the limitations of the laboratory network (see subsection 6.4), including providing same-day results. However, point-of-care testing is not available at all facilities or may not be cost-effective at health-care facilities with low patient volumes. When testing is not available on site, specimen referral systems can provide access to the diagnostics network by moving specimens from the collection facility (also known as the referring facility) to a facility with the necessary capacity (the testing or referral laboratory). Alternative specimen types, such as dried blood spot specimens, can also be used to further increase access. Moving the specimen removes the burden of people living with HIV having to travel to the laboratory for testing. In this way, the specimen referral network extends the reach and coverage of the diagnostics network. The same system for referring specimens is also often used for returning paper results, which may be sent even if electronic results are available. Various specimen referral systems can be found at different levels of a tiered health system, in different regions of a country and across disease programmes. Together, these systems should be harmonized, connected and efficiently coordinated to form the overall specimen referral network, which, in turn, is a vital part of a diagnostics network. A specimen referral system or network has five main goals (Box 5). A specimen referral system comprises various components that are critical to ensure a successful and efficient system (Fig. 8): • management and leadership – ideally, someone in the health ministry should supervise the overall referral Box 5. Five main goals of a specimen referral system • Contribute to increased access to diagnostics where on-site services do not exist by referring the specimen to the testing laboratory • Maintain and improve the quality of specimens delivered to the testing laboratory by proper specimen management in transit, including cold- chain requirements • Ensure the safety and security of all individuals and the environment involved with specimen referrals by proper management in transit, including packaging and handling • Meet the timeliness requirements of the specimen reaching the testing laboratory and the paper result reaching the facility, clinician, patient and necessary files • Enhance the cost-efficiency of the diagnostic network through harmonization and coordination MANAGEMENT AND LEADERSHIP DATA SYSTEMS AND MONITORING AND EVALUATION EQUIPMENT LOGISTICS HUMAN RESOURCES FINANCING TRANSPORT Fig. 8. Components to ensure a successful and efficient specimen referral system 26 26 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis network, ensure that it is supporting the needs of the diagnostics network and advocate for necessary resources throughout the network; • human resources – these are the personnel at the referring facilities, referral laboratories, regional health teams, transporters, etc. that are involved in the entire referral process and returning results; • financing – the funding that is necessary for the overall referral network, incorporating all aspects of the network as well as specimen types and disease areas; • transport – this includes the type of vehicle (motorcycle or four-wheeled vehicle) and service provider (professional courier or clinical implementing partner) and ideal combinations thereof to service all facilities as necessary; • logistics – this includes the overall logistical system, such as scheduling and routing and depends on many factors, such as timeliness requirements for managing specimens and returning the results; • equipment – such as necessary packaging materials, contributes to specimen quality and biosafety during referrals; and • data systems and monitoring and evaluation – the system to collect data, analyse them and use the analysis for decision-making and continual quality improvement. Three key components of the specimen referral system can be challenging and may require additional consideration and focus: transport, logistics and data systems and monitoring and evaluation. Design of transport and logistics systems is closely related and may or may not be managed by the same organization or company. Although these components are only two in the overall system, they require additional technical expertise that is usually not a core capacity of health-care facility or laboratory staff. Three considerations for these important components include: • Type of vehicle. The type of vehicles used depends on resources, distances, terrain and carrying capacity. If the transport and logistics systems are outsourced, the service provider will likely decide the vehicle type. Examples include (listed by highest to lowest prevalence): motorcycles, four-wheeled vehicles, bicycles, boats, horses, on foot, airplanes and unmanned aerial vehicles (also known as drones). Key considerations when choosing a vehicle type include: – specimen types (dried blood spot specimens, whole blood, plasma, etc.) and requirements per test (cold chain or specimens tested for highly contagious diseases may require additional packaging, which may not fit on or be suitable for certain vehicle types such as drones); – level or tier of system used, distance travelled and type of terrain covered; and – demand and volumes at the referring facilities to understand the carrying capacity required. • Transport service provider. The service provider, which could be the health ministry, an implementing partner or a private company, is the one who operates the transport and usually employs the vehicle operators. When choosing a service provider, key considerations include: – paying the provider and the sustainability of the system; – the availability of local private sector transporters or third-party logistics providers, such as Riders for Health, DHL, G4Sor the national postal service, and the ability to contract with a third party logistics provider; – which entity will manage, own and operate the vehicles: health-care facility ownership versus provided to the facility through the health ministry or vehicles belonging to the government, partners or a private company; – which entity will manage and employ the vehicle operators (rider, driver, etc.); – specimens accompanies by a person during transit; and – dedicating the system solely to transporting specimens and results: vehicles such as ambulances, whose primary purpose is not specimen transport, should not be the only mode of transport of specimens available and used. • Logistics, scheduling and routing. At its core, a specimen referral system is a logistics system. Specimens need to be moved physically from collection points to first-line diagnostic testing sites or hubs and then possibly specialized testing sites and the results returned in reverse. Key logistics considerations include the following. – Health facilities, collection points, hubs and referral laboratories should be mapped using geocodes. Then current referral needs, links and pathways for each specimen type should be mapped out, based on testing algorithms and capacity and between each relevant level or tier of the health system, including the community or health post level, if considered. The mapping exercise should be redone when the overall diagnostics network changes, such as further decentralization of equipment or integration. – Consider whether to have a fixed schedule for pick- ups and returning results versus on-demand services. – Frequencies of pick-up should be based on patient volumes and need, specimen collection, specimen type, specimen stability and machine capability at the testing laboratory. For example, whole-blood and plasma specimens require same-day, rapid transport and storage at the correct temperature. 27HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Box 6. Considerations for data systems and monitoring and evaluation This is a critical component to the systems and network but often weak and overlooked. A standardized monitoring and evaluation framework for specimen transport is required to assess and compare the performance of the often fragmented systems. Key considerations for this component include the following. • The monitoring and evaluation framework and standardized indicators should be based on the five goals of a specimen referral system (Box 5) and included in the national specimen referral guidelines. • Data collection tools should be in place or introduced, including registers and logbooks, chain-of-custody forms (showing every time a specimen or result changes hands), transport logs, reporting forms, questions within supervisory checklists, etc. • Indicators may be aspirational, but once the specimen transport network and necessary data systems are in place, the feasibility of collecting each should be assessed. • Reporting processes should be outlined and feedback mechanisms used. • Detailed turnaround time is important to collect, including each step between the collection of the specimen from the patient to the time the result is filed in the patient’s records. • Continual quality improvement should be emphasized, including corrective actions. © W H O 28 28 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis – In a hub-and-spoke system, the hub may be a testing facility and/or a facility to consolidate and store specimens on the way to a higher level versus point to point, in which the specimen goes directly from the referring facility to the testing laboratory without consolidation at a hub on the way. For viral load testing using plasma specimens, a hub system should be set up in which each hub is equipped with refrigerators, freezers and centrifuges to process the specimens and ensure the integrity of the specimens. – Consider the ability of the referring facility or hub to prepare and store specimens. – Consider the ability to reconsider administrative boundaries if it is more logistically efficient: whether a specimen can be referred to a laboratory in a different administrative region if it is closer than the pre-assigned laboratory. – Can the system be integrated with other types of specimens? – Is the delivery of paper results included, if necessary? – What are the cut-off times for specimen reception (time by which specimens need to reach the hub for further processing or storage or laboratory) and the earliest arrival and pick-up time at the referring facilities? – For shared (non-dedicated) vehicles, schedules need to be carefully planned to not disrupt other activities. Best practices. Although there are many ways to design, implement and monitor a specimen referral system, countries are currently adopting key best practices, including the following. • Management: the health ministry must lead, coordinate and supervise the overall specimen referral network, regardless of the transport mechanism used or funding. • National guidelines are developed for specimen referrals as well as a laboratory handbook, which describes individual procedures for collection, packaging, storage and transport depending on the specimen type and test requested. • Monitoring: a robust monitoring and evaluation framework should include standardized indicators. • Network approach: the design of the specimen referral network must work within the diagnostics network and be optimized periodically to improve efficiency and costs. • Specimen types should be integrated with disease programme activities, where this is possible and logistically efficient. • Transport and referral procedures must be well documented for each specimen type and all personnel at all levels properly trained, including: specimen collection, storage, documentation, packaging and dispatch, transport, specimen receipt, results dispatch and results receipt. • Biosafety and quality: provide appropriate personal protective equipment, spill kits and proper packaging materials, including safe and secure shipping containers as necessary for each specimen type. Integration. The diagnostics network can be integrated to use a specimen referral system and network for multiple specimen types or disease programmes. In this case, usually integration is easiest and most efficient from the referring facility to the first hub or first-line diagnostic location. The overall national specimen referral network should always be fully integrated, meaning that it should cater for all specimen types and diseases. However, the transport and logistics necessary to achieve this integration may require incorporating some separate systems for certain tests or specimens based on routing and/or laboratory locations and specimen management requirements. For example, the nucleic acid–based laboratory for HIV testing may differ from the tuberculosis culture laboratory, and so separate routes and logistics may be necessary for specific aspects of the specimen transport networks. Further, depending on the specimen type and other factors, the transport mechanisms used on each route may differ. For example, for specimens that require very timely, same-day transport, or require temperature controls, public transport with no control over timeliness or temperatures may not be appropriate. If there is an outbreak investigation, the specimens may not be able to wait for and use the routine transport mechanism. Transport and logistics service provider options. The national specimen referral network will likely include a combination of the options listed in Table 10 depending on the level of the health system and local geography of a region. Box 7. Links to tools and resources for specimen referrals • Global Laboratory Initiative (GLI) Specimen Referral Toolkit. Geneva: Stop TB Partnership; 2019 (http://www.stoptb.org/wg/gli/srt.asp). • GLI guide to TB specimen referral systems and integrated networks. Geneva: Stop TB Partnership; 2019 (http://www.stoptb.org/wg/gli/assets/ documents/GLI_Guide_specimens_web_ready. pdf). • Guidance for developing a specimen transport and referral system for viral load and infant virologic HIV diagnosis testing networks. Addis Ababa: African Society for Laboratory Medicine; 2015 (http://www.aslm.org/?wpdmdl=18275). 29HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 10. Options for selecting transport and logistics service providers Se lf- ru n – op er at ed b y th e he al th m in is tr y di re ct ly o r a cl in ic al im pl em en ti ng p ar tn er ; a ll ca n ea si ly ca rr y re su lt s or o th er s up pl ie s fo r no a dd it io na l c os t Type or example Benefits Challenges Best-use case Dedicated health ministry courier system Likely share existing health ministry resources, such as staffing, to run and manage the system to save on the overall costs required Transport and logistics expertise is generally not a core competency within the health ministry Use in countries with high referral volumes where outsourcing is difficult and health ministry capacity to manage a complex transport and logistics network is high Dedicated partner- run courier system Will share some existing partner resources, such as staffing, to run and manage the system to save on the overall costs required Transport and logistics expertise is generally not a core competency – to run these systems, additional staff must be hired just for this one system, which is not cost- effective Use in countries with high referral volumes where outsourcing is difficult and health ministry capacity to manage a complex transport and logistics network is low Hand-carried by facility staff Often carried out by laboratory staff so biosafety and quality control are well understood Takes limited staff out of the health facility and away from their main responsibilities; more expensive than sending a package on its own Use where specimen referral volumes are very low and erratic Use of non- dedicated health ministry vehicles Used by programme officials to conduct supervisory visits and to deliver supplies and commodities. Some programmes have also used the vehicles to transport specimens and results Often do not visit the collection sites frequently enough for timely transport; with shared priorities, specimens are not always transported in a timely and quality-controlled manner; the use of ambulances is not recommended, since this form of transport is unpredictable and interferes with regular duties Use for health posts or facilities that only collect specimens when an outreach health team is visiting, since they can bring back the specimens with them to the laboratory Use of public transport, not accompanied (such as buses, trains, boat and aircraft) Play a major role in both rural and urban transport with extensive nationwide access and coverage; used by private courier companies and national postal systems to send letters, packages and money; less expensive to send a package unaccompanied than with a facility staff member Usually have to bring packages to depot; special permission may be needed to transport potentially infectious material; schedules may not be adhered to strictly; specimens and test results may not be properly handled due to lack of training, limited personnel and lack of clear roles and responsibilities; may not have a system in place to track specimens Use where there are reputable bus companies with regular schedules, professional staff and a central depot where health facility staff can collect and dispatch specimens O ut so ur ce d – al l h av e lo gi st ic s ex pe rt is e an d w ill m an ag e tr an sp or t Dedicated professional courier (NGO, social enterprise, private), such as Riders for Health Ability to design a dedicated system including in hard-to- reach or underserved areas; result return or carrying other supplies on scheduled routes at no additional charge Total costs may appear to be higher since the system is all-inclusive (includes vehicles, transport, drivers and riders, operating costs, etc.) and run by a third-party (resources, such as health ministry or partner staffing, will not be shared but will be at an additional cost) Use in countries with limited or undeveloped road infrastructure and transport providers Non-dedicated private professional courier, such as FedEx or DHL Specialize in collecting and delivering packages, on- demand or regularly scheduled pick-ups, documentation and tracking of shipments Not all are able or willing to transport potentially infectious biological specimens; costs may be higher; coverage and flexibility may be limited; may not be a cost-effective way to return results Use where speed, security, documentation, tracking, name and signature of receiving person, specialization and individualization of express services are sufficiently important to warrant the extra cost; best coverage in major cities National postal service, non- dedicated courier (public or semi- private) Usually a parastatal entity, which may be easier for the health ministry to contract with than a private courier; mandate to be present across an entire country; typically on a predictable schedule Availability of and accessibility to local post offices; adherence to schedules; specimens requiring strict transit time or careful temperature control may be challenging unless a guaranteed service is offered (such as express mail) Use where the national postal system is strong and has good coverage; otherwise, use only for less stringent and longer shelf-life specimens such as dried blood spot specimens 30 30 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 7.2 INFANT DIAGNOSIS AND VIRAL LOAD SPECIMEN COLLECTION BUNDLES More than 10 individual commodities are required for collecting whole-blood specimens for plasma separation or dried blood spot specimens from patients for nucleic acid–based testing (either diagnosis or viral load). Some of these items, such as the dried blood spot filter paper collection cards used to collect blood specimens, are specialized and only recommended from specific suppliers. Other items, such as gauze and alcohol swabs, are generic. In the early stages of establishing infant diagnosis testing programmes, countries needed to procure these items individually, which made ordering and facility distribution a complex endeavour. Further, stock-outs of any single item could compromise the quality of specimens or prevent the collection and/or processing of blood specimens altogether. Drawing from the experience with infant diagnosis, for ease of procurement and distribution and to ensure the quality of commodities, suppliers have developed plasma and dried blood spot specimen collection bundles for viral load testing as well. Plasma and dried blood spot specimen collection bundles contain individual and single-use collection kits that include all required items and commodities to draw, dry (for dried blood spot specimens) and transport a specimen from the facility to the laboratory. Contents of specimen collection bundles Table 11 lists the items included in single-use EDTA blood collection kits (100 tests per bundle) to obtain a plasma specimen for viral load testing using venepuncture. These specimens can then either (1) be shipped directly to the testing laboratory for processing (centrifugation) into plasma and tested or (2) be separated into plasma by centrifugation at the health-care facility and transferred to another tube, which is then sent to the laboratory under appropriate storage conditions for testing. Table 12 lists the items included in single-use DBS specimen collection kits with perforated dried blood spot cards (20 or 50 tests per bundle) that can be used for both infant diagnosis and viral load testing. Table 11. Whole-blood and plasma specimen collection bundles No. Item Quantity Specifications 1 Swab alcohol, 70% isopropyl 1 Swab alcohol WBCL 2 Swab gauze 8 ply non-sterile 10 × 10cm 1 Swab gauze 8 ply non-sterile 100 × 100mm 3 Gloves examination latex powder free 2 Gloves examination latex, powder free, medium 4 Bandage fabric 1 Bandage fabric 5 Bag autoclave clear biohazard 415 × 600 mm (1 per bundle) 1 Bag autoclavable clear print biohazard 415 × 600 mm 6 5 mL EDTA-treated evacuated tube 1 Tube 5 mL K2EDTA lavender 13 × 100 mm 7 Vacuum tube needle holder 1 Speedy quick release holder 8 Vacuum tube needle, 20G 1 Vacuum multiple use draw needle 21G × 1.5" 38 × 0.8 mm Green Sterile 9 Tourniquet (one per bundle) 1 Tourniquet disposable without clip, latex-free, synthetic rubber band, non-sterile 10 Packing box 1 Box plain white with liner 385 × 310 × 145mm 11 Pasteur transfer pipette (optional)a 1 Pasteur transfer pipette 1 mL fine tip, individual sterile pack (can be requested at additional cost) a Used to transfer the plasma aliquot after centrifugation and before transport to the laboratory. 31HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 12. DBS specimen collection bundles No. Item Quantity Infant diagnosis Viral load Specifications 1 DBS collection instructions 1 DBS collection instructions card 2 Powder-free gloves 2 Examination gloves powder free latex – medium 3 Alcohol gauze pad 2 Single-use individually wrapped alcohol-impregnated medical gauze swab 4 Lancets 1 Single-use retractable lancet with non-adjustable 2 mm penetration depth blade (not needle-type) 5 Gauze swab 1 Swab gauze non-sterile 8 ply 50 mm × 50 mm 6 EDTA capillary tube 1 100 µl EDTA capillary tube, plastic, with 70 µl markings 7 DBS Filter paper S&S 903 1 Whatman 903 card or Munktell TNF, perforated 8 Drying rack for DBS card 1 Drying rack for Whatman 903 card 9 Silica desiccant pack 3 Indicating silica gel 1-gram sachet 10 Plastic bags 1 Low gas permeable double Ziploc bag (150 mm × 180 mm) with white write-on area 11 Packing and repacking 1 Packing and repacking: five pieces per bundle 12 Packing box 1 Buff board box to contain all bundle contents, with tuck-in lid 13 Lab requisition form (optional) 1 Customized early infant diagnosis/viral load requisition form in a pad of 50s or 100s in duplicate copies 14 Barcode stickers (optional) 1 Customized early infant diagnosis/viral load barcode stickers Source: M. Rioja, Clinton Health Access Initiative. 32 32 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis A specific dried blood spot specimen collection bundle for viral load testing contains similar items, with the addition of an EDTA microcapillary tube added (Fig. 8). The EDTA microcapillary tube serves to ensure that the required volume per spot is accurately collected. Viral load testing is a quantitative measure that heavily relies on the quantity of blood inputted into the assay, regardless of specimen type. Limited evidence suggests that free drops of blood applied directly to a dried blood spot card can produce accurate test results compared with plasma specimens. Therefore, using a fixed volume or graduated measuring microcapillary tube or pipette may support increased accuracy of dried blood spot specimen preparation. Health-care workers should be appropriately trained on the differences in the specimen collection technique and process for infant diagnosis and viral load dried blood spots. Source: M. Rioja, Clinton Health Access Initiative. What are the expected benefits of using sample collection bundles? The following are benefits of specimen collection bundles for a national programme: • simpler and more standardized forecasting, procurement and supply chain management (instead of ordering individual items from various manufacturers); • ensured availability of necessary items in the correct proportions and reduced waste; • simpler and more rapid scaling up of testing services at new sites since all materials to train and test are packaged together; and • bundles result in lower costs for all components compared with individual itemized procurement. The following are benefits of specimen collection bundles for health facilities: • quality assurance of items in bundles if the supplier has a proven track record, which is especially critical for certain items that must adhere strictly to quality standards (such as lancets and powder-free gloves) to ensure that proper care is delivered to the patient and that the specimen is prepared correctly (thus mitigating the risk of the specimen being rejected at the laboratory) and the safety of the end-user ensured; • reduced risk or repurposing of items such as gloves for other services, thereby reducing stock-outs and waste of individual items; • easier sharing or distribution of individually packed single-use bundles to satellite sites with lower patient testing demand; • simplified stock audit, monitoring, inventory and distribution of supplies; and • simplified workflow in the clinic because of individually packed single-use bundles, enabling health-care workers to reach into the box and grab one bag that has everything they need to collect a specimen. Which suppliers offer specimen collection bundles? Such bundles are readily available for procurement through at least two suppliers that source items and components directly from individual manufacturers: • LASEC (https://www.lasec.com/diagnostics); and • LabMate (https://www.labmate.co.za). Conclusion By ensuring that all items needed are available to health- care workers or laboratory technicians in a single kit or box, bundled products for nucleic acid–based test sampling have simplified and standardized the supply chain for such commodities and reduced the occurrence of testing delays resulting from the stock-out or misappropriation of a single item. Many countries have become experienced in using these bundles and, as a result, have less waste and order lower buffer stock. Although the bundles themselves are a cost-effective alternative to bulk individual commodity purchasing, the significant reductions in waste contribute to additional cost savings for countries. Further, the use of bundled specimen collection products has contributed significantly to the scaling up of infant diagnosis and viral load testing services in several resource-limited countries. 33HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 7.3 OPERATIONAL INTERVENTIONS: USING VIRAL LOAD TESTING TO DIAGNOSE INFANTS Infant diagnosis testing has expanded considerably in the past decade in low- and middle-income settings, but access remains limited. In 2017, only 51% of HIV-exposed infants received an early infant diagnostic test within the first two months of life (11), as recommended by WHO (2) . Several challenges have limited the scaling up of this critical test for a highly vulnerable population. Infant diagnosis has primarily been offered at centralized testing laboratories, requiring transport of dried blood spot specimens that can often take weeks and often months before the results are returned to clinicians and caregivers for clinical action. The delays can be caused by several issues, including: • the need to batch infant specimens until a full run can be performed, to ensure that testing is cost-effective and cost-saving; • low infant diagnosis volumes limit the number of devices and laboratories capable of testing, which can be far from health-care facilities, and create a challenging procurement environment that has often lead to stock- outs of reagents in the laboratories; • in the past, and sometimes still, infant diagnosis reagents can be more expensive than other HIV nucleic acid–based tests, such as viral load; and • since this type of specimen, dried blood spots, is also often used for viral load testing and infant volumes are as low as a few needed tests per month, specimen collection materials have and can be reappropriated for viral load specimen collection, occasionally resulting in stock-outs when an infant specimen may be needed. Qualitative infant diagnosis assays have primarily been used to diagnose HIV among HIV-exposed infants in resource-limited settings. The nucleic acid–based technique (quantitative PCR) used for viral load testing is very similar, often the same, for infant testing or qualitative assays. HIV DNA PCR is a commonly used synonym for HIV infant diagnosis testing; however, several technologies currently on the market do not specifically target HIV DNA. The primary specimen type for nucleic acid–based infant diagnosis is whole blood, which can contain proviral DNA, intracellular RNA and extracellular RNA. Similarly to when using whole-blood dried blood spot specimens for viral load testing (see subsection 6.1), whole blood for qualitative infant diagnosis assays generally results in the detection of the variety of HIV nucleic acids. Since both HIV DNA and RNA are present, virological testing or HIV nucleic acid amplification testing are now more accurate terms for infant PCR testing than HIV DNA PCR. Current considerations The 2010 WHO recommendations on the diagnosis of HIV infection in infants and children (74) and 2016 WHO consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection (2) recommend that virological testing to diagnose HIV infection among infants be performed using HIV DNA PCR on whole-blood specimens or dried blood spot specimens, HIV RNA PCR on plasma or dried blood spot or ultrasensitive p24 antigen on plasma or dried blood spot. Further, guidelines in high- income countries, including guidelines in the United States of America (75) , recommend HIV RNA testing to diagnose HIV infection among infants. Existing research has suggested that HIV RNA (often quantitative) testing may yield comparable results to assays specifically detecting DNA (76–79). However, questions remain about the technical and clinical feasibility of using RNA and/or quantitative testing for diagnosis given the increased maternal and infant exposure to antiretroviral therapy through programmes to prevent the mother- to-child transmission of HIV, option B+ and “treat all” policies, since all previous studies were conducted before 2003 and the option B+ era. Updated data Two studies have recently been conducted to better understand the performance and potential role of using HIV quantitative (viral load) assays using dried blood spot specimens for diagnosing HIV among infants younger than 18 months (80,81) . These studies were conducted in current settings with high rates of maternal and infant exposure to drugs. In Mozambique, 95% of mothers and infants were on antiretroviral therapy or receiving antiretroviral prophylaxis, respectively. While in Uganda, 75% of mothers were receiving antiretroviral therapy and 65% of infants were receiving antiretroviral prophylaxis. In the study conducted in Mozambique, the sensitivity and specificity of using the viral load assay to detect infection were 100.0% and 99.9%, respectively. The positive and negative predictive values were 99% (95% CI: 94.3– 100.0%) and 100% (95% CI: 99.6–100.0%). In the study conducted in Uganda, the sensitivity and specificity of using the viral load assay to detect infection were 98.9% and 98.8%, respectively. One key consideration in both studies was that the dried blood spot specimens were prepared using the buffer and specimen preparation techniques traditionally used to prepare infant diagnosis specimens. Current WHO recommendations and these data further indicate that viral load can be used as a diagnostic assay for infants. In fact, some technologies specifically aim to 34 34 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 13. Sensitivity and specificity of viral load assays in Mozambique Study setting Sample type Sample size Sensitivity (95% CI)a Specificity (95% CI)a Mozambique (80) Plasma 1021 100% (96.2–100.0%) 99.9% (99.4–100.0%) Uganda (81) Plasma 520 98.9% (96.7–99.6%) 98.8% (96.6–99.6%) a Sensitivity and specificity using a treatment failure threshold of 1000 copies/mL. target only HIV RNA and yet have been shown to have high sensitivity and specificity, comparable to gold standard technologies, and achieved WHO prequalification. Although some manufacturers have already sought dual claims, it would be ideal for manufacturers to seek regulatory approval within their current and/or future intended use claims for their viral load assays, to support implementation of this technique. Programmatic considerations There are potentially some advantages to using viral load or dual claim assays as a diagnostic assay for infants, including: • optimizing laboratory work flow, where infant diagnosis and viral load samples could be batched together, reducing the need to wait for full infant diagnosis batches; • reducing the risk of giving lower priority to infant diagnosis at the facility and laboratory levels as the viral load programmes scale up; • streamlining forecasting and quantification for infant diagnosis and viral load testing; • simplifying procurement, supply chain management and distribution of infant diagnosis and viral load specimen collection commodities; • saving money resulting from price parity between viral load and infant diagnosis tests and increased efficiency of laboratory operations and procurement processes; and • improving care, since a viral load result could be provided for an infant living with HIV at the time of diagnosis. Conclusions Creating more efficient, streamlined and clinically supportive diagnostic systems is critical to improving care. Using viral load assays with a validated dual intended use claim to also support infant diagnosis should be considered to alleviate some of the current challenges and improve infant diagnosis. 7.4 NOVEL POINT-OF-CARE TOOLS FOR EARLY INFANT DIAGNOSIS OF HIV A decade of investment in conventional laboratory networks has expanded access to early infant diagnosis testing, but only 51% of HIV-exposed infants were tested for HIV infection before two months of age in 2015 (82). The advent of point-of-care early infant diagnosis technologies (10) is a breakthrough that creates the opportunity to increase coverage of early infant diagnosis testing. It will enable same-day test results, enable treatment to be initiated earlier and address some of the key limitations of conventional early infant diagnosis networks – especially long turnaround times for tests and high rates of loss to follow-up. Significant progress has been made in ensuring the quality of new point-of-care early infant diagnosis technologies. The following regulatory approvals and technical evaluations have been made for point-of-care early infant diagnosis (countries often consider these approvals when procuring diagnostic technologies): • CE-IVD (Conformité Européene in vitro diagnostics). Four point-of-care early infant diagnosis technologies have received CE-IVD: AlereTMq HIV-1/2 Detect, Cepheid Xpert®HIV-1 Qual and Diagnostics for the Real World’s SAMBA I HIV-1 Qual Test and SAMBA II HIV-1 Qual Whole Blood Test. • WHO prequalification: Two point-of-care early infant diagnosis technologies have met WHO requirements: AlereTMq HIV 1/2 Detect (83) and Cepheid Xpert®HIV-1 Qual (84) received WHO prequalification on 13 June 2016. Independent technical evaluations: the Point-of-care Early Infant Diagnosis Consortium comprised a group of principal investigators across six countries conducting technical 35HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis field evaluations of point-of-care early infant diagnosis technologies to expedite the release of independent performance data to accelerate national approval processes and in-country implementation. The results from nine technical field evaluations were consolidated across the six countries (Table 14). A total of 3383 specimens were tested using the AlereTMq HIV-1/2 Detect, and 4401 specimens were tested using the Cepheid Xpert®HIV-1 Qual (85). WHO recommendations The 2016 WHO consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection (2) recommend that nucleic acid testing technologies that are developed and validated for use at or near the point of care can be used for early infant HIV testing. Point-of-care early infant diagnosis provides the opportunity to reduce test turnaround times, limit patient loss along the HIV testing cascade, reduce infant mortality and enable task shifting to lower cadres of health-care workers at decentralized facilities (2). Current use Several countries are implementing point-of-care early infant diagnosis technologies. For example, Malawi, Mozambique and South Africa reported results from point- of-care early infant diagnosis pilot projects in 2016 showing significantly shorter test turnaround times for results and increased rates of initiation of antiretroviral therapy compared with conventional laboratory systems (86–88) . Considering the high and early mortality rate of untreated infants living with HIV (89,90), point-of-care early infant diagnosis could also reduce observed infant mortality. Based on the CE-IVD and WHO PQ approvals, robust results from independent technical field evaluations, procurement eligibility, the WHO recommendation for the use of point- of-care early infant diagnosis and initial results on patient impact from implementation pilots, countries should begin planning to implement point-of-care early infant diagnosis Table 14. Technical evaluations of point-of-care early infant diagnosis technologies Study setting Sample type Sample size Sensitivity (95% CI) Specificity (95% CI) AlereTMq HIV-1/2 Detect WHO PQ CDC/NHLS Whole blood 98.67% (95.27– 99.84%) 100.00% (97.59– 100.00%) Early Infant Diagnosis Consortium Whole blood 99.00% (96.45– 99.88%) 99.97% (99.83– 100.00%) Cepheid Xpert® HIV-1 Qual WHO PQ CDC/NHLS Whole blood 98.86% (93.83– 99.97%) 100.00% (97.55– 100.00%) Early Infant Diagnosis Consortium Whole blood 96.79% (92.68– 98.95%) 99.91% (99.76– 99.97%) WHO PQ CDC/NHLS Dried blood spots 99.34% (96.40– 100.00%) 100.00% (97.60– 100.00%) by incorporating it into national HIV care and treatment guidelines, national strategic plans, PEPFAR country operational plans, grant applications to the Global Fund to Fight AIDS, Tuberculosis and Malaria and HIV programme budgets. Conclusions Sufficient evidence has been generated on the performance of these assays in the intended field settings to support rapid national regulatory approval and initiation of scale- up. Performance was consistent between laboratory and field settings and across countries Further technical evaluations of these technologies are unlikely to add value but may instead delay implementation and timely diagnoses of infants living with HIV, a critical and vulnerable population. National regulatory agencies are encouraged to not delay adoption by conducting further evaluations but instead adopt a rapid and streamlined registration and national approval process for immediate implementation. 7.5 OPERATIONAL INTERVENTIONS: UPDATED CONSIDERATIONS FOR A COMPREHENSIVE QUALITY MANAGEMENT PACKAGE FOR POINT-OF-CARE TESTING WITHIN NATIONAL HEALTH PROGRAMMES The introduction and implementation of point-of-care technologies and the ability to decentralize testing has greatly improved access to diagnostic services. Since 2015, new WHO recommendations have been published (2) . In 2016, WHO conditionally recommended nucleic acid testing technologies that are developed and validated for use at or near the point of care for early infant HIV testing. Further, CD4 cell count testing at the point of care can be used to give priority for urgent linkage to care and antiretroviral therapy initiation. Finally, several point-of-care or near- 36 36 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis point-of-care technologies have been prequalified since 2015 for early infant diagnosis, CD4, HIV viral load, hepatitis C viral load, cervical cancer screening and HIV and syphilis (91). Point-of-care testing has been found to facilitate rapid and decentralized delivery of health services. A systematic review of using point-of-care CD4 to support antiretroviral therapy initiation (92) showed significantly improved linkage to HIV care and timeliness of antiretroviral therapy initiation. Further, recent published studies in Malawi and Mozambique have shown significantly reduced test turnaround times and increased antiretroviral therapy initiation rates when using point-of-care testing for early infant diagnosis (93,94). This decentralization of both qualitative and quantitative testing has presented both opportunities and challenges as countries monitor an increasing number of devices and operators across a decentralized testing network. This has required expanding traditional external quality assessment schemes to reach an unprecedented number of health facilities, and in many cases, considering novel mechanisms to support the quality management process. The principles presented throughout the publication on improving the quality of HIV-related point-of-care testing (95) remain highly relevant. However, it is now critical to update considerations for countries and implementing partners, since experiences with point-of-care technologies and quality assurance mechanisms have developed. As more experience has been gained, a more comprehensive approach to quality assuring point-of-care technologies is critical to ensure reliable and accurate testing. Several alternative options to quality assurance that should form a comprehensive package along with traditional proficiency testing include: • in-training and ongoing competency assessment; • internal quality controls; • proficiency testing panels; • alternative external quality assessment, if traditional proficiency testing panels are not available: o paper-based and online o duplicate specimen testing/reverse testing; • data management through connectivity; and • site training and mentorship Each quality assurance mechanism may touch on different steps within the testing cascade; however, once consolidated into a package, they provide a comprehensive and inclusive approach. Importance of a comprehensive quality management package for point-of-care technologies Quality management of diagnostics is critical to the overall quality of care by ensuring reliable and accurate test results. Pre-market quality assessments of in vitro diagnostics, such as WHO prequalification, provide information on product safety, quality and performance, manufacturing reliability and quality management systems. Further, stringent regulatory authorities aim to assess high-quality products for their intended use. Together, these processes ensure that only high-quality products are eligible for procurement. However, ongoing quality assurance and quality control are necessary to ensure the accuracy and precision of the results produced by diagnostic testing to prevent misdiagnosis. Internal controls and standards are meant to eliminate differences in random and systematic errors between each specimen and between specimens and known standards. External quality assessment proficiency testing schemes specifically assess the performance of a laboratory or health-care facility in accurately testing stabilized specimens of known value or result. The results of these assessments should alert national programmes to a problem, at which point action can be taken to identify the cause and potential remediations. This is valuable for understanding the performance levels of individual facilities but also in reviewing the overall national laboratory network. Further, data monitoring of the invalid rates, daily controls and utilization patterns of device-based technologies through connectivity can provide critical information on testing quality, recurring device or operator errors and the need for refresher training or specific mentorship. A comprehensive quality management package can identify gaps and bring them to the attention of laboratory programme managers. Good quality assurance programmes enable testing sites and laboratory programmes to work together to prevent, detect and correct problems throughout the entire testing cascade and to monitor all aspects of a testing programme for continual and high-quality testing services. The comprehensive quality management programme should bring together a series of activities that can together touch on all aspects of testing, including: • identifying patients; • collecting specimens; • handling specimens; • ensuring specimen and reagent storage conditions and expiry dates; 37HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis • applying specimens; • ensuring the performance of technology; • applying reagents, if necessary; • ensuring technical procedures; • interpreting results; and • recording results, Such a comprehensive quality management package for point-of-care testing is meant to complement the suggested and ongoing national laboratory-wide pre- and post-market surveillance activities outlined in other WHO publications (96–99). Implementation considerations for developing a quality management package A strong and comprehensive quality management package for point-of-care testing requires quality activities in addition to proficiency testing panels. Establishing a comprehensive package with some of the alternative strategies discussed here will enable coverage of the entire testing cascade and provide more regular monitoring of decentralized testing. At a minimum, national programmes should consider proficiency testing panels, encouraging suppliers to develop robust internal control systems, in-service training and competency assessments, data management through connectivity as well as regular and planned site training and mentorship. In an effort to provide a comprehensive quality management programme, national health policies must be developed that consider the available resources to ensure sustained adoption for the implementation of quality assurance in each context. In addition to exploring different models, countries must consider the timing and frequency of quality assurance activities, the content of each activity and the subsequent cost of conducting these activities. All these parameters have important quality and cost implications, and using country-specific policies and data to inform these decisions is critical. Some of the parameters to help understand implementation are listed below. In addition, routine programmatic quality mechanisms are still critical to ensure consistent procurement and introduction of high-quality technologies. Regular lot testing, service and maintenance and post-market surveillance are necessary structures of an overall laboratory quality system that this comprehensive quality management package for point-of-care testing should complement (98,99). No quality management programme is complete without reviewing data and taking clear and consistent preventive and corrective action when necessary. This is a critical component of the programme that must be clearly planned and determined to ensure that issues are addressed and operators are given the necessary support to continue providing testing and results in a high-quality manner. 38 38 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 8. CONCLUSIONS Increasing scale-up of treatment monitoring approaches through viral load testing as well as infant diagnosis will be critical to ensure high-quality care and treatment as well as programmatic success. Considering the optimal diagnostic network, specimen types, interventions and strategies in each country and across national, regional and partner stakeholders will support this effort, enhance collaboration and maximize diagnostic investment into clear clinical impact. © W H O 39HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis REFERENCES 1. Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection: recommendations for a public health approach. Geneva: World Health Organization; 2013 (https://www.who.int/hiv/pub/ guidelines/arv2013/en, accessed 8 July 2019). 2. Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection: recommendations for a public health approach – second edition 2016. Geneva: World Health Organization; 2016 (https://www. who.int/hiv/pub/arv/arv-2016/en, accessed 8 July 2019). 3. What’s new in treatment monitoring: viral load and CD4 testing. Geneva: World Health Organization; 2017 (https:// www.who.int/hiv/pub/arv/treatment-monitoring-info-2017/ en, accessed 8 July 2019). 4. Guidelines for managing advanced HIV disease and rapid initiation of antiretroviral therapy. Geneva: World Health Organization; 2017 (https://www.who.int/hiv/ pub/guidelines/advanced-HIV-disease/en, accessed 8 July 2019). 5. HIV market report: the state of the HIV treatment, testing, and prevention markets in low- and middle-income countries, 2017–2022. Boston: Clinton Health Access Initiative; 2018. 6. Landmark HIV diagnostic access program will save $150m and help achieve new global goals on HIV. Geneva: UNAIDS; 2014 (https://www.unaids.org/en/resources/ presscentre/pressreleaseandstatementarchive/2014/ september/20140925prviralload, accessed 8 July 2019). 7. Hologic Global Access Initiative: A revolutionary approach to diagnostic testing for resource-limited settings. Marlborough (MA): Hologic; 2019 (https://www.hologic. com/globalaccessinitiative, accessed 8 July 2019). 8. Cepheid announces expanded access to Xpert family of virology tests in global regions with the greatest need. Sunnyvale (CA): Cepheid; 2018 (http://www.cepheid. com/en/about/news-events/press-releases/319-cepheid- announces-expanded-access-to-xpert-family-of-virology- tests-in-global-regions-with-the-greatest-need, accessed 8 July 2019). 9. Putting HIV and HCV to the test: a product guide for point-of-care CD4 tests and laboratory-based and point-of- care HIV and HCV viral load tests. Geneva: Médecins Sans Frontières; 2017. 10. HIV/AIDS diagnostics technology landscape. 5th ed. Geneva: Unitaid; 2015 (http://www.unitaid.org/assets/ UNITAID_HIV_Nov_2015_Dx_Landscape-1.pdf, accessed 8 July 2019). 11. Miles to go: closing gaps, breaking barriers, righting injustices. Geneva: UNAIDS; 2018 (https://www.unaids. org/en/resources/documents/2018/global-aids-update, accessed 8 July 2019). 12. HIV diagnosis and ARV use in HIV-exposed infants: a programmatic update. Geneva: World Health Organization; 2018 (https://www.who.int/hiv/pub/paediatric/diagnosis- arv-infants/en, accessed 8 July 2019). 13. 90–90–90: an ambitious treatment target to help end the AIDS epidemic. Geneva: UNAIDS; 2017 (https:// www.unaids.org/en/resources/documents/2017/90-90-90, accessed 8 July 2019). 14. Population-based HIV impact assessments [website]. New York: ICAP at Columbia University, 2019 (https://phia. icap.columbia.edu, accessed 8 July 2019). 15. Making viral load routine: successes and challenges in the implementation of routine HIV viral load monitoring. Geneva: Médecins Sans Frontières; 2016. 16. Vojnov L, Venter F, Sacks J, Ehrenkranz P, Kiyaga C, Reynolds S et al. Next steps in HIV treatment monitoring: a call to action to ensure the use of viral load test results. In preparation. 17. HIV viral load scale-up tools [website]. Addis Ababa: ASLM; 2019 (http://www.aslm.org/hiv-viral-load-testing/ hiv-viral-load-scale-tools, accessed 8 July 2019). 18. Tools for scaling-up viral load monitoring [website]. Washington (DC): USAID; 2019 (https://aidsfree.usaid.gov/ news-events/tools-scaling-viral-load-monitoring, accessed 8 July 2019). 19. AIDSFree viral load and early infant diagnosis knowledge base [online database]. Washington (DC): USAID; 2019 (https://aidsfree.usaid.gov/resources/vl-eid, accessed 8 July 2019). 20. Abbott RealTime HIV-1 instructions for use. Chicago: Abbott Laboratories; 2014. 40 40 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 21. Public report: Abbott RealTime HIV-1. Geneva: World Health Organization; 2016 (https://www.who.int/ diagnostics_laboratory/evaluations/pq-list/hiv-vrl/180423_ amended_final_pqpr_0145_027_00_v11.pdf?ua=1, accessed 8 July 2019). 22. m-PIMA™ HIV-1/2 VL cartridge guide. Chicago: Abbott Laboratories; 2019. 23. Public report: m-PIMA HIV-1/2 VL. Geneva: World Health Organization; 2019 (https://www.who.int/ diagnostics_laboratory/evaluations/pq-list/190408_ pqdx_0359_032_00_pqpr_mpima.pdf?ua=1, accessed 8 July 2019). 24. GENERIC HIV Charge Virale instructions for use. Collingwood (NJ); Biocentric; 2019. 25. NucliSENS EasyQ® HIV-1 v2.0 instructions for use. Marcy-l’Étoile: bioMérieux; 2010. 26. Public report: NucliSENS EasyQ® HIV-1 v2.0. Geneva: World Health Organization; 2017 (https://www.who. int/diagnostics_laboratory/evaluations/pq-list/hiv-vrl/ pqdx_0127_016_00_public_report_v3.pdf?ua=1, accessed 8 July 2019). 27. ExaVir™ Load instructions for use, version 3. Uppsala: Cavidi; 2011. 28. Xpert® HIV-1 Viral Load instructions for use. Sunnyvale (CA): Cepheid; 2017. 29. Public report: Xpert® HIV-1 viral load. Geneva: World Health Organization; 2017 (https://www.who.int/ diagnostics_laboratory/evaluations/pq-list/hiv-vrl/170830_ amended_final_pqpr_0192_0193_0194_0195_070_00_ v3.pdf?ua=1, accessed 8 July 2019). 30. Aptima™ HIV-1 Quant Dx Assay instructions for use. Marlborough (MA): Hologic; 2017. 31. Public report: Aptima™ HIV-1 Quant Dx Assay. Geneva: World Health Organization; 2018 (https://www.who.int/ diagnostics_laboratory/evaluations/pq-list/180207_final_ pqpr_0236_078_00_v5_IFU_v4.pdf?ua=1, accessed 8 July 2019). 32. artus® HI Virus -1 RG RT-PCR Kit handbook. Hilden: Qiagen; 2013. 33. artus® HI Virus -1 QS-RGQ Kit handbook. Hilden: Qiagen; 2013 34. COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0 instructions for use. Basel: Roche; 2017. 35. Public report: COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0. Geneva: World Health Organization; 2018 (https://www.who.int/diagnostics_laboratory/evaluations/ pq-list/hiv-vrl/160530_amended_final_public_report_ pqdx_0200_046_00_taqman96.pdf?ua=1, accessed 8 July 2019). 36. cobas® HIV-1 Quantitative Nucleic Acid Test for Use on the cobas® 4800 System instructions for use. Basel: Roche; 2018. 37. cobas® HIV-1 Quantitative Nucleic Acid Test for Use on the cobas® 6800/8800 Systems instructions for use. Basel: Roche; 2017. 38. HIV Real-TM Quant DX handbook. Como: Sacace Biotechnologies; 2015. 39. VERSANT® HIV-1 RNA 1.5 Assay instructions for use. Munich: Siemens; 2017. 40. Bonner K, Siemieniuk RA, Boozary A, Roberts T, Fajardo E, Cohn J. Expanding access to HIV viral load testing: a systematic review of RNA stability in EDTA tubes and PPT beyond current time and temperature thresholds. PLoS One. 2014;9:e113813. 41. Hardie DR, Korsman SN, Ameer S, Vojnov L, Hsiao N-Y. Reliability of plasma HIV viral load testing beyond 24 hours: insights gained from a study in a routine diagnostic laboratory. PLoS One. (in press). 42. Gullett JC, Nolte FS. Quantitative nucleic acid amplification methods for viral infections. Clin Chem. 2015;61:72–8. 43. Undetectable: how viral load monitoring can improve HIV treatment in developing countries. Geneva: Médecins Sans Frontières; 2012. 44. Quinn TC, Wawer MJ, Sewankambo N, Serwadda D, Li C, Wabwire-Mangen F et al. Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group. N Engl J Med. 2000;342:921–9. 45. Gray RH, Wawer MJ, Brookmeyer R, Sewankambo NK, Serwadda D, Wabwire-Mangen F et al. Probability of HIV-1 transmission per coital act in monogamous, heterosexual, HIV-1-discordant couples in Rakai, Uganda. Lancet. 2001;357:1149–53. 46. Avettand-Fènoël V, Hocqueloux L, Ghosn J, Cheret A, Frange P, Melard A et al. Total HIV-1 DNA, a marker of viral reservoir dynamics with clinical implications. Clin Microbiol Rev. 2016;29:859–80. 41HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 47. Parkin NT. Measurement of HIV-1 viral load for drug resistance surveillance using dried blood spots: literature review and modeling of contribution of DNA and RNA. AIDS Rev. 2014;16:160–71. 48. Lee TH, Stromberg RR, Heitman JW, Sawyer L, Hanson CV, Busch MP. Distribution of HIV type 1 (HIV-1) in blood components: detection and significance of high levels of HIV- 1 associated with platelets. Transfusion. 1998;38:580–8. 49. Smit PW, Sollis KA, Fiscus S, Ford N, Vitoria M, Essajee S et al. Systematic review of the use of dried blood spots for monitoring HIV viral load and for early infant diagnosis. PLoS One. 2014;9:e86461. 50. Vojnov L, Carmona S, Zeh C, Markby J, Boeras D, Prescott MR et al. The performance of using dried blood spot specimens for HIV-1 viral load testing: a systematic review and meta-analysis. In preparation. 51. Holodniy M, Mole L, Yen-Lieberman B, Margolis D, Starkey C, Carroll R et al. Comparative stabilities of quantitative human immunodeficiency virus RNA in plasma from samples collected in VACUTAINER CPT, VACUTAINER PPT, and standard VACUTAINER tubes. J Clin Microbiol. 1995;33:1562–6. 52. Ginocchio CC, Wang XP, Kaplan MH, Mulligan G, Witt D, Romano JW et al. Effects of specimen collection, processing, and storage conditions on stability of human immunodeficiency virus type 1 RNA levels in plasma. J Clin Microbiol. 1997;35:2886–93. 53. Holodniy M, Rainen L, Herman S, Yen-Lieberman B. Stability of plasma human immunodeficiency virus load in VACUTAINER PPT plasma preparation tubes during overnight shipment. J Clin Microbiol. 2000;38:323–6. 54. Elbeik T, Nassos P, Kipnis P, Haller B, Ng VL. Evaluation of the VACUTAINER PPT Plasma Preparation Tube for use with the Bayer VERSANT assay for quantification of human immunodeficiency virus type 1 RNA. J Clin Microbiol. 2005;43:3769–71. 55. Giordano M, Kelleher T, Colonno RJ, Lazzarin A, Squires K. The effects of the Roche AMPLICOR HIV- 1 MONITOR UltraSensitive Test versions 1.0 and 1.5 viral load assays and plasma collection tube type on determination of response to antiretroviral therapy and the inappropriateness of cross-study comparisons. J Clin Virol. 2006;35:420–5. 56. Griffith BP, Mayo DR. Increased levels of HIV RNA detected in samples with viral loads close to the detection limit collected in plasma preparation tubes (PPT). J Clin Virol. 2006;35:197–200. 57. Wan H, Seth A, Rainen L, Fernandes H. Coamplification of HIV-1 proviral DNA and viral RNA in assays used for quantification of HIV-1 RNA. J Clin Microbiol. 2010;48:2186–90. 58. Salimnia H, Moore EC, Crane LR, Macarthur RD, Fairfax MR. Discordance between viral loads determined by Roche COBAS AMPLICOR human immunodeficiency virus type 1 monitor (version 1.5): standard and ultrasensitive assays caused by freezing patient plasma in centrifuged Becton- Dickinson Vacutainer brand plasma preparation tubes. J Clin Microbiol. 2005;43:4635–9. 59. García-Bujalance S, Ladrón de Guevara C, González- García J, Arribas JR, Zamora F, Gutiérrez A. Elevation of viral load by PCR and use of plasma preparation tubes for quantification of human immunodeficiency virus type 1. J Microbiol Methods. 2007;69:384–6. 60. Rebeiro PF, Kheshti A, Bebawy SS, Stinnette SE, Erdem H, Tang YW et al. Increased detectability of plasma HIV-1 RNA after introduction of a new assay and altered specimen-processing procedures. Clin Infect Dis. 2008;47:1354–7. 61. Kran AM, Jonassen TØ, Sannes M, Jakobsen K, Lind A, Maeland A et al. Overestimation of human immunodeficiency virus type 1 load caused by the presence of cells in plasma from plasma preparation tubes. J Clin Microbiol. 2009;47:2170–4. 62. Kraft CS, Binongo JN, Burd EM, Eaton ME, McCloskey CB, Fernandes H et al. Successful use of Plasma Preparation Tubes™ (PPTs) in the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 test. J Clin Virol. 2013;57:77–9. 63. Fernandes H, Morosyuk S, Abravaya K, Ramanathan M, Rainen L. Evaluation of effect of specimen-handling parameters for plasma preparation tubes on viral load measurements obtained by using the Abbott RealTime HIV- 1 load assay. J Clin Microbiol. 2010;48:2464–8. 64. Adachi D, Benedet M, Tang JW, Taylor GD. Comparative evaluation of Roche’s COBAS Ampliprep/COBAS TaqMan HIV-1 Test v2.0 and Abbott’s RealTime m2000sp/rt HIV-1 assay on PPTs and EDTA samples. J Clin Virol. 2014;60:78–9. 65. Cloherty G, Swanson P, Lucic D, Dieckhaus K, Anthony P, Cataline P et al. Clinical implications of elevated HIV- 1 viral load results obtained from samples stored frozen in vacutainer plasma preparation tubes. J Virol Methods. 2014;204:91–2. 66. Goedhals D, Scott LE, Moretti S, Cooper MA, Opperman WJ, Rossouw I. Evaluation of the use of plasma preparation tubes for HIV viral load testing on the COBAS AmpliPrep/ COBAS TaqMan HIV-1 version 2.0. J Virol Methods. 2013;187:248–50. 42 42 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 67. BD Vacutainer® PPT instructions for use. Franklin Lakes (NJ): Becton, Dickinson and Company; 2015. 68. K2E K2EDTA Sep. Kremsmünster: Greiner Bio-One; 2019 (https://shop.gbo.com/en/row/products/preanalytics/ venous-blood-collection/vacuette-tube/edta/k2e-k2edta- sep, accessed 8 July 2019). 69. K2EDTA Gel Tube. Negeri Sembilan: TUD: 2019 (http:// www.tud.my/product_info_901003-100013.htm, accessed 8 July 2019). 70. Putting HIV and HCV to the test. Geneva: Médecins Sans Frontières; 2017 (https://www.msf.org/sites/msf.org/ files/putting_hiv_and_hcv_to_the_test.pdf, accessed ( July2019). 71. Sacks JA, Fong Y, Perez Gonzalez M, Andreotti M, Baliga S, Garrett N et al. Performance of Cepheid GeneXpert HIV-1 viral load plasma assay to accurately detect treatment failure: a clinical meta-analysis. AIDS. (in press). 72. Sacks JA, Fong Y, Gonzalez MP, Andreotti M, Baliga S, Garrett N et al. Performance of cepheid GeneXpert HIV- 1 viral load plasma assay to accurately detect treatment failure: a clinical meta-analysis. AIDS. doi: 10.1097/ QAD.0000000000002303. [Epub ahead of print] 73. Information note: considerations for adoption and use of multidisease testing devices in integrated laboratory networks. Geneva: World Health Organization; 2017 (https://www.who.int/tb/publications/2017/ considerations_multidisease_testing_devices_2017/en, accessed 8 July 2019). 74. WHO recommendations on the diagnosis of HIV infection in infants and children. Geneva: World Health Organization; 2010 (https://apps.who.int/iris/ handle/10665/44275, accessed 8 July 2019). 75. Recommendations for the use of antiretroviral drugs in pregnant women with HIV infection and interventions to reduce perinatal HIV transmission in the United States. Washington (DC): United States Department of Health and Human Services; 2018 (https://aidsinfo.nih.gov/guidelines/ html/3/perinatal/509/diagnosis-of-hiv-infection-in-infants- and-children, accessed 8 July 2019). 76. Delamere C, Burgard M, Mayaux MJ, Blanche S, Doussin A, Ivanoff S et al. HIV-1 RNA detection in plasma for the diagnosis of infection in neonates. J Acquir Immune Defic Syndr Hum Retrovirol. 1997;15:121–5. 77. Nesheim S, Palumbo P, Sullivan K, Lee F, Vink P, Abrams E et al. Quantitative RNA testing for diagnosis of HIV-infected infants. J Acquir Immune Defic Syndr Hum Retrovirol. 2003;32:192·5. 78. Simonds RJ, Brown TM, Thea DM, Orloff SL, Steketee RW, Lee FK et al. Sensitivity and specificity of a qualitative RNA detection assay to diagnose HIV infection in young infants. AIDS. 1998;12:1545–9. 79. Young N, Shaffer N, Chaowanachan T, Chotpitayasunondh T, Vanparapar N, Mock PA et al. Early diagnosis of HIV-1 infected infants in Thailand using RNA and DNA PCR assays sensitive to non-B subtypes; J Acquir Immune Defic Syndr Hum Retrovirol. 2000;24:401–7. 80. Vubil A, Nhachigule C, Loquiha O, Meggi B, Mabunda N, Bollinger T et al. Viral load assay performs comparably to early infant diagnosis assay to diagnose infants with HIV. J Init AIDS Soc. In preparation. 81. Kiyaga C, Fong Y, Okiira C, Kushemererwa GE, Kayongo I, Tadeo I et al. Viral load assay performed well when used as a diagnostic for infants. 11th International Workshop on HIV Pediatrics, Mexico City, Mexico, 19–20 July 2019. 82. Global Plan towards the elimination of new HIV infections among children by 2015 and keeping their mothers alive. Geneva: UNAIDS; 2011 (https://www. unaids.org/en/resources/documents/2011/20110609_ JC2137_Global-Plan-Elimination-HIV-Children_en.pdf, accessed 8 July 2019). 83. Public report: Alere™q HIV-1/2 Detect. Geneva: World Health Organization; 2016 (http://www.who. int/diagnostics_laboratory/evaluations/pq-list/ hiv- vrl/160613PQPublicReport_0226-032-00AlereHIVDetect_ v2.pdf?ua=1, accessed 8 July 2019). 84. Public report: Cepheid Xpert®HIV-1 Qual. Geneva: World Health Organization; 2016 (http://www.who.int/ diagnostics_laboratory/evaluations/pq-list/ hiv-vrl/16 0613PQPublicReport_0259-0700-00_XpertQualHIV_ v2.pdf?ua=1, accessed 8 July 2019). 85. Carmona S, Wedderburn C, Macleod W, Hsaio M, Jani I, Kroon M et al. Field performance of point-of-care HIV testing for early infant diagnosis: pooled analysis from six countries for the EID Consortium. ASLM2016, Cape Town, South Africa, 3–8 December 2016 (https://eidconsortium. org/Files/EID%20Poster%20v5%20Low%20res.pdf, accessed 8 July 2019). 86. Jani IV, Meggi B, Mabunda N, Vubil A, Tobaiwa O, Bollinger T et al. Effect of point-of-care early infant diagnosis on retention of patients and rates of antiretroviral therapy initiation on primary health care clinics: a cluster- randomized trial in Mozambique. 21st International AIDS Conference, Durban, South Africa, 18–22 July 2016. 87. Mwenda R. Impact of point-of-care EID testing into the national EID program: pilot experiences from Malawi. 21st International AIDS Conference, Durban, South Africa, 18–22 July 2016. 43HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 88. Technau K, Sherman G, Bhowan K, Murnane P, Coovadia AH, Kuhn L. Comparing point of care to laboratory HIV PCR testing at birth in a hospital setting in Johannesburg, South Africa (Abstract O-14). 8th International Workshop on HIV Pediatrics, Durban, South Africa, 15–16 July 2016. 89. Bourne DE, Thompson M, Brody LL, Cotton M, Draper B, Laubscher R et al. Emergence of a peak in early infant mortality due to HIV/AIDS in South Africa. AIDS. 2009;23:101–6. 90. Newell ML, Coovadia H, Cortina-Borja M, Rollins N, Gaillard P, Dabis F. Mortality of infected and uninfected infants born to HIV-infected mothers in Africa: a pooled analysis. Lancet. 2004;364:1236–43. 91. WHO list of prequalified in vitro diagnostic products. Geneva: World Health Organization; 2019 https://www. who.int/diagnostics_laboratory/evaluations/PQ_list/en, accessed 8 July 2019). 92. Vojnov L, Markby J, Boeke C, Harris L, Ford N, Peter T. POC CD4 testing improves linkage to HIV care and timeliness of ART initiation in a public health approach: a systematic review and meta-analysis. PLoS One. 2016;11:e0155256. 93. Mwenda R, Fong Y, Magombo T, Saka E, Midiani D, Mwase C et al. Significant patient impact observed upon implementation of point-of-care early infant diagnosis technologies in an observational study in Malawi. Clin Infect Dis. 2018;67:701–7. 94. Jani IV, Meggi B, Loquiha O, Tobaiwa O, Mudenyanga C, Zitha A et al. Effect of point-of-care early infant diagnosis on antiretroviral therapy initiation and retention of patients. AIDS. 2018;32:1453–63. 95. Improving the quality of HIV-related point-of-care testing: ensuring the reliability and accuracy of test results. Geneva: World Health Organization; 2015 (https://www. who.int/hiv/pub/toolkits/handbook-point-of-care-testing/ en, accessed 8 July 2019). 96. Prequalification of in vitro diagnostics. Geneva: World Health Organization; 2019 (https://www.who.int/ diagnostics_laboratory/evaluations/en, accessed 8 July 2019). 97. Procurement of in vitro diagnostics. Geneva: World Health Organization; 2019 (https://www.who.int/ diagnostics_laboratory/procurement/en, accessed 8 July 2019). 98. Post-market surveillance for in vitro diagnostics (IVDs). Geneva: World Health Organization; 2019 (https://www. who.int/diagnostics_laboratory/postmarket/en, accessed 8 July 2019). 99. Quality assurance of in vitro diagnostics and laboratory technology. Geneva: World Health Organization; 2019 (https://www.who.int/diagnostics_laboratory/quality/en, accessed 8 July 2019). 44 44 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis ANNEX 1. INFANT DIAGNOSIS ALGORITHM HIV-exposed newborn (0-2 days) Consider NATa,b Negative Negative Immediately start ARTc Repeat NAT to confirm infection Infant/child is infected HIV-exposed infant or child (4-6 weeks to 18 months) Conduct NATb (at 4-6 weeks or at the earliest opportunity thereafter) Positive Positive Immediately start ARTc Repeat NAT to confirm infection HIV infection not detected but if infant/child is breastfed the risk of acquiring HIV infection remains until complete cessation of breastfeedingd Regular clinical monitoring Conduct NATb (at 9 months) Antibody testing at 18 months of age or 3 months after cessation of breastfeeding, whichever is laterf Infant/child is infected HIV unlikely unless still breastfedinge Negative a Based on 2016 WHO Consolidated ARV Guidelines, addition of NAT at birth to the existing testing algorithm can be considered. b POC NAT can be used to diagnose HIV infection as well as to confirm positive results. c Start ART without delay. At the same time, retest to confirm infection. As maternal treatment is scaled up and MTCT transmission rates decrease, false-positive results are expected to increase: retesting after a first positive NAT is hence important to avoid unnecessary treatment, particularly in settings with lower transmission rates. If the second test is negative, a third NAT should be performed before interrupting ART. d For children who were never breastfed, additional testing following a negative NAT at 4–6 weeks is included in this algorithm to account for potential false-negative NAT results. e The risk of HIV transmission remains as long as breastfeeding continues. If the 9-month test is conducted earlier than 3 months after cessation of breastfeeding, infection acquired in the last days of breastfeeding may be missed. Retesting at 18 months or 3 months after cessation of breastfeeding (whichever is later) should be carried out for final assessment of HIV status. f If breastfeeding extends beyond 18 months, the final diagnosis of HIV status can only be assessed at the end of breastfeeding. If breastfeeding ends before 18 months, the final diagnosis of HIV status with antibody testing can only be assessed at 18 months. Antibody testing should be undertaken at least 3 months after cessation of breastfeeding (to allow for development of HIV antibodies). For infants younger than 18 months of age NAT should be performed to confirm infection. If the infant is older than 18 months, negative antibody testing confirms that the infant is uninfected; positive antibody testing confirms infant is infected. Source: HIV diagnosis and ARV use in HIV-exposed infants: a programmatic update (12) .

For more information, contact: World Health Organization Department of HIV/AIDS 20, avenue Appia 1211 Geneva 27 Switzerland E-mail: hiv-aids@who.int www.who.int/hiv ISBN 978 92 4 151621 1

HIV TREATMENT AND CARE HIV MOLECULAR DIAGNOSTICS TOOLKIT TO IMPROVE ACCESS TO VIRAL LOAD TESTING AND INFANT DIAGNOSIS TOOLKIT JULY 2019 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis ISBN 978-92-4-151621-1 © World Health Organization 2019 Some rights reserved. This work is available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 IGO licence (CC BY-NC-SA 3.0 IGO; https://creativecommons.org/licenses/by-nc-sa/3.0/igo). Under the terms of this licence, you may copy, redistribute and adapt the work for non-commercial purposes, provided the work is appropriately cited, as indicated below. In any use of this work, there should be no suggestion that WHO endorses any specific organization, products or services. The use of the WHO logo is not permitted. If you adapt the work, then you must license your work under the same or equivalent Creative Commons licence. If you create a translation of this work, you should add the following disclaimer along with the suggested citation: “This translation was not created by the World Health Organization (WHO). WHO is not responsible for the content or accuracy of this translation. The original English edition shall be the binding and authentic edition”. Any mediation relating to disputes arising under the licence shall be conducted in accordance with the mediation rules of the World Intellectual Property Organization. Suggested citation. HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis. Geneva: World Health Organization; 2019. Licence: CC BY-NC-SA 3.0 IGO. Cataloguing-in-Publication (CIP) data. CIP data are available at http://apps.who.int/iris. Sales, rights and licensing. To purchase WHO publications, see http://apps.who.int/bookorders. To submit requests for commercial use and queries on rights and licensing, see http://www.who.int/about/licensing. Third-party materials. If you wish to reuse material from this work that is attributed to a third party, such as tables, figures or images, it is your responsibility to determine whether permission is needed for that reuse and to obtain permission from the copyright holder. The risk of claims resulting from infringement of any third-party-owned component in the work rests solely with the user. General disclaimers. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of WHO concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted and dashed lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by WHO in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by WHO to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall WHO be liable for damages arising from its use. Cover photo: © WHO Layout by L’IV Com Sàrl Printed in Switzerland HIV MOLECULAR DIAGNOSTICS TOOLKIT TO IMPROVE ACCESS TO VIRAL LOAD TESTING AND INFANT DIAGNOSIS TOOLKIT – JULY 2019 22 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis CONTENTS Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1. Introduction: molecular diagnostics for HIV viral load testing and infant diagnosis . . . . . . . . . . . . . . . . . . . 4 2. Viral load test result utilization to support clinical management of people living with HIV on antiretroviral therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3. Estimated reach of and access to viral load testing using traditional plasma specimens . . . . . . . . . . . . . . . . 10 4. Specimen stability for HIV viral load testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 5. Technical background: molecular testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 6. Alternative specimen types and technologies for consideration when liquid plasma cannot be used widely for viral load testing due to infrastructure, transport or other constraints. . . . . . . . . . . . . . . . . . . . . 18 6.1. Alternative specimen types and technologies: dried blood spot specimens for HIV viral load testing . . . . . . . . . . 18 6.2. Alternative specimen types and technologies: dried plasma spot specimens for HIV viral load testing . . . . . . . . 20 6.3. Alternative specimen types and technologies: plasma preparation tubes for HIV viral load testing . . . . . . . . . . . 21 6.4. Alternative specimen types and technologies: point-of-care and near-point-of-care tools for HIV viral load testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 7. Operational interventions and considerations in scaling up viral load and infant diagnosis . . . . . . . . . . . . 25 7.1. Specimen transport options for molecular diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 7.2. Infant diagnosis and viral load specimen collection bundles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 7.3. Operational interventions: using viral load as a diagnostic for infants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 7.4. Novel point-of-care tools for early infant diagnosis of HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 7.5. Operational interventions: updated considerations for a comprehensive quality management package for point-of-care testing within national health programmes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Annex 1. Infant diagnosis algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 © W H O 3HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis ACKNOWLEDGEMENTS Several key stakeholders have provided significant input throughout the development process of this publication, including the following. – Robert Luo, Kameko Nichols and Neil Parkin – African Society for Laboratory Medicine: Charles Kiyaga and Anafi Mataka – Clinton Health Access Initiative: Paolo Maggiore, Maria Rosezoil Rioja and Jilian Sacks – Elizabeth Glaser Pediatric AIDS Foundation: Jennifer Cohn – United States Agency for International Development: Dianna Edgil, Matthew Wattleworth and Jason Williams – WHO Regional Office for Africa: Fatim Cham Jallow and Fausta Mosha – WHO Essential Medicines and Health Products Programme: Mercedes Perez and Ute Ströher In particular: – The Clinton Health Access Initiative and the United States Agency for International Development supported the development of Section 3. – Robert Luo supported the development of Section 5 and subsection 6.1. – Kameko Nichols supported the development of subsection 7.1. – The Clinton Health Access Initiative supported the development of subsection 7.2. © W H O 44 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 1. INTRODUCTION: MOLECULAR DIAGNOSTICS FOR HIV VIRAL LOAD TESTING AND INFANT DIAGNOSIS Treatment failure monitoring Monitoring people receiving antiretroviral therapy is important to ensure successful treatment, identify adherence problems and determine whether antiretroviral therapy regimens should be switched in case of treatment failure. In 2013, WHO recommended viral load testing as the preferred monitoring approach to diagnose and confirm antiretroviral therapy failure (1) . Compared with clinical or immunological monitoring, viral load provides an early and more accurate indication of treatment failure. Measuring viral load can help to distinguish between drug resistance and non-adherence when coupled with robust enhanced adherence counselling. Further, viral load can serve as a proxy measure for the risk of transmission and effectiveness of prevention interventions at both the individual and population levels. Updated 2016 WHO guidelines recommend routine viral load monitoring be carried out at 6 months, 12 months after initiation of antiretroviral therapy and then every 12 months thereafter if the person is stable on antiretroviral therapy (2) . If viral load is not routinely available, CD4 count and clinical monitoring should be used to assess treatment failure. Further, dried blood spot specimens using venous or capillary whole blood can be used to determine the HIV viral load. A threshold of 1000 copies/mL should be used to determine treatment failure when using dried blood spot specimens, as similarly defined for testing using plasma. Treatment failure is defined by a persistently detectable viral load exceeding 1000 copies/mL (2) : that is, two consecutive viral load measurements within a three-month interval with adherence support between measurements after at least six months of starting a new antiretroviral therapy regimen (Box 1). In addition, viral load may support differentiated service delivery strategies for people living with HIV, including those who are stable on antiretroviral therapy (2) . Stable individuals are defined as those who have received antiretroviral therapy for at least one year and have no adverse drug reactions that require regular monitoring, no current illnesses or pregnancy, are not currently breastfeeding and have good understanding of lifelong adherence and evidence of treatment success (two consecutive viral load measurements below 1000 copies/mL). The package of care for stable individuals can include less frequent clinic visits and medication pickup, community-based care and cessation of CD4 count monitoring if viral load testing is available. Many national guidelines now recommend and are scaling up access to viral load testing for treatment monitoring (Fig. 1). The proportion of yearly viral load tests performed has increased significantly since 2013 (Fig. 2) (5). About 15 million viral load tests were conducted in 2017, and projections suggest that nearly 29 million tests may be performed in 2022. Despite increasing volumes, the total coverage of the demand of viral load testing remained below 60% in 2017. As national viral load test volumes are large and continue to grow, this will add significantly more costs to national testing budgets. Fortunately, several recent pricing commitments have been negotiated to support viral load testing expansion and access (6–8) . Numerous technologies, both laboratory-based and near- point-of-care assays, currently exist to support the scaling up of viral load testing and infant diagnosis. Several additional technologies are being developed (9,10). Box 1. Assessing advanced HIV disease Since CD4 count is the best predictor of disease status and immediate risk of death, it should be used to identify people with advanced HIV disease. Everyone entering or re-entering care should receive a CD4 test at treatment baseline and as clinically indicated for people who are clinically unstable or have symptoms of advanced HIV disease (2,3). Further, it is strongly recommended that people with advanced HIV disease (CD4 count below 200 cells/mm3 or WHO stage 3 or 4) receive a package of care (4). 5HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 0 1,700 3,400850 Kilometers National policy on routine viral load testing for monitoring ART and level of implementation for adults and adolescents in low- and middle-income countries (situation as of July 2019) Fully implemented Partially implemented Not implemented Targeted viral load testing only No policy on viral load testing Data not reported Fast-Track countries High-income countries Not applicable Source: Global AIDS Monitoring (UNAIDS/WHO/UNICEF) and WHO HIV Country Intelligence Tool, 2019 Fig. 1. National policy on routine viral load testing for monitoring ART and level of implementation for adults and adolescents in low- and middle-income countries (situation s of July 2019) Fig. 2. Estimated viral load forecast in low- and middle-income countries globally Source: 2018 CHAI HIV Market Report. 14.7M 17.3M 20.9M 23.6M 26.4M 28.5M 10.2M 9.6M 7.7M 6.7M 5.M 3.9M 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0 M 5 M 10 M 15 M 20 M 25 M 30 M 35 M 2017 2018 2019 2020 2021 2022 Co ve ra ge VL T es ts Viral Load Demand Forecast Forecasted Demand Unmet Need Coverage 66 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Infant diagnosis Infant diagnosis consists of testing throughout the exposure period of HIV-exposed infants. Depending on the age, this can comprise either nucleic acid–based testing or serological testing. More specifically, early infant diagnosis refers specifically to nucleic acid-based testing of infants within two months of birth. See Annex 1 for the infant diagnosis algorithm. Coverage of early infant diagnosis (testing within two months of birth) has remained stagnant in recent years, with about 51% of HIV-exposed infants receiving a nucleic acid test within the first two months of life in 2018 (11). The proportions of HIV-exposed infants tested at nine months or at the end of the exposure period have been difficult to gather. Current forecasts for nucleic acid testing suggest moderate growth and sustained volumes through 2022 (Fig. 3) (5). About 1.4 million infant nucleic acid tests were performed in 2017, with more than 2 million projected to be needed for 2022. Since 2010, several key recommendations have been made to support access to and expanded scale-up of infant diagnosis (2,12). • An indeterminate range should be used to improve the accuracy of all nucleic acid–based infant diagnosis assays (strong recommendation, moderate-quality evidence). • Among infants with an initial positive nucleic acid test result, it is strongly recommended that antiretroviral therapy be started without delay and, at the same time, a second specimen be collected to confirm the initial positive test (strong recommendation, low-quality evidence). • It is strongly recommended that children (18 months or older) with suspected HIV infection or HIV exposure have HIV serological testing performed according to the standard diagnostic HIV algorithm used for adults to determine final diagnosis (strong recommendation, high-quality evidence). • In generalized epidemic settings, infants and children with unknown HIV status who are admitted for inpatient care or attending malnutrition or TB clinics should be routinely tested for HIV (strong recommendation, low- quality evidence). • In generalized epidemic settings, infants and children with unknown HIV status should be offered HIV testing in outpatient or immunization clinics (conditional recommendation, low-quality evidence). • Nucleic acid–testing technologies that are developed and validated for use at or near to the point of care can be used for infant HIV testing (conditional recommendation, low-quality evidence). • Addition of nucleic acid testing at birth to existing infant diagnosis approaches can be considered to identify HIV infection among HIV-exposed infants (conditional recommendation, low-quality evidence). • Consideration should be given to replace serological testing at nine months of age with nucleic acid–based testing. Fig. 3. Estimated infant diagnosis forecast in low- and middle-income countries globally Source: 2018 CHAI HIV Market Report. 1.4M 1.6M 1.7M 1.9M 2.M 2.2M 922K 921K 912K 875K 750K 541K 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 0.0 M 0.5 M 1.0 M 1.5 M 2.0 M 2.5 M 3.0 M 2017 2018 2019 2020 2021 2022 Co ve ra ge EI D T es ts R un EID Demand Forecast Forecasted Demand Unmet Need Coverage 7HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 2. USING VIRAL LOAD TEST RESULTS TO SUPPORT THE CLINICAL MANAGEMENT OF PEOPLE LIVING WITH HIV RECEIVING ANTIRETROVIRAL THERAPY The 2016 WHO consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection (2) provided a strong recommendation for using viral load testing routinely as the preferred antiretroviral drug monitoring tool. WHO recommends viral load testing at six months after initiating antiretroviral therapy, at 12 months and then annually thereafter to enable early detection of treatment failure, prevent drug resistance, identify people with high viral loads with poor adherence and avoid inappropriate switching of treatment regimens (2). In 2014, UNAIDS launched the 90–90–90 treatment targets to be accomplished by 2020, aimed at helping to end the AIDS epidemic as a public health threat. The third 90 aims to ensure that 90% of the people receiving antiretroviral therapy have suppressed viral loads (13). Antiretroviral therapy and treatment adherence provides remarkable and sustained clinical benefits, even among people with advanced HIV disease. Evidence from national viral load dashboards and the population-based HIV impact assessment (14) results suggests that the suppression rates among people living with HIV on antiretroviral therapy are generally 85–92% (Fig. 4). It is important to understand viral suppression rates at the population level to identify potential hot-spots of Fig. 4. Viral load suppression rates in selected countries Suppressed Non-suppressed Lesotho Malawi Swaziland United Republic of Tanzania Zambia Zimbabwe Kenya Uganda 88.3% 90.8% 91.9% 87.7% 89.2% 86.5% 86.4% 92.2% Population based HIV impact assessments, 2015-2017 National viral load dashboards: 2017 85.0% South Africa Namibia 91.3% Cameroon 80.0% Côte d’Ivoire 75.9% 88 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis transmission, inform national targets and provide targeted programmatic quality improvement efforts, but perhaps more importantly, at the patient level to provide enhanced care and support to people living with HIV. Clinically stable people with undetectable viral loads can be provided with differentiated service delivery options that reduce clinic visits and allow for three- or six-monthly drug prescriptions. Further, viral load testing is critical to ensure that those with detectable viral loads greater than 1000 copies/mL are provided enhanced adherence counselling and more closely monitored to determine whether they need to switch to second-line treatment (Fig. 5). The treatment monitoring algorithm is meant to support clinicians and patients in determining whether elevated viral loads or suspicion of treatment failure is caused by drug resistance or poor adherence. It is undesirable for both people living with HIV and programmes to unnecessarily switch people to more expensive and less-well-tolerated second-line regimens when they are simply non-adherent, primarily because adherence issues will not necessarily improve upon switching. However, continuing on a failing drug regimen when the root cause is drug resistance can lead to further drug resistance, additional immune deterioration and possibly clinical effects. Are viral load test results being used to make clinical decisions? Viral load testing has been significantly scaled up in recent years, from 7 million tests in 2013 to 15 million tests in 2017. However, performing viral load tests should not be the main consideration of viral load programmes. Programmes may want to also focus on how viral load test results are used to inform clinical decision-making. Médecins Sans Frontières carried out an in-depth analysis of the execution of key steps in the viral load treatment monitoring algorithm across six countries and 149 clinical sites supported by their programmes (15) . Among people with an elevated initial viral load (18% mean), an average of 68% received at least one enhanced adherence counselling session, 52% received a second follow-up viral load test, 34% re-suppressed (<1000 copies/mL) and 33% of those eligible switched to second-line treatment. These results are further supported by a preliminary analysis of publicly available data published on national dashboards in three countries in eastern Africa (16). Despite Fig. 5. WHO treatment failure monitoring algorithm 9HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis an increase in viral load testing coverage and encouraging viral suppression proportions, less than 10% of the people with an elevated first viral load went through the viral load algorithm to receive a second follow-up viral load test to determine the need for switching to a second-line regimen (Fig. 6). This trend has remained consistent across years. Key considerations Diagnostic tests are not of significant value unless the test results are used clinically. The laboratory–clinical interface may be the most difficult yet the most critical and rewarding mechanism for improving patient management. To create effective health services that provide optimal care and treatment to people living with HIV, programmes must revitalize and invest in the laboratory–clinical interface and ensure that the right training, tools and environment are available to improve the uptake and use of all diagnostic results in a timely manner. Several tools currently exist to better support the clinical uptake of viral load test results and could be adapted and adopted by national programmes (17–19) . Scaling up successful viral load programmes requires using all test results and integrating them into clinical services to optimize patient care and programmatic success. Fig. 6. Viral load tests conducted in three countries in eastern Africa, 2012–2016 VL VL>1000 2nd viral load 1 500 000 1 000 000 500 000 N um be r o f v ira l l oa d te st s 0 2012 2013 2014 2015 2016 10 10 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 3. ESTIMATED REACH OF AND ACCESS TO VIRAL LOAD TESTING USING TRADITIONAL PLASMA SPECIMENS The 2016 WHO consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection (2) recommend viral load as the preferred monitoring approach to diagnose and confirm treatment failure and plasma specimens as the preferred specimen type for viral load testing. Although significant scale-up has occurred across resource-limited countries with a high burden of HIV infection since the initial recommendation in 2013, several significant barriers have limited full access. In particular, the use of liquid plasma using EDTA tubes (see Section 4) can be limited because of strict specimen storage stability times and temperatures, within which the specimens would need to be transported to the testing laboratory or an intermediary hub for processing. Further, there is limited cold-chain availability between many health-care facilities and testing sites in resource-limited settings. However, traditional EDTA plasma specimens have significant potential for viral load testing. Even within the specimen storage stability times and temperatures, many people still have access to viral load using this type of specimen. An analysis was recently conducted to better understand the radius around testing laboratories or intermediary hubs within which people may access to viral load testing using traditional EDTA plasma specimens. The analysis was conducted across four countries: Eswatini, Nigeria, Rwanda and Zimbabwe. Several assumptions were included, such as: • vehicles travelling at 50 km/h; • straight-line measurements from the health-care facility to the testing laboratory or intermediary hub plus a 17% circuity factor; • testing laboratories or intermediary hubs considered as the final point for separating plasma; • the last specimen collected each day had a maximum wait of two hours at the health-care facility before pick-up and transport to reach the testing laboratory or intermediary hub within the stipulated manufacturer storage stability time; and • this analysis does not incorporate alternative plasma specimen types or consider on-site centrifugation and associated specimen storage and transport. Illustrative plasma radius analysis Across the four countries, just under half of all health- care facilities are near enough to the testing laboratory or intermediary hub to transport traditional EDTA plasma specimens within the time stipulated by the manufacturer. This translates to more than 50% or nearly 1 million people Table 1. Access to viral load testing using traditional EDTA plasma Country Access to viral load testing using traditional EDTA plasma Facilities People Eswatini 260/350 (74%) 250 000/320 000 (78%) Nigeria 750/2 600 (29%) 450 000/1 200 000 (38%) Rwanda 505/550 (92%) 148 000/165 000 (90%) Zimbabwe 700/1 500 (47%) 120 000/190 000 (63%) Total 2 215/5 000 (44%) 968 000/1 875 000 (52%) 11HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis across the four countries analysed having access to viral load testing using traditional EDTA plasma. Even in such a geographically large country as Nigeria, nearly 40% of the people receiving antiretroviral therapy and needing viral load testing would have access using traditional EDTA plasma. Geographically smaller countries, such as Eswatini and Rwanda, may have fewer laboratory facilities but can provide access to viral load testing using traditional EDTA plasma specimens to nearly 80% or more of the people accessing antiretroviral therapy. The high access of viral load testing using traditional EDTA plasma is likely because most viral load testing laboratories are in major urban centres. Likewise, the largest antiretroviral therapy centres where people seek care are often also located in major urban centres. This analysis highlights the link in which more than 50% of the people receiving antiretroviral therapy who need viral load testing are within a specimen transport time of a few hours from the testing laboratory or intermediary hub (Fig. 7). Fig. 7. Illustrative example of plasma access radius around testing laboratories in Zimbabwe Conclusions This illustrative analysis provides a snapshot highlighting the potential access to viral load testing using the preferred plasma specimen. The proportion of people who can access viral load testing using traditional EDTA plasma specimens can vary across settings, depending on several factors including the number of laboratories, road infrastructure and size of the country. Efforts should be made and maximized to ensure access to viral load testing using the preferred plasma specimen. It might be helpful to conduct similar in-depth analyses across countries to determine the facilities and people who may be able to access viral load testing using traditional plasma specimens. Current infrastructure may not always enable the use of traditional EDTA plasma in many settings because of poor roads and infrastructure, large distances, ad hoc specimen transport, etc. Therefore, for the facilities and people without access to viral load testing using traditional EDTA plasma, alternatives could be considered to ensure viral load access, including improved infrastructure, specimen transport networks and alternative specimen types and technologies. This molecular diagnostics toolkit will provide background information and data on several of these alternative strategies to ensure a complementary approach to expanding access. 12 12 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 4. SPECIMEN STABILITY FOR HIV VIRAL LOAD TESTING The 2016 WHO consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection (2) recommend viral load as the preferred monitoring approach to diagnose and confirm treatment failure and plasma specimens as the preferred specimen type for viral load testing. Although significant scale-up has occurred across resource-limited countries with a high burden of HIV infection since the initial recommendation in 2013, several significant barriers have limited full access to viral load testing. In particular, using liquid plasma and using ethylenediaminetetraacetic acid (EDTA) or plasma preparation tubes (see subsection 6.1) can be limited because of strict specimen storage stability times, within which the specimens would need to be transported to the testing facility or an intermediary hub for processing. Highlighted in Table 2, these are the maximum times according to the storage temperature stipulated by the manufacturers from whole-blood specimen collection to plasma separation. Extending storage times before processing beyond these recommendations could affect performance and risks providing incorrect results to clinicians and patients. Table 2. Manufacturer-stated whole-blood stability details Assay Maximum time from whole-blood specimen collection to plasma separation Room temperature (temperature) Refrigeration (temperature) Abbott RealTime HIV-1 (20,21) 24 hours (15–30°C) 48 hours (2–8°C) Abbott m-PIMA HIV-1/2 VL (22,23) 48 hours (18–28°C) NR Biocentric Generic HIV Charge Virale (24) 24 hours (2–25°C) 24 hours (2–25°C) bioMérieux NucliSENS EasyQ® HIV-1 (25,26) NR 24 hours (2–8°C) Cavidi ExaVir Load (27) 4–6 hours (no temperature specified) Cepheid Xpert HIV-1 Viral Load (28,29) 8 hours (15–30°C) 72 hours (2–8°C) Hologic Aptima HIV-1 Quant Dx (30,31) 24 hours (2–30°C) 24 hours (2–30°C) Qiagen artus HI Virus-1 RG (32) 6 hours (no temperature specified) Qiagen artus HI Virus-1 QS-RGQ (33) 6 hours (no temperature specified) Roche COBAS TaqMan HIV-1 (34,35) 24 hours (2–25°C) 24 hours (2–25°C) Roche cobas HIV-1 for cobas 4800 System (36) 24 hours (2–25°C) 24 hours (2–25°C) Roche cobas HIV-1 for cobas 6800/8800 Systems (37) 24 hours (2–25°C) 24 hours (2–25°C) Sacace HIV Real-TM Quant Dx (38) NR 12 hours (2–8°C) Siemens VERSANT HIV-1 RNA 1.5 (39) 6 hours (15–25°C) 24 hours (2–8°C) © W H O NR: not reported. 13HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Once whole-blood specimens are separated into plasma, they can be frozen for long periods of time before testing. However, health-care facilities may lack centrifuges, freezers and/or the associated necessary skills to fully leverage these extended stability times after plasma separation. Whole-blood stability for HIV viral load systematic review A systematic review of nine studies entitled “Expanding access to HIV viral load testing: RNA stability in EDTA tubes and plasma preparation tubes beyond current time and temperature thresholds” was published in 2014 (40) .The systematic review highlighted three key findings. • Whole blood and plasma were stable up to 168 hours after specimen collection when refrigerated. • Whole blood was stable up to 72 hours after specimen collection when stored at 25°C. • Plasma was stable up to 48 hours after specimen collection (plasma preparation tubes) or plasma separation (EDTA) when stored at 25°C. Some important limitations to be considered, however, are that all studies included laboratory analyses rather than active realistic storage and transport times and temperatures and all were conducted in the United States or Europe. Further, only a few relevant studies were available for inclusion, and most had small sample sizes. In addition, few studies included samples that had suppressed viral loads (<1000 copies/mL), making the results difficult to interpret within this range. However, a recently published study observed elevated viral load results of undetectable viral load specimens when plasma was stored beyond 72 hours (41) . Interestingly, 20% of the undetectable viral load results became low-level viraemic at any room temperature or refrigeration. Further, 51% of undetectable specimens became low-level viraemic if the plasma was not centrifuged again before testing after 48 hours of storage. Conclusions Fortunately, since the systematic review was published, several manufacturers have now lengthened their room temperature stability intended claims to allow for 24 hours from whole-blood specimen collection to plasma separation. Although the systematic review suggests that specimens are stable beyond the manufacturer intended claims, countries and laboratories would be responsible for viral load test results under such off-label conditions. Additional research and manufacturer support to extending whole-blood stability intended use claims should be encouraged since broader specimen stability would support the expansion of viral load access using the preferred specimen type: plasma. © W H O 14 14 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 5. TECHNICAL BACKGROUND: NUCLEIC ACID– BASED TESTING What is viral load testing? HIV viral load testing is a way to measure the number of viruses present in a blood sample. Whole blood consists of cellular components (white blood cells, red blood cells and platelets) and cell-free plasma. Nucleic acid–based testing is done using a nucleic acid amplification test, which determines the number of copies of HIV per millilitre of plasma. Nucleic acid amplification tests work by amplifying either HIV genetic material or a probe that binds to HIV (42). The test then uses a chemical reaction to measure the amount of amplification seen during the test, which corresponds to the quantity of HIV present in the sample. The most common type of viral load test is a quantitative polymerase chain reaction (qPCR). Other types of viral load testing include transcription-mediated amplification and branched DNA testing (2). Usefulness of viral load testing • HIV viral load monitoring is important to ensure successful antiretroviral therapy. Viral load monitoring is the preferred approach to diagnose and confirm treatment failure (2) . • Viral load testing provides clients with knowledge, control and motivation to understand their HIV infection and adhere to their treatment (43). • Having low or undetectable viral loads reduce the risk of disease progression and HIV transmission (44,45). © W H O 15HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 3. HIV DNA and RNA in whole blood Blood: cellular portion Blood: plasma portion HIV DNA and RNA White blood cells (such as CD4 cells): HIV DNA is contained inside the cells along with copies of HIV that contain HIV RNA during HIV replication. HIV has also been found to be associated with platelets, most likely on the cell surface, although the virus is not found inside platelets or red blood cells. HIV RNA is found in free virus in plasma. HIV DNA should not be present in any significant proportions, although small amounts of DNA may be found in plasma from cells that have broken open or if cells have carried over into the plasma from insufficient separation of whole blood. Sample type Whole blood contains both the cellular component of blood as well as the plasma. Whole blood contains both HIV DNA, intracellular RNA and cell-free RNA and has been used for early infant diagnosis of HIV and HIV drug resistance testing. Plasma is the preferred sample type for viral load testing, which aims to detect the number of copies of HIV RNA per millilitre of plasma. Plasma can also be used for HIV drug resistance testing if adequate HIV RNA (>400 copies/mL) is present. Testing methods Whole blood is tested either in liquid form or from a dried blood spot. Viral load testing using whole blood may be inaccurate if significant quantities of HIV DNA and/ or intracellular RNA are detected by the assay in addition to the cell-free (plasma) RNA the assay is designed to detect. Plasma is typically tested in liquid form but can also be tested from a dried plasma spot. HIV DNA versus HIV RNA HIV is an RNA virus comprising RNA and proteins. During its replication cycle, the genetic material of HIV exists in both RNA and DNA forms. HIV DNA is the genetic material of HIV that is found inside cells of the body infected by HIV. In whole blood, HIV DNA is mostly found inside white blood cells called CD4 cells, which are an important part of the immune system. HIV integrates its DNA into the DNA of the CD4 cells so it can use the cells to make more copies of itself. In this form, it is known as HIV proviral DNA (46–48). HIV RNA is most commonly found in plasma, which is the part of whole blood after removing all of the cells. Whole blood is typically separated into plasma and its cellular components by centrifuging the blood. HIV exists as an RNA virus in plasma before it infects cells, as intracellular RNA inside cells as copies of the virus are being made and in plasma again once these viral copies are released (6–8). When HIV is suppressed by antiretroviral therapy, HIV DNA remains present inside cells and occasionally as intracellular RNA, but little to no HIV RNA can be detected in plasma since the medicines prevent viral replication. However, when HIV is not suppressed, most of the HIV nucleic acid is typically present as HIV RNA in plasma, with additional intracellular RNA from active viral replication, and a smaller proportion present as HIV DNA inside cells (7). How does viral load testing work? Nucleic acid testing for HIV can detect both HIV DNA and RNA that are present in a sample. Some assays have been designed to preferentially detect DNA or RNA, but since HIV DNA and RNA are copies of the same genetic material, they can also be hard to distinguish. However, viral load testing is designed to measure the amount of HIV RNA in plasma. Plasma is therefore the preferred sample type for viral load testing; however, alternative specimen types and technologies exist to support expanded access to viral load testing, including dried blood spots prepared using whole blood (2,49). Dried blood spot specimens can allow for longer transport and storage times; however, using whole blood results often in detecting HIV proviral DNA, intracellular RNA and cell-free RNA. Together, this can result in excessive quantification of viral load results. Table 3 explains the differences between the two major components of whole blood (DNA and RNA) in HIV viral load testing. © W H O 16 16 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Box 2. Timing versus technologies for diagnosing HIV among infants The nucleic acid–based technique (qPCR) used for viral load testing is very similar, often the same, for infant testing or qualitative assays. HIV “DNA PCR” is a commonly used synonym for HIV infant diagnosis testing. However, it is important to distinguish between the technology used for testing (such as PCR versus an HIV antibody test) and the time frame of testing. Early infant diagnosis specifically refers to nucleic acid–based testing at birth or in the first two months of life, whereas infant diagnosis refers to testing during the exposure period including the nine-month nucleic acid test. Infant diagnosis is often done on whole blood, either in liquid form or on a dried blood spot. These assays can detect HIV DNA, intracellular RNA and cell-free RNA. This is not a problem and will even improve the sensitivity of the assay, since the presence of any HIV genetic material in the blood can indicate HIV infection. Since both HIV DNA and RNA are present, virological testing or HIV nucleic acid amplification testing are more accurate terms for infant PCR testing than HIV DNA PCR. Box 3. Key viral load terms Suppressed viral load: viral load measurements below 1000 copies/mL. An unsuppressed or elevated viral load is a measurement above 1000 copies/mL (2). Undetectable viral load: the absence of any HIV found in a blood specimen by viral load testing. Table 4 shows the detection limits of commercially available viral load assays. © W H O 17HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 4. Summary of HIV viral load assays Manufacturer and test name Sample type Limit of detection (copies/mL) Maximum time from whole-blood specimen collection to plasma separation Regulatory approval Early infant diagnosis testing Abbott: RealTime HIV-1 (20,21)1,2 m-PIMATM HIV-1/2 VL (22,23) Plasma DBS Plasma 40 839 800 24 h at 15–30°C, 48 h at 2–8°C 48 h at 18–28°C CE, FDA, WHO CE, WHO CE, WHO Available, separate test Available, separate test Biocentric GENERIC HIV Charge Virale (24) Plasma 390 24 h at 2–25°C CE Available, separate test bioMérieux NucliSENS EasyQ® HIV-1 v2.0 (25,26) Plasma DBS 25 802 24 h at 2–8°C CE, WHO CE, WHO N/A Cavidi ExaVirTM Load (27) Plasma 200 4–6 h, no temperature specified CE N/A Cepheid Xpert® HIV-1 Viral Load (28,29) Plasma 40 8 h at 15–30°C, 24 h at 15–25°C, 72 h at 2–8°C CE, WHO Available, separate test Hologic Aptima™ HIV-1 Quant Dx (30,31) Plasma 30 24 h at 2–30°C CE, FDA, WHO Same Test Qiagen: artus® HI Virus-1 RG (32) artus® HI Virus-1 QS-RGQ (33) Plasma Plasma 60 45 6 h, no temperature specified 6 h, no temperature specified CE CE N/A N/A Roche: COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0 (34,35) cobas® HIV-1 for cobas® 4800 System (36) cobas® HIV-1 for cobas® 6800/8800 Systems (37) Plasma PSC Plasma PSC Plasma PSC 20 738 20 599 13.2 790 24 h at 2–25°C 24 h at 2–25°C 24h at 2-25°C CE, FDA, WHO CE, WHO CE CE, FDA CE Available, Separate Test Same Test Available, Separate Test Sacace HIV Real-TM Quant DX (38) Plasma 48 IU/mL 12 h at 2–8°C CE N/A Siemens VERSANT® HIV-1 RNA 1.5 (39) Plasma 37 6 h at 15–25°C, 24 h at 2–8°C CE N/A 1 Abbott Laboratories (2014). Abbott RealTime HIV-1 Instructions for Use. 2 WHO Prequalification of Diagnostics Programme (2016). Public Report: Abbott RealTime HIV-1. Available at https://www.who.int/diagnostics_laboratory/evaluations/pq-list/hiv- vrl/180423_amended_final_pqpr_0145_027_00_v11.pdf?ua=1 © W H O h: hours; CE: Conformité Européenne, conforming to European Union regulations; FDA: United States Food and Drug Administration approval; WHO: WHO prequalification of in vitro diagnostics; DBS: dried blood spot; PSC: dried plasma spot from a plasma separation card; N/A: not currently available; IU: international units. 18 18 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 6. ALTERNATIVE SPECIMEN TYPES AND TECHNOLOGIES FOR CONSIDERATION WHEN LIQUID PLASMA CANNOT BE USED WIDELY FOR VIRAL LOAD TESTING BECAUSE OF INFRASTRUCTURE, TRANSPORT OR OTHER CONSTRAINTS 6.1 ALTERNATIVE SPECIMEN TYPES AND TECHNOLOGIES: DRIED BLOOD SPOT SPECIMENS FOR HIV VIRAL LOAD TESTING Although plasma specimens are the standard for viral load testing, their use is restricted by the limited ambient temperature stability of viral biomarkers in whole blood and plasma during storage and transport and the limited cold-chain availability between many health-care facilities in resource-limited settings. Dried blood spot specimens for HIV testing are well established in resource-limited settings and have been routinely used for collecting and shipping infant HIV diagnosis specimens for testing by PCR in centralized laboratories. They are beneficial since they do not require centrifuges, refrigerators or freezers at the specimen collection site, can be stored and transported for weeks at ambient temperature and require a simple finger-prick or heel-stick blood specimen that can be prepared by lower cadres of health-care facility staff. Similar benefits could be achieved by using dried blood spot specimens for viral load testing programmes in resource-limited settings. The required storage and shipping conditions may differ when dried blood spot specimens are used for drug resistance testing. Dried blood spot specimens for viral load testing using nucleic acid–based detection methods use whole blood as the input specimen, which can result in extraction and © W H O 19HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 5. Summarized results from technical evaluation meta-analysis Assay Sample size Sensitivity (95% CI)a Specificity (95% CI)a Abbott RealTime HIV-1, one-spotb 700 88.26% (49.64–98.28) 99.07% (68.38–99.98) Abbott RealTime HIV-1, two-spot 2004 93.13% (83.72–97.27) 91.11% (82.35–95.75) Biocentric Generic HIV Charge Virale 531 94.86% (71.14–99.28) 55.16% (35.01–73.75) bioMérieux NucliSENS EasyQ® HIV-1 1062 82.95% (78.38–86.71) 95.06% (89.29–97.80) Hologic Aptima 382 87.52% (77.93–93.30) 87.18% (59.01–96.98) Roche COBAS TaqMan HIV-1 Free Virus Elution 3076 94.77% (84.59–98.36) 93.93% (71.95–98.94) Roche COBAS TaqMan HIV-1 SPEX 3190 98.23% (95.85–99.26) 48.49% (22.63–75.18) Siemens VERSANT HIV-1 RNA 144 90.97% (69.20–97.83) 87.76% (75.28–94.41) a Sensitivity and specificity using a treatment failure threshold of 1000 copies/mL. b As a change notification, a laboratory evaluation of dried blood spot specimens using the CE-marked protocol was not conducted within WHO prequalification review. detection of proviral DNA and intracellular RNA in addition to the primary biomarker target of free viral RNA circulating in the plasma. Together, this may result in excessive quantification of the viral load result. Limited progress has been made in ensuring the quality of using dried blood spot specimens for HIV viral load testing through international regulatory approval. Dried blood spot specimen regulatory approvals and technical evaluations (countries often consider these approvals when procuring or selecting diagnostic technologies): • CE-IVD (Conformité Européenne in vitro diagnostics): two technologies have received CE- IVD for using dried blood spot specimens for viral load testing: Abbott RealTime HIV-1 and bioMérieux NucliSENS EasyQ® HIV-1; and • WHO prequalification: two technologies have met WHO requirements: bioMérieux NucliSENS EasyQ® HIV-1 in January 2017 and Abbott RealTime HIV-1 (21) on 24 August 2017. The limit of detection of the Abbott RealTime HIV-1 assay using dried blood spot specimens is 839 copies/mL (21) . Independent technical evaluations: the results from 40 technical evaluations of dried blood spot specimens across over 25 countries examining six commercially available viral load testing technologies were included in a comprehensive clinical meta-analysis, which resulted in more than 10 000 paired dried blood spot–plasma data points (Table 5) (50). WHO recommendations The 2016 WHO consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection (2) recommend that dried blood spot specimens using venous or capillary whole blood can be used to determine the HIV viral load. A threshold of 1000 copies/mL should be used to determine treatment failure when using dried blood spot specimens, as defined for testing in plasma. Although plasma specimens are preferred for viral load testing, dried blood spot specimens are recommended for use in settings where logistical, infrastructural or operational barriers prevent routine viral load monitoring using plasma specimens. Current use Dried blood spot specimens provide a way to improve the coverage and reach of viral load testing where the preparation and transport of plasma specimens may be limited by cold-chain requirements or transport challenges. Several countries are currently implementing dried blood spot specimens to support viral load access and scale-up. In 2018, more than 2 million viral load tests were run using dried blood spot specimens across six countries with a high burden of HIV infection. Further, some countries have begun implementing the use of DBS specimens for viral load testing using protocols recommended by manufacturers despite its off-label use. © W H O 20 20 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Conclusions Sufficient evidence has been generated on the performance of dried blood spot specimens for viral load testing to support rapid national regulatory approval and initiation of scale-up. Further technical evaluations of these technologies are unlikely to add value but may instead delay implementation and timely treatment monitoring. However, it is essential that suppliers seek regulatory approval and WHO prequalification of such alternative specimen types to support country scale-up and access to viral load testing. 6.2 ALTERNATIVE SPECIMEN TYPES AND TECHNOLOGIES: DRIED PLASMA SPOT SPECIMENS FOR HIV VIRAL LOAD TESTING An additional alternative to using liquid plasma for viral load testing is dried plasma spot specimens. These specimens use the same or similar filter paper as dried blood spot specimens for viral load or infant diagnosis; however, with the application of plasma instead of whole blood. Plasma separation cards and simple devices are also currently in development or recently available on the market to support the expansion of viral load testing using plasma specimens. Dried plasma spot specimens for HIV testing are an alternative specimen type developed similarly to the well- established dried blood spot specimens (subsection 6.1) that have been routinely used for collecting and shipping infant HIV diagnosis specimens for testing by PCR in centralized laboratories. Although they require centrifugation or collection of plasma for spotting on the card, they can be stored and transported for weeks at ambient temperature. An advantage of dried plasma spot specimens is that plasma separation and use removes the detection and quantification of intracellular RNA and proviral DNA often observed with whole-blood specimens; however, the smaller input specimen volume may limit the perfect comparability with liquid plasma specimens. Table 6. Summarized results from technical evaluation meta-analysis Assay Sample size Sensitivity (95% CI)a Specificity (95% CI)a All technologies 1872 92.54% (87.85–95.52%) 95.15% (87.41–98.23%) Abbott RealTime HIV-1 245 99.39% (95.78–99.91%) 85.37% (75.97–91.50%) Biocentric Generic HIV Charge Virale 148 98.12% (56.78–99.95%) 75.00% (46.90–91.06%) bioMérieux NucliSENS EasyQ® HIV-1 173 77.78% (53.53–91.40%) 99.35% (95.57–99.91%) Roche COBAS TaqMan HIV-1 1077 93.05% (87.75–96.16%) 94.90% (78.59–98.95%) a Sensitivity and specificity using a treatment failure threshold of 1000 copies/mL. Typically, plasma prepared for dried plasma spot specimens or plasma separation cards or devices is derived from whole blood taken in EDTA tubes or plasma preparation tubes (see subsection 6.3). Manufacturers should, therefore, include one or both tubes types in their intended use claims and regulatory approval documentation. Most viral load assays currently on the market include one or both tube types. Independent technical evaluations: the results from 17 technical evaluations across 12 countries and looking at four commercially available technologies were included in a comprehensive meta-analysis, which resulted in nearly 2000 paired dried plasma spot–plasma data points (Table 6) (50). The performance of dried plasma spot specimens across all technologies was comparable to using traditional liquid plasma. As expected, since the input specimen type, plasma, was used, limited upward and downward misclassification was observed. WHO recommendations The 2016 WHO consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection (2) recommend viral load as the preferred monitoring approach to diagnose and confirm treatment failure and prefer plasma specimens for viral load testing. A threshold of 1000 copies/mL can be used to determine treatment failure when using any specimens, including dried plasma spot specimens, as defined for testing in plasma. Current use Dried plasma spot specimens provide a way to improve the coverage and reach of viral load testing, where storage and transport of liquid plasma specimens may be limited by cold-chain requirements or transport challenges. However, preparation of dried plasma spot specimens requires centrifugation to separate plasma from whole blood. This can be done either at the point of specimen collection, if feasible, or within a few hours of specimen collection, depending on manufacturer guidelines, by a hub or regional laboratory. 21HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Conclusions Sufficient evidence has been generated on the performance of dried plasma spot specimens for viral load testing to support the initiation of scale-up, if desired within national operational plans to support country scale-up and access to viral load testing. Further technical evaluations of these technologies are unlikely to add value but may instead delay implementation and timely treatment monitoring. However, information focusing on the feasibility and operational best practices of using dried plasma spot specimens within viral load scale-up plans has been limited. 6.3 ALTERNATIVE SPECIMEN TYPES AND TECHNOLOGIES: PLASMA PREPARATION TUBES FOR HIV VIRAL LOAD TESTING The gold standard plasma specimen for viral load testing is generally collected using whole blood in an EDTA (ethylenediaminetetraacetic acid anti-coagulant) tube (purple or lavender cap). As highlighted in Section 4, whole blood in EDTA tubes must be transported and plasma separated within 6–24 hours, depending on the manufacturer. This can be restrictive for many countries and health-care facilities. However, some alternative plasma specimens can be considered. Plasma preparation tubes as well as plasma collected on cards, such as dried plasma spots (subsection 6.2) and plasma separation cards can also be considered to support scale-up. Unlike standard EDTA blood collection tubes, plasma preparation tubes can facilitate simpler handling and storage of plasma for nucleic acid–based testing. Plasma preparation tubes use the same EDTA anticoagulant but contain a gel that separates the plasma from blood cells after centrifugation. After the blood is collected, the plasma preparation tube is spun in a centrifuge within 24 hours and a gel barrier inside the plasma preparation tube separates the plasma from the rest of the whole blood so the plasma can be used for HIV viral load testing. The same plasma specimen volume is used for the viral load assay; therefore, limits of detection are generally synonymous with EDTA plasma. Plasma preparation tube regulatory approvals: • CE-IVD (Conformité Européenne in vitro diagnostics): seven technologies have received CE-IVD for using plasma preparation tubes for viral load testing: Abbott RealTime HIV-1, Cepheid Xpert® HIV-1 Viral Load, Hologic AptimaTM HIV-1 Quant Dx, Roche COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0, Roche cobas® HIV-1 for cobas® 4800, Roche cobas® HIV-1 for cobas® 6800/8800 and Siemens VERSANT® HIV-1 RNA 1.5. • WHO prequalification: four technologies have met WHO requirements: Abbott RealTime HIV-1, Cepheid Xpert® HIV-1 Viral Load, Hologic AptimaTM HIV-1 Quant Dx and Roche COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0. • FDA (United States Food and Drug Administration): four technologies have received FDA approval for using plasma preparation tubes for viral load testing: Abbott RealTime HIV-1, Hologic AptimaTM HIV-1 Quant Dx, Roche COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0 and Roche cobas® HIV-1 for cobas® 6800/8800. Systematic review and best practices: a systematic review was conducted to examine the accuracy of plasma preparation tubes for HIV viral load testing. The review identified 16 peer-reviewed published studies from 1995 to 2014 that compared plasma preparation tubes to standard EDTA blood collection tubes on HIV viral load assays approved by a stringent regulatory authority. Although the earliest studies demonstrated that plasma preparation tubes could be used with no significant differences in viral load results (51–54) , later studies demonstrated elevated viral loads from plasma preparation tubes, especially at viral loads less than 5000 copies/mL (55–57) . The increase in viral load results likely resulted from the leakage of HIV nucleic acids, such as proviral HIV DNA and intracellular RNA present in the cellular component of whole blood, which moved back through the gel barrier into the plasma. Additional studies found that this issue could be resolved by either aliquoting the plasma into a second tube quickly after the initial centrifugation (58–60) or repeating centrifugation after transport of the plasma preparation tubes to the laboratory before aliquoting and testing (61,62). Four published studies evaluated plasma preparation tubes on currently available viral load assays (Abbott RealTime HIV-1 and Roche COBAS AmpliPREP/COBAS TaqMan HIV- 1 Test, v2.0) (63–66). The three studies using the Abbott viral load assay showed no significant change in viral load results regardless of whether the plasma preparation tubes were frozen and thawed or transported after initial centrifugation and before testing. The three studies using a Roche assay found elevated viral load results if the plasma preparation tubes were frozen or transported without a second centrifugation before testing. These viral load results were found to be between zero and several thousand copies/ mL higher than plasma prepared from a standard EDTA collection tube, with the difference being most noticeable for plasma viral loads less than 1000 copies/mL. Consequently, manufacturer instructions recommend an additional centrifugation step before testing using the Roche assay. For both the Abbott and Roche assays, aliquoting the plasma into a secondary tube after initial centrifugation also ensured accurate viral load results (Table 7). 22 22 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 7. Published handling methods for commercially available plasma preparation tubes and viral load assays Product Published plasma preparation tube handling methods providing accurate viral load results Abbott RealTime HIV- 1 (63–65) • Aliquoting plasma into new tube after initial centrifugation • Freezing plasma preparation tubes at –20°C after initial centrifugation and thawing before testing, without the necessity for another centrifugation step • Transporting plasma preparation tubes after initial centrifugation between sites before testing, without the necessity for another centrifugation step Roche COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0 (64–66) • Aliquoting plasma into new tube after initial centrifugation • Repeat centrifugation after transport or freezing of plasma preparation tubes to ensure complete the separation of the cellular and plasma components of blood before testing Note: In the absence of repeat centrifugation after freezing and thawing of plasma preparation tubes or after transport of plasma preparation tubes, some viral load results were observed to be erroneously high. Repeat centrifugation is not necessary if the plasma has already been aliquoted into a new tube before freezing or transport. BD Vacutainer® PPTTM (67) • Centrifuge for at least 10 minutes at 1100 × g at room temperature, within 6 hours of collecting whole blood to prepare plasma. • Follow assay manufacturer instructions for storage and transport: typically plasma preparation tubes can be stored at ambient temperature for one day or refrigerated at 4°C for up to five days; if longer storage is desired, the plasma should be frozen. Table 8. Advantages and challenges associated with plasma preparation tubes Advantages Challenges • Fewer manual sampling handling steps than standard EDTA tubes • Reduced risk of sample contamination and laboratory errors if plasma is not aliquoted into a new tube • Ability to store plasma for longer periods of time than uncentrifuged whole blood, which can facilitate longer transport times to the laboratory • Higher cost of plasma preparation tubes than standard EDTA tubes • Programmatic complexities involving supply chain logistics, staff training and proper implementation of plasma preparation tubes • Centrifuges are required on-site for immediate plasma separation • Primary tube sampling is not always possible • Inaccurate viral load results may be seen if manufacturer-specific instructions are not followed: for example, a repeat centrifugation step may be necessary before testing • Sample bundling currently unavailable All studies evaluated only BD Vacutainer® PPTTM. Additional plasma preparation tubes exist (also referred to as EDTA with gel separate tubes: Grenier (68) or TUD (69) ); however, no studies have been published. Further, other regulatory-approved and/or WHO-prequalified viral load assays (such as the Cepheid Xpert HIV-1 and Hologic Aptima HIV-1 Quant Dx assay) that include plasma preparation tubes in their instructions for use do not provide any further specific guidance on how they should be used (Table 8). Conclusions Plasma preparation tubes allow plasma to be prepared, stored and transported in the same tube used to collect venous whole blood. Plasma preparation tubes provide equivalent viral load results to plasma from standard EDTA tubes if their proper handling is followed according to manufacturer instructions and guidance from independently published studies. Centrifugation of plasma preparation tubes and/or aliquoting of plasma into a separate tube before viral load testing has been shown to prevent spuriously elevated viral load results. However, not all viral load assays have clear instructions or peer- reviewed evaluations published on their use of plasma preparation tubes, and centrifuges (and the associated skills) are necessary at the point of specimen collection. Plasma preparation tubes may be worth considering in settings in which simpler plasma preparation, reduced cross- contamination risk, and the need for longer sample transport times can facilitate the scaling up of viral load testing. © W H O 23HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 6.4 ALTERNATIVE SPECIMEN TYPES AND TECHNOLOGIES: POINT-OF-CARE AND NEAR-POINT-OF-CARE TOOLS FOR HIV VIRAL LOAD TESTING Technologies developed for use at or near the point of care may also be considered for viral load testing. These technologies can be decentralized and used at the point of care. Point-of-care technologies do not require consistent electricity, temperature-controlled rooms or routine calibration, are relatively easy to use, are automated, have no or minimal third-party commodity requirements and can be operated by non-laboratory professionals. Near-point- of-care technologies are similar but may require the use of consistent electricity and/or temperature-controlled rooms. Further, most technologies currently available require plasma specimens. Significant progress has been made in ensuring the quality of new point-of-care viral load technologies. Point-of-care and near-point-of-care viral load regulatory approvals and technical evaluations (countries often consider these approvals when procuring or selecting diagnostic technologies): • CE-IVD (Conformité Européenne in vitro diagnostics): four technologies have received CE-IVD: Abbott™ m-PIMA HIV-1/2 VL, Cepheid Xpert® HIV-1 Viral Load and Diagnostics for the Real World’s SAMBA I HIV-1 Semi-Quantitative Plasma Test and SAMBA II HIV- 1 Semi-Quantitative Plasma Test; and • WHO prequalification: two technologies have met WHO requirements: Abbott™ m-PIMA HIV-1/2 VL (23) and Cepheid Xpert® HIV-1 Viral Load (29)1 received WHO prequalification on 8 April 2019 and 20 July 2017, respectively. The Abbott™ m-PIMA HIV-1/2 VL assay requires 50 µl of venous EDTA plasma and can detect HIV-1 groups M, N, and O and HIV-2. The limit of detection is 800 copies/mL (23) . The Cepheid Xpert® HIV-1 Viral Load assay requires 1 mL of plasma (can be derived from ACD, EDTA or PPT-EDTA blood specimen tubes) and can detect HIV-1 groups M, N, and O. The limit of detection is 40 copies/mL (29) . Additional specifications of these and products in development are available (10,70). Independent technical evaluations: the results from 13 technical field evaluations of the Cepheid Xpert® HIV-1 Viral Load assay were consolidated across 11 countries into a meta-analysis (Table 9) (71). 1 The Cepheid Xpert® HIV-1 Viral Load assay can be used with a variety of Xpert devices at or near the point of care, from the 1-module EDGE to the 16-module Xpert. © W H O 24 24 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 9. Summarized results from WHO prequalification and independent technical evaluations Assay Evaluator Sample type Sample size Sensitivity (95% CI)a Specificity (95% CI)a Abbott™ m-PIMA HIV- 1/2 VLb WHO prequalification/ United States Centers for Disease Control and Prevention Plasma 421 95.1% (91.7–97.5%) (23) 99.4% (96.8–99.9%) (23) Cepheid Xpert® HIV-1 Viral Load WHO prequalification/ United States Centers for Disease Control and Prevention Plasma 439 94.14% (90.37–96.76%) (29) 98.50% (95.68–99.69%) (29) Meta-analysis Plasma 3790 96.47% (95.10–97.47%) (72) 96.59% (92.90–98.39%) (72) a Sensitivity and specificity using a treatment failure threshold of 1000 copies/mL. b No meta-analysis has yet been prepared because of a lack of published independent technical evaluations. Considerations As of 2019, WHO does not have a recommendation for the consideration of point-of-care viral load technologies; however, this will be reviewed in 2020. Considering some of the challenges in scaling up viral load testing, both clinically and logistically, point-of-care viral load testing may support broader access to viral load, deliver results to clinicians and patients more quickly and accelerate decision-making through same-day testing. Box 4. Setting priorities for viral load testing Several population groups could be considered and given priority for point-of-care viral load testing when overall volumes may overwhelm such technologies. • Pregnant and breastfeeding women, especially around the time of delivery, may benefit from faster result delivery and clinical decision-making to prevent mother-to-child transmission. • Infants and other children living with HIV, who typically are at higher risk of treatment failure and drug resistance because of exposure to maternal antiretroviral therapy and postnatal prophylaxis, may benefit from more rapid delivery of results and more attentive treatment monitoring. • Further, people re-entering care, those who for whom treatment failure is suspected and those with advanced HIV disease may benefit from more rapid delivery of results and clinical decision- making. In addition, several point-of-care technologies are also polyvalent or multi-disease technologies capable of testing different conditions using disease-specific tests on the same platform. Significant existing device footprint may allow for programmatic and diagnostic integration to expand access to viral load testing (73). Conclusions Sufficient evidence has been generated on the performance of some point-of-care viral load assays to support rapid national regulatory approval and the initiation of scale- up. Further technical evaluations of these technologies are unlikely to add value but may instead delay implementation. Studies of the impact on patient management and care, operational feasibility, acceptability and cost–effectiveness are ongoing. However, countries need to individually determine the contextual importance, utility and range of point-of-care viral load assays within their patient care and diagnostic networks. 25HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 7. OPERATIONAL INTERVENTIONS AND CONSIDERATIONS IN SCALING UP VIRAL LOAD TESTING AND INFANT DIAGNOSIS 7.1 OPTIONS FOR TRANSPORTING SPECIMENS FOR NUCLEIC ACID–BASED DIAGNOSTICS Laboratories and testing capacity within a diagnostics network are not present onsite at every health-care facility patients attend. The testing and analysis usually offered at centralized laboratories are critical for managing people living with HIV, such as providing viral load testing and infant diagnosis of HIV, but accessing these services can be a challenge. Testing at or closer to the point of care is one solution to address the limitations of the laboratory network (see subsection 6.4), including providing same-day results. However, point-of-care testing is not available at all facilities or may not be cost-effective at health-care facilities with low patient volumes. When testing is not available on site, specimen referral systems can provide access to the diagnostics network by moving specimens from the collection facility (also known as the referring facility) to a facility with the necessary capacity (the testing or referral laboratory). Alternative specimen types, such as dried blood spot specimens, can also be used to further increase access. Moving the specimen removes the burden of people living with HIV having to travel to the laboratory for testing. In this way, the specimen referral network extends the reach and coverage of the diagnostics network. The same system for referring specimens is also often used for returning paper results, which may be sent even if electronic results are available. Various specimen referral systems can be found at different levels of a tiered health system, in different regions of a country and across disease programmes. Together, these systems should be harmonized, connected and efficiently coordinated to form the overall specimen referral network, which, in turn, is a vital part of a diagnostics network. A specimen referral system or network has five main goals (Box 5). A specimen referral system comprises various components that are critical to ensure a successful and efficient system (Fig. 8): • management and leadership – ideally, someone in the health ministry should supervise the overall referral Box 5. Five main goals of a specimen referral system • Contribute to increased access to diagnostics where on-site services do not exist by referring the specimen to the testing laboratory • Maintain and improve the quality of specimens delivered to the testing laboratory by proper specimen management in transit, including cold- chain requirements • Ensure the safety and security of all individuals and the environment involved with specimen referrals by proper management in transit, including packaging and handling • Meet the timeliness requirements of the specimen reaching the testing laboratory and the paper result reaching the facility, clinician, patient and necessary files • Enhance the cost-efficiency of the diagnostic network through harmonization and coordination MANAGEMENT AND LEADERSHIP DATA SYSTEMS AND MONITORING AND EVALUATION EQUIPMENT LOGISTICS HUMAN RESOURCES FINANCING TRANSPORT Fig. 8. Components to ensure a successful and efficient specimen referral system 26 26 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis network, ensure that it is supporting the needs of the diagnostics network and advocate for necessary resources throughout the network; • human resources – these are the personnel at the referring facilities, referral laboratories, regional health teams, transporters, etc. that are involved in the entire referral process and returning results; • financing – the funding that is necessary for the overall referral network, incorporating all aspects of the network as well as specimen types and disease areas; • transport – this includes the type of vehicle (motorcycle or four-wheeled vehicle) and service provider (professional courier or clinical implementing partner) and ideal combinations thereof to service all facilities as necessary; • logistics – this includes the overall logistical system, such as scheduling and routing and depends on many factors, such as timeliness requirements for managing specimens and returning the results; • equipment – such as necessary packaging materials, contributes to specimen quality and biosafety during referrals; and • data systems and monitoring and evaluation – the system to collect data, analyse them and use the analysis for decision-making and continual quality improvement. Three key components of the specimen referral system can be challenging and may require additional consideration and focus: transport, logistics and data systems and monitoring and evaluation. Design of transport and logistics systems is closely related and may or may not be managed by the same organization or company. Although these components are only two in the overall system, they require additional technical expertise that is usually not a core capacity of health-care facility or laboratory staff. Three considerations for these important components include: • Type of vehicle. The type of vehicles used depends on resources, distances, terrain and carrying capacity. If the transport and logistics systems are outsourced, the service provider will likely decide the vehicle type. Examples include (listed by highest to lowest prevalence): motorcycles, four-wheeled vehicles, bicycles, boats, horses, on foot, airplanes and unmanned aerial vehicles (also known as drones). Key considerations when choosing a vehicle type include: – specimen types (dried blood spot specimens, whole blood, plasma, etc.) and requirements per test (cold chain or specimens tested for highly contagious diseases may require additional packaging, which may not fit on or be suitable for certain vehicle types such as drones); – level or tier of system used, distance travelled and type of terrain covered; and – demand and volumes at the referring facilities to understand the carrying capacity required. • Transport service provider. The service provider, which could be the health ministry, an implementing partner or a private company, is the one who operates the transport and usually employs the vehicle operators. When choosing a service provider, key considerations include: – paying the provider and the sustainability of the system; – the availability of local private sector transporters or third-party logistics providers, such as Riders for Health, DHL, G4Sor the national postal service, and the ability to contract with a third party logistics provider; – which entity will manage, own and operate the vehicles: health-care facility ownership versus provided to the facility through the health ministry or vehicles belonging to the government, partners or a private company; – which entity will manage and employ the vehicle operators (rider, driver, etc.); – specimens accompanies by a person during transit; and – dedicating the system solely to transporting specimens and results: vehicles such as ambulances, whose primary purpose is not specimen transport, should not be the only mode of transport of specimens available and used. • Logistics, scheduling and routing. At its core, a specimen referral system is a logistics system. Specimens need to be moved physically from collection points to first-line diagnostic testing sites or hubs and then possibly specialized testing sites and the results returned in reverse. Key logistics considerations include the following. – Health facilities, collection points, hubs and referral laboratories should be mapped using geocodes. Then current referral needs, links and pathways for each specimen type should be mapped out, based on testing algorithms and capacity and between each relevant level or tier of the health system, including the community or health post level, if considered. The mapping exercise should be redone when the overall diagnostics network changes, such as further decentralization of equipment or integration. – Consider whether to have a fixed schedule for pick- ups and returning results versus on-demand services. – Frequencies of pick-up should be based on patient volumes and need, specimen collection, specimen type, specimen stability and machine capability at the testing laboratory. For example, whole-blood and plasma specimens require same-day, rapid transport and storage at the correct temperature. 27HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Box 6. Considerations for data systems and monitoring and evaluation This is a critical component to the systems and network but often weak and overlooked. A standardized monitoring and evaluation framework for specimen transport is required to assess and compare the performance of the often fragmented systems. Key considerations for this component include the following. • The monitoring and evaluation framework and standardized indicators should be based on the five goals of a specimen referral system (Box 5) and included in the national specimen referral guidelines. • Data collection tools should be in place or introduced, including registers and logbooks, chain-of-custody forms (showing every time a specimen or result changes hands), transport logs, reporting forms, questions within supervisory checklists, etc. • Indicators may be aspirational, but once the specimen transport network and necessary data systems are in place, the feasibility of collecting each should be assessed. • Reporting processes should be outlined and feedback mechanisms used. • Detailed turnaround time is important to collect, including each step between the collection of the specimen from the patient to the time the result is filed in the patient’s records. • Continual quality improvement should be emphasized, including corrective actions. © W H O 28 28 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis – In a hub-and-spoke system, the hub may be a testing facility and/or a facility to consolidate and store specimens on the way to a higher level versus point to point, in which the specimen goes directly from the referring facility to the testing laboratory without consolidation at a hub on the way. For viral load testing using plasma specimens, a hub system should be set up in which each hub is equipped with refrigerators, freezers and centrifuges to process the specimens and ensure the integrity of the specimens. – Consider the ability of the referring facility or hub to prepare and store specimens. – Consider the ability to reconsider administrative boundaries if it is more logistically efficient: whether a specimen can be referred to a laboratory in a different administrative region if it is closer than the pre-assigned laboratory. – Can the system be integrated with other types of specimens? – Is the delivery of paper results included, if necessary? – What are the cut-off times for specimen reception (time by which specimens need to reach the hub for further processing or storage or laboratory) and the earliest arrival and pick-up time at the referring facilities? – For shared (non-dedicated) vehicles, schedules need to be carefully planned to not disrupt other activities. Best practices. Although there are many ways to design, implement and monitor a specimen referral system, countries are currently adopting key best practices, including the following. • Management: the health ministry must lead, coordinate and supervise the overall specimen referral network, regardless of the transport mechanism used or funding. • National guidelines are developed for specimen referrals as well as a laboratory handbook, which describes individual procedures for collection, packaging, storage and transport depending on the specimen type and test requested. • Monitoring: a robust monitoring and evaluation framework should include standardized indicators. • Network approach: the design of the specimen referral network must work within the diagnostics network and be optimized periodically to improve efficiency and costs. • Specimen types should be integrated with disease programme activities, where this is possible and logistically efficient. • Transport and referral procedures must be well documented for each specimen type and all personnel at all levels properly trained, including: specimen collection, storage, documentation, packaging and dispatch, transport, specimen receipt, results dispatch and results receipt. • Biosafety and quality: provide appropriate personal protective equipment, spill kits and proper packaging materials, including safe and secure shipping containers as necessary for each specimen type. Integration. The diagnostics network can be integrated to use a specimen referral system and network for multiple specimen types or disease programmes. In this case, usually integration is easiest and most efficient from the referring facility to the first hub or first-line diagnostic location. The overall national specimen referral network should always be fully integrated, meaning that it should cater for all specimen types and diseases. However, the transport and logistics necessary to achieve this integration may require incorporating some separate systems for certain tests or specimens based on routing and/or laboratory locations and specimen management requirements. For example, the nucleic acid–based laboratory for HIV testing may differ from the tuberculosis culture laboratory, and so separate routes and logistics may be necessary for specific aspects of the specimen transport networks. Further, depending on the specimen type and other factors, the transport mechanisms used on each route may differ. For example, for specimens that require very timely, same-day transport, or require temperature controls, public transport with no control over timeliness or temperatures may not be appropriate. If there is an outbreak investigation, the specimens may not be able to wait for and use the routine transport mechanism. Transport and logistics service provider options. The national specimen referral network will likely include a combination of the options listed in Table 10 depending on the level of the health system and local geography of a region. Box 7. Links to tools and resources for specimen referrals • Global Laboratory Initiative (GLI) Specimen Referral Toolkit. Geneva: Stop TB Partnership; 2019 (http://www.stoptb.org/wg/gli/srt.asp). • GLI guide to TB specimen referral systems and integrated networks. Geneva: Stop TB Partnership; 2019 (http://www.stoptb.org/wg/gli/assets/ documents/GLI_Guide_specimens_web_ready. pdf). • Guidance for developing a specimen transport and referral system for viral load and infant virologic HIV diagnosis testing networks. Addis Ababa: African Society for Laboratory Medicine; 2015 (http://www.aslm.org/?wpdmdl=18275). 29HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 10. Options for selecting transport and logistics service providers Se lf- ru n – op er at ed b y th e he al th m in is tr y di re ct ly o r a cl in ic al im pl em en ti ng p ar tn er ; a ll ca n ea si ly ca rr y re su lt s or o th er s up pl ie s fo r no a dd it io na l c os t Type or example Benefits Challenges Best-use case Dedicated health ministry courier system Likely share existing health ministry resources, such as staffing, to run and manage the system to save on the overall costs required Transport and logistics expertise is generally not a core competency within the health ministry Use in countries with high referral volumes where outsourcing is difficult and health ministry capacity to manage a complex transport and logistics network is high Dedicated partner- run courier system Will share some existing partner resources, such as staffing, to run and manage the system to save on the overall costs required Transport and logistics expertise is generally not a core competency – to run these systems, additional staff must be hired just for this one system, which is not cost- effective Use in countries with high referral volumes where outsourcing is difficult and health ministry capacity to manage a complex transport and logistics network is low Hand-carried by facility staff Often carried out by laboratory staff so biosafety and quality control are well understood Takes limited staff out of the health facility and away from their main responsibilities; more expensive than sending a package on its own Use where specimen referral volumes are very low and erratic Use of non- dedicated health ministry vehicles Used by programme officials to conduct supervisory visits and to deliver supplies and commodities. Some programmes have also used the vehicles to transport specimens and results Often do not visit the collection sites frequently enough for timely transport; with shared priorities, specimens are not always transported in a timely and quality-controlled manner; the use of ambulances is not recommended, since this form of transport is unpredictable and interferes with regular duties Use for health posts or facilities that only collect specimens when an outreach health team is visiting, since they can bring back the specimens with them to the laboratory Use of public transport, not accompanied (such as buses, trains, boat and aircraft) Play a major role in both rural and urban transport with extensive nationwide access and coverage; used by private courier companies and national postal systems to send letters, packages and money; less expensive to send a package unaccompanied than with a facility staff member Usually have to bring packages to depot; special permission may be needed to transport potentially infectious material; schedules may not be adhered to strictly; specimens and test results may not be properly handled due to lack of training, limited personnel and lack of clear roles and responsibilities; may not have a system in place to track specimens Use where there are reputable bus companies with regular schedules, professional staff and a central depot where health facility staff can collect and dispatch specimens O ut so ur ce d – al l h av e lo gi st ic s ex pe rt is e an d w ill m an ag e tr an sp or t Dedicated professional courier (NGO, social enterprise, private), such as Riders for Health Ability to design a dedicated system including in hard-to- reach or underserved areas; result return or carrying other supplies on scheduled routes at no additional charge Total costs may appear to be higher since the system is all-inclusive (includes vehicles, transport, drivers and riders, operating costs, etc.) and run by a third-party (resources, such as health ministry or partner staffing, will not be shared but will be at an additional cost) Use in countries with limited or undeveloped road infrastructure and transport providers Non-dedicated private professional courier, such as FedEx or DHL Specialize in collecting and delivering packages, on- demand or regularly scheduled pick-ups, documentation and tracking of shipments Not all are able or willing to transport potentially infectious biological specimens; costs may be higher; coverage and flexibility may be limited; may not be a cost-effective way to return results Use where speed, security, documentation, tracking, name and signature of receiving person, specialization and individualization of express services are sufficiently important to warrant the extra cost; best coverage in major cities National postal service, non- dedicated courier (public or semi- private) Usually a parastatal entity, which may be easier for the health ministry to contract with than a private courier; mandate to be present across an entire country; typically on a predictable schedule Availability of and accessibility to local post offices; adherence to schedules; specimens requiring strict transit time or careful temperature control may be challenging unless a guaranteed service is offered (such as express mail) Use where the national postal system is strong and has good coverage; otherwise, use only for less stringent and longer shelf-life specimens such as dried blood spot specimens 30 30 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 7.2 INFANT DIAGNOSIS AND VIRAL LOAD SPECIMEN COLLECTION BUNDLES More than 10 individual commodities are required for collecting whole-blood specimens for plasma separation or dried blood spot specimens from patients for nucleic acid–based testing (either diagnosis or viral load). Some of these items, such as the dried blood spot filter paper collection cards used to collect blood specimens, are specialized and only recommended from specific suppliers. Other items, such as gauze and alcohol swabs, are generic. In the early stages of establishing infant diagnosis testing programmes, countries needed to procure these items individually, which made ordering and facility distribution a complex endeavour. Further, stock-outs of any single item could compromise the quality of specimens or prevent the collection and/or processing of blood specimens altogether. Drawing from the experience with infant diagnosis, for ease of procurement and distribution and to ensure the quality of commodities, suppliers have developed plasma and dried blood spot specimen collection bundles for viral load testing as well. Plasma and dried blood spot specimen collection bundles contain individual and single-use collection kits that include all required items and commodities to draw, dry (for dried blood spot specimens) and transport a specimen from the facility to the laboratory. Contents of specimen collection bundles Table 11 lists the items included in single-use EDTA blood collection kits (100 tests per bundle) to obtain a plasma specimen for viral load testing using venepuncture. These specimens can then either (1) be shipped directly to the testing laboratory for processing (centrifugation) into plasma and tested or (2) be separated into plasma by centrifugation at the health-care facility and transferred to another tube, which is then sent to the laboratory under appropriate storage conditions for testing. Table 12 lists the items included in single-use DBS specimen collection kits with perforated dried blood spot cards (20 or 50 tests per bundle) that can be used for both infant diagnosis and viral load testing. Table 11. Whole-blood and plasma specimen collection bundles No. Item Quantity Specifications 1 Swab alcohol, 70% isopropyl 1 Swab alcohol WBCL 2 Swab gauze 8 ply non-sterile 10 × 10cm 1 Swab gauze 8 ply non-sterile 100 × 100mm 3 Gloves examination latex powder free 2 Gloves examination latex, powder free, medium 4 Bandage fabric 1 Bandage fabric 5 Bag autoclave clear biohazard 415 × 600 mm (1 per bundle) 1 Bag autoclavable clear print biohazard 415 × 600 mm 6 5 mL EDTA-treated evacuated tube 1 Tube 5 mL K2EDTA lavender 13 × 100 mm 7 Vacuum tube needle holder 1 Speedy quick release holder 8 Vacuum tube needle, 20G 1 Vacuum multiple use draw needle 21G × 1.5" 38 × 0.8 mm Green Sterile 9 Tourniquet (one per bundle) 1 Tourniquet disposable without clip, latex-free, synthetic rubber band, non-sterile 10 Packing box 1 Box plain white with liner 385 × 310 × 145mm 11 Pasteur transfer pipette (optional)a 1 Pasteur transfer pipette 1 mL fine tip, individual sterile pack (can be requested at additional cost) a Used to transfer the plasma aliquot after centrifugation and before transport to the laboratory. 31HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 12. DBS specimen collection bundles No. Item Quantity Infant diagnosis Viral load Specifications 1 DBS collection instructions 1 DBS collection instructions card 2 Powder-free gloves 2 Examination gloves powder free latex – medium 3 Alcohol gauze pad 2 Single-use individually wrapped alcohol-impregnated medical gauze swab 4 Lancets 1 Single-use retractable lancet with non-adjustable 2 mm penetration depth blade (not needle-type) 5 Gauze swab 1 Swab gauze non-sterile 8 ply 50 mm × 50 mm 6 EDTA capillary tube 1 100 µl EDTA capillary tube, plastic, with 70 µl markings 7 DBS Filter paper S&S 903 1 Whatman 903 card or Munktell TNF, perforated 8 Drying rack for DBS card 1 Drying rack for Whatman 903 card 9 Silica desiccant pack 3 Indicating silica gel 1-gram sachet 10 Plastic bags 1 Low gas permeable double Ziploc bag (150 mm × 180 mm) with white write-on area 11 Packing and repacking 1 Packing and repacking: five pieces per bundle 12 Packing box 1 Buff board box to contain all bundle contents, with tuck-in lid 13 Lab requisition form (optional) 1 Customized early infant diagnosis/viral load requisition form in a pad of 50s or 100s in duplicate copies 14 Barcode stickers (optional) 1 Customized early infant diagnosis/viral load barcode stickers Source: M. Rioja, Clinton Health Access Initiative. 32 32 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis A specific dried blood spot specimen collection bundle for viral load testing contains similar items, with the addition of an EDTA microcapillary tube added (Fig. 8). The EDTA microcapillary tube serves to ensure that the required volume per spot is accurately collected. Viral load testing is a quantitative measure that heavily relies on the quantity of blood inputted into the assay, regardless of specimen type. Limited evidence suggests that free drops of blood applied directly to a dried blood spot card can produce accurate test results compared with plasma specimens. Therefore, using a fixed volume or graduated measuring microcapillary tube or pipette may support increased accuracy of dried blood spot specimen preparation. Health-care workers should be appropriately trained on the differences in the specimen collection technique and process for infant diagnosis and viral load dried blood spots. Source: M. Rioja, Clinton Health Access Initiative. What are the expected benefits of using sample collection bundles? The following are benefits of specimen collection bundles for a national programme: • simpler and more standardized forecasting, procurement and supply chain management (instead of ordering individual items from various manufacturers); • ensured availability of necessary items in the correct proportions and reduced waste; • simpler and more rapid scaling up of testing services at new sites since all materials to train and test are packaged together; and • bundles result in lower costs for all components compared with individual itemized procurement. The following are benefits of specimen collection bundles for health facilities: • quality assurance of items in bundles if the supplier has a proven track record, which is especially critical for certain items that must adhere strictly to quality standards (such as lancets and powder-free gloves) to ensure that proper care is delivered to the patient and that the specimen is prepared correctly (thus mitigating the risk of the specimen being rejected at the laboratory) and the safety of the end-user ensured; • reduced risk or repurposing of items such as gloves for other services, thereby reducing stock-outs and waste of individual items; • easier sharing or distribution of individually packed single-use bundles to satellite sites with lower patient testing demand; • simplified stock audit, monitoring, inventory and distribution of supplies; and • simplified workflow in the clinic because of individually packed single-use bundles, enabling health-care workers to reach into the box and grab one bag that has everything they need to collect a specimen. Which suppliers offer specimen collection bundles? Such bundles are readily available for procurement through at least two suppliers that source items and components directly from individual manufacturers: • LASEC (https://www.lasec.com/diagnostics); and • LabMate (https://www.labmate.co.za). Conclusion By ensuring that all items needed are available to health- care workers or laboratory technicians in a single kit or box, bundled products for nucleic acid–based test sampling have simplified and standardized the supply chain for such commodities and reduced the occurrence of testing delays resulting from the stock-out or misappropriation of a single item. Many countries have become experienced in using these bundles and, as a result, have less waste and order lower buffer stock. Although the bundles themselves are a cost-effective alternative to bulk individual commodity purchasing, the significant reductions in waste contribute to additional cost savings for countries. Further, the use of bundled specimen collection products has contributed significantly to the scaling up of infant diagnosis and viral load testing services in several resource-limited countries. 33HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 7.3 OPERATIONAL INTERVENTIONS: USING VIRAL LOAD TESTING TO DIAGNOSE INFANTS Infant diagnosis testing has expanded considerably in the past decade in low- and middle-income settings, but access remains limited. In 2017, only 51% of HIV-exposed infants received an early infant diagnostic test within the first two months of life (11), as recommended by WHO (2) . Several challenges have limited the scaling up of this critical test for a highly vulnerable population. Infant diagnosis has primarily been offered at centralized testing laboratories, requiring transport of dried blood spot specimens that can often take weeks and often months before the results are returned to clinicians and caregivers for clinical action. The delays can be caused by several issues, including: • the need to batch infant specimens until a full run can be performed, to ensure that testing is cost-effective and cost-saving; • low infant diagnosis volumes limit the number of devices and laboratories capable of testing, which can be far from health-care facilities, and create a challenging procurement environment that has often lead to stock- outs of reagents in the laboratories; • in the past, and sometimes still, infant diagnosis reagents can be more expensive than other HIV nucleic acid–based tests, such as viral load; and • since this type of specimen, dried blood spots, is also often used for viral load testing and infant volumes are as low as a few needed tests per month, specimen collection materials have and can be reappropriated for viral load specimen collection, occasionally resulting in stock-outs when an infant specimen may be needed. Qualitative infant diagnosis assays have primarily been used to diagnose HIV among HIV-exposed infants in resource-limited settings. The nucleic acid–based technique (quantitative PCR) used for viral load testing is very similar, often the same, for infant testing or qualitative assays. HIV DNA PCR is a commonly used synonym for HIV infant diagnosis testing; however, several technologies currently on the market do not specifically target HIV DNA. The primary specimen type for nucleic acid–based infant diagnosis is whole blood, which can contain proviral DNA, intracellular RNA and extracellular RNA. Similarly to when using whole-blood dried blood spot specimens for viral load testing (see subsection 6.1), whole blood for qualitative infant diagnosis assays generally results in the detection of the variety of HIV nucleic acids. Since both HIV DNA and RNA are present, virological testing or HIV nucleic acid amplification testing are now more accurate terms for infant PCR testing than HIV DNA PCR. Current considerations The 2010 WHO recommendations on the diagnosis of HIV infection in infants and children (74) and 2016 WHO consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection (2) recommend that virological testing to diagnose HIV infection among infants be performed using HIV DNA PCR on whole-blood specimens or dried blood spot specimens, HIV RNA PCR on plasma or dried blood spot or ultrasensitive p24 antigen on plasma or dried blood spot. Further, guidelines in high- income countries, including guidelines in the United States of America (75) , recommend HIV RNA testing to diagnose HIV infection among infants. Existing research has suggested that HIV RNA (often quantitative) testing may yield comparable results to assays specifically detecting DNA (76–79). However, questions remain about the technical and clinical feasibility of using RNA and/or quantitative testing for diagnosis given the increased maternal and infant exposure to antiretroviral therapy through programmes to prevent the mother- to-child transmission of HIV, option B+ and “treat all” policies, since all previous studies were conducted before 2003 and the option B+ era. Updated data Two studies have recently been conducted to better understand the performance and potential role of using HIV quantitative (viral load) assays using dried blood spot specimens for diagnosing HIV among infants younger than 18 months (80,81) . These studies were conducted in current settings with high rates of maternal and infant exposure to drugs. In Mozambique, 95% of mothers and infants were on antiretroviral therapy or receiving antiretroviral prophylaxis, respectively. While in Uganda, 75% of mothers were receiving antiretroviral therapy and 65% of infants were receiving antiretroviral prophylaxis. In the study conducted in Mozambique, the sensitivity and specificity of using the viral load assay to detect infection were 100.0% and 99.9%, respectively. The positive and negative predictive values were 99% (95% CI: 94.3– 100.0%) and 100% (95% CI: 99.6–100.0%). In the study conducted in Uganda, the sensitivity and specificity of using the viral load assay to detect infection were 98.9% and 98.8%, respectively. One key consideration in both studies was that the dried blood spot specimens were prepared using the buffer and specimen preparation techniques traditionally used to prepare infant diagnosis specimens. Current WHO recommendations and these data further indicate that viral load can be used as a diagnostic assay for infants. In fact, some technologies specifically aim to 34 34 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis Table 13. Sensitivity and specificity of viral load assays in Mozambique Study setting Sample type Sample size Sensitivity (95% CI)a Specificity (95% CI)a Mozambique (80) Plasma 1021 100% (96.2–100.0%) 99.9% (99.4–100.0%) Uganda (81) Plasma 520 98.9% (96.7–99.6%) 98.8% (96.6–99.6%) a Sensitivity and specificity using a treatment failure threshold of 1000 copies/mL. target only HIV RNA and yet have been shown to have high sensitivity and specificity, comparable to gold standard technologies, and achieved WHO prequalification. Although some manufacturers have already sought dual claims, it would be ideal for manufacturers to seek regulatory approval within their current and/or future intended use claims for their viral load assays, to support implementation of this technique. Programmatic considerations There are potentially some advantages to using viral load or dual claim assays as a diagnostic assay for infants, including: • optimizing laboratory work flow, where infant diagnosis and viral load samples could be batched together, reducing the need to wait for full infant diagnosis batches; • reducing the risk of giving lower priority to infant diagnosis at the facility and laboratory levels as the viral load programmes scale up; • streamlining forecasting and quantification for infant diagnosis and viral load testing; • simplifying procurement, supply chain management and distribution of infant diagnosis and viral load specimen collection commodities; • saving money resulting from price parity between viral load and infant diagnosis tests and increased efficiency of laboratory operations and procurement processes; and • improving care, since a viral load result could be provided for an infant living with HIV at the time of diagnosis. Conclusions Creating more efficient, streamlined and clinically supportive diagnostic systems is critical to improving care. Using viral load assays with a validated dual intended use claim to also support infant diagnosis should be considered to alleviate some of the current challenges and improve infant diagnosis. 7.4 NOVEL POINT-OF-CARE TOOLS FOR EARLY INFANT DIAGNOSIS OF HIV A decade of investment in conventional laboratory networks has expanded access to early infant diagnosis testing, but only 51% of HIV-exposed infants were tested for HIV infection before two months of age in 2015 (82). The advent of point-of-care early infant diagnosis technologies (10) is a breakthrough that creates the opportunity to increase coverage of early infant diagnosis testing. It will enable same-day test results, enable treatment to be initiated earlier and address some of the key limitations of conventional early infant diagnosis networks – especially long turnaround times for tests and high rates of loss to follow-up. Significant progress has been made in ensuring the quality of new point-of-care early infant diagnosis technologies. The following regulatory approvals and technical evaluations have been made for point-of-care early infant diagnosis (countries often consider these approvals when procuring diagnostic technologies): • CE-IVD (Conformité Européene in vitro diagnostics). Four point-of-care early infant diagnosis technologies have received CE-IVD: AlereTMq HIV-1/2 Detect, Cepheid Xpert®HIV-1 Qual and Diagnostics for the Real World’s SAMBA I HIV-1 Qual Test and SAMBA II HIV-1 Qual Whole Blood Test. • WHO prequalification: Two point-of-care early infant diagnosis technologies have met WHO requirements: AlereTMq HIV 1/2 Detect (83) and Cepheid Xpert®HIV-1 Qual (84) received WHO prequalification on 13 June 2016. Independent technical evaluations: the Point-of-care Early Infant Diagnosis Consortium comprised a group of principal investigators across six countries conducting technical 35HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis field evaluations of point-of-care early infant diagnosis technologies to expedite the release of independent performance data to accelerate national approval processes and in-country implementation. The results from nine technical field evaluations were consolidated across the six countries (Table 14). A total of 3383 specimens were tested using the AlereTMq HIV-1/2 Detect, and 4401 specimens were tested using the Cepheid Xpert®HIV-1 Qual (85). WHO recommendations The 2016 WHO consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection (2) recommend that nucleic acid testing technologies that are developed and validated for use at or near the point of care can be used for early infant HIV testing. Point-of-care early infant diagnosis provides the opportunity to reduce test turnaround times, limit patient loss along the HIV testing cascade, reduce infant mortality and enable task shifting to lower cadres of health-care workers at decentralized facilities (2). Current use Several countries are implementing point-of-care early infant diagnosis technologies. For example, Malawi, Mozambique and South Africa reported results from point- of-care early infant diagnosis pilot projects in 2016 showing significantly shorter test turnaround times for results and increased rates of initiation of antiretroviral therapy compared with conventional laboratory systems (86–88) . Considering the high and early mortality rate of untreated infants living with HIV (89,90), point-of-care early infant diagnosis could also reduce observed infant mortality. Based on the CE-IVD and WHO PQ approvals, robust results from independent technical field evaluations, procurement eligibility, the WHO recommendation for the use of point- of-care early infant diagnosis and initial results on patient impact from implementation pilots, countries should begin planning to implement point-of-care early infant diagnosis Table 14. Technical evaluations of point-of-care early infant diagnosis technologies Study setting Sample type Sample size Sensitivity (95% CI) Specificity (95% CI) AlereTMq HIV-1/2 Detect WHO PQ CDC/NHLS Whole blood 98.67% (95.27– 99.84%) 100.00% (97.59– 100.00%) Early Infant Diagnosis Consortium Whole blood 99.00% (96.45– 99.88%) 99.97% (99.83– 100.00%) Cepheid Xpert® HIV-1 Qual WHO PQ CDC/NHLS Whole blood 98.86% (93.83– 99.97%) 100.00% (97.55– 100.00%) Early Infant Diagnosis Consortium Whole blood 96.79% (92.68– 98.95%) 99.91% (99.76– 99.97%) WHO PQ CDC/NHLS Dried blood spots 99.34% (96.40– 100.00%) 100.00% (97.60– 100.00%) by incorporating it into national HIV care and treatment guidelines, national strategic plans, PEPFAR country operational plans, grant applications to the Global Fund to Fight AIDS, Tuberculosis and Malaria and HIV programme budgets. Conclusions Sufficient evidence has been generated on the performance of these assays in the intended field settings to support rapid national regulatory approval and initiation of scale- up. Performance was consistent between laboratory and field settings and across countries Further technical evaluations of these technologies are unlikely to add value but may instead delay implementation and timely diagnoses of infants living with HIV, a critical and vulnerable population. National regulatory agencies are encouraged to not delay adoption by conducting further evaluations but instead adopt a rapid and streamlined registration and national approval process for immediate implementation. 7.5 OPERATIONAL INTERVENTIONS: UPDATED CONSIDERATIONS FOR A COMPREHENSIVE QUALITY MANAGEMENT PACKAGE FOR POINT-OF-CARE TESTING WITHIN NATIONAL HEALTH PROGRAMMES The introduction and implementation of point-of-care technologies and the ability to decentralize testing has greatly improved access to diagnostic services. Since 2015, new WHO recommendations have been published (2) . In 2016, WHO conditionally recommended nucleic acid testing technologies that are developed and validated for use at or near the point of care for early infant HIV testing. Further, CD4 cell count testing at the point of care can be used to give priority for urgent linkage to care and antiretroviral therapy initiation. Finally, several point-of-care or near- 36 36 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis point-of-care technologies have been prequalified since 2015 for early infant diagnosis, CD4, HIV viral load, hepatitis C viral load, cervical cancer screening and HIV and syphilis (91). Point-of-care testing has been found to facilitate rapid and decentralized delivery of health services. A systematic review of using point-of-care CD4 to support antiretroviral therapy initiation (92) showed significantly improved linkage to HIV care and timeliness of antiretroviral therapy initiation. Further, recent published studies in Malawi and Mozambique have shown significantly reduced test turnaround times and increased antiretroviral therapy initiation rates when using point-of-care testing for early infant diagnosis (93,94). This decentralization of both qualitative and quantitative testing has presented both opportunities and challenges as countries monitor an increasing number of devices and operators across a decentralized testing network. This has required expanding traditional external quality assessment schemes to reach an unprecedented number of health facilities, and in many cases, considering novel mechanisms to support the quality management process. The principles presented throughout the publication on improving the quality of HIV-related point-of-care testing (95) remain highly relevant. However, it is now critical to update considerations for countries and implementing partners, since experiences with point-of-care technologies and quality assurance mechanisms have developed. As more experience has been gained, a more comprehensive approach to quality assuring point-of-care technologies is critical to ensure reliable and accurate testing. Several alternative options to quality assurance that should form a comprehensive package along with traditional proficiency testing include: • in-training and ongoing competency assessment; • internal quality controls; • proficiency testing panels; • alternative external quality assessment, if traditional proficiency testing panels are not available: o paper-based and online o duplicate specimen testing/reverse testing; • data management through connectivity; and • site training and mentorship Each quality assurance mechanism may touch on different steps within the testing cascade; however, once consolidated into a package, they provide a comprehensive and inclusive approach. Importance of a comprehensive quality management package for point-of-care technologies Quality management of diagnostics is critical to the overall quality of care by ensuring reliable and accurate test results. Pre-market quality assessments of in vitro diagnostics, such as WHO prequalification, provide information on product safety, quality and performance, manufacturing reliability and quality management systems. Further, stringent regulatory authorities aim to assess high-quality products for their intended use. Together, these processes ensure that only high-quality products are eligible for procurement. However, ongoing quality assurance and quality control are necessary to ensure the accuracy and precision of the results produced by diagnostic testing to prevent misdiagnosis. Internal controls and standards are meant to eliminate differences in random and systematic errors between each specimen and between specimens and known standards. External quality assessment proficiency testing schemes specifically assess the performance of a laboratory or health-care facility in accurately testing stabilized specimens of known value or result. The results of these assessments should alert national programmes to a problem, at which point action can be taken to identify the cause and potential remediations. This is valuable for understanding the performance levels of individual facilities but also in reviewing the overall national laboratory network. Further, data monitoring of the invalid rates, daily controls and utilization patterns of device-based technologies through connectivity can provide critical information on testing quality, recurring device or operator errors and the need for refresher training or specific mentorship. A comprehensive quality management package can identify gaps and bring them to the attention of laboratory programme managers. Good quality assurance programmes enable testing sites and laboratory programmes to work together to prevent, detect and correct problems throughout the entire testing cascade and to monitor all aspects of a testing programme for continual and high-quality testing services. The comprehensive quality management programme should bring together a series of activities that can together touch on all aspects of testing, including: • identifying patients; • collecting specimens; • handling specimens; • ensuring specimen and reagent storage conditions and expiry dates; 37HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis • applying specimens; • ensuring the performance of technology; • applying reagents, if necessary; • ensuring technical procedures; • interpreting results; and • recording results, Such a comprehensive quality management package for point-of-care testing is meant to complement the suggested and ongoing national laboratory-wide pre- and post-market surveillance activities outlined in other WHO publications (96–99). Implementation considerations for developing a quality management package A strong and comprehensive quality management package for point-of-care testing requires quality activities in addition to proficiency testing panels. Establishing a comprehensive package with some of the alternative strategies discussed here will enable coverage of the entire testing cascade and provide more regular monitoring of decentralized testing. At a minimum, national programmes should consider proficiency testing panels, encouraging suppliers to develop robust internal control systems, in-service training and competency assessments, data management through connectivity as well as regular and planned site training and mentorship. In an effort to provide a comprehensive quality management programme, national health policies must be developed that consider the available resources to ensure sustained adoption for the implementation of quality assurance in each context. In addition to exploring different models, countries must consider the timing and frequency of quality assurance activities, the content of each activity and the subsequent cost of conducting these activities. All these parameters have important quality and cost implications, and using country-specific policies and data to inform these decisions is critical. Some of the parameters to help understand implementation are listed below. In addition, routine programmatic quality mechanisms are still critical to ensure consistent procurement and introduction of high-quality technologies. Regular lot testing, service and maintenance and post-market surveillance are necessary structures of an overall laboratory quality system that this comprehensive quality management package for point-of-care testing should complement (98,99). No quality management programme is complete without reviewing data and taking clear and consistent preventive and corrective action when necessary. This is a critical component of the programme that must be clearly planned and determined to ensure that issues are addressed and operators are given the necessary support to continue providing testing and results in a high-quality manner. 38 38 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 8. CONCLUSIONS Increasing scale-up of treatment monitoring approaches through viral load testing as well as infant diagnosis will be critical to ensure high-quality care and treatment as well as programmatic success. Considering the optimal diagnostic network, specimen types, interventions and strategies in each country and across national, regional and partner stakeholders will support this effort, enhance collaboration and maximize diagnostic investment into clear clinical impact. © W H O 39HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis REFERENCES 1. Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection: recommendations for a public health approach. Geneva: World Health Organization; 2013 (https://www.who.int/hiv/pub/ guidelines/arv2013/en, accessed 8 July 2019). 2. Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection: recommendations for a public health approach – second edition 2016. Geneva: World Health Organization; 2016 (https://www. who.int/hiv/pub/arv/arv-2016/en, accessed 8 July 2019). 3. What’s new in treatment monitoring: viral load and CD4 testing. Geneva: World Health Organization; 2017 (https:// www.who.int/hiv/pub/arv/treatment-monitoring-info-2017/ en, accessed 8 July 2019). 4. Guidelines for managing advanced HIV disease and rapid initiation of antiretroviral therapy. Geneva: World Health Organization; 2017 (https://www.who.int/hiv/ pub/guidelines/advanced-HIV-disease/en, accessed 8 July 2019). 5. HIV market report: the state of the HIV treatment, testing, and prevention markets in low- and middle-income countries, 2017–2022. Boston: Clinton Health Access Initiative; 2018. 6. Landmark HIV diagnostic access program will save $150m and help achieve new global goals on HIV. Geneva: UNAIDS; 2014 (https://www.unaids.org/en/resources/ presscentre/pressreleaseandstatementarchive/2014/ september/20140925prviralload, accessed 8 July 2019). 7. Hologic Global Access Initiative: A revolutionary approach to diagnostic testing for resource-limited settings. Marlborough (MA): Hologic; 2019 (https://www.hologic. com/globalaccessinitiative, accessed 8 July 2019). 8. Cepheid announces expanded access to Xpert family of virology tests in global regions with the greatest need. Sunnyvale (CA): Cepheid; 2018 (http://www.cepheid. com/en/about/news-events/press-releases/319-cepheid- announces-expanded-access-to-xpert-family-of-virology- tests-in-global-regions-with-the-greatest-need, accessed 8 July 2019). 9. Putting HIV and HCV to the test: a product guide for point-of-care CD4 tests and laboratory-based and point-of- care HIV and HCV viral load tests. Geneva: Médecins Sans Frontières; 2017. 10. HIV/AIDS diagnostics technology landscape. 5th ed. Geneva: Unitaid; 2015 (http://www.unitaid.org/assets/ UNITAID_HIV_Nov_2015_Dx_Landscape-1.pdf, accessed 8 July 2019). 11. Miles to go: closing gaps, breaking barriers, righting injustices. Geneva: UNAIDS; 2018 (https://www.unaids. org/en/resources/documents/2018/global-aids-update, accessed 8 July 2019). 12. HIV diagnosis and ARV use in HIV-exposed infants: a programmatic update. Geneva: World Health Organization; 2018 (https://www.who.int/hiv/pub/paediatric/diagnosis- arv-infants/en, accessed 8 July 2019). 13. 90–90–90: an ambitious treatment target to help end the AIDS epidemic. Geneva: UNAIDS; 2017 (https:// www.unaids.org/en/resources/documents/2017/90-90-90, accessed 8 July 2019). 14. Population-based HIV impact assessments [website]. New York: ICAP at Columbia University, 2019 (https://phia. icap.columbia.edu, accessed 8 July 2019). 15. Making viral load routine: successes and challenges in the implementation of routine HIV viral load monitoring. Geneva: Médecins Sans Frontières; 2016. 16. Vojnov L, Venter F, Sacks J, Ehrenkranz P, Kiyaga C, Reynolds S et al. Next steps in HIV treatment monitoring: a call to action to ensure the use of viral load test results. In preparation. 17. HIV viral load scale-up tools [website]. Addis Ababa: ASLM; 2019 (http://www.aslm.org/hiv-viral-load-testing/ hiv-viral-load-scale-tools, accessed 8 July 2019). 18. Tools for scaling-up viral load monitoring [website]. Washington (DC): USAID; 2019 (https://aidsfree.usaid.gov/ news-events/tools-scaling-viral-load-monitoring, accessed 8 July 2019). 19. AIDSFree viral load and early infant diagnosis knowledge base [online database]. Washington (DC): USAID; 2019 (https://aidsfree.usaid.gov/resources/vl-eid, accessed 8 July 2019). 20. Abbott RealTime HIV-1 instructions for use. Chicago: Abbott Laboratories; 2014. 40 40 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 21. Public report: Abbott RealTime HIV-1. Geneva: World Health Organization; 2016 (https://www.who.int/ diagnostics_laboratory/evaluations/pq-list/hiv-vrl/180423_ amended_final_pqpr_0145_027_00_v11.pdf?ua=1, accessed 8 July 2019). 22. m-PIMA™ HIV-1/2 VL cartridge guide. Chicago: Abbott Laboratories; 2019. 23. Public report: m-PIMA HIV-1/2 VL. Geneva: World Health Organization; 2019 (https://www.who.int/ diagnostics_laboratory/evaluations/pq-list/190408_ pqdx_0359_032_00_pqpr_mpima.pdf?ua=1, accessed 8 July 2019). 24. GENERIC HIV Charge Virale instructions for use. Collingwood (NJ); Biocentric; 2019. 25. NucliSENS EasyQ® HIV-1 v2.0 instructions for use. Marcy-l’Étoile: bioMérieux; 2010. 26. Public report: NucliSENS EasyQ® HIV-1 v2.0. Geneva: World Health Organization; 2017 (https://www.who. int/diagnostics_laboratory/evaluations/pq-list/hiv-vrl/ pqdx_0127_016_00_public_report_v3.pdf?ua=1, accessed 8 July 2019). 27. ExaVir™ Load instructions for use, version 3. Uppsala: Cavidi; 2011. 28. Xpert® HIV-1 Viral Load instructions for use. Sunnyvale (CA): Cepheid; 2017. 29. Public report: Xpert® HIV-1 viral load. Geneva: World Health Organization; 2017 (https://www.who.int/ diagnostics_laboratory/evaluations/pq-list/hiv-vrl/170830_ amended_final_pqpr_0192_0193_0194_0195_070_00_ v3.pdf?ua=1, accessed 8 July 2019). 30. Aptima™ HIV-1 Quant Dx Assay instructions for use. Marlborough (MA): Hologic; 2017. 31. Public report: Aptima™ HIV-1 Quant Dx Assay. Geneva: World Health Organization; 2018 (https://www.who.int/ diagnostics_laboratory/evaluations/pq-list/180207_final_ pqpr_0236_078_00_v5_IFU_v4.pdf?ua=1, accessed 8 July 2019). 32. artus® HI Virus -1 RG RT-PCR Kit handbook. Hilden: Qiagen; 2013. 33. artus® HI Virus -1 QS-RGQ Kit handbook. Hilden: Qiagen; 2013 34. COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0 instructions for use. Basel: Roche; 2017. 35. Public report: COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0. Geneva: World Health Organization; 2018 (https://www.who.int/diagnostics_laboratory/evaluations/ pq-list/hiv-vrl/160530_amended_final_public_report_ pqdx_0200_046_00_taqman96.pdf?ua=1, accessed 8 July 2019). 36. cobas® HIV-1 Quantitative Nucleic Acid Test for Use on the cobas® 4800 System instructions for use. Basel: Roche; 2018. 37. cobas® HIV-1 Quantitative Nucleic Acid Test for Use on the cobas® 6800/8800 Systems instructions for use. Basel: Roche; 2017. 38. HIV Real-TM Quant DX handbook. Como: Sacace Biotechnologies; 2015. 39. VERSANT® HIV-1 RNA 1.5 Assay instructions for use. Munich: Siemens; 2017. 40. Bonner K, Siemieniuk RA, Boozary A, Roberts T, Fajardo E, Cohn J. Expanding access to HIV viral load testing: a systematic review of RNA stability in EDTA tubes and PPT beyond current time and temperature thresholds. PLoS One. 2014;9:e113813. 41. Hardie DR, Korsman SN, Ameer S, Vojnov L, Hsiao N-Y. Reliability of plasma HIV viral load testing beyond 24 hours: insights gained from a study in a routine diagnostic laboratory. PLoS One. (in press). 42. Gullett JC, Nolte FS. Quantitative nucleic acid amplification methods for viral infections. Clin Chem. 2015;61:72–8. 43. Undetectable: how viral load monitoring can improve HIV treatment in developing countries. Geneva: Médecins Sans Frontières; 2012. 44. Quinn TC, Wawer MJ, Sewankambo N, Serwadda D, Li C, Wabwire-Mangen F et al. Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group. N Engl J Med. 2000;342:921–9. 45. Gray RH, Wawer MJ, Brookmeyer R, Sewankambo NK, Serwadda D, Wabwire-Mangen F et al. Probability of HIV-1 transmission per coital act in monogamous, heterosexual, HIV-1-discordant couples in Rakai, Uganda. Lancet. 2001;357:1149–53. 46. Avettand-Fènoël V, Hocqueloux L, Ghosn J, Cheret A, Frange P, Melard A et al. Total HIV-1 DNA, a marker of viral reservoir dynamics with clinical implications. Clin Microbiol Rev. 2016;29:859–80. 41HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 47. Parkin NT. Measurement of HIV-1 viral load for drug resistance surveillance using dried blood spots: literature review and modeling of contribution of DNA and RNA. AIDS Rev. 2014;16:160–71. 48. Lee TH, Stromberg RR, Heitman JW, Sawyer L, Hanson CV, Busch MP. Distribution of HIV type 1 (HIV-1) in blood components: detection and significance of high levels of HIV- 1 associated with platelets. Transfusion. 1998;38:580–8. 49. Smit PW, Sollis KA, Fiscus S, Ford N, Vitoria M, Essajee S et al. Systematic review of the use of dried blood spots for monitoring HIV viral load and for early infant diagnosis. PLoS One. 2014;9:e86461. 50. Vojnov L, Carmona S, Zeh C, Markby J, Boeras D, Prescott MR et al. The performance of using dried blood spot specimens for HIV-1 viral load testing: a systematic review and meta-analysis. In preparation. 51. Holodniy M, Mole L, Yen-Lieberman B, Margolis D, Starkey C, Carroll R et al. Comparative stabilities of quantitative human immunodeficiency virus RNA in plasma from samples collected in VACUTAINER CPT, VACUTAINER PPT, and standard VACUTAINER tubes. J Clin Microbiol. 1995;33:1562–6. 52. Ginocchio CC, Wang XP, Kaplan MH, Mulligan G, Witt D, Romano JW et al. Effects of specimen collection, processing, and storage conditions on stability of human immunodeficiency virus type 1 RNA levels in plasma. J Clin Microbiol. 1997;35:2886–93. 53. Holodniy M, Rainen L, Herman S, Yen-Lieberman B. Stability of plasma human immunodeficiency virus load in VACUTAINER PPT plasma preparation tubes during overnight shipment. J Clin Microbiol. 2000;38:323–6. 54. Elbeik T, Nassos P, Kipnis P, Haller B, Ng VL. Evaluation of the VACUTAINER PPT Plasma Preparation Tube for use with the Bayer VERSANT assay for quantification of human immunodeficiency virus type 1 RNA. J Clin Microbiol. 2005;43:3769–71. 55. Giordano M, Kelleher T, Colonno RJ, Lazzarin A, Squires K. The effects of the Roche AMPLICOR HIV- 1 MONITOR UltraSensitive Test versions 1.0 and 1.5 viral load assays and plasma collection tube type on determination of response to antiretroviral therapy and the inappropriateness of cross-study comparisons. J Clin Virol. 2006;35:420–5. 56. Griffith BP, Mayo DR. Increased levels of HIV RNA detected in samples with viral loads close to the detection limit collected in plasma preparation tubes (PPT). J Clin Virol. 2006;35:197–200. 57. Wan H, Seth A, Rainen L, Fernandes H. Coamplification of HIV-1 proviral DNA and viral RNA in assays used for quantification of HIV-1 RNA. J Clin Microbiol. 2010;48:2186–90. 58. Salimnia H, Moore EC, Crane LR, Macarthur RD, Fairfax MR. Discordance between viral loads determined by Roche COBAS AMPLICOR human immunodeficiency virus type 1 monitor (version 1.5): standard and ultrasensitive assays caused by freezing patient plasma in centrifuged Becton- Dickinson Vacutainer brand plasma preparation tubes. J Clin Microbiol. 2005;43:4635–9. 59. García-Bujalance S, Ladrón de Guevara C, González- García J, Arribas JR, Zamora F, Gutiérrez A. Elevation of viral load by PCR and use of plasma preparation tubes for quantification of human immunodeficiency virus type 1. J Microbiol Methods. 2007;69:384–6. 60. Rebeiro PF, Kheshti A, Bebawy SS, Stinnette SE, Erdem H, Tang YW et al. Increased detectability of plasma HIV-1 RNA after introduction of a new assay and altered specimen-processing procedures. Clin Infect Dis. 2008;47:1354–7. 61. Kran AM, Jonassen TØ, Sannes M, Jakobsen K, Lind A, Maeland A et al. Overestimation of human immunodeficiency virus type 1 load caused by the presence of cells in plasma from plasma preparation tubes. J Clin Microbiol. 2009;47:2170–4. 62. Kraft CS, Binongo JN, Burd EM, Eaton ME, McCloskey CB, Fernandes H et al. Successful use of Plasma Preparation Tubes™ (PPTs) in the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 test. J Clin Virol. 2013;57:77–9. 63. Fernandes H, Morosyuk S, Abravaya K, Ramanathan M, Rainen L. Evaluation of effect of specimen-handling parameters for plasma preparation tubes on viral load measurements obtained by using the Abbott RealTime HIV- 1 load assay. J Clin Microbiol. 2010;48:2464–8. 64. Adachi D, Benedet M, Tang JW, Taylor GD. Comparative evaluation of Roche’s COBAS Ampliprep/COBAS TaqMan HIV-1 Test v2.0 and Abbott’s RealTime m2000sp/rt HIV-1 assay on PPTs and EDTA samples. J Clin Virol. 2014;60:78–9. 65. Cloherty G, Swanson P, Lucic D, Dieckhaus K, Anthony P, Cataline P et al. Clinical implications of elevated HIV- 1 viral load results obtained from samples stored frozen in vacutainer plasma preparation tubes. J Virol Methods. 2014;204:91–2. 66. Goedhals D, Scott LE, Moretti S, Cooper MA, Opperman WJ, Rossouw I. Evaluation of the use of plasma preparation tubes for HIV viral load testing on the COBAS AmpliPrep/ COBAS TaqMan HIV-1 version 2.0. J Virol Methods. 2013;187:248–50. 42 42 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 67. BD Vacutainer® PPT instructions for use. Franklin Lakes (NJ): Becton, Dickinson and Company; 2015. 68. K2E K2EDTA Sep. Kremsmünster: Greiner Bio-One; 2019 (https://shop.gbo.com/en/row/products/preanalytics/ venous-blood-collection/vacuette-tube/edta/k2e-k2edta- sep, accessed 8 July 2019). 69. K2EDTA Gel Tube. Negeri Sembilan: TUD: 2019 (http:// www.tud.my/product_info_901003-100013.htm, accessed 8 July 2019). 70. Putting HIV and HCV to the test. Geneva: Médecins Sans Frontières; 2017 (https://www.msf.org/sites/msf.org/ files/putting_hiv_and_hcv_to_the_test.pdf, accessed ( July2019). 71. Sacks JA, Fong Y, Perez Gonzalez M, Andreotti M, Baliga S, Garrett N et al. Performance of Cepheid GeneXpert HIV-1 viral load plasma assay to accurately detect treatment failure: a clinical meta-analysis. AIDS. (in press). 72. Sacks JA, Fong Y, Gonzalez MP, Andreotti M, Baliga S, Garrett N et al. Performance of cepheid GeneXpert HIV- 1 viral load plasma assay to accurately detect treatment failure: a clinical meta-analysis. AIDS. doi: 10.1097/ QAD.0000000000002303. [Epub ahead of print] 73. Information note: considerations for adoption and use of multidisease testing devices in integrated laboratory networks. Geneva: World Health Organization; 2017 (https://www.who.int/tb/publications/2017/ considerations_multidisease_testing_devices_2017/en, accessed 8 July 2019). 74. WHO recommendations on the diagnosis of HIV infection in infants and children. Geneva: World Health Organization; 2010 (https://apps.who.int/iris/ handle/10665/44275, accessed 8 July 2019). 75. Recommendations for the use of antiretroviral drugs in pregnant women with HIV infection and interventions to reduce perinatal HIV transmission in the United States. Washington (DC): United States Department of Health and Human Services; 2018 (https://aidsinfo.nih.gov/guidelines/ html/3/perinatal/509/diagnosis-of-hiv-infection-in-infants- and-children, accessed 8 July 2019). 76. Delamere C, Burgard M, Mayaux MJ, Blanche S, Doussin A, Ivanoff S et al. HIV-1 RNA detection in plasma for the diagnosis of infection in neonates. J Acquir Immune Defic Syndr Hum Retrovirol. 1997;15:121–5. 77. Nesheim S, Palumbo P, Sullivan K, Lee F, Vink P, Abrams E et al. Quantitative RNA testing for diagnosis of HIV-infected infants. J Acquir Immune Defic Syndr Hum Retrovirol. 2003;32:192·5. 78. Simonds RJ, Brown TM, Thea DM, Orloff SL, Steketee RW, Lee FK et al. Sensitivity and specificity of a qualitative RNA detection assay to diagnose HIV infection in young infants. AIDS. 1998;12:1545–9. 79. Young N, Shaffer N, Chaowanachan T, Chotpitayasunondh T, Vanparapar N, Mock PA et al. Early diagnosis of HIV-1 infected infants in Thailand using RNA and DNA PCR assays sensitive to non-B subtypes; J Acquir Immune Defic Syndr Hum Retrovirol. 2000;24:401–7. 80. Vubil A, Nhachigule C, Loquiha O, Meggi B, Mabunda N, Bollinger T et al. Viral load assay performs comparably to early infant diagnosis assay to diagnose infants with HIV. J Init AIDS Soc. In preparation. 81. Kiyaga C, Fong Y, Okiira C, Kushemererwa GE, Kayongo I, Tadeo I et al. Viral load assay performed well when used as a diagnostic for infants. 11th International Workshop on HIV Pediatrics, Mexico City, Mexico, 19–20 July 2019. 82. Global Plan towards the elimination of new HIV infections among children by 2015 and keeping their mothers alive. Geneva: UNAIDS; 2011 (https://www. unaids.org/en/resources/documents/2011/20110609_ JC2137_Global-Plan-Elimination-HIV-Children_en.pdf, accessed 8 July 2019). 83. Public report: Alere™q HIV-1/2 Detect. Geneva: World Health Organization; 2016 (http://www.who. int/diagnostics_laboratory/evaluations/pq-list/ hiv- vrl/160613PQPublicReport_0226-032-00AlereHIVDetect_ v2.pdf?ua=1, accessed 8 July 2019). 84. Public report: Cepheid Xpert®HIV-1 Qual. Geneva: World Health Organization; 2016 (http://www.who.int/ diagnostics_laboratory/evaluations/pq-list/ hiv-vrl/16 0613PQPublicReport_0259-0700-00_XpertQualHIV_ v2.pdf?ua=1, accessed 8 July 2019). 85. Carmona S, Wedderburn C, Macleod W, Hsaio M, Jani I, Kroon M et al. Field performance of point-of-care HIV testing for early infant diagnosis: pooled analysis from six countries for the EID Consortium. ASLM2016, Cape Town, South Africa, 3–8 December 2016 (https://eidconsortium. org/Files/EID%20Poster%20v5%20Low%20res.pdf, accessed 8 July 2019). 86. Jani IV, Meggi B, Mabunda N, Vubil A, Tobaiwa O, Bollinger T et al. Effect of point-of-care early infant diagnosis on retention of patients and rates of antiretroviral therapy initiation on primary health care clinics: a cluster- randomized trial in Mozambique. 21st International AIDS Conference, Durban, South Africa, 18–22 July 2016. 87. Mwenda R. Impact of point-of-care EID testing into the national EID program: pilot experiences from Malawi. 21st International AIDS Conference, Durban, South Africa, 18–22 July 2016. 43HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis 88. Technau K, Sherman G, Bhowan K, Murnane P, Coovadia AH, Kuhn L. Comparing point of care to laboratory HIV PCR testing at birth in a hospital setting in Johannesburg, South Africa (Abstract O-14). 8th International Workshop on HIV Pediatrics, Durban, South Africa, 15–16 July 2016. 89. Bourne DE, Thompson M, Brody LL, Cotton M, Draper B, Laubscher R et al. Emergence of a peak in early infant mortality due to HIV/AIDS in South Africa. AIDS. 2009;23:101–6. 90. Newell ML, Coovadia H, Cortina-Borja M, Rollins N, Gaillard P, Dabis F. Mortality of infected and uninfected infants born to HIV-infected mothers in Africa: a pooled analysis. Lancet. 2004;364:1236–43. 91. WHO list of prequalified in vitro diagnostic products. Geneva: World Health Organization; 2019 https://www. who.int/diagnostics_laboratory/evaluations/PQ_list/en, accessed 8 July 2019). 92. Vojnov L, Markby J, Boeke C, Harris L, Ford N, Peter T. POC CD4 testing improves linkage to HIV care and timeliness of ART initiation in a public health approach: a systematic review and meta-analysis. PLoS One. 2016;11:e0155256. 93. Mwenda R, Fong Y, Magombo T, Saka E, Midiani D, Mwase C et al. Significant patient impact observed upon implementation of point-of-care early infant diagnosis technologies in an observational study in Malawi. Clin Infect Dis. 2018;67:701–7. 94. Jani IV, Meggi B, Loquiha O, Tobaiwa O, Mudenyanga C, Zitha A et al. Effect of point-of-care early infant diagnosis on antiretroviral therapy initiation and retention of patients. AIDS. 2018;32:1453–63. 95. Improving the quality of HIV-related point-of-care testing: ensuring the reliability and accuracy of test results. Geneva: World Health Organization; 2015 (https://www. who.int/hiv/pub/toolkits/handbook-point-of-care-testing/ en, accessed 8 July 2019). 96. Prequalification of in vitro diagnostics. Geneva: World Health Organization; 2019 (https://www.who.int/ diagnostics_laboratory/evaluations/en, accessed 8 July 2019). 97. Procurement of in vitro diagnostics. Geneva: World Health Organization; 2019 (https://www.who.int/ diagnostics_laboratory/procurement/en, accessed 8 July 2019). 98. Post-market surveillance for in vitro diagnostics (IVDs). Geneva: World Health Organization; 2019 (https://www. who.int/diagnostics_laboratory/postmarket/en, accessed 8 July 2019). 99. Quality assurance of in vitro diagnostics and laboratory technology. Geneva: World Health Organization; 2019 (https://www.who.int/diagnostics_laboratory/quality/en, accessed 8 July 2019). 44 44 HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis ANNEX 1. INFANT DIAGNOSIS ALGORITHM HIV-exposed newborn (0-2 days) Consider NATa,b Negative Negative Immediately start ARTc Repeat NAT to confirm infection Infant/child is infected HIV-exposed infant or child (4-6 weeks to 18 months) Conduct NATb (at 4-6 weeks or at the earliest opportunity thereafter) Positive Positive Immediately start ARTc Repeat NAT to confirm infection HIV infection not detected but if infant/child is breastfed the risk of acquiring HIV infection remains until complete cessation of breastfeedingd Regular clinical monitoring Conduct NATb (at 9 months) Antibody testing at 18 months of age or 3 months after cessation of breastfeeding, whichever is laterf Infant/child is infected HIV unlikely unless still breastfedinge Negative a Based on 2016 WHO Consolidated ARV Guidelines, addition of NAT at birth to the existing testing algorithm can be considered. b POC NAT can be used to diagnose HIV infection as well as to confirm positive results. c Start ART without delay. At the same time, retest to confirm infection. As maternal treatment is scaled up and MTCT transmission rates decrease, false-positive results are expected to increase: retesting after a first positive NAT is hence important to avoid unnecessary treatment, particularly in settings with lower transmission rates. If the second test is negative, a third NAT should be performed before interrupting ART. d For children who were never breastfed, additional testing following a negative NAT at 4–6 weeks is included in this algorithm to account for potential false-negative NAT results. e The risk of HIV transmission remains as long as breastfeeding continues. If the 9-month test is conducted earlier than 3 months after cessation of breastfeeding, infection acquired in the last days of breastfeeding may be missed. Retesting at 18 months or 3 months after cessation of breastfeeding (whichever is later) should be carried out for final assessment of HIV status. f If breastfeeding extends beyond 18 months, the final diagnosis of HIV status can only be assessed at the end of breastfeeding. If breastfeeding ends before 18 months, the final diagnosis of HIV status with antibody testing can only be assessed at 18 months. Antibody testing should be undertaken at least 3 months after cessation of breastfeeding (to allow for development of HIV antibodies). For infants younger than 18 months of age NAT should be performed to confirm infection. If the infant is older than 18 months, negative antibody testing confirms that the infant is uninfected; positive antibody testing confirms infant is infected. Source: HIV diagnosis and ARV use in HIV-exposed infants: a programmatic update (12) .

For more information, contact: World Health Organization Department of HIV/AIDS 20, avenue Appia 1211 Geneva 27 Switzerland E-mail: hiv-aids@who.int www.who.int/hiv ISBN 978 92 4 151621 1

TRATAMENTO DO VIH E CUIDADOS KIT DE FERRAMENTAS DE DIAGNÓSTICO MOLECULAR DO VIH PARA MELHORAR O ACESSO AOS TESTES DE CARGA VIRAL E DIAGNÓSTICO PRECOCE PEDIÁTRICO KIT DE FERRAMENTAS JULHO DE 2019 Kit de ferramentas de diagnóstico molecular do VIH para melhorar o acesso aos testes de carga viral e diagnóstico precoce pediátrico [HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis] ISBN 978-92-4-000417-7 (versão electrónica) ISBN 978-92-4-000418-4 (versão impressa) © Organização Mundial da Saúde 2020 Alguns direitos reservados. Este trabalho poderá ser disponibilizado através da licença da Creative Commons Attribution- NonCommercial-ShareAlike 3.0 IGO (CC BY-NC-SA 3.0 IGO; https://creativecommons.org/licenses/by-nc-sa/3.0/igo Nos termos desta licença, é possível copiar, redistribuir e adaptar o trabalho para fins não comerciais, desde que dele se faça a devida menção, como abaixo se indica. Em nenhuma circunstância, deve este trabalho sugerir que a OMS aprova uma determinada organização, produtos ou serviços. O uso do logótipo da OMS não é autorizado. Para adaptação do trabalho, é preciso obter a mesma licença de Creative Commons ou equivalente. Numa tradução deste trabalho, é necessário acrescentar a seguinte isenção de responsabilidade, juntamente com a citação sugerida: “Esta tradução não foi criada pela Organização Mundial da Saúde (OMS). A OMS não é responsável, nem pelo conteúdo, nem pelo rigor desta tradução. A edição original em inglês será a única autêntica e vinculativa”. Qualquer mediação relacionada com litígios resultantes da licença deverá ser conduzida em conformidade com o Regulamento de Mediação da Organização Mundial da Propriedade Intelectual. Citação sugerida. Kit de ferramentas de diagnóstico molecular do VIH para melhorar o acesso aos testes de carga viral e diagnóstico precoce pediátrico [HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis]. Genebra: Organização Mundial da Saúde; 2020. Licença: CC BY-NC-SA 3.0 IGO. Dados da catalogação na publicação (CIP). Os dados da CIP estão disponíveis em http://apps.who.int/iris. Vendas, direitos e licenciamento. Para comprar as publicações da OMS, ver http://apps.who.int/bookorders. Para apresentar pedidos para uso comercial e esclarecer dúvidas sobre direitos e licenças, consultar http://www.who.int/about/licensing. Materiais de partes terceiras. Para utilizar materiais desta publicação, tais como quadros, figuras ou imagens, que sejam atribuídos a uma parte terceira, compete ao utilizador determinar se é necessária autorização para esse uso e obter a devida autorização do titular dos direitos de autor. O risco de pedidos de indemnização resultantes de irregularidades pelo uso de componentes da autoria de uma parte terceira é da responsabilidade exclusiva do utilizador. Isenção geral de responsabilidade. As denominações utilizadas nesta publicação e a apresentação do material nela contido não significam, por parte da Organização Mundial da Saúde, nenhum julgamento sobre o estatuto jurídico ou as autoridades de qualquer país, território, cidade ou zona, nem tampouco sobre a demarcação das suas fronteiras ou limites. As linhas ponteadas e tracejadas nos mapas representam de modo aproximativo fronteiras sobre as quais pode não existir ainda acordo total. A menção de determinadas companhias ou do nome comercial de certos produtos não implica que a Organização Mundial da Saúde os aprove ou recomende, dando-lhes preferência a outros análogos não mencionados. Salvo erros ou omissões, uma letra maiúscula inicial indica que se trata dum produto de marca registado. A OMS tomou todas as precauções razoáveis para verificar a informação contida nesta publicação. No entanto, o material publicado é distribuído sem nenhum tipo de garantia, nem expressa nem implícita. A responsabilidade pela interpretação e utilização deste material recai sobre o leitor. Em nenhum caso se poderá responsabilizar a OMS por qualquer prejuízo resultante da sua utilização. Foto da capa: © WHO Arranjo gráfico de L’IV Com Sàrl Impresso na Suíça KIT DE FERRAMENTAS DE DIAGNÓSTICO MOLECULAR DO VIH PARA MELHORAR O ACESSO AOS TESTES DE CARGA VIRAL E DIAGNÓSTICO PRECOCE PEDIÁTRICO KIT DE FERRAMENTAS – JULHO DE 2019 22 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce ÍNDICE Agradecimentos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1. Introdução: diagnóstico molecular para os testes da carga viral do VIH e diagnóstico precoce pediátrico . . . . . . . . 4 2. Uso dos resultados dos testes da carga viral para apoiar o tratamento clínico das pessoas que vivem com o VIH . . . 7 3. Alcance e acesso estimado aos testes da carga viral usando amostras de plasma tradicionais . . . . . . . . . . . . . . . . . 10 4. Estabilidade das amostras para os testes da carga viral . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 5. Antecedentes técnicos: testes moleculares . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 6. Tipos alternativos de amostras e tecnologias a considerar quando não é possível usar plasma líquido para os testes da carga viral devido a constrangimentos de infraestruturas, transporte ou outros 18 . . . . . . . . . . . . . . . . 18 6.1. Tipos alternativos de amostras e tecnologias: amostras de gotas de sangue seco para os testes da carga viral do VIH. .................................................................................................................................................18 6.2 Tipos alternativos de amostras e tecnologias: amostras de gotas de plasma seco para os testes da carga viral do VIH 20 .............................................................................................................................................20 6.3 Tipos alternativos de amostras e tecnologias: tubos de preparação do plasma para os testes da carga viral do VIH ..............21 6.4 Tipos alternativos de amostras e tecnologia: ferramentas nos pontos de cuidados ou na sua proximidade para os testes da carga viral do VIH ........................................................................................................................................ 33\ 7. Intervenções operacionais e considerações sobre o reforço da carga viral e diagnóstico pediátrico . . . . . . . . . . . . . 25 7.1. Opções de transporte das amostras para o diagnóstico molecular. .........................................................................................25 7.2 Pacotes para o diagnóstico pediátrico e colheita de amostras para a carga viral. ................................................................... 30 7.3 Intervenções operacionais: testes da carga viral para diagnóstico pediátrico. ......................................................................... 33 7.4 Novas ferramentas nos pontos de cuidados para o diagnóstico pediátrico precoce do VIH. ................................................... 34 7.5 Intervenções operacionais: considerações actualizadas sobre um pacote abrangente de gestão da qualidade dos testes nos pontos de cuidados, no âmbito dos programas nacionais de saúde ........................................... 36 8. Conclusões . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Referências . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Anexo 1. Algoritmo do diagnóstico precoce pediátrico .......................................................................................................44 © W H O 3Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce AGRADECIMENTOS Várias partes interessadas importantes deram um significativo contributo ao longo do processo de elaboração desta publicação, incluindo as seguintes. – Robert Luo, Kameko Nichols and Neil Parkin – African Society for Laboratory Medicine: Charles Kiyaga and Anafi Mataka – Clinton Health Access Initiative: Paolo Maggiore, Maria Rosezoil Rioja and Jilian Sacks – Elizabeth Glaser Pediatric AIDS Foundation: Jennifer Cohn – United States Agency for International Development: Dianna Edgil, Matthew Wattleworth and Jason Williams – WHO Regional Office for Africa: Fatim Cham Jallow and Fausta Mosha – WHO Essential Medicines and Health Products Programme: Mercedes Perez and Ute Ströher Em particular: – Clinton Health Access Initiative e United States Agency for International Development (secção 3). – Robert Luo apoiou o desenvolvimento da secção 5 and subsecção 6.1. – Kameko Nichols apoiou o desenvolvimento da secção 7.1. – The Clinton Health Access Initiative apoiou o desenvolvimento da subsecção 7.2. © W H O 44 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce 1. INTRODUÇÃO: DIAGNÓSTICO MOLECULAR PARA TESTES DA CARGA VIRAL DO VIH E DIAGNÓSTICO PRECOCE PEDIÁTRICO Monitorização do insucesso no tratamento A monitorização das pessoas que recebem terapia antirretroviral é importante para garantir o êxito do tratamento, identificar problemas de adesão e determinar se os regimes terapêuticos antirretrovirais devem ser mudados no caso de insucesso do tratamento. Em 2013, a OMS recomendava o uso dos testes da carga viral como a abordagem ideal de monitorização, para diagnosticar e confirmar o insucesso da terapia antirretroviral (1). Em comparação com a monitorização clínica ou imunológica, a carga viral fornece uma indicação precoce e mais rigorosa do insucesso do tratamento. A medição da carga viral pode ajudar a distinguir entre a resistência aos medicamentos e a não adesão, quando combinada com um aconselhamento firme e reforçado sobre a adesão. Para além disso, a carga viral pode servir como medida representativa do risco de transmissão e eficácia das intervenções de prevenção, tanto a nível individual como populacional. As orientações actualizadas da OMS de 2016 recomendam que a monitorização de rotina da carga viral se faça aos 6 meses, 12 meses após o início da terapia antirretroviral e, depois, de 12 em 12 meses, se a pessoa estiver estável na terapia antirretroviral (2). Se a carga viral não estiver rotineiramente disponível, deve usar-se a contagem de CD4 e a monitorização clínica para avaliar o insucesso do tratamento. Além disso, podem usar-se amostras de sangue seco, usando sangue total venoso ou capilar, para determinar a carga viral do VIH. Para determinar o insucesso do tratamento, quando se usam amostras de sangue seco, deve usar-se um limiar de 1000 cópias/ml, tal como definido para os testes no plasma. Insucesso no tratamento é definido como uma carga viral persistentemente detectável que exceda 1000 cópias/ml (2) , isto é, duas medições consecutivas da carga viral com um intervalo de 3 meses, com apoio à adesão entre as medições após, pelo menos, seis meses do início de um novo regime de terapia antirretroviral (Caixa 1). Além disso, a carga viral pode apoiar estratégias de prestação de serviços diferenciados para as pessoas que vivem com o VIH, incluindo as que estão estáveis com a terapia antirretroviral (2). Pessoas estáveis são aquelas que receberam terapia antirretroviral durante, pelo menos, um ano e não tiveram quaisquer reacções adversas aos medicamentos que requeiram uma monitorização regular, não têm doenças, nem estão grávidas, não estão a amamentar e compreendem bem as vantagens de uma adesão vitalícia, além de apresentarem evidências do sucesso do tratamento (duas medições consecutivas da carga viral abaixo de 1000 cópias/ml). O pacote de cuidados para indivíduos estáveis pode incluir consultas menos frequentes, recolha de medicamentos, cuidados comunitários e a cessação da monitorização por contagem de CD4, se estiver disponível o teste da carga viral. Há muitas orientações nacionais que presentemente recomendam e estão a reforçar o acesso aos testes da carga viral para a monitorização do tratamento (Fig. 1). A percentagem de testes da carga viral que se fazem anualmente tem aumentado significativamente desde 2013 (Fig. 2) (5). Em 2017, foram realizados aproximadamente, 15 milhões de testes da carga viral e as projecções sugerem que, em 2022, se poderão fazer quase 29 milhões de testes. Apesar deste aumento do volume de testes, a cobertura total da procura de testes da carga viral continuou abaixo dos 60%, em 2017. O maior volume de testes da carga viral e a continuação do seu crescimento a nível nacional representam um aumento significativo dos encargos do orçamento nacional com os testes. Felizmente, foram negociados recentemente compromissos sobre preços em apoio à expansão e acesso a esses testes (6–8). Existem actualmente numerosas tecnologias, não só laboratoriais como também ensaios na proximidade dos pontos de cuidados, para apoiar o aumento dos testes da carga viral e o diagnóstico precoce pediátrico, estando a ser desenvolvidas novas tecnologias (9,10). Diagnóstico pediátrico O diagnóstico pediátrico consiste na realização de testes durante todo durante todo o período de exposição da criança ao VIH. Consoante a idade, o diagnóstico pode basearse em testes de ácido nucleico ou testes serológicos. Mais especificamente, o diagnóstico pediátrico precoce refere-se especificamente ao teste de ácido nucleico em crianças nos primeiros dois meses de vida. Para conhecer o algoritmo do diagnóstico pediátrico, ver o Anexo 1. Caixa 1. Avaliar a doença avançada do VIH Uma vez que a contagem de CD4 é o melhor indicador do estado da doença e do imediato risco de morte, deve ser usada para identificar as pessoas em um estado avançado da infecção por VIH. Todas as pessoas que comecem ou recomecem os cuidados devem fazer um teste de CD4 no início do tratamento, como é clinicamente indicado para as pessoas que estejam clinicamente instáveis ou apresentem sintomas de doença avançada por VIH (2,3). Além disso, recomenda-se firmemente que as pessoas em estado avançado de doença por VIH (contagem de CD4 inferior a 200 células/mm3 ou fase 3 ou 4 da OMS) recebam um pacote de cuidados (4). 5Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce 0 1,700 3,400850 Kilometers Integralmente implementada Parcialmente implementada Não implementada Dirigida só para o teste da carga viral Sem política para o teste da carga viral Dados não comunicados Países mais rápidos Países de altos rendimentos Não aplicável Fonte: Global AIDS Monitoring (UNAIDS/WHO/UNICEF) and WHO HIV Country Intelligence Tool, 2019. Fig. 1. Política nacional para o testes de rotina da carga viral para monitorizar a TAR e nível de implementação em adultos e adolescentes em países de baixos-médios rendimentos Fig. 2. Previsão da carga viral estimada nos países de baixos-médios rendimentos em todo o mundo Fonte: 2018 CHAI HIV Market. 14.7M 17.3M 20.9M 23.6M 26.4M 28.5M 10.2M 9.6M 7.7M 6.7M 5.0M 3.9M 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0 M 5 M 10 M 15 M 20 M 25 M 30 M 35 M 2017 2018 2019 2020 2021 2022 Co be rt ur a CV Procura prevista Necessidade não satisfeita Cobertura 66 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce A cobertura do diagnóstico pediátrico precoce (teste nos primeiros dois meses de vida) tem permanecido estável nos últimos anos, com cerca de 51% das crianças expostas ao VIH recebendo um teste de ácido nucleico nos primeiros dois meses de vida em 2018 (11). A percentagem de crianças expostas ao VIH que fizeram o teste aos nove meses ou no final do período de exposição tem sido difícil de calcular. As previsões actuais relativamente ao teste do ácido nucleico sugerem um crescimento moderado e volumes sustentados até 2022 (Fig. 3) (5). Em 2017, realizaram-se, aproximadamente, 1,4 milhões de testes do ácido nucleico em bebés, sendo projectados mais de 2 milhões para 2022. Desde 2010, têm sido feitas várias recomendações para apoiar o acesso e o reforço alargado ao diagnóstico pediátrico (2,12). • Deve usar-se uma amplitude indeterminada, para melhorar o rigor de todos os testes de diagnóstico pediátrico baseados no ácido nucleico (recomendação firme, evidências de qualidade moderada). • Nos bebés com um resultado inicial positivo no teste do ácido nucleico, recomenda-se firmemente que a terapia antirretroviral se inicie sem demora e, ao mesmo tempo, seja colhida uma segunda amostra para confirmar o teste inicial positivo (recomendação firme, evidências de baixa qualidade). • Recomenda-se firmemente que as crianças (com 18 meses ou mais) com suspeita de infecção por VIH ou exposição ao VIH façam testes serológicos do VIH, de acordo com o algoritmo normal de diagnóstico do VIH usado nos adultos, para determinar o diagnóstico final (recomendação firme, evidências de alta qualidade). • Em contextos de epidemia generalizada, os recém-nascidos e as crianças pequenas com estado de VIH desconhecido que sejam internados para cuidados ou consultem clínicas de malnutrição ou de TB devem fazer o teste do VIH de rotina (recomendação firme, evidências de baixa qualidade). • Em contextos de epidemia generalizada, os recém-nascidos e as crianças pequenas com estado de VIH desconhecido devem fazer o teste do VIH em clínicas de ambulatório ou de vacinação (recomendação condicional, evidências de baixa qualidade). • As tecnologias dos testes de ácido nucleico que sejam desenvolvidas e validadas para serem usadas nas pontos de cuidados ou na sua proximidade podem ser usadas para o teste de VIH em crianças (recomendação condicional, evidências de baixa qualidade). • A adição do teste do ácido nucleico à nascença às abordagens já existentes para o diagnóstico pediátrico pode ser considerada, para identificar a infecção pelo VIH em crianças expostas ao VIH (recomendação condicional, evidências de baixa qualidade). • Deve considerar-se a substituição do teste serológico aos nove meses de idade pelo teste do ácido nucleico. Fig. 3. Previsão estimada do diagnóstico pediátrico nos países de baixos e médios rendimentos em todo o mundo 1.4 M 1.6M 1.7M 1.9M 2.0M 2.2M 922K 921K 912K 875K 750K 541K 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 0.0M 0.5M 1.0M 1.5M 2.0M 2.5M 3.0M 2017 2018 2019 2020 2021 2022 Co be rt ur a Procura prevista Necessidade não satisfeita Cobertura Te st es d e D PP Fonte: 2018 CHAI HIV Market 7Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce 2. USO DOS RESULTADOS DOS TESTES DA CAR PEDIÁTRAICO VIRAL PARA APOIAR O TRATAMENTO CLÍNICO DAS PESSSOAS QUE VIVEM COM O VIH E FAZEM TERAPIA ANTIRRETROVIRAL As orientações consolidadas da OMS de 2016 sobre o uso de antirretrovirais para tratar e evitar a infecção pelo VIH (2) apresenta a firme recomendação de se usar o teste da carga viral regularmente como ferramenta ideal de monitorização dos antirretrovirais. A OMS recomenda a realização do teste da carga viral aos seis meses depois do início da terapia antirretroviral, aos 12 meses e, depois, anualmente, para permitir a detecção precoce do insucesso do tratamento, prevenir a resistência aos medicamentos, identificar as pessoas com cargas virais elevadas e fraca adesão e evitar uma mudança inapropriada do regime de tratamento (2). Em 2014, a ONUSIDA lançou as metas de tratamento 90–90–90 que deveriam ser atingidas em 2020 e se destinavam a ajudar a erradicar a epidemia da SIDA enquanto ameaça para a saúde pública. A terceira meta 90 pretende garantir que 90% das pessoas que fazem terapia antirretroviral terão suprimido as cargas virais (13). A terapia antirretroviral e a adesão ao tratamento oferece benefícios clínicos notáveis e sustentados, mesmo nas pessoas com doença por VIH já avançada. As evidências das tabelas nacionais sobre a carga viral e os resultados da avaliação do impacto sobre o VIH baseado nas populações (14) sugerem que as taxas de supressão nas pessoas que vivem com o VIH e fazem a terapia antirretroviral se situam geralmente entre 85–92% (Fig. 4). É importante compreender as taxas de supressão viral a nível da população para identificar potenciais focos de transmissão, informar as metas nacionaise fazer esforços orientados para a melhoria da qualidade dos programas, mas talvez seja mais importante, a nível do doente, oferecer cuidados melhorados e apoio às pessoas que vivem com o VIH. As pessoas clinicamente estáveis com cargas virais não detectáveis podem optar pela prestação de serviços diferenciados que reduzem o número de visitas às clínicas e permitem a prescrição de medicamentos para três a seis meses. Para além disso, o teste da carga viral é fundamental para assegurar que as pessoas que vivem com cargas virais detectáveis superiores a 1000 cópias/ml Fig. 4. Taxas de supressão da carga viral em alguns países Suprimida Não suprimida Lesoto Maláui Eswatini República Unida da Tanzânia Zâmbia Zimbabué 88.3% 90.8% 91.9 % 87.7% 89.2% 86.5% Population based HIV impact assessments, 2015–2017 Namíbia 91.3% Camarões 80.0% Côte d’Ivoire 75.9% Quénia Uganda 86.4% 92.2% Tabelas nacionais da carga viral: 2017 85.0% África do Sul 88 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce recebam aconselhamento mais enfático para a adesão e sejam monitorizadas mais de perto para determinar se precisam de mudar para um tratamento de segunda linha (Fig. 5). O algoritmo de monitorização do tratamento destina-se a ajudar os clínicos e os doentes a determinarem se as cargas virais elevadas ou a suspeita de insucesso do tratamento são causadas pela resistência aos medicamentos ou pela fraca adesão. Não é desejável, nem para as pessoas que vivem com o VIH, nem para os programas, que se faça uma mudança desnecessária para regimes de segunda linha mais dispendiosos e menos bem tolerados, quando as pessoas simplesmente não aderem aos tratamentos, em primeiro lugar, porque os problemas da adesão não irão necessariamente ser resolvidos com a mudança. Contudo, continuar um regime que não resulta, quando a causa de fundo é a resistência aos medicamentos pode conduzir a maior resistência medicamentosa, maior deterioração da imunidade e possivelmente a efeitos clínicos. Os resultados dos testes da carga viral estão a ser usados para tomar decisões clínicas? Os testes da carga viral têm aumentado significativamente nos últimos anos, de 7 milhões de testes, em 2013, para 15 milhões, em 2017. No entanto, a realização de testes da carga viral não deveria ser a principal preocupação dos programas da carga viral. Os programas deverão igualmente concentrar-se no modo como os resultados desses testes são usados para informar a tomada de decisões clínicas. Os Médicos Sem Fronteiras realizaram uma análise aprofundada da execução dos principais passos no algoritmo de monitorização do tratamento da carga viral em seis países e 149 unidades de saúde apoiadas pelos seus programas (15). Entre as pessoas com uma carga viral inicial elevada (média de 18%), uma média de 68% assistiu, pelo menos, a uma sessão de aconselhamento para uma maior adesão, 52% fizeram um segundo teste de seguimento da carga viral, 34% voltaram a suprimir (<1000 cópias/ml) e 33% das pessoas elegíveis mudaram para um tratamento de segunda linha. Estes resultados são ainda apoiados por uma análise preliminar dos dados publicamente disponíveis e que foram divulgados nas tabelas nacionais de três países da África Oriental (16). Apesar de um aumento na cobertura dos testes da carga viral e da percentagem animadora da supressão viral, foram menos de 10% as pessoas com carga viral elevada no primeiro teste que passaram pelo algoritmo da carga viral para fazerem um segundo teste de seguimento, a fim de se determinar a necessidade de mudarem para um regime de segunda linha (Fig. 6). Esta tendência tem permanecido constante ao longo dos anos. Fig. 5. Algoritmo de monitorização da OMS para o insucesso do tratamento Monitorização orientada da carga viral (suspeita de falha clínica ou imunológica) Carga viral de rotina (detecção precoce de falha virológica) Teste da carga viral Carga viral> 1000 cópias/ml Avaliação das preocupações com a adesão Repetir o teste da carga viral após 3-6 meses Carga viral < 1000 cópias/ml Manter terapia de primeira linha Mudar para terapia de segunda linha Carga viral >1000 cópias/ml 9Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce Principais considerações Os testes de diagnóstico só terão algum valo significativo, se os resultados forem usados clinicamente. A interface laboratório- clínicas pode ser o mecanismo mais difícil, mas também o mais crítico e gratificante, para melhorar o tratamento do doente. Para criar serviços de saúde eficazes que prestem os melhores cuidados e tratamento às pessoas que vivem com o VIH, os programas terão de revitalizar e investir na interface laboratório-clínicas e garantir a disponibilidade adequada de formação, instrumentos e ambiente para melhorar a utilização em devido tempo de todos os resultados do diagnóstico. Existem actualmente vários instrumentos para melhorar a utilização clínica dos resultados dos testes da carga viral e que poderão ser adaptados e adoptados pelos programas nacionais (17-19). O reforço dos programas da carga viral bem sucedidos exige que sejam usados todos os resultados dos testes, que devem ser integrados nos serviços clínicos, para optimizar os cuidados aos doentes e o êxito dos programas. Fig. 6. Testes da carga viral realizados em três países da África Oriental, 2012–2016 Carga viral Carga viral > 1000 2e Carga viral 1 500 000 1 000 000 500 000 N úm er o da te st es d a ca rg a vi ra l 0 2012 2013 2014 2015 2016 10 10 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce 3. ALCANCE E ACESSO ESTIMADO AOS TESTES DA CARGA VIRAL USANDO AMOSTRAS DE PLASMA TRADICIONAIS As orientações consolidadas da OMS de 2016 sobre o uso de medicamentos antirretrovirais para tratar e prevenir a infecção pelo VIH (2) recomendam a carga viral como a abordagem ideal de monitorização para diagnosticar e confirmar o insucesso do tratamento e as amostras de plasma como o tipo ideal de amostras para os testes da carga viral. Embora, desde a recomendação inicial, em 2013, se tenha registado um reforço significativo nos países de recursos limitados e com pesado fardo de infecção pelo VIH, o acesso integral tem sido limitado por inúmeros obstáculos. Em particular, a utilização de plasma usando tubos de EDTA (ver Secção 4) pode ser limitada, devido à rigidez dos tempos de estabilidade e temperaturas de armazenamento das amostras que são transportadas para o laboratório de referência ou para um centro intermédio para processamento. Para além disso, também é limitada a disponibilidade de cadeias de frio entre as várias unidades de saúde e os pontos de testagem, em contextos de recursos limitados. Contudo, as amostras tradicionais de plasma EDTA têm um significativo potencial para os testes da carga viral. Mesmo dentro dos tempos de estabilidade e temperaturas de armazenamento das amostras, muitas pessoas ainda têm acesso aos testes da carga viral usando este tipo de amostra. Recentemente, foi feita uma análise para se entender melhor o raio em torno dos laboratórios de referência ou centros intermédios dentro do qual as pessoas podem aceder aos testes da carga viral, usando amostras tradicionais de plasma EDTA. Essa análise foi feita em quatro países: Eswatini, Nigéria, Ruanda e Zimbabué. Foram assumidos vários pressupostos, nomeadamente: • Veículos viajando a 50 km/h; • Medições em linha recta desde a unidade de saúde até ao laboratório de referência ou centro intermédio mais um factor de circuito de 17%; • Laboratórios de referência ou centros intermédios considerados como o ponto final para a separação do plasma; • A última amostra colhida todos os dias teve uma espera máxima de duas horas na unidade de saúde, antes de ser recolhida e transportada para o laboratório de testes ou centro intermédio dentro do tempo de estabilidade no armazenamento estipulado pelo fabricante; e • Esta análise não incorpora outros tipos alternativos de amostras de plasma, nem considera a centrifugação no local, nem o respectivo armazenamento e transporte. Tabela 1. Acesso aos testes da carga viral usando plasma EDTA tradicional Pais Acesso aos testes da carga viral usando plasma EDTA tradicional Unidades Pessoas Eswatini 260/350 (74%) 250 000/320 000 (78%) Nigéria 750/2 600 (29%) 450 000/1 200 000 (38%) Ruanda 505/550 (92%) 148 000/165 000 (90%) Zimbabué 700/1 500 (47%) 120 000/190 000 (63%) Total 2 215/5 000 (44%) 968 000/1 875 000 (52%) 11Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce Análise ilustrativa do raio para o plasma Nos quatro países, pouco menos de metade de todas as unidades de saúde estão suficientemente perto do laboratório de testes ou do centro intermédio para transportar as amostras tradicionais de plasma EDTA dentro do tempo estipulado pelo fabricante. Isso representa mais de 50% ou quase 1 milhão de pessoas nos quatro países analisados que têm acesso a testes da carga viral usando plasma EDTA tradicional. Mesmo num país geograficamente tão grande como a Nigéria, quase 40% das pessoas em terapia antirretroviral e que precisam de testes da carga viral teriam acesso usando plasma EDTA tradicional. Países geograficamente mais pequenos, como Eswatini e Ruanda, podem ter menos laboratórios, mas podem fornecer acesso a testes da carga viral, usando amostras de plasma EDTA tradicional, a quase 80% ou mais das pessoas em terapia antirretroviral. O elevado acesso a testes da carga viral usando plasma EDTA tradicional é possível, porque a maioria dos laboratórios de referência da carga viral se situam nos principais centros urbanos. Do mesmo modo, os maiores centros de terapia antirretroviral a que as pessoas recorrem estão localizados, muitas vezes, nos principais centros urbanos. Esta análise salienta o facto de mais de 50% das pessoas em terapia antirretroviral e que precisam de fazer testes da carga viral residirem a algumas horas de distância do laboratório de testes ou centro intermédio, permitindo assim o transporte das amostras dentro do tempo estabelecido (Fig. 7). Conclusões Esta análise ilustrativa constitui um retrato do potencial acesso aos testes da carga viral, usando a amostra de plasma como preferencial preferencial. A percentagem de pessoas que têm acesso a testes da carga viral usando amostras de plasma EDTA tradicional pode variar conforme os locais, dependendo de vários factores, como o número de laboratórios, as infraestruturas rodoviárias e o tamanho do país. Devem fazer-se todos os esforços possíveis para garantir o acesso a testes da carga viral usando as amostras de plasma que são ideais. Poderá ser útil realizar análises aprofundadas semelhantes em todos os países, para determinar as unidades de saúde e as pessoas que conseguem ter acesso aos testes da carga viral usando amostras de plasma tradicionais. As actuais infraestruturas poderão nem sempre permitir o uso do plasma EDTA tradicional em muitos locais, devido ao mau estado das estradas e das infraestruturas, grandes distâncias, transporte improvisado de amostras, etc. Por conseguinte, para as unidades e pessoas que não têm acesso a testes da carga viral usando o plasma EDTA tradicional, podem ser encontradas alternativas para garantir esse acesso, incluindo melhores infraestruturas, redes de transporte de amostras e tipos de amostras e tecnologias diferentes. Este kit de ferramentas de diagnóstico molecular fornecerá informação básica e dados sobre várias dessas estratégias alternativas para garantir uma abordagem complementar à expansão do acesso. Fig. 7. Exemplo ilustrativo do raio de acesso do plasma aos laboratórios de referência do Zimbabué 12 12 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce 4. ESTABILIDADE DAS AMOSTRAS PARA OS TESTES DA CARGA VIRAL DO VIH As orientações consolidadas da OMS de 2016 sobre o uso de antirretrovirais para tratar e prevenir a infecção pelo VIH (2) recomendam a carga viral como a abordagem ideal de monitorização, para diagnosticar e confirmar o insucesso do tratamento e as amostras de plasma como o tipo de amostra ideal para os testes da carga viral. Embora, desde a recomendação inicial de 2013, se tenha registado um reforço significativo em países de recursos limitados com um pesado fardo de infecções pelo VIH, existem vários obstáculos significativos que têm limitado o acesso integral aos testes da carga viral. Em particular, o uso de plasma líquido e o uso do ácido etilenodiamino tetra-acético (EDTA) ou tubos de preparação do plasma (ver subsecção 6.1) podem ser limitados devido à rigidez dos tempos de estabilidade no armazenamento das amostras, dentro dos quais as amostras devem ser transportadas para o laboratório de referência ou para um centro intermédio, para processamento. A Tabela 2 apresenta os tempos máximos, de acordo com a temperatura de armazenamento estipulada pelos fabricantes, desde a colheita das amostras de sangue total até à separação do plasma. Prolongar o tempo de armazenamento antes do processamento para além destas recomendações poderá afectar o desempenho e os riscos, fornecendo resultados incorrectos aos clínicos e aos doentes. Depois de separadas em plasma, as amostras podem ser congeladas durante longos períodos de tempo, antes de serem analisadas. Contudo, as unidades de saúde podem não ter centrífugas, congeladores e/ou as necessárias competências para prolongarem esses tempos de estabilidade depois da separação do plasma. Tabela 2. Informações do fabricante sobre a estabilidade do sangue total Teste Tempo máximo da colheita de amostras de sangue total para separação de plasma Temperatura ambiente (temperatura) Refrigeração (temperatura) Abbott RealTime HIV-1 (20,21) 24 heures (15 à 30°C) (temperatura) 48 horas (2–8°C) Abbott m-PIMA HIV-1/2 VL (22,23) 48 horas (18–28°C) NR Biocentric Generic HIV Charge Virale (24) 24 horas (2–25°C) 24 horas (2–25°C) bioMérieux NucliSENS EasyQ® HIV-1 (25,26) NR 24 horas (2–8°C) Cavidi ExaVir Load (27) 4–6 horas (sem temperatura especificada) Cepheid Xpert HIV-1 Viral Load (28,29) 8 horas (15–30°C) 72 horas (2–8°C) Hologic Aptima HIV-1 Quant Dx (30,31) 24 horas (2–30°C) 24 horas (2–30°C) Qiagen artus HI Virus-1 RG (32) 6 horas (sem temperatura especificada) Qiagen artus HI Virus-1 QS-RGQ (33) 6 horas (sem temperatura especificada) Roche COBAS TaqMan HIV-1 (34,35) 24 horas (2–25°C) 24 horas (2–25°C) Roche cobas HIV-1 for cobas 4800 System (36) 24 horas (2–25°C) 24 horas (2–25°C) Roche cobas HIV-1 for cobas 6800/8800 Systems (37) 24 horas (2–25°C) 24 horas (2–25°C) Sacace HIV Real-TM Quant Dx (38) NR 12 horas (2–8°C) Siemens VERSANT HIV-1 RNA 1.5 (39) 6 horas (15–25°C) 24 horas (2–8°C) © W H O NR: não reportado. 13Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce Estabilidade do sangue total para uma revisão sistemática da carga viral do VIH Em 2014, foi publicada uma revisão sistemática de nove estudos intitulada “Expandir o acesso a testes da carga viral do VIH: Estabilidade do ARN em tubos de EDTA e tubos de preparação de plasma para além dos limites actuais de tempo e temperatura” (40). Esta revisão sistemática apontava três achados principais: • O sangue total e o plasma eram estáveis até 168 horas depois; • O sangue total era estável até 72 horas depois da colheita da amostra, quando armazenado a 25°C; • O plasma era estável até 48 horas depois da colheita da amostra (tubos de preparação de plasma) ou da separação do plasma (EDTA), quando armazenado a 25°C. No entanto, algumas limitações importantes a considerar são que todos os estudos incluíam análises laboratoriais e não os tempos e as temperaturas realistas de armazenamento e transporte e todos foram realizadas nos Estados Unidos ou na Europa. Por outro lado, apenasestavam disponíveis alguns estudos relevantes para inclusão e na maioria deles o tamanho das amostras era pequeno. Para além disso, poucos estudos incluíam amostras que tivessem as cargas virais suprimidas (<1000 cópias/ml), tornando difícil a interpretação dos resultados dentro dessa amplitude. Contudo, um estudo recentemente publicado observava resultados elevados de carga viral em amostras de carga viral indetectável, quando o plasma era armazenado para além das 72 horas (41) . Curiosamente, 20% dos resultados da carga viral indetectável revelavam viremia de baixo nível a qualquer temperatura ambiente ou refrigeração. Além disso, 51% das amostras indetectáveis tornavam-se virémicas de baixo nível, se o plasma não fosse de novo centrifugado antes dos testes após 48 horas de armazenamento. Conclusões Felizmente, desde a publicação da revisão sistemática, vários fabricantes prolongaram para 24 horas a recomendação para a estabilidade à temperatura ambiente, desde a colheita das amostras de sangue total até à separação do plasma. Embora a revisão sistemática sugira que as amostras são estáveis para além das indicações do fabricante, os países e os laboratórios são responsáveis pelos resultados dos testes da carga viral nessas condições não oficiais. Outras investigações e o apoio dos fabricantes à extensão das recomendações de estabilidade do sangue total para o uso pretendido devem ser encorajadas, uma vez que uma estabilidade mais alargada das amostras sustentaria a expansão do acesso aos testes da carga viral, usando o tipo de amostra ideal: o plasma. © W H O 14 14 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce 5. CONTEXTO TÉCNICO: TESTES BASEADOS NO ÁCIDO NUCLEICO O que são testes da carga viral? Os testes da carga viral do VIH são uma forma de quantificar o número de vírus presentes numa amostra de sangue. O sangue total é constituído por componentes celulares (glóbulos brancos, glóbulos vermelhos e plaquetas) e plasma livre de células. Os testes de ácido nucleico fazem-se usando um teste de amplificação do ácido nucleico, que determina o número de cópias de VIH por mililitro de plasma. Os testes de amplificação do ácido nucleico funcionam amplificando, quer o material genético do VIH, quer uma sonda ligada ao VIH (42). O teste usa depois uma reacção química para medir a quantidade de amplificação observada durante o teste, que corresponde à quantidade de VIH presente na amostra. O tipo mais comum de teste da carga viral é a reacção em cadeia da polimerase quantitativa (qPCR). Outros tipos de testes da carga viral são a amplificação mediada por transcrição e os testes de ADN ramificado (2). Utilidade dos testes da carga viral • A monitorização da carga viral do VIH é importante para garantir o êxito da terapia antirretroviral. A monitorização da carga viral é a abordagem ideal para diagnosticar e confirmar o insucesso do tratamento (2). • Os testes da carga viral fornecem conhecimentos, controlo e motivação aos utentes para que estes compreendam a sua infecção pelo VIH e adiram ao tratamento (43). • Progressão da doença e a transmissão do VIH (44,45). © W H O 15Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce Tabela 3. AND e ARN do VIH no sangue total Blood: cellular portion Blood: plasma portion AND e ARN do VIH Glóbulos brancos (como as células CD4): o ADN do VIH está contido no interior das células juntamente com cópias do VIH que contêm ARN do VIH durante a replicação do VIH. Também se descobriu que o VIH está associado às plaquetas, muito provavelmente à superfície das células, embora o vírus não se encontre no interior das plaquetas ou glóbulos vermelhos. O ARN do VIH encontra-se no vírus livre do plasma. O ADN do VIH não deveria estar presente em quantidades significativas, embora se possam encontrar pequenas quantidades de ADN no plasma de células que se abriram ou que foram transferidas para o plasma por insuficiente separação do sangue total. Tipo de amostra O sangue total contém tanto a componente celular do sangue como o plasma. O sangue total contém tanto o ADN do VIH, como o ARN intracelular e ARN livre de células e tem sido usado para o diagnóstico pediátrico precoce do VIH e os testes de resistência do VIH aos medicamentos. O plasma é o tipo de amostra ideal para os testes da carga viral, que pretende detectar o número de cópias de ARN do VIH por mililitro de plasma. Este também pode ser usado para o teste de resistência do VIH aos medicamentos, se estiver presente o ARN adequado do VIH (>400 cópias/ml). Métodos de teste O sangue total é analisado tanto na forma líquida como na de gota de sangue seco. O teste da carga viral que usa o sangue total poderá não ser rigoroso, se o teste detectar quantidades significativas de ADN do VIH e/ou ARN intracelular, além do ARN livre de células (plasma) que o teste deveria detectar. O plasma é, normalmente, analisado na forma líquida mas pode também ser analisado a partir de uma gota de plasma seco. ADN do VIH versus ARN do VIH O VIH é um vírus do ARN que compreende o ARN e proteínas. Durante o seu ciclo de replicação, o material genético do VIH existe tanto em forma de ARN como de ADN. O ADN do VIH é o material genético do VIH que se encontra no interior das células do corpo infectadas pelo VIH. No sangue total, o ADN do VIH encontra-se principalmente no interior dos glóbulos brancos chamados de células CD4, que são uma parte importante do sistema imunitário. O VIH integra o seu ADN no ADN das células CD4 e, por isso, pode usar as células para fazer mais cópias de si próprio. Nesta forma, é conhecido como ADN pró-viral do VIH (46–48). O ARN do VIH encontra-se. normalmente, no plasma, que é a parte do sangue total, depois de removidas todas as células. O sangue total é, normalmente, separado em plasma e seus componentes celulares, através da centrifugação do sangue. O VIH existe como vírus do ARN no plasma, antes de infectar as células, como ARN intracelular dentro das células, à medida que são feitas cópias do vírus e de novo no plasma, quando essas cópias do vírus são libertadas (6–8). Quando o VIH é suprimido pela terapia antirretroviral, o ADN do VIH continua presente nas células e ocasionalmente como ARN intracelular, mas no plasma é detectado pouco ou nenhum ARN do VIH, uma vez que os medicamentos impedem a replicação viral. Contudo, quando o VIH não é suprimido, a maior parte do ácido nucleico do VIH fica. normalmente, presente no plasma como ARN do VIH, com ARN intracelular adicional da replicação viral activa, ficando uma percentagem mais pequena presente no interior das células como ADN do VIH (7). Como funcionam os testes da carga viral? Os testes do ácido nucleico do VIH podem detectar tanto o ADN do VIH como o ARN que estão presentes numa amostra. Alguns testes foram concebidos para detectar preferencialmente o ADN ou o ARN, mas como o ADN e o ARN do VIH são cópias do mesmo material genético, também podem ser difíceis de distinguir. No entanto, os testes da carga viral destinam-se a medir a quantidade de ARN do VIH no plasma. O plasma é, portanto, o tipo de amostra ideal para os testes da carga viral; contudo, existem tipos alternativos de amostras e tecnologias para apoiar a expansão do acesso aos testes, incluindo gotas de sangue seco que são preparadas usando o sangue total (2,49). As amostras de gotas de sangue seco permitem tempos mais longos de armazenamento e transporte; todavia, o uso do sangue total resulta, muitas vezes, na detecção de ADN próviral do VIH, ARN intracelular e ARN livre de células. Em conjunto, isso pode resultar numa quantificação excessiva dos resultados da carga viral. A Tabela 3 explica as diferenças entre os dois principais componentes do sangue total (ADN e ARN) nos testes da carga viral do VIH. © W H O 16 16 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce Caixa 2. Tempo versus tecnologias para diagnosticar o VIH em bebés A técnica baseada no ácido nucleico (qPCR) usada nos testes da carga viral é muito semelhante e, muitas vezes, a mesma, tanto para os testes pediátricos como para os testes qualitativos. O “DNA PCR” do VIH é um sinónimo geralmente usado para os testes de diagnóstico pediátrico do VIH. No entanto, é importante distinguir entre a tecnologia usada para os testes (tais como PCR versus teste de anticorpos do VIH) e o intervalo de tempo dos testes. O diagnóstico pediátrico precoce refere-se especificamente ao teste do ácido nucleico à nascença ou nos primeiros dois meses de vida, enquanto o diagnóstico pediátrico se refere aos testes efectuados durante o período de exposição, incluindo o teste de ácido nucleico aos nove meses. O diagnóstico pediátrico é normalmente feito no sangue total, quer na forma líquida, quer em gota de sangue seco. Estes testes podem detectar o ADN do VIH, o ARN intracelular e o ARN livre de células. Isso não é um problema e até melhora a sensibilidade do teste, uma vez que a presença de material genético do VIH no sangue pode indicar infecção pelo VIH. Uma vez que tanto o ADN como o ARN do VIH estão presentes, o teste virológico ou o teste de amplificação do ácido nucleico do VIH são termos mais rigorosos para o PCR do bebé do que o PCR do ADN do VIH. Caixa 3. Principal terminologia da carga viral Carga viral suprimida: medições da carga viral abaixo de 1000 cópias/ml. Uma carga viral não suprimida ou elevada é uma medição acima de 1000 cópias/ml (2). Carga viral indetectável: ausência de VIH detectado numa amostra de sangue através do teste da carga viral. A Tabela 4 mostra os limites de detecção dos testes da carga viral disponíveis no comércio viral. © W H O 17Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce Tabela 4. Resumo dos testes da carga viral do VIH Nome do fabricante e do teste Nome do fabricante e do teste Tipo de amostra Limite de detecção (cópias/mL) Tempo máximo desde a colheita da amostra de sangue total até à separação do plasma Aprovação da entidade reguladora Teste de diagnóstico pediátrico precoce Abbott: RealTime HIV-1 (20,21)1,2 m-PIMATM HIV-1/2 VL (22,23) Plasma DBS Plasma 40 839 800 24 h at 15–30°C, 48 h entre 2 et 8°C 48 h at 18–28°C CE, FDA, OMS CE, OMS CE, OMS Disponível, teste separado Disponível, teste separado Biocentric GENERIC HIV Charge Virale (24) Plasma 390 24 h at 2–25°C CE Disponível, teste separado bioMérieux NucliSENS EasyQ® HIV- 1 v2.0 (25,26) Plasma DBS 25 802 24 h entre 2–8°C CE, OMS CE, OMS N/D Cavidi ExaVirTM Load (27) Plasma 200 4–6 h, sem temperatura especificada CE N/D Cepheid Xpert® HIV-1 Viral Load (28,29) Plasma 40 8 h at 15–30°C, 24 h at 15–25°C, 72 h entre 2–8°C CE, OMS Disponível, teste separado Hologic Aptima™ HIV-1 Quant Dx (30,31) Plasma 30 24 h at 2–30°C CE, FDA, OMS Même test Qiagen: artus® HI Virus-1 RG (32) artus® HI Virus-1 QS-RGQ (33) Plasma Plasma 60 45 6 h, sem temperatura especificada 6 h, sem temperatura especificada CE CE N/D N/D Roche: COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0 (34,35) cobas® HIV-1 for cobas® 4800 System (36) cobas® HIV-1 for cobas® 6800/8800 Systems (37) Plasma PSC Plasma PSC Plasma PSC 20 738 20 599 13.2 790 24 h at 2–25°C 24 h at 2–25°C 24h at 2–25°C CE, FDA, OMS CE, OMS CE CE, FDA CE Disponível, teste separado Mesmo teste Disponível, teste separado Sacace HIV Real-TM Quant DX (38) Plasma 48 IU/mL 12 h at 2–8°C CE N/D Siemens VERSANT® HIV-1 RNA 1.5 (39) Plasma 37 6 h at 15–25°C, 24 h at 2–8°C CE N/D © W H O 1 1 Abbott Laboratories (2014). Abbott RealTime HIV-1 Instructions for Use. 2 WHO Prequalification of Diagnostics Programme (2016). Public Report: Abbott RealTime HIV-1. Disponible à l’adresse suivante : https://www.who.int/ diagnostics_laboratory/evaluations/pq-list/hiv-vrl/180423_amended_final_pqpr_0145_027_00_v11.pdf. h: horas; CE: Conformidade Europeia, cumprindo os regulamentos da União Europeia; FDA: aprovação da Administração dos Estados Unidos para os Alimentos e Medicamentos; OMS: pré-qualificação da OMS do diagnóstico in vitro; DBS: gota de sangue seco; PSC: gota de plasma seco de um cartão de separação do plasma; N/D: actualmente não disponível; IU: unidades internacionais. 18 18 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce 6. TIPOS ALTERNATIVOS DE AMOSTRAS E TECNOLOGIAS A CONSIDERAR QUANDO NÃO FOR POSSÍVEL USAR PLASMA LÍQUIDO DE MODO GENERALIZADO PARA OS TESTES DA CARGA VIRAL, DEVIDO A CONSTRANGIMENTOS COM INFRAESTRUTURAS, TRANSPORTES OU OUTROSINFRASTRUCTURES, AU TRANSPORT OU AUTRES 6.1 TIPOS ALTERNATIVOS DE AMOSTRAS E TECNOLOGIAS: AMOSTRAS DE GOTA Embora as amostras de plasma sejam as normais para os testes da carga viral, o seu uso é limitado pelas exigências de estabilidade da temperatura ambiente dos marcadores biológicos virais no sangue total e no plasma durante o armazenamento e transporte e a limitada disponibilidade de cadeias de frio entre muitas unidades de saúde nos locais com escassos recursos. As amostras de gotas de sangue seco para os testes do VIH estão bem estabelecidas em locais de recursos limitados e têm sido regularmente usadas para a colheita e transporte de amostras para o diagnóstico pediátrico do VIH, através de testes por PCR, em laboratórios centralizados. Essas amostras são benéficas, porque não exige centrífugas, frigoríficos ou congeladores no local da sua colheita, podem ser armazenadas e transportadas durante semanas à temperatura ambiente e requerem apenas uma simples picada no dedo ou no calcanhar para colher uma amostra de sangue, que pode ser preparada por quadros inferiores do pessoal das unidades de saúde. Benefícios semelhantes podem ser obtidos, usando amostras de gotas de sangue seco para os programas de teste da carga viral em locais de recursos limitados. As condições necessárias para o armazenamento e transporte podem ser diferentes, quando se usam as amostras de sangue seco para os testes de resistência aos medicamentos. As amostras de gotas de sangue seco para os testes da carga viral usando métodos de detecção baseados no ácido nucleico usam o sangue total como a amostra de entrada, o que pode resultar na extracção e detecção de ADN pró-viral e ARN intracelular, além da meta do biomarcador primário do ARN viral livre que circula no plasma. Em conjunto, isto pode resultar numa quantificação excessiva do resultado da carga viral. Têm sido limitados os progressos feitos na garantia da qualidade do uso de amostras de gotas de sangue seco nos testes da carga viral do VIH, através da aprovação das entidades reguladoras internacionais. Aprovações da regulação e avaliações técnicas das amostras de gotas de sangue seco (os países consideram muitas vezes essas aprovações, quando compram ou seleccionam tecnologias de diagnóstico): © W H O 19Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce Tabela 5. Síntese dos resultados da meta-análise das avaliações técnicas Teste Tamanho da amostra Sensibilidade (95% CI)a Especificidade (95% CI)a Abbott RealTime HIV-1, one-spotb 700 88.26% (49.64–98.28) 99.07% (68.38–99.98) Abbott RealTime HIV-1, two-spot 2004 93.13% (83.72–97.27) 91.11% (82.35–95.75) Biocentric Generic HIV Charge Virale 531 94.86% (71.14–99.28) 55.16% (35.01–73.75) bioMérieux NucliSENS EasyQ® HIV-1 1062 82.95% (78.38–86.71) 95.06% (89.29–97.80) Hologic Aptima 382 87.52% (77.93–93.30) 87.18% (59.01–96.98) Roche COBAS TaqMan HIV-1 Free Virus Elution 3076 94.77% (84.59–98.36) 93.93% (71.95–98.94) Roche COBAS TaqMan HIV-1 SPEX 3190 98.23% (95.85–99.26) 48.49% (22.63–75.18) Siemens VERSANT HIV-1 RNA 144 90.97% (69.20–97.83) 87.76% (75.28–94.41) a La Sensibilidade e especificidade usando um limiar de insucesso do tratamento de 1000 cópias/ml. b Como uma notificação da mudança, uma avaliação laboratorial de amostras de gotas de sangue seco usando o protocolo indicado pela CE não foi realizado no seio da análise de pré-qualificação da OMS. • CE-IVD (Conformidade Europeia do diagnóstico in vitro): duas tecnologias receberam CE- IVD para usarem amostras de gotas de sangue seco nos testes da carga viral: Abbott RealTime HIV- 1 e bioMérieux NucliSENS EasyQ® HIV-1; e • Pré-qualificação da OMS: há duas tecnologias que cumpriram os requisites da OMS: bioMérieux NucliSENS EasyQ® HIV-1, em Janeiro de 2017 e Abbott RealTime HIV-1 (21) , em 24 de Agosto de 2017. O limite de detecção do teste Abbott RealTime HIV-1 usando amostras de gotas de sangue seco é 839 cópias/ml (21). Avaliações técnicas independentes: os resultados de 40 avaliações técnicas de amostras de gotas de sangue seco em mais de 25 países, examinando seis tecnologias de teste da carga viral disponíveis no mercado, foram incluídas numa meta-análise clínica abrangente, que resultou em mais de 10 000 pontos de dados emparelhados de gotas de sangue seco– plasma (Tabela 5) (50). Recomendações da OMS As orientações consolidadas da OMS de 2016 sobre o uso de antirretrovirais no tratamento e prevenção da infecção pelo VIH (2) , recomendam que as amostras de gotas de sangue seco, usando sangue total venoso ou capilar, possam ser usadas para determinar a carga viral do VIH. Deve usar-se um limite de 1000 cópias/ml para determinar o insucesso do tratamento, quando se usam amostras de gotas de sangue seco, como está definido para os testes no plasma. Embora as amostras de plasma sejam as preferidas para os testes da carga viral, recomenda-se o uso de amostras de gotas de sangue seco nos locais em que os obstáculos logísticos, infraestruturais ou operacionais impedem uma monitorização regular da carga viral usando amostras de plasma. Uso actual As amostras de gotas de sangue seco constituem uma forma de melhorar a cobertura e o alcance dos testes da carga viral, quando a preparação e o transporte de amostras de plasma podem ser limitadas pelos requisitos da cadeia de frio ou os problemas de transporte. Há vários países que estão actualmente a usar amostras de gotas de sangue seco para permitir o acesso e reforço dos testes da carga viral. Em 2018, fizeram-se mais de 2 milhões de testes da carga viral usando amostras de gotas de sangue seco, em seis países com um pesado fardo de infecções pelo VIH. Além disso, alguns países começaram a implementar o uso de amostras de sangue seco nos testes da carga viral, usando os protocolos recomendados pelos fabricantes, apesar de a sua utilização não estar indicada no rótulo. Conclusões Existem evidências suficientes sobre o desempenho das amostras de gotas de sangue seco nos testes da carga viral que permitem apoiar uma rápida aprovação das entidades nacionais reguladoras e o início do reforço. Para além disso, não é provável que as avaliações técnicas destas tecnologias acrescentem algum valor, mas, pelo contrário, podem retardar a implementação e a monitorização do tratamento em devido tempo. Contudo, é essencial que os fornecedores procurem a aprovação dos reguladores e a pré-qualificação da OMS para esses tipos alternativos de amostras, em apoio ao reforço e acesso do país aos testes da carga viral. © W H O 20 20 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce 6.2 TIPOS ALTERNATIVOS DE AMOSTRAS E TECNOLOGIAS: PLASMA AMOSTRAS DE GOTAS DE PLASMA SECO PARA OS TESTES DA CARGA VIRAL DO VIH Uma outra alternativa ao uso de plasma líquido para os testes da carga viral são as amostras de gotas de plasma seco. Estas amostras usam o mesmo papel de filtro ou semelhante que as amostras de gota de sangue seco para o diagnóstico da carga viral ou pediátrico; contudo, com a aplicação do plasma em vez do sangue total, estão agora em desenvolvimento ou passaram a estar recentemente disponíveis no mercado cartões de separação do plasma e dispositivos simples, para apoiar a expansão dos testes da carga viral usando amostras de plasma. As amostras de gotas de plasma seco para os testes do VIH são um tipo de amostra alternativo desenvolvido de modo semelhante às amostras bem conhecidas de gotas de sangue seco (sub-secção 6.1) que têm sido regularmente utilizadas para colher e transportar amostras para o diagnóstico pediátrico do VIH, que são submetidas a testes por PCR em laboratórios centralizados. Embora exijam centrifugação ou colheita do plasma para a colocação de gotas no cartão, podem ser armazenadas e transportadas durante semanas à temperatura ambiente. Uma vantagem das amostras de gotas de plasma seco é que a separação e a utilização do plasma retira a detecção e quantificação do ARN intracelular e do ADN pró-viral muitas vezes observados com as amostras de sangue total; contudo, o menor volume de amostra analisada pode limitar uma comparabilidade perfeita com as amostras de plasma líquido. Normalmente, o plasma preparado para amostras de gotas de plasma seco ou cartões ou dispositivos de separação do plasma deriva do sangue total colhido em tubos de EDTA ou tubos de preparação do plasma (ver sub-secção 6.3). Os fabricantes deverão, por isso, incluir um ou ambos os tipos de tubos nas suas recomendações para o uso pretendido e na documentação para aprovação das entidades reguladoras. A maioria dos testes da carga viral actualmente no mercado incluem um ou ambos os tipos de tubos. Tabela 6. Síntese dos resultados da meta-análise da avaliação técnica Teste Tamanho da amostra Sensibilidade (95% CI)a Especificidade (95% CI)a All technologies 1872 92.54% (87.85-95.52%) 95.15% (87.41-98.23%) Abbott RealTime HIV-1 245 99.39% (95.78-99.91%) 85.37% (75.97-91.50%) Biocentric Generic HIV Charge Virale 148 98.12% (56.78-99.95%) 75.00% (46.90-91.06%) bioMérieux NucliSENS EasyQ® HIV-1 173 77.78% (53.53-91.40%) 99.35% (95.57-99.91%) Roche COBAS TaqMan HIV-1 1077 93.05% (87.75-96.16%) 94.90% (78.59-98.95%) aSensibilidade especificidade usando um limite de insucesso do tratamento de 1000 cópias/ml. Avaliações técnicas independentes: os resultados de 17 avaliações técnicas em 12 países e considerando quatro tecnologias disponíveis no mercado foram incluídos numa meta- análise abrangente, que resultou em quase 2000 pontos de dados emparelhados de gotas de plasma seco– plasma (Tabela 6) (50). O desempenho das amostras de gotas de plasma seco em todas as tecnologias foi comparável ao uso do plasma líquido tradicional. Como previsto, desde que o tipo de amostra utilizada, o plasma, foi usado, observaram-se menos erros de classificação, tanto para cima como para baixo. Recomendações da OMS As orientações consolidadas da OMS de 2016 sobre o uso de antirretrovirais no tratamento e prevenção da infecção pelo VIH (2) recomendam a carga viral como abordagem ideal de monitorização para diagnosticar e confirmar o insucesso do tratamento e preferem as amostras de plasma para os testes da carga viral. Pode usar-se um limite de 1000 cópias/ml para determinar o insucesso do tratamento, quando se usa qualquer uma das amostras, incluindo as amostras de gotas de plasma seco, conforme definição para testes no plasma. Uso actual As amostras de gotas de plasma seco constituem uma forma de melhorar a cobertura e o alcance dos testes da carga viral, podendo o armazenamento e o transporte das amostras de plasma líquido ser limitados por requisitos da cadeia de frio ou problemas de transporte. No entanto, a preparação de amostras de gotas de plasma seco requer centrifugação, para separar o plasma do sangue total. Isso pode ser feito, quer no ponto da colheita da amostra, se possível, quer algumas horas após a colheita da amostra, consoante as orientações do fabricante, por um laboratório central ou regional. 21Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce Conclusões Existem evidências suficientes sobre o desempenho das amostras de gotas de plasma seco nos testes da carga viral para apoiarem o início do reforço, se assim for desejado, no seio dos planos operacionais, para apoiar o reforço e o acesso do país aos testes da carga viral. Para além disso, não é provável que as avaliações técnicas destas tecnologias acrescentem algum valor, podendo, pelo contrário, retardar a implementação e a monitorização do tratamento em devido tempo. Contudo, a informação centrada na viabilidade e nas melhores práticas operacionais no uso de amostras de gotas de plasma seco no seio dos planos de reforço da carga viral tem sido limitada. 6.3 TIPOS ALTERNATIVOS DE AMOSTRAS E TECNOLOGIAS: TUBOS DE PREPARAÇÃO DO PLASMA PARA OS TESTES DA CARGA VIRAL DO VIH A amostra de plasma padrão ideal para os testes da carga viral é, normalmente, colhida, usando sangue total num tubo (com tampa de cor lilás ou lavanda) de EDTA (anticoagulante de ácido etilenodiaminotetraacético). Como se refere na Secção 4, o sangue total em tubos de EDTA deve ser transportado e o plasma separado em 6–24 horas, conforme as indicações do fabricante. Isso pode ser restritivo para muitos países e unidades de saúde. Contudo, podem ser consideradas algumas amostras de plasma alternativas. Os tubos de preparação do plasma, assim como o plasma colhido em cartões, tais como gotas de plasma seco (sub- secção 6.2) e os cartões de separação do plasma podem também ser considerados para apoiar o reforço. Ao contrário dos tubos padrão de EDTA para a colheita de sangue, os tubos de preparação do plasma podem facilitar um manuseamento e um armazenamento mais simples do plasma para os testes baseados no ácido nucleico. Os tubos de preparação do plasma usam o mesmo anticoagulante EDTA, mas contêm um gel que separa o plasma dos glóbulos sanguíneos após a centrifugação. Depois de colhido o sangue, o tubo de preparação do plasma é centrifugado dentro das 24 horas aceitáveis e uma barreira de gel dentro do tubo de preparação do plasma separa o plasma do resto do sangue total, para que o plasma possa ser usado no teste da carga viral do VIH. O mesmo volume da amostra do plasma é usado para o teste da carga viral; por isso, os limites da detecção são, geralmente, sinónimos de plasma EDTA. Aprovação regulatória dos tubos de preparação do plasma. • CE-IVD (Conformidade Europeia para o diagnóstico in vitro): shá ete tecnologias que receberam CE-IVD para utilização de tubos de preparação do plasma para os testes da carga viral: Abbott RealTime HIV-1, Cepheid Xpert® HIV-1 Viral Load, Hologic AptimaTM HIV-1 Quant Dx, Roche COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0, Roche cobas® HIV-1 for cobas® 4800, Roche cobas® HIV-1 for cobas® 6800/8800 e Siemens VERSANT® HIV-1 RNA 1.5. • Pré-qualificação da OMS: há quatro tecnologias que cumpriram os requisitos da OMS: Abbott RealTime HIV-1, Cepheid Xpert® HIV-1 Viral Load, Hologic AptimaTM HIV-1 Quant Dx and Roche COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0. • FDA (Administração dos Estados Unidos para os Alimentos e Medicamentos): há quatro tecnologias que receberam aprovação da FDA para utilização de tubos de preparação do plasma nos testes da carga viral: Abbott RealTime HIV-1, Hologic AptimaTM HIV-1 Quant Dx, Roche COBAS® AmpliPREP/COBAS® TaqMan® HIV-1. Revisão sistemática e melhores práticas: efectuou-se uma revisão sistemática para examinar o rigor dos tubos de preparação do plasma para os testes da carga viral do VIH. Essa revisão identificou 16 estudos revistos por pares e que foram publicados entre 1995 e 2014, comparando os tubos de preparação do plasma com os tubos padrão EDTA para colheita de sangue, com vista a testes da carga viral do VIH em ensaios aprovados por uma exigente autoridade reguladora. Embora os primeiros estudos demonstrassem que os tubos de preparação do plasma podiam ser utilizados sem qualquer diferença significativa nos resultados da carga viral (51–54) , estudos posteriores revelaram cargas virais elevadas em tubos de preparação de plasma, especialmente em cargas virais inferiores a 5000 cópias/ml (55–57). O aumento dos resultados da carga viral é provavelmente explicado pela fuga de ácidos nucleicos do VIH, tais como ADN pró-viral do VIH e ARN intracelular presentes nos componentes celulares do sangue total, que retornavam para o plasma através da barreira de gel. Outros estudos demonstraram que este problema pode ser resolvido, quer com alíquotas do plasma para um segundo tubo, rapidamente depois da centrifugação inicial (58–60) , quer repetindo a centrifugação depois do transporte dos tubos de preparação do plasma para o laboratório, antes das alíquotas e dos testes (61,62). Quatro estudos publicados avaliaram os tubos de preparação do plasma nos testes da carga viral actualmente disponíveis (Abbott RealTime HIV-1 and Roche COBA AmpliPREP/COBAS TaqMan HIV- 1 Test, v2.0) (63–66). Os três estudos que usaram o ensaio da carga viral de Abbott não revelaram qualquer alteração significativa nos resultados da carga viral, independentemente de os tubos de preparação do plasma terem sido congelados e descongelados ou transportados após a centrifugação inicial e antes dos testes. Os três estudos que usaram um ensaio Roche revelaram resultados elevados da carga viral, se os tubos de preparação do plasma tivessem sido congelados ou transportados sem uma segunda centrifugação antes do teste. Estes resultados da carga viral encontravam-se entre zero e vários milhares de cópias/ml mais elevadas do que no plasma preparado num tubo padrão de colheita de EDTA, com a diferença mais notória nas cargas virais do plasma inferiores a 1000 cópias/ml. Consequentemente, as instruções do fabricante recomendam uma fase de centrifugação adicional, antes do teste com o ensaio Roche. Tanto para os ensaios Abbott como Roche, introduzir alíquotas ao plasma num tubo secundário após a centrifugação inicial também garante resultados rigorosos da carga viral (Tabela 7). 22 22 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce Tabela 7. Métodos de manuseamento publicados para os tubos de preparação de plasma disponíveis no mercado e testes da carga viral Produtos Métodos publicados de manuseio de tubos de preparação de plasma que fornecem resultados precisos de carga viral Abbott RealTime HIV-1 (63–65) • Alíquotas de plasma num novo tubo, após centrifugação inicial. • Congelação dos tubos de preparação de plasma a –20°C após centrifugação inicial e descongelação antes do teste, sem necessidade de outra fase de centrifugação • Fransporte dos tubos de preparação do plasma, após centrifugação inicial, entre os locais, antes do teste, sem necessidade de outra fase de centrifugação Roche COBAS® AmpliPREP/ COBAS® TaqMan® HIV-1 Test, v2.0 (64–66) • Alíquotas de plasma num novo tubo, após centrifugação inicial • Repetição da centrifugação, após transporte ou congelação dos tubos de preparação do plasma, para garantir a completa separação dos componentes das células e do plasma do sangue antes do teste. Nota: Na ausência de centrifugação repetida após congelação e descongelação dos tubos de preparação do plasma ou depois do seu transporte, observou-se que alguns resultados da carga viral eram erroneamente elevados. Não é necessário repetir a centrifugação, se o plasma já tiver sido distribuído por alíquotas num novo tubo, antes da congelação ou transporte. BD Vacutainer® PPTTM (67) • Centrifugar durante, pelo menos, 10 minutos a 1100 × g à temperatura ambiente, dentro de 6 horas após a colheita do sangue total, para preparar o plasma. • Seguir as instruções de armazenamento e transporte do fabricante: normalmente, os tubos de preparação do plasma podem ser armazenados à temperatura ambiente durante um dia ou refrigerados a 4°C até cinco dias; se se pretender um armazenamento mais longo, o plasma terá de ser congelado. Tabela 8. Vantagens e desvantagens associadas aos tubos de preparação do plasma Vantagens Desvantagens • Mitar tempos mais longos de transporte até ao laboratórioenos passos no manuseamento das amostras do que com os tubos EDTA padrão • Menor risco de contaminação das amostras e de erros laboratoriais, se o plasma não for introduzido em alíquotas num novo tubo • Capacidade para armazenar plasma durante períodos de tempo mais longos do que o sangue total não centrifugado, o que pode facil • Custo mais elevado dos tubos de preparação do plasma do que os tubos EDTA padrão • Complexidades programáticas envolvendo a logística da cadeia de abastecimento, formação do pessoal e a devida implementação dos tubos de preparação do plasma • São necessárias centrifugadoras no local para separação imediata do plasma • A amostragem primária nos tubos nem sempre é possível • Os resultados da carga viral poderão não ser rigorosos, se não forem cumpridas as instruções específicas do fabricante: por exemplo, pode ser necessária a repetição da centrifugação antes do teste • A junção das amostras está actualmente indisponível Todos os estudos avaliaram apenas BD Vacutainer® PPTTM. Existem outros tubos de preparação do plasma (também referidos como EDTA, com tubos de gel separados: Grenier (68) ou TUD (69) ; contudo, não foram publicados quaisquer estudos. Além disso, outros testes da carga viral aprovados pelo regulador e/ou préqualificados pela OMS (tais como Cepheid Xpert HIV-1 e Hologic Aptima HIV-1 Quant Dx), que incluem tubos de preparação de plasma nas suas instruções de utilização, não fornecem quaisquer outras orientações específicas sobre o modo de utilização (Tabela 8). Conclusões Os tubos de preparação de plasma permitem que este seja preparado, armazenado e transportado no mesmo tubo usado para colher sangue total venoso. Os tubos de preparação de plasma fornecem resultados da carga viral equivalentes aos do plasma dos tubos EDTA padrão, se forem cumpridas as instruções e orientações de manuseamento do fabricante contidas em estudos independentes publicados. Está provado que a centrifugação dos tubos de preparação do plasma e/ou a introdução de alíquotas de plasma num tubo separado, antes do teste da carga viral, evita resultados da carga viral artificialmente elevados. Contudo, nem todos os testes da carga viral têm instruções claras ou avaliações de pares publicadas sobre o seu uso de tubos de preparação de plasma e são necessárias centrífugas (e capacidades associadas) no ponto de colheita das amostras. O uso de tubos de preparação de plasma poderá ser considerado nos locais em que a preparação mais simples do plasma, a redução do risco de contaminação cruzada e a necessidade de tempos de transporte mais alargados possam facilitar o reforço dos testes da carga viral. 6.4 TIPOS ALTERNATIVOS DE AMOSTRAS E TECNOLOGIAS: FERRAMENTAS NOS PONTOS DE CUIDADOS OU NA SUA PROXIMIDADE PARA OS TESTES DA CARGA VIRAL DO VIH As tecnologias desenvolvidas para utilização nos pontos de cuidados ou perto deles podem também ser consideradas para os testes da carga viral. Essas tecnologias podem ser descentralizadas e usadas nos pontos de cuidados. As tecnologias nos pontos de cuidados não exigem corrente eléctrica © W H O 23Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce O teste Cepheid Xpert® HIV-1 Viral Load requer 1 ml de plasma (pode ser derivado de tubos de amostra de sangue ACD, EDTA ou PPT-EDTA) e pode detectar os grupos M, N e O do VIH-1. O limite de detecção é 40 cópias/ml (29). Estão disponíveis outras especificações destes testes e produtos em desenvolvimento (10,70). Avaliações técnicas independentes: os resultados de 13 avaliações técnicas no terreno do teste da carga viral Cepheid Xpert® HIV-1 foram consolidados em 11 países numa meta-análise (Tabela 9) (71). Considerações Em 2019, a OMS não tinha recomendações para a consideração de tecnologias da carga viral nos pontos de cuidados; contudo, essa situação será revista em 2020. Considerando alguns dos desafios do reforço dos testes da carga viral, tanto clínica como logisticamente, os testes da carga viral nos pontos de cuidados pode facilitar um maior acesso à carga viral, entregar mais rapidamente os resultados aos clínicos e doentes e acelerar a tomada de decisões através de testes realizados no próprio dia. Além disso, várias tecnologias nos pontos de cuidados também são polivalentes, ou são tecnologias para várias doenças, capazes de testar diferentes condições, usando testes para doenças específicas na mesma plataforma. Uma presença significativa dos dispositivos existentes pode permitir a integração dos programas e dos diagnósticos de modo a expandir o acesso aos testes da carga viral (73). © W H O estável, salas de temperatura controlada, nem calibração regular, são relativamente fáceis de usar, são automatizadas, têm poucas ou nenhumas exigências de produtos de partes terceiras e podem ser operadas por profissionais não laboratoriais. As tecnologias próximas dos pontos de cuidados são semelhantes mas podem exigir o uso de electricidade estável e/ou salas de temperatura controlada. Além disso, a maioria das tecnologias actualmente disponíveis requer amostras de plasma. Têm sido feitos progressos significativos na garantia da qualidade das novas tecnologias para a carga viral nos pontos de cuidados. Aprovações dos reguladores e avaliações técnicas da carga viral nos pontos de cuidados ou na sua proximidade (os países consideram, muitas vezes, estas aprovações, quando compram ou seleccionam tecnologias de diagnóstico): • CE-IVD (Conformidade Europeia do diagnóstico in vitro): quatro tecnologias receberam CE-IVD: Abbott™ m-PIMA HIV-1/2 VL, Cepheid Xpert® HIV-1 Viral Load and Diagnostics for the Real World’s SAMBA I HIV-1 Semi- Quantitative Plasma Test e SAMBA II HIV- 1 Semi-Quantitative Plasma Test; e • Pré-qualificação da OMS: duas tecnologias cumpriram os requisitos da OMS: Abbott™ m-PIMA HIV-1/2 VL VL (23) e Cepheid Xpert® HIV-1 Viral Load (29) 1, receberam a pré- qualificação da OMS, respectivamente, em 8 de Abril de 2019 e 20 de Julho de 2017. O teste Abbott™ m-PIMA HIV-1/2 VL requer 50 μl de plasma EDTA venoso e pode detectar os grupos M, N e O do VIH-1 e VIH- 2. O limite da detecção é 800 cópias/ml (23). 24 24 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce Tabela 9. Síntese dos resultados da pré-qualificação da OMS e avaliações técnicas independentes Teste Avaliador Tipo de amostra Tamanho da amostra Sensibilidade (95% CI)aa Especificidade (95% CI)aa Abbott™ m-PIMA HIV-1/2 VLb Pré-qualificaço da OMS/ Centros dos EUA para o Controlo e Prevenção das Doenças Plasma 421 95.1% (91.7–97.5%) (23) 99.4% (96.8–99.9%) (23) Cepheid Xpert® HIV-1 Viral Load Pré-qualificaço da OMS/ Centros dos EUA para o Controlo e Prevenção das Doenças Plasma 439 94.14% (90.37–96.76%) (29) 98.50% (95.68–99.69%) (29) Meta-análise Plasma 3790 96.47% (95.10-97.47%) (72) 96.59% (92.90–98.39%) (72) a Sensibilidade e especificidade usando um limiar de insucesso do tratamento de 1000 cópias/ml. b Não foi ainda preparada uma meta-análise devido à falta de avaliações técnicas independentes publicadas.. Conclusões Existem evidências suficientes sobre o desempenho de alguns testes da carga viral nos pontos de cuidados para apoiar uma rápida aprovação dos reguladores nacionais e o início do reforço. Outras avaliações técnicas dessas tecnologias não deverão representar uma mais valia, mas, pelo contrário, retardar a implementação. Caixa 4. Estabelecer prioridades para os testes da carga viral Há vários grupos populacionais que podem ser considerados e receber prioridade para os testes da carga viral nos pontos de cuidados, quando os volumes globais podem sobrecarregar essas tecnologias. • As mulheres grávidas e lactantes, especialmente quando estão próximas do parto, podem beneficiar com a entrega mais rápida dos resultados e da tomada de decisões, para evitar a transmissão da mãe para o filho. • Os bebés e outras crianças que vivem com o VIH, que, normalmente, estão em maior risco de insucesso do tratamento e resistência aos medicamentos, devido à sua exposição à terapia antirretroviral materna e profilaxia pós-natal, podem também beneficiar com a entrega mais rápida dos resultados e uma monitorização mais atenta do tratamento. • Além disso, as pessoas que recomeçam os cuidados, aquelas em que há suspeita de insucesso do tratamento e as que têm doença avançada pelo VIH podem beneficiar com a entrega mais rápida dos resultados e da tomada de decisões clínicas. Estão em curso estudos sobre o impacto no tratamento e cuidados aos doentes, a viabilidade operacional, a aceitabilidade e a relação custo-eficácia. No entanto, os países terão de determinar individualmente a importância contextual, a utilidade e a amplitude dos testes da carga viral nos pontos de cuidados, no âmbito das suas redes de cuidados e diagnóstico. 25Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce 7. INTERVENÇÕES OPERACIONAIS E CONSIDERAÇÕES SOBRE O REFORÇO DOS TESTES DA CARGA VIRAL E DO DIAGNÓSTICO PEDIÁTRICO 7.1 OPÇÕES DE TRANSPORTE DAS AMOSTRASPARA DIAGNÓSTICO BASEADO NO ÁCIDO NUCLEICO Os laboratórios e as capacidades para efectuar testes no seio de uma rede de diagnóstico não estão localmente presentes em todas as unidades de saúde a que os doentes recorrem. Os testes e análises normalmente oferecidas nos laboratórios centralizados são fundamentais para tratar as pessoas que vivem com o VIH, nomeadamente a oferta de testes da carga viral e do diagnóstico pediátrico precoce do VIH, mas o acesso a esses serviços pode revelar-se um desafio. Os testes nos pontos de cuidados ou na sua proximidade podem ser uma solução para superar as limitações da rede de laboratórios (ver subsecção 6.4), incluindo a entrega dos resultados no próprio dia. Contudo, os testes nos pontos de cuidados não estão disponíveis em todas as unidades ou podem não ter uma boa relação custo-eficácia nas unidades de saúde com poucos doentes. Quando os testes não estão disponíveis no local, os sistemas de transferência das amostras podem proporcionar o acesso à rede de diagnóstico, enviando as amostras da unidade que as colheu (também conhecida como unidade requisitante) para uma unidade com a necessária capacidade (o laboratório de testes ou de referência). Tipos de amostras alternativos, tais como amostras de gotas de sangue seco, podem igualmente ser usadas para facilitar mais o acesso. Transferir as amostras significa um alívio para as pessoas que vivem com o VIH e teriam de viajar até ao laboratório, para fazerem os testes. Desta forma, a rede de transferência de amostras alarga o alcance e a cobertura da rede de diagnóstico. O mesmo sistema de transferir amostras também é, muitas vezes, usado para devolver os resultados em papel, os quais podem ser enviados, mesmo que esses resultados estejam informatizados. Vários sistemas de transferência de amostras podem ser encontrados em vários níveis de um sistema de saúde hierarquizado, em diferentes regiões de um país e nos programas relativos às doenças. Em conjunto, estes sistemas devem ser harmonizados, interligados e eficientemente coordenados para constituir a rede global de transferência de amostras, que, por sua vez, é uma parte vital de uma rede de diagnóstico. Um sistema ou rede de transferência de amostras tem cinco objectivos (Caixa 5). Um sistema de transferência de amostras compreende várias componentes, que são cruciais para garantir um sistema bem sucedido e eficiente (Fig. 8): • Gestão e liderança – idealmente, alguém do ministério da saúde deverá supervisionar a rede global de transferência, garantir que ela apoia as necessidades da rede de diagnóstico e procurar os recursos necessários em toda a rede; Caixa 5. Os cinco principais objectivos de um sistema de transferência de amostras • Contribuir para o aumento do acesso ao diagnóstico onde não existam serviços no local, transferindo as amostras para um laboratório de testes • Manter e melhorar a qualidade das amostras enviadas ao laboratório de testes, gestão adequada das amostras em trânsito, incluindo a necessidade de uma cadeia de frio • Garantir a segurança de todos os indivíduos e do ambiente durante a transferência das amostras, através de uma gestão adequada em trânsito, incluindo o acondicionamento e o manuseamento • Cumprir os requisitos de tempo que a amostra deve levar até chegar ao laboratório e o tempo de entrega dos resultados em papel à unidade, aos clínicos, aos doentes e ficheiros necessários • Melhorar a relação custo-eficácia da rede de diagnóstico, através da harmonização e coordenaçãode diagnostic en assurant l’harmonisation et la coordination des procédures GESTÃO E LIDERANÇA SISTEMAS DE DADOS E MONITORIZAÇÃO E AVALIAÇÃO EQUIPAMENTO LOGÍSTICA RECURSOS HUMANOS FINANCIAMENTO TRANSPORTE Fig. 8. Componentes que garantem um sistema de transferência de amostras bem sucedido e eficiente 26 26 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce • Recursos humanos – são os funcionários das unidades requisitantes, laboratórios de referência, equipas de saúde regionais, transportadores, etc., que estão envolvidos em todo o processo de transferência e devolução dos resultados; • Financiamento – os fundos necessários para a rede global de transferência, englobando todos os aspectos da rede, assim como os tipos de amostras e áreas de doenças; • Transporte – inclui o tipo de veículo (mota ou carro) e o prestador de serviços (correio expresso ou parceiro clínico da implementação) e as respectivas combinações ideais para servir todas as unidades, consoante as necessidades; • Logística – inclui todo o sistema logístico, nomeadamente o calendário e a rota, e depende de vários factores, tais como os requisitos de tempo para tratar as amostras e devolver os resultados; • Equipamento – os materiais de acondicionamento necessários que contribuem para a qualidade e biossegurança da amostra durante a transferência; e • Sistemas de dados e monitorização e avaliação – sistemas para recolher dados, analisá-los e usar a análise para a tomada de decisões e melhoria contínua da qualidade. Três componentes essenciais do sistema de transferência de amostras podem ser problemáticas e exigir consideração e atenção adicionais: transporte, logística e sistemas de dados e monitorização e avaliação. A estrutura dos sistemas de transporte e logística está intimamente relacionada e pode ou não ser gerida pela mesma organização ou empresa. Embora essas componentes sejam apenas duas em todo o sistema, requerem conhecimentos técnicos adicionais que, normalmente, não são uma capacidade essencial do pessoal das unidades de saúde ou dos laboratórios. Três considerações para estas importantes componentes são: • Tipo de veículo. O tipo de veículo usado depende dos recursos, distâncias, terreno e capacidade de carga. Se os sistemas de transporte e logística forem externalizados, será, provavelmente, o prestador de serviços que decidirá o tipo de veículo. Alguns exemplos são (ordenados da prevalência mais alta para a mais baixa): motociclos, veículos de quatro rodas, bicicletas, barcos, cavalos, a pé, aeronaves e veículos aéreos não tripulados (também conhecidos como drones). As principais considerações ao escolher o tipo de veículo incluem: – Tipo de amostras (amostras de gotas de sangue seco, sangue total, plasma, etc.) e requisitos para os testes (cadeia de frio ou amostras de doenças altamente contagiosas para teste podem exigir embalagens adicionais, que podem não caber nem ser adequadas para certos tipos de veículos, como os drones); – Nível ou escalão do sistema usado, distância percorrida e tipo de terreno; e – Procura e volumes nas unidades requisitantes para compreender a capacidade de carga necessária. • Prestador de serviços de transporte. O prestador de serviços, que pode ser o ministério da saúde, um parceiro da implementação ou uma empresa privada, é aquele que opera o transporte e, normalmente emprega os operadores do veículo. Ao escolher um prestador de serviços, há que ter em consideração o seguinte: – Pagamento ao prestador e sustentabilidade do sistema; – Disponibilidade de transportadores locais do sector privado ou prestadores de logística de partes terceiras, tais como Riders for Health, DHL, G4S ou o serviço postal nacional, e a capacidade para fazer contratos com um prestador de logística de parte terceira; – Que entidade gere, detém e opera os veículos: propriedade da unidades de saúde versus fornecidos à unidade através do ministério da saúde ou veículos pertencentes ao governo, parceiros ou empresa privada; – Que entidade gere e emprega os operadores do veículo (motociclista, motorista, etc.); – Amostras acompanhadas por uma pessoa durante o transporte; e – Reservar o sistema apenas para o transporte de amostras e resultados: veículos como ambulâncias, cuja missão principal não é o transporte de amostras, não devem ser o único meio de transporte de amostras disponível e utilizado. • Logística, calendário e rota. No fundo, um sistema de transferência de amostras é um sistema logístico. As amostras tem de ser deslocadas fisicamente desde o ponto de colheita até aos locais ou centros de teste de diagnóstico de primeira linha e, depois, possivelmente, para locais de teste especializados, e os resultados devolvidos. As principais considerações sobre logística incluem o seguinte: – As unidades de saúde, pontos de colheita, centros e laboratórios de referência devem ser mapeados usando códigos geográficos. Em seguida, as actuais necessidades de transferência, ligações e vias para cada tipo de amostra devem ser mapeados, com base nos algoritmos e capacidade de teste e entre cada nível ou escalão relevante do sistema de saúde, incluindo a comunidade ou o nível do posto de saúde, se considerado. O exercício de mapeamento deve ser refeito, quando a rede global de diagnóstico muda, designadamente se houver descentralização do equipamento ou integração. – Considerar se é necessário ter um calendário fixo para a recolha das amostras e a devolução dos resultados versus serviços a pedido. – A frequência das recolhas deve basear-se no volume e necessidades dos doentes, colheita de amostras, tipo de amostra, estabilidade da amostra e capacidade das máquinas do laboratório de teste. Por exemplo, as amostras de sangue total e plasma requerem transporte e armazenamento rápidos no mesmo dia à temperatura correcta. – 27Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce Caixa 6. Considerações para os sistemas de dados e monitorização e avaliação Esta é uma componente crucial para os sistemas e para a rede, mas, muitas vezes, é frágil e subestimada. É necessário um quadro normalizado de monitorização e avaliação do transporte das amostras, para avaliar e comparar o desempenho dos sistemas, os quais, muitas vezes, se encontram fragmentados. As principais considerações sobre esta componente são as seguintes: • O quadro de monitorização e avaliação e os indicadores normalizados devem basear-se nos cinco objectivos de um sistema de transferência de amostras (Caixa 5) e ser incluídos nas orientações nacionais sobre essa transferência. • Devem existir ou ser introduzidos instrumentos de recolha de dados, incluindo registos e diários, formulários da cadeia de custódia (anotando cada vez que uma amostra ou resultado muda de mãos), registo do transporte, formulários de notificação, questões incluídas nas listas de verificação da supervisão, etc. • Os indicadores podem ser ambiciosos, mas quando a rede de transporte de amostras e os sistemas de dados necessários estiverem instituídos, deve ser avaliada a viabilidade de recolher cada um deles. • Os processos de notificação devem ser sublinhados e devem ser usados mecanismos de feedback. • É importante estabelecer o tempo de devolução dos resultados, incluindo cada passo dado desde a colheita da amostra no doente até ao momento em que o resultado é registado na sua ficha. • É preciso ter em atenção a melhoria contínua da qualidade, incluindo as medidas correctivas. © W H O 28 28 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce – Num sistema de pontos de trânsito e partida (hub-and spoke), o ponto de trânsito pode ser uma unidade de testes e/ou uma unidade para consolidar e armazenar amostras que vão a caminho de um nível superior, em vez de irem de ponto a ponto, isto é, directamente da unidade requisitante para o laboratório de referência, sem consolidação num ponto de trânsito intermédio. Para um teste da carga viral que use amostras de plasma, deve ser criado um ponto de trânsito que esteja equipado com frigoríficos, congeladores e centrífugas, para processar amostras e garantir a sua integridade. – Considerar a capacidade da unidade ou centro requisitante para preparar e armazenar amostras. – Estudar a possibilidade de reconsiderar as fronteiras administrativas, se isso for mais eficiente do ponto de vista logístico: decidir se a amostra poderá ser enviada para um laboratório de uma região administrativa diferente, se esta estiver mais próxima do que o laboratório pré-indicado. – O sistema pode ser integrado cm outros tipos de amostras? – A entrega de resultados em papel está contemplada, caso seja necessário? – Qual é o tempo-limite para a recepção das amostras (momento em que as amostras devem chegar ao centro, para continuação do processamento ou armazenamento, ou ao laboratório) e o tempo mais rápido de chegada e recolha nas unidades requisitantes? – Em relação aos veículos (não dedicados), os horários devem ser cuidadosamente planeados para não perturbar outras actividades. Melhores práticas. Embora haja muitas formas de planear, implementar e monitorizar um sistema de transferência de amostras, os países estão presentemente a adoptar as principais melhores práticas, incluindo as seguintes: • Gestão: o ministério da saúde deve liderar, coordenar e supervisionar a rede global de transferência de amostras, independentemente do mecanismo de transporte usado ou do financiamento. • Para a transferência de amostras são desenvolvidas orientações nacionais, bem como um manual laboratorial que descreve os procedimentos individuais para a colheita, acondicionamento, armazenamento e transporte, consoante o tipo de amostra e o teste requisitado. • Monitorização: um quadro robusto de monitorização e avaliação deve incluir indicadores normalizados. • Estratégia das redes: o planeamento da rede de transferência das amostras deverá ser feito no seio da rede de diagnóstico e optimizado periodicamente para melhorar a eficiência e os custos. • Os tipos de amostras devem ser integrados nas actividades dos programas das doenças, quando tal for possível e logisticamente eficiente. • Os procedimentos de transporte e transferência devem estar bem documentados para cada tipo de amostra e todo o pessoal de todos os níveis deverá receber formação apropriada, incluindo: colheita de amostras, armazenamento, documentação, acondicionamento e expedição, transporte, recebimento da amostra, transporte e recebimento dos resultados. • Biossegurança e qualidade: fornecer equipamento de protecção individual apropriado, material estanque apropriado para acondicionamento, incluindo contentores seguros, conforme necessário, para cada tipo de amostra. Integração. A rede de diagnóstico pode ser integrada para usar um sistema e rede de transferência de amostras para vários tipos de amostras ou programas de doenças. Neste caso, é mais fácil e mais eficiente fazer a integração a partir da unidade requisitante para o primeiro ponto de transferência ou local do diagnóstico de primeira linha. A rede nacional global de transferência de amostras deve ser sempre totalmente integrada, o que significa que deve tratar todos os tipos de amostras e doenças. Contudo, o transporte e a logística necessários para conseguir essa integração poderá exigir que se incorporem alguns sistemas separados para certos testes ou amostras, com base na rota e/ou local dos laboratórios, e requisitos de gestão das amostras. Por exemplo, o teste laboratorial do VIH baseado no ácido nucleico poderá diferir da cultura laboratorial para a tuberculose e, portanto, poderão ser necessárias rotas e logísticas separadas para aspectos específicos das redes de transporte de amostras. Para além disso, conforme o tipo da amostra e outros factores, os mecanismos de transporte usados em cada rota podem ser diferentes. Por exemplo, para amostras que requeiram transporte muito rápido, transporte no próprio dia ou que requeiram controlo da temperatura, os transportes públicos sem horários certos nem controlo de temperatura podem não ser apropriados. Se houver investigação de um surto, as amostras poderão não conseguir esperar para usar o mecanismo de transporte de rotina. Caixa 7. Hiperligações para ferramentas e recursos para a transferência de amostras • Global Laboratory Initiative (GLI) Specimen Referral Toolkit. Geneva: Stop TB Partnership; 2019 (http://www. stoptb.org/wg/gli/srt.asp). • GLI guide to TB specimen referral systems and integrated networks. Geneva: Stop TB Partnership; 2019 (http:// www.stoptb.org/wg/gli/assets/ documents/GLI_Guide_ specimens_web_ready. pdf). • Guidance for developing a specimen transport and referral system for viral load and infant virologic HIV diagnosis testing networks. Addis Ababa: African Society for Laboratory Medicine; 2015 (http://www.aslm. org/?wpdmdl=18275).org/?wpdmdl=18275). 29Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce Tabela 10. Opções para seleccionar prestadores de serviços de transporte e logística A ut o- ge ri do e o pe ra do d ir ec tl am en te p el o m in is té ri o da s aú de o u po r um p ar ce ir o cl in ic o da im pl em en ta çã o; to do s el es p od em f ac ilm en te t ra ns po rt ar o s re su lt ad os o u ou tr o m at er ia l, se m q ua lq ue r cu st o ad ic io na l Tipo ou exemplo Benefícios Desafios Casos de melhor uso Sistema dedicado de correio urgente do ministério da saúde Provável partilha dos recursos do ministério da saúde, como o pessoal, para operar e gerir o sistema, poupando assim nos custos totais envolvidos De modo geral, o ministério da saúde não tem especialistas em transportes e logística Usado em países com grande volume de transferências, caso em que a externalização é difícilí e a capacidade do ministério para gerir uma rede complexa de transporte e logística é elevada Sistema dedicado de correio urgente operado por parceiros Partilha de alguns recursos de parceiros, tais como o pessoal, para operar e gerir o sistema, poupando assim nos custos totais envolvidos De modo geral, o ministério da saúde não tem especialistas em transportes e logística – para operar esses sistemas é necessário contratar pessoas especializadas só para esse sistema, o que não é economicamente favorável Usado em países com grande volume de transferências, caso em que a externalização é difícilí e a capacidade do ministério para gerir uma rede complexa de transporte e logística é baixa Transporte em mão por pessoal da unidade Muitas vezes, executados pelo pessoal de laboratório, para se compreender bem a questão da biossegurança e controlo da qualidade Retira os poucos recursos humanos da unidade de saúde e afasta-os das suas principais funções; mais dispendioso do que enviar só uma encomenda Usado quando o volume de transferência de amostras é muito baixo e errático Uso de veículos não dedicados do ministério da saúde Usado pelos responsáveis dos programas para efectuarem visitas de supervisão e para entregar materiais e produtos. Alguns programas também têm utilizado os veículos para transportar as amostras e os resultados Muitas vezes, não comparecem nos locais de colheita com frequência suficiente para um transporte rápido; com prioridades partilhadas, as amostras nem sempre são transportadas em tempo oportuno, nem com o devido controlo de qualidade; o uso de ambulâncias não é recomendado, visto que é uma forma de transporte imprevisível e interfere com os deveres regulares Usados para postos ou unidades de saúde que apenas colhem amostras quando há a visita de uma equipa de saúde de proximidade, visto que esta pode trazer com ela as amostras para o laboratório Uso de transportes públicos, sem acompanhamento (autocarros, comboios, barcos e aeronaves) Desempenham um importante papel nos transportes rurais e urbanos com vasto acesso e cobertura em todo o país; usados por empreses privadas de correio urgente e sistemas postais nacionais, para enviarem cartas, encomendas e dinheiro; menos dispendioso para enviar uma encomenda não acompanhada do que com um funcionário da unidade Normalmente, é preciso levar as embalagens ao depósito; pode ser necessária uma autorização especial para o transporte de matérias potencialmente perigosas; os horários podem não ser rigorosamente cumpridos; as amostras e os resultados dos testes podem não ser devidamente tratados por falta de formação, falta de pessoal e inexistência de funções e responsabilidades claras; pode não existir um sistema para acompanhar as amostras Usados quando existem companhias rodoviárias com a reputação de cumprir horários, pessoal profissional e um depósito central onde o pessoal da unidade de saúde pode levant Ex te rn al iz ad o – to do s sã o es pe ci al is ta s em lo gí st ic a e ge re m o t ra ns po rt e Correio urgente profissional (ONG, empresa social, privado), como os Riders for Health Capacidade para planear um sistema dedicado, inclusive em zonas de difícil acesso ou mal servidas; devolução de resultados ou transporte de outro material em rotas regulares, sem encargos adicionais A despesa total parece ser mais elevada, visto que o sistema inclui tudo (veículos, transporte, motoristas e motociclistas, custos operacionais, etc.) e é operado por uma parte terceira (recursos, como o pessoal do ministério da saúde ou de parceiros, não serão partilhados, tendo um custo adicional) Usado em países com infraestruturas rodoviárias e prestadores de transportes limitados ou pouco Correio urgente profissional privado não dedicado, como FedEx ou DHL Especializado em recolha e entrega de encomendas, a pedido ou por recolha regularmente programada, documentação e acompanhamento da expedição Nem todos estão dispostos a transportar amostras biológicas potencialmente infecciosas; os custos podem ser mais elevados; a cobertura e a flexibilidade podem ser limitadas; pode não ser uma forma económica de devolver os resultados. Usado quando a rapidez, segurança, documentação, acompanhamento, nome e assinatura do destinatário, especialização e individualização dos serviços expresso são suficientemente importantes para justificar o custo extra; a melhor cobertura existe nas grandes Serviço postal nacional, correio urgente não dedicado (público ou semi-privado) Normalmente, uma entidade para- estatal, que pode ser mais fácil o ministério da saúde contratar do que uma empresa privada de correio urgente; mandato presente em todo o país; normalmente num horário previsível postos de correio locais; cumprimento dos horários; as amostras que exigem um tempo de trânsito rigoroso ou um cuidadoso controlo da temperatura podem constituir um problema, a não ser que seja oferecido um serviço garantido (como o correio expresso) Usado onde o sistema postal nacional é forte e tem boa cobertura; caso contrário, usado apenas para amostras menos urgentes e com maior prazo de validade, como as amostra de gotas 30 30 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce Opções de prestadores de serviços de transporte e logística. A rede nacional de transporte de amostras poderá incluir uma combinação das opções enumeradas na Tabela 10, conforme o nível do sistema de saúde e a geografia local de uma região. 7.2 PACOTES PARA O DIAGNÓSTICO PEDIÁTRICO E COLHEITA DE AMOSTRAS PARA A CARGA VIRAL São necessários mais de 10 produtos individuais para colher amostras de sangue total destinadas à separação do plasma ou amostras de gotas de sangue seco dos doentes para os testes de ácido nucleico (quer para diagnóstico, quer para carga viral). Alguns desses materiais, como os cartões de papel de filtro para colheita de gotas de sangue seco usados para colher amostras de sangue, são especializados e apenas recomendados de determinados fornecedores. Outros materiais, como gaze ou compressas com álcool, são genéricos. Nas primeiras fases da criação de programas de testes de diagnóstico pediátrico, os países tinham de comprar cada um desses produtos individualmente, o que tornava uma missão complexa a sua encomenda e distribuição às unidades de saúde. Por outro lado, as rupturas de stocks de qualquer um desses produtos podia comprometer a qualidade das amostras ou impedir completamente a colheita e/ou o processamento das amostras de sangue. Aprendendo com a experiência do diagnóstico pediátrico precoce, para maior facilidade de compra e distribuição e para garantir a qualidade dos produtos, os fornecedores conceberam pacotes de colheita de amostras de plasma e gotas de sangue seco para os testes da carga viral. Esses pacotes contêm kits de colheita individuais e de utilização única que incluem todo o material e produtos necessários para colher, secar (para amostras de gotas de sangue seco) e transportar uma amostra da unidade de saúde para o laboratório. Conteúdo dos pacotes de colheita de amostras A Tabela 11 enumera os itens incluídos nos kits de colheita de sangue em tubos de EDTA de utilização única (100 testes por pacote), para obter uma amostra de plasma destinada a testes da carga viral, usando a punção venosa. Posteriormente, estas amostras podem ser (1) enviadas directamente para o laboratório de referência para processamento (centrifugação) em plasma e testadas ou (2) ser separadas em plasma por centrifugação na unidade de saúde e transferidas para outro tubo, sendo depois enviadas para o laboratório devidamente acondicionadas para os testes. Tabela 11. Pacotes para colheita de amostras de sangue total e plasma No Item Quantidade Especificações 1 Compressas com .lcool, 70% 1 Compressas com álcool WBCL 2 Compressas de gaze não estéreis com 8 dobras 10 x 10 cm 1 Compressas de gaze com 8 dobras não estéreis 100 × 100mm 3 Gants d’examen en latex non poudrés 2 Luvas de látex sem pó para exame, tamanho médio 4 Pansement 1 Tecido de ligaduras 5 Bag autoclave clear biohazard saco de autoclave com símbolo de biossegurança 415 × 600 mm (1 por pacote) 1 Bag autoclavable clear print biohazard saco de autoclave com símbolo de biossegurança 415 × 600 mm 6 Tubo de 5 ml de EDTA vácuo 1 Tubo de 5 mL K2EDTA lavanda 13 × 100 mm 7 Porta-agulhas em tubo de vácuo 1 Suporte Speedy de libertação rápida 8 Agulha em tubo de vácuo, 21G 1 Agulha de colheita em vácuo de múltipla utilização 21G × 1.5” 38 × 0.8mm Verde Estéril 9 Torniquete (um por pacote) 1 Torniquete descartável sem clip, sem latex, banda de borracha sintética, não estéril 10 Caixa de acondicionamento 1 Caixa branca com revestimento 385 × 310 × 145mm 11 Pipeta de transferência Pasteur (opcional)a 1 Pipeta de transferência Pasteur 1 mL ponta fina, embalagem individual estéril (pode ser requisitada com custo adicional) a Agulha de colheita em vácuo de múltipla utilização 21G × 1.5” 38 × 0.8 mm Verde Estéril 31Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce Tabela 12. Pacotes para colheita de amostras de DBS N.o Item Quantidade Diagnóstico pediátrico Carga viral Especificações 1 Instruções para a colheita de DBS 1 Ficha de instruções para a colheita de DBS 2 Luvas sem pó 2 Luvas de exame em çátex sem pó – tamanho médio 3 Penso de gaze com álcool 2 Penso de gaze impregnado com álcool, de utilização única e individualmente embalado 4 Lancetas 1 Lanceta retráctil de utilização única com lâmina de 2 mm não ajustável para penetração em profundidade (não do tipo agulha) 5 Compressa de gaze 1 Compressas de gaze não esterilizadas 8 dobras 50 mm × 50 mm 6 Tubo capilar de EDTA 1 χ Tubo capilar de EDTA 100 μl, de plástico, com marcações de 70μl 7 Papel de filtro S&S 903 para DBS 1 Cartão Whatman 903 ou Munktell TNF, perfurado 8 Suporte de secagem para cartões DBS 1 Support de séchage pour carte Whatman 903 9 Embalagem de dessecante de sílica 3 Saqueta de 1 grama de silica gel 10 Sacos de plástico 1 Saco Ziploc duplo de baixa permeabilidade a gases (150 mm × 180 mm) com espaço branco para escrever 11 Embalagem e reembalagem 1 Embalagem e reembalagem: cinco peças por pacote 12 Caixa de acondicionamento 1 Caixa de cartão amarelado para conter todo o conteúdo dos pacotes, com tampa de enfiar 13 Formulário de requisição laboratorial (opcional) 1 Formulário de requisição personalizado para o diagnóstico pediátrico precoce/carga viral num bloco de 50 ou 100 em cópias duplicadas 14 Auto-colantes para código de barras (opcional) 1 Auto-colantes para código de barras personalizados para diagnóstico pediátrico precoce/carga viral Fonte: M. Rioja, Clinton Health Access 32 32 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce A Tabela 12 enumera os itens incluídos nos kits de colheita de amostras de gotas de sangue seco de utilização única com cartões para gotas de sangue seco perfurados (20 ou 50 testes por pacote), os quais podem ser usados para os testes de diagnóstico pediátrico precoce ou testes da carga viral. Um pacote específico de colheita de amostras de gotas de sangue seco para os testes da carga viral contém itens semelhantes, com adição de um tubo microcapilar de EDTA (Fig. 8). O tubo de EDTA microcapilar serve para garantir que é colhido rigorosamente o volume necessário por gota. O teste da carga viral é uma medida quantitativa que depende muito da quantidade de sangue usado no teste, independentemente do tipo de amostra. Algumas evidências sugerem que gotas livres de sangue aplicadas directamente a um cartão de gota de sangue seco pode produzir resultados de teste rigorosos, em comparação com as amostras de plasma. Por conseguinte, usar um volume fixo ou um tubo ou pipeta microcapilar de medição graduados pode ajudar a um maior rigor na preparação da amostra de gota de sangue seco. Os profissionais de saúde devem ser devidamente formados sobre as diferenças das técnicas e processos de colheita de amostras para o diagnóstico pediátrico e gotas de sangue seco para a carga viral. Source : M. Rioja, Clinton Health Access Initiative. Que benefícios se esperam da utilização dos pacotes de colheita de amostras? São os seguintes os benefícios dos pacotes de colheita de amostras para um programa nacional: • Previsão, compra e gestão da cadeia de abastecimento mais simples e mais normalizada (em vez de encomendar cada um dos itens a vários fabricantes); • Disponibilidade garantida dos itens necessários nas proporções correctas e menor desperdício; • Reforço mais simples e mais rápido dos serviços de testes em novos locais, visto que todos os materiais para formação e testes são embalados em conjunto; e • Os pacotes saem mais baratos para todos os componentes, em comparação com a compra individual de cada item. São os seguintes os benefícios dos pacotes de colheita de amostras para as unidades de saúde: • Garantia de qualidade dos itens contidos nos pacotes, se o fornecedor tiver um histórico comprovado, o que é especialmente importante para certos itens que têm de cumprir escrupulosamente os padrões de qualidade (tais como, lancetas e luvas sem pó), a fim de garantir a prestação de cuidados adequados aos doentes e a preparação correcta das amostras (mitigando assim o risco de rejeição das amostras pelo laboratório) e garantir a segurança do utilizador final; • Menores riscos ou reorientação dos itens, tais como luvas, para outros serviços, reduzindo assim a ruptura de stocks e o desperdício de determinados itens; • Partilha ou distribuição mais fácil de pacotes de utilização única individualmente acondicionados para postos-satélite, com menos pedidos de testes; e • Simplificação do fluxo do trabalho na clínica, devido aos pacotes de utilização única individualmente acondicionados, permitindo aos agentes de saúde abrir a caixa e retirar um saco que tem tudo aquilo de que precisam para colher uma amostra. Que fornecedores oferecem pacotes de colheita de amostras? Os pacotes estão prontamente disponíveis para compra através de, pelo menos, dois fornecedores que recebem os itens directamente de determinados fabricantes: • LASEC (https://www.lasec.com/diagnostics); • LabMate Conclusão Garantindo que todos os itens necessários estarão disponíveis aos agentes de saúde ou aos técnicos de laboratório num único kit ou embalagem, os pacotes de produtos para a colheita de amostras destinadas a testes do ácido nucleico simplificaram e normalizaram a cadeia de abastecimento desses produtos e reduziram a ocorrência de demoras na realização dos testes, que resultariam de rupturas de stocks ou apropriação indevida de um determinado item. Muitos países já têm experiência na utilização destes pacotes e, consequentemente, têm menos desperdício e encomendam menos existências de segurança. Embora os pacotes em si constituam uma alternativa económica às compras por grosso de produtos isolados, a redução significativa de 33Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce desperdícios contribui também para maior poupança por parte dos países. Por outro lado, a utilização de produtos de colheita de amostras em pacotes tem contribuído significativamente para melhorar os serviços de diagnóstico pediátrico e de testes da carga viral, em vários países de recursos limitados. 7.3 INTERVENÇÕES OPERACIONAIS: TESTES DA CARGA VIRAL PARA O DIAGNÓSTICO PEDIÁTRICO PRECOCE Os testes de diagnóstico pediátrico têm-se expandido consideravelmente na última década nas regiões de baixos e médios rendimentos, mas o seu acesso continua a ser limitado. Em 2017, apenas 51% das crianças expostas ao VIH fizeram um teste de diagnóstico precoce nos primeiros dois meses de vida (11) , conforme a recomendação da OMS (2). Há vários problemas que têm limitado o reforço deste importante teste numa população altamente vulnerável. O diagnóstico pediátrico precoce foi inicialmente oferecido em laboratórios de testes centralizados, o que exigia o transporte de amostras de gotas de sangue seco e podia, muitas vezes, levar semanas e, por vezes, meses para o envio dos resultados aos clínicos e cuidadores, para acção clínica. As demoras podem ser causadas por vários motivos, incluindo: • A necessidade de fazer lotes de amostras pediátricas, antes de se proceder a uma análise completa, para garantir que os testes terão uma boa relação custo-eficácia e resultarão em poupanças; • Pequenos volumes de diagnóstico pediátrico limitam o número de dispositivos e laboratórios com capacidade para efectuar testes, os quais se poderão localizar longe das unidades de saúde e criar um contexto problemático de compras que, muitas vezes, gera ruptura de stocks de reagentes nos laboratórios; • No passado e, por vezes, ainda hoje, os reagentes para os testes pediátricos podem ser mais caros do que outros testes de VIH baseados no ácido nucleico, tais como os da carga viral; e • Como este tipo de amostra, gotas de sangue seco, é também muitas vezes usado nos testes da carga viral e o número mensal de bebés que precisam de fazer testes é reduzido, os materiais para colheita de amostras têm sido e podem ser reutilizados na colheita de amostras para os testes da carga viral, o que, ocasionalmente, resulta em ruptura de stocks, quando pode ser necessária uma amostra pediátrica. Os testes quantitativos de diagnóstico pediátrico começaram por ser usados para diagnosticar o VIH em crianças expostos ao VIH, em contextos de recursos limitados. A técnica baseada no ácido nucleico (PCR quantitativa) usada nos testes da carga viral é muito semelhante, muitas vezes a mesma, que para os testes pediátricos ou ensaios qualitativos. A PCR do ADN do VIH é um sinónimo normalmente usado para os testes de diagnóstico pediátrico precoce do VIH; contudo, há presentemente várias tecnologias no mercado que não se destinam especificamente ao ADN do VIH. O principal tipo de amostra para o diagnóstico pediátrico baseado no ácido nucleico é o sangue total, que pode conter ADN pró-viral, ARN intracelular e ARN extracelular. Do mesmo modo que usando amostras de gotas de sangue seco do sangue total para os testes da carga viral (ver subsecção 6.1), o sangue total para os testes qualitativos de diagnóstico pediátrico geralmente resulta na detecção da variedade dos ácidos nucleicos do VIH. Uma vez que tanto o ADN como o ARN do VIH estão presentes, os testes virológicos ou os testes de amplificação do ácido nucleico do VIH são agora termos mais rigorosos para os testes pediátricos por PCR do que a PCR do ADN do VIH. Considerações actuais As recomendações da OMS de 2010 sobre o diagnóstico da infecção pelo VIH em bebés e crianças pequenas (74) as orientações consolidadas da OMS de 2016 sobre o uso de antirretrovirais no tratamento e prevenção da infecção pelo VIH (2) recomendam que se façam testes virológicos para diagnosticar a infecção pelo VIH em bebés, usando PCR do ADN do VIH em amostras de sangue total ou em amostras de gotas de sangue seco, PCR do ARN do VIH no plasma ou gota de sangue seco ou antigénio p24 ultra-sensitivo no plasma ou gota de sangue seco. Por outro lado, as orientações em países de altos rendimentos, incluindo as orientações dos Estados Unidos da América (75) , recomendam os testes do ARN do VIH para diagnosticar a infecção pelo VIH nos bebés. A investigação existente sugere que os testes de ARN do VIH (normalmente, quantitativos) podem apresentar resultados comparáveis aos testes que, especificamente, detectam o ADN (76–79). No entanto, persistem dúvidas acerca da viabilidade técnica e clínica da utilização do ARN e/ou testes quantitativos para o diagnóstico, dada a maior exposição de mães e bebés à terapia antirretroviral, através dos programas de prevenção e transmissão vertical do VIH, opção B+ e das políticas “Tratar Todos”, visto que todos os estudos anteriores se realizaram antes de 2003 e da era da opção B+. Dados actualizados Recentemente, foram realizados dois estudos destinados a um melhor entendimento do desempenho e potencial papel de se usarem os testes quantitativos do VIH (carga viral), usando amostras de gotas de sangue seco para diagnosticar o VIH em crianças menores de 18 meses (80,81). Esses estudos foram realizados em contextos actuais, com elevadas taxas de exposição materna e infantil a medicamentos. Em Moçambique, 95% das mães e dos bebés recebiam, respectivamente, terapia antirretroviral ou profilaxia antirretroviral, enquanto, no Uganda, 75% das mães recebiam terapia antirretroviral e 65% dos bebés recebiam profilaxia antirretroviral. No estudo realizado em Moçambique, a sensibilidade e a especificidade de usar o teste da carga viral para detectar a infecção foram, respectivamente, de 100.0% e 99.9%. Os valores 34 34 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce Table 13. Sensibilidade e especificidade dos testes da carga viral em Moçambique e no Uganda Pais Configuração do estudo Tipo de amostra Sensibilidade (CI 95%)a Especificidade (CI 95%)a Mozambique (80) Plasma 1021 100% (96.2-100.0%) 99.9% (99.4-100.0%) Ouganda (81) Plasma 520 98.9% (96.7-99.6%) 98.8% (96.6-99.6%) a Sensibilidade e especificidade usando um limiar de insucesso do tratamento de 1000 cópias/ml preditivos positivos e negativos foram de 99% (95% CI: 94.3– 100.0%) e 100% (95% CI: 99.6–100.0%). No estudo realizado no Uganda, a sensibilidade e a especificidade de usar o teste da carga viral para detectar a infecção foram, respectivamente, de 98.9% e 98.8%. Uma importante consideração em ambos os estudos foi que as amostras de gotas de sangue seco foram preparadas usando as técnicas de preparação de tampão e amostras tradicionalmente usadas para preparar as amostras para diagnóstico pediátrico. As actuais recomendações da OMS e estes dados confirmam que a carga viral pode ser usada como teste de diagnóstico pediátrico. De facto, algumas tecnologias têm como alvo específico apenas o ARN do VIH e, no entanto, está demonstrado que têm alta sensibilidade e especificidade, comparáveis às das melhores tecnologias padrão, e conquistaram a pré-qualificação da OMS. Embora alguns fabricantes já tenham solicitado dupla aplicação, o ideal seria que pedissem aprovação das entidades reguladoras no âmbito das suas pretensões para uso presente e/ou futuro dos seus testes de carga viral, para apoiar a implementação desta técnica. Considerações programáticas Existem algumas vantagens potenciais em usar testes da carga viral ou de dupla aplicação como teste de diagnóstico pediátrico, incluindo: • optimizar o fluxo do trabalho laboratorial, podendo reunir-se no mesmo lote amostras para diagnóstico pediátrico precoce e para a carga viral, reduzindo a necessidade de esperar por lotes completos para o diagnóstico pediátrico; • reduzir o risco de atribuir uma prioridade mais baixa ao diagnóstico pediátrico a nível da unidade de saúde e do laboratório, à medida que os programas da carga viral são reforçados; • agilizar a previsão e a quantificação dos testes de diagnóstico pediátrico e da carga viral; • simplificar as compras, a gestão da cadeia de abastecimento e a distribuição de produtos para a colheita de amostras de diagnóstico pediátrico e da carga viral; • poupar dinheiro como resultado da paridade de preços entre os testes da carga viral e de diagnóstico pediátrico e aumentar a eficiência das operações laboratoriais e dos processos de compras; e • melhorar os cuidados, uma vez que um resultado da carga viral poderá ser fornecido para um bebé que viva com o VIH no momento do diagnóstico. Conclusões Criar sistemas de diagnóstico mais eficientes, racionalizados e clinicamente favoráveis é essencial para melhorar os cuidados. Deve considerar-se o uso de testes da carga viral com dupla utilização pretendida validada, para apoiar também o diagnóstico pediátrico e atenuar alguns dos actuais problemas, melhorando o diagnóstico pediátrico. 7.4 NOVAS FERRAMENTAS NOS PONTOS DE CUIDADOS PARA O DIAGNÓSTICO PEDIÁTRICO PRECOCE DO VIH Uma década de investimento em redes de laboratórios convencionais expandiu o acesso aos testes de diagnóstico pediátrico precoce, mas apenas 51% das crianças expostos ao VIH fizeram testes do VIH antes dos dois meses de idade, em 2015 (82). O advento das tecnologias de diagnóstico pediátrico precoce nos pontos de cuidados (10) ruma novidade que cria a oportunidade de aumentar a cobertura dos testes de diagnóstico pediátrico precoce. Permitirá a obtenção dos resultados no próprio dia, permitindo que o tratamento seja iniciado mais cedo e resolvendo algumas das principais limitações das redes convencionais de diagnóstico pediátrico precoce, especialmente os longos tempos de entrega de resultados dos testes e as elevadas taxas de perda no seguimento. Têm sido feitos progressos significativos na garantia da qualidade das novas tecnologias de diagnóstico pediátrico precoce nos pontos de cuidados. Foram feitas as seguintes aprovações reguladoras e avaliações técnicas para o diagnóstico pediátrico precoce nos pontos de cuidados (os países consideram, muitas vezes, estas aprovações, quando adquirem tecnologias de diagnóstico): • CE-IVD (Conformidade Europeia diagnóstico in vitro). Há quatro tecnologias de diagnóstico pediátrico precoce nos 35Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce pontos de cuidados que receberam CE-IVD: AlereTM m-PIMA HIV-1/2 Detect, Cepheid Xpert® HIV-1 Qual e Diagnostics for the Real World’s SAMBA I HIV-1 Qual Test e SAMBA II HIV-1 Qual Whole Blood Test. • Pré-qualificação da OMS: Há duas tecnologias de diagnóstico pediátrico precoce nos pontos de cuidados que cumpriram os requisitos da OMS: AlereTM m-PIMA HIV 1/2 Detect (83) e Cepheid Xpert® HIV-1 Qual (84) receberam a pré-qualificação da OMS em 13 de Junho de 2016. Avaliações técnicas independentes: o consórcio Diagnóstico Pediátrico Precoce nos Pontos de Cuidados era constituído por um grupo de investigadores principais de seis países que realizou avaliações técnicas no terreno das tecnologias de diagnóstico pediátrico precoce nos pontos de cuidados para acelerar a revelação de dados de desempenho independentes, com vista a acelerar os processos nacionais de aprovação e a implementação nos países. Os resultados de nove avaliações técnicas no terreno foram consolidados nos seis países (Tabela 14). Foi analisado um total de 3383 amostras, usando AlereTM m-PIMA HIV-1/2 Detect, e 4401 amostras foram analisadas usando Cepheid Xpert® HIV-1 Qual (85). Recomendações da OMS As orientações consolidadas da OMS de 2016 sobre o uso de antirretrovirais no tratamento e prevenção da infecção pelo VIH (2) recomendam que as tecnologias de teste de ácido nucleico que são desenvolvidas e validadas para utilização nos pontos de cuidados ou na sua proximidade possam ser usadas em testes de diagnóstico pediátrico precoce do VIH. O diagnóstico pediátrico precoce nos pontos de cuidados constitui uma oportunidade para reduzir os tempos de entrega dos resultados, limitar a desistência dos doentes ao longo do ciclo de testes do VIH, reduzir a mortalidade infantil e permitir a delegação de tarefas em quadros inferiores de agentes de saúde em unidades de saúde descentralizadas (2). Tableau 14. Avaliações técnicasdas tecnologias de diagnóstico pediátrico precoce nos pontos de cuidados Configuração do estudo Tipo de amostra Tamanho da amostra Sensibilidade (CI 95%)a Especificidade (CI 95%)a AlereTM m-PIMA HIV-1/2 Detect WHO PQ CDC/NHLS Sang total 98.67% (95.27-99.84%) 100.00% (97.59-100.00%) Consórcio de diagnóstico pediátrico precoce Sang total 99.00% (96.45-99.88%) 99.97% (99.83-100.00%) Cepheid Xpert® HIV-1 Qual WHO PQ CDC/NHLS Sang total 98.86% (93.83-99.97%) 100.00% (97.55-100.00%) Consórcio de diagnóstico pediátrico precoce Sang total 96.79% (92.68-98.95%) 99.91% (99.76-99.97%) WHO PQ CDC/NHLS Gouttes de sang séché 99.34% (96.40-100.00%) 100.00% (97.60-100.00%) Uso actual Há vários países que estão a implementar tecnologias de diagnóstico pediátrico precoce nos pontos de cuidados. Por exemplo, o Maláui, Moçambique e África do Sul comunicaram resultados de projectos-piloto de diagnóstico pediátrico precoce nos pontos de cuidados em 2016, os quais mostravam tempos de entrega de resultados significativamente mais curtos e maiores taxas de início da terapia antirretroviral, em comparação com os sistemas laboratoriais convencionais (86–88). Considerando a elevada taxa de mortalidade precoce de crianças não tratadas que vivem com o VIH (89,90), o diagnóstico pediátrico precoce nos pontos de cuidados poderá também reduzir a mortalidade infantil observada. Com base nas aprovações da CE-IVD e WHO PQ, os resultados robustos de avaliações técnicas independentes no terreno, a elegibilidade para compras, a recomendação da OMS para a utilização do diagnóstico pediátrico precoce nos pontos de cuidados e os resultados iniciais do impacto nos doentes da implementação dos projectos-piloto, os países deverão começar a planear a implementação do diagnóstico pediátrico precoce nos pontos de cuidados, incorporando-o nas orientações nacionais sobre cuidados e tratamento do VIH, plano estratégico nacional, planos operacionais nacionais do PEPFAR, candidaturas a financiamento do Fundo Mundial de Luta contra a SIDA, Tuberculose e Paludismo e orçamentos-programa do VIH. Conclusões Existem evidências suficientes sobre o desempenho destes testes nos contextos pretendidos no terreno, para apoiar a rápida aprovação do regulador nacional e o início do reforço. O desempenho foi consistente entre o laboratório e os contextos no terreno, assim como entre os países. Novas avaliações técnicas destas tecnologias não deverão acrescentar qualquer valor, podendo, pelo contrário, retardar a implementação e o diagnóstico atempado de crianças que vivem com o VIH, uma população crítica e vulnerável. Os organismos reguladores nacionais são encorajados a não protelarem a adopção fazendo novas avaliações, mas antes a adoptarem um processo rápido e eficiente de registo e aprovação nacional para implementação imediata. 36 36 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce 7.5 INTERVENÇÕES OPERACIONAIS: CONSIDERAÇÕES ACTUALIZADAS SOBRE UM PACOTE ABRANGENTE DE GESTÃO DA QUALIDADE DOS TESTES NOS PONTOS DE CUIDADOS NO ÂMBITO DOS PROGRAMAS NACIONAIS DE SAÚDE A introdução e a implementação de tecnologias nos pontos de cuidados e a capacidade para descentralizar os testes melhorou substancialmente o acesso aos serviços de diagnóstico. Desde 2015, foram publicadas novas recomendações da OMS (2). Em 2016, a OMS recomendava condicionalmente as tecnologias de testes com ácido nucleico, que são desenvolvidas e validadas para utilização nos pontos de cuidados ou na sua proximidade, para os testes pediátricos precoces do VIH. Por outro lado, o teste da contagem de células CD4 nos pontos de cuidados pode ser usado para dar prioridade ao urgente início dos cuidados e da terapia antirretroviral. Finalmente, há várias tecnologias nos pontos de cuidados ou na sua proximidade, que foram pré- qualificadas desde 2015, para o diagnóstico pediátrico precoce, CD4, carga viral do VIH, carga viral da hepatite C, rastreio do cancro cervical e VIH e sífilis (91). Está demonstrado que os testes administrados nos pontos de cuidados facilitam uma prestação de serviços de saúde rápida e descentralizada. Uma revisão sistemática do uso de CD4 nos pontos de cuidados para iniciar a terapia antirretroviral (92) revelou uma ligação significativamente melhor aos cuidados do VIH e à rapidez do início da terapia antirretroviral. Para além disso, estudos recentemente publicados no Maláui e em Moçambique revelaram tempos de entrega dos resultados dos testes significativamente mais reduzidos e maiores taxas de início da terapia, quando os testes de diagnóstico pediátrico precoce são feitos nos pontos de cuidados (93,94). A descentralização dos testes qualitativos e quantitativos apresentou tanto oportunidades como desafios, uma vez que os países monitorizam um número cada vez maior de dispositivos e operadores em toda uma rede de testes descentralizados. Isso exigiu a expansão dos esquemas tradicionais de avaliação externa da qualidade de modo a alcançar um número sem precedentes de unidades de saúde e, em muitos casos, a possibilidade novos mecanismos de apoio ao processo de gestão da qualidade. Os princípios apresentados ao longo de toda a publicação sobre a melhoria da qualidade dos testes do VIH realizados nos pontos de cuidados (95) continuam a ser altamente relevantes. Todavia, é agora crucial actualizar as considerações para os países e parceiros da implementação, uma vez que as experiências com as tecnologias nos pontos de cuidados e os mecanismos de garantia de qualidade têm evoluído. À medida que se adquire mais experiência, mais importante se torna uma abordagem abrangente à garantia de qualidade das tecnologias nos pontos de cuidados, para assegurar que os testes são fidedignos e rigorosos. Há várias opções alternativas para a garantia de qualidade que devem constituir um pacote abrangente, juntamente com os testes tradicionais de proficiência e que incluem: • Avaliação de competências durante a formação e em exercício; • Controlo interno da qualidade; • Painéis de testes de proficiência; • Avaliação externa alternativa da qualidade, se não existirem painéis de testes de proficiência: ≈ em papel e online ≈ testes de amostras duplicadas/testes de transcriptase reversa; • Gestão de dados através da conectividade; e • Formação e tutoria no local. Cada mecanismo de garantia de qualidade pode visar diferentes passos do ciclo de testes; contudo, uma vez consolidados num pacote, constituem uma abordagem abrangente e inclusiva. Importância de um pacote abrangente de gestão da qualidade para as tecnologias nos pontos de cuidados A gestão da qualidade dos diagnósticos é fundamental para a qualidade global dos cuidados, assegurando resultados dos testes fidedignos e rigorosos. As avaliações de qualidade dos diagnósticos in vitro, antes da sua introdução no mercado, como a pré-qualificação da OMS, fornecem informação sobre a segurança, qualidade e desempenho do produto, fiabilidade do fabrico e sistemas de gestão da qualidade. Além disso, as exigências das autoridades reguladoras visam avaliar produtos de alta qualidade para o seu uso pretendido. Em conjunto, esses processos garantem que apenas são elegíveis para aquisição produtos de alta qualidade. No entanto, é necessário que existam uma garantia e um controlo da qualidade contínuos, para assegurar o rigor e a precisão dos resultados produzidos pelos testes de diagnóstico, a fim de evitar erros de diagnóstico. Os controlos internos e os padrões pretendem eliminar as diferenças de erros sistemáticos e aleatórios em cada amostra e entre amostras e padrões conhecidos. Os esquemas de testes de proficiência para a avaliação externa da qualidade avaliam especificamente o desempenho de um laboratório ou unidade de saúde em testes rigorosos de amostras estabilizadas de valor ou resultados conhecidos. Os resultados dessas avaliações alertam os programas nacionais para um problema, em que ponto se devem tomar medidas para identificar a causa e potenciais 37Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce acções de remediação. Isso é importante para entender os níveis de desempenho de cada uma das unidades e também para analisar a rede global dos laboratórios nacionais. Além disso, a monitorização dos dados de taxas inválidas, os controlos diários e os padrões de utilização de tecnologias baseadas em dispositivos através da conectividade podem fornecer informação essencial sobre a qualidade dos testes, erros recorrentes dos dispositivos ou dos operadores e a necessidade de cursos de reciclagem ou tutoria específica. Um pacote abrangente de gestão da qualidade poderá identificar falhas e levá-las ao conhecimento dos directores dos programas laboratoriais. Bons programas de garantia de qualidade permitem que os locais de testes e os programas laboratoriais trabalhem coordenadamente para evitar, detectar e corrigir os problemas ao longo de todo o ciclo de testes, assim como monitorizar todos os aspectos de um programa de testes para serviços de testes contínuos e de boa qualidade. O programa abrangente de gestão da qualidade deve reunir uma série de actividades que, em conjunto, podem analisar todos os aspectos dos testes, incluindo: • Identificação de doentes; • Colheita de amostras; • Tratamento das amostras; • Garantia das condições de armazenamento de amostras e; • reagentes e prazos de validade; • Aplicação de amostras; • Garantia do desempenho da tecnologia; • Aplicação de reagentes, se necessário; • Garantia de procedimentos técnicos; techniques; • Interpretação dos resultados; e • Registo dos resultados. Um tal pacote abrangente de gestão da qualidade dos testes nos pontos de cuidados destina-se a complementar as actividades de vigilância pré e pós-mercado sugeridas e em curso em todos os laboratórios nacionais e que estão recomendadas noutras publicações da OMS (96–99). Considerações de implementação para a criação de um pacote de gestão da qualidade Um pacote de gestão da qualidade forte e abrangente para os testes realizados nos pontos de cuidados requer actividades de qualidade, para além dos painéis de testes de proficiência. Criar um pacote abrangente com algumas das estratégias alternativas aqui discutidas permitirá a cobertura de todo o ciclo de testes e proporcionará uma monitorização mais regular dos testes descentralizados. No mínimo, os programas nacionais deverão considerar painéis de testes de proficiência, encorajando os fornecedores a criarem sistemas de controlo interno robustos, formação em serviço e avaliação de competências, gestão de dados através da conectividade e formação e tutoria no local regulares e planeadas. Num esforço para criar um programa abrangente de gestão da qualidade, terão de ser elaboradas políticas nacionais de saúde que considerem os recursos disponíveis para garantir a adopção sustentada da implementação da garantia de qualidade em todos os contextos. Além de explorarem diferentes modelos, os países deverão considerar os momentos e a frequência das actividades de garantia da qualidade, o conteúdo de cada actividade e os encargos a assumir com essas actividades. Todos estes parâmetros têm importantes implicações na qualidade e nos custos, sendo fundamental usar as políticas e os dados específicos dos países para fundamentar essas decisões. Alguns dos parâmetros que ajudam a compreender a implementação estão enumerados abaixo. Para além disso, os mecanismos regulares da qualidade dosprogramas continuam a ser essenciais para garantircompras consistentes e a introdução de tecnologias de altaqualidade. O teste regular dos lotes, serviços e manutençãoe vigilância pós-mercado são estruturas necessárias de umsistema global de qualidade laboratorial que este pacoteabrangente de gestão da qualidade dos testes nos pontos decuidados deverá complementar (98,99). Nenhum programa de gestão da qualidade ficará completo semuma análise dos dados e tomando medidas preventivas ecorrectivas claras e consistentes, sempre que necessário. Esta éuma componente crucial do programa que deve ser claramenteplaneada e determinada, para garantir que os problemas serãoestudados e os operadores terão o necessário apoio paracontinuarem a fornecer testes e resultados de elevada qualidade. 38 38 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce 8. CONCLUSÕES Intensificar o reforço das abordagens de monitorização do tratamento através dos testes da carga viral, assim como o diagnóstico pediátrico precoce, será fundamental para garantir cuidados e tratamentos de boa qualidade e o êxito dos programas. Considerar uma boa rede de diagnósticos, tipos de amostras, intervenções e estratégias em cada um dos países e em todas as partes interessadas nacionais, regionais e parceiros contribuirá para apoiar este esforço, melhorar a colaboração e maximizar o investimento nos diagnósticos para um impacto clínico visível. © W H O 39Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce REFERÊNCIAS 1. Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection: recommendations for a public health approach. Geneva: World Health Organization; 2013 (https://www.who.int/hiv/pub/guidelines/arv2013/en, accessed 8 July 2019). 2. Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection: recommendations for a public health approach – second edition 2016. Geneva: World Health Organization; 2016 (https://www.who.int/hiv/pub/arv/ arv-2016/en, accessed 8 July 2019). 3. What’s new in treatment monitoring: viral load and CD4 testing. Geneva: World Health Organization; 2017 (https:// www.who.int/hiv/pub/arv/treatment-monitoring-info-2017/en, accessed 8 July 2019). 4. Guidelines for managing advanced HIV disease and rapid initiation of antiretroviral therapy. Geneva: World Health Organization; 2017 (https://www.who.int/hiv/pub/guidelines/ advanced-HIV-disease/en, accessed 8 July 2019). 5. HIV market report: the state of the HIV treatment, testing, and prevention markets in low- and middle-income countries, 2017–2022. Boston: Clinton Health Access Initiative; 2018. 6. Landmark HIV diagnostic access program will save $150m and help achieve new global goals on HIV. Geneva: UNAIDS; 2014 (https://www.unaids.org/en/resources/ presscentre/pressreleaseandstatementarchive/2014/ september/20140925prviralload, accessed 8 July 2019). 7. Hologic Global Access Initiative: A revolutionary approach to diagnostic testing for resource-limited settings. Marlborough (MA): Hologic; 2019 (https://www.hologic.com/ globalaccessinitiative, accessed 8 July 2019). 8. Cepheid announces expanded access to Xpert family of virology tests in global regions with the greatest need. Sunnyvale (CA): Cepheid; 2018 (http://www.cepheid.com/en/about/news- events/press-releases/319-cepheid-announces-expanded-access- to-xpert-family-of-virology-tests-in-global-regions-with-the- greatest-need, accessed 8 July 2019). 9. Putting HIV and HCV to the test: a product guide for point-of- care CD4 tests and laboratory-based and point-of-care HIV and HCV viral load tests. Geneva: Médecins Sans Frontières; 2017. 10. HIV/AIDS diagnostics technology landscape. 5th ed. Geneva: Unitaid; 2015 (http://www.unitaid.org/assets/UNITAID_HIV_ Nov_2015_Dx_Landscape-1.pdf, accessed 8 July 2019). 11. Miles to go: closing gaps, breaking barriers, righting injustices. Geneva: UNAIDS; 2018 (https://www.unaids.org/en/ resources/documents/2018/global-aids-update, accessed 8 July 2019). 12. HIV diagnosis and ARV use in HIV-exposed infants: a programmatic update. Geneva: World Health Organization; 2018 (https://www.who.int/hiv/pub/paediatric/diagnosis-arv-infants/ en, accessed 8 July 2019). 13. 90–90–90: an ambitious treatment target to help end the AIDS epidemic. Geneva: UNAIDS; 2017 (https://www.unaids.org/ en/resources/documents/2017/90-90-90, accessed 8 July 2019). 14. Population-based HIV impact assessments [website]. New York: ICAP at Columbia University, 2019 (https://phia.icap. columbia.edu, accessed 8 July 2019). 15. Making viral load routine: successes and challenges in the implementation of routine HIV viral load monitoring. Geneva: Médecins Sans Frontières; 2016. 16. Vojnov L, Venter F, Sacks J, Ehrenkranz P, Kiyaga C, Reynolds S et al. Next steps in HIV treatment monitoring: a call to action to ensure the use of viral load test results. In preparation. 17. HIV viral load scale-up tools [website]. Addis Ababa: ASLM; 2019 (http://www.aslm.org/hiv-viral-load-testing/hiv-viral-load- scale-tools, accessed 8 July 2019). 18. Tools for scaling-up viral load monitoring [website]. Washington (DC): USAID; 2019 (https://aidsfree.usaid.gov/news- events/tools-scaling-viral-load-monitoring, accessed 8 July 2019). 19. AIDSFree viral load and early infant diagnosis knowledge base [online database]. Washington (DC): USAID; 2019 (https:// aidsfree.usaid.gov/resources/vl-eid, accessed 8 July 2019). 20. Abbott RealTime HIV-1 instructions for use. Chicago: Abbott Laboratories; 2014. 21. Public report: Abbott RealTime HIV-1. Geneva: World Health Organization; 2016 (https://www.who.int/diagnostics_ laboratory/evaluations/pq-list/hiv-vrl/180423_amended_final_ pqpr_0145_027_00_v11.pdf?ua=1, accessed 8 July 2019). 22. m-PIMA™ HIV-1/2 VL cartridge guide. Chicago: Abbott Laboratories; 2019. 40 40 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce 23. Public report: m-PIMA HIV-1/2 VL. Geneva: World Health Organization; 2019 (https://www.who.int/diagnostics_ laboratory/evaluations/pq-list/190408_pqdx_0359_032_00_ pqpr_mpima.pdf?ua=1, accessed 8 July 2019). 24. GENERIC HIV Charge Virale instructions for use. Collingwood (NJ); Biocentric; 2019. 25. NucliSENS EasyQ® HIV-1 v2.0 instructions for use. Marcy- l’Étoile: bioMérieux; 2010. 26. Public report: NucliSENS EasyQ® HIV-1 v2.0. Geneva: World Health Organization; 2017 (https://www.who.int/diagnostics_ laboratory/evaluations/pq-list/hiv-vrl/pqdx_0127_016_00_ public_report_v3.pdf?ua=1, accessed 8 July 2019). 27. ExaVir™ Load instructions for use, version 3. Uppsala: Cavidi; 2011. 28. Xpert® HIV-1 Viral Load instructions for use. Sunnyvale (CA): Cepheid; 2017. 29. Public report: Xpert® HIV-1 viral load. Geneva: World Health Organization; 2017 (https://www.who.int/diagnostics_ laboratory/evaluations/pq-list/hiv-vrl/170830_amended_final_ pqpr_0192_0193_0194_0195_070_00_v3.pdf?ua=1, accessed 8 July 2019). 30. Aptima™ HIV-1 Quant Dx Assay instructions for use. Marlborough (MA): Hologic; 2017. 31. Public report: Aptima™ HIV-1 Quant Dx Assay. Geneva: World Health Organization; 2018 (https://www.who.int/ diagnostics_laboratory/evaluations/pq-list/180207_final_ pqpr_0236_078_00_v5_IFU_v4.pdf?ua=1, accessed 8 July 2019). 32. artus® HI Virus -1 RG RT-PCR Kit handbook. Hilden: Qiagen; 2013. 33. artus® HI Virus -1 QS-RGQ Kit handbook. Hilden: Qiagen; 2013 34. COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0 instructions for use. Basel: Roche; 2017. 35. Public report: COBAS® AmpliPREP/COBAS® TaqMan® HIV-1 Test, v2.0. Geneva: World Health Organization; 2018 (https://www.who.int/diagnostics_laboratory/evaluations/ pq-list/hiv-vrl/160530_amended_final_public_report_ pqdx_0200_046_00_taqman96.pdf?ua=1, accessed 8 July 2019). 36. cobas® HIV-1 Quantitative Nucleic Acid Test for Use on the cobas® 4800 System instructions for use. Basel: Roche; 2018. 37. cobas® HIV-1 Quantitative Nucleic Acid Test for Use on the cobas® 6800/8800 Systems instructions for use. Basel: Roche; 2017. 38. HIV Real-TM Quant DX handbook. Como: Sacace Biotechnologies; 2015. 39. VERSANT® HIV-1 RNA 1.5 Assay instructions for use. Munich: Siemens; 2017. 40. Bonner K, Siemieniuk RA, Boozary A, Roberts T, Fajardo E, Cohn J. Expanding access to HIV viral load testing: a systematic review of RNA stability in EDTA tubes and PPT beyond current time and temperature thresholds. PLoS One. 2014;9:e113813. 41. Hardie DR, Korsman SN, Ameer S, Vojnov L, Hsiao N-Y. Reliability of plasma HIV viral load testing beyond 24 hours: insights gained from a study in a routine diagnostic laboratory. PLoS One. (in press). 42. Gullett JC, Nolte FS. Quantitative nucleic acid amplification methods for viral infections. Clin Chem. 2015;61:72–8. 43. Undetectable: how viral load monitoring can improve HIV treatment in developing countries. Geneva: Médecins Sans Frontières; 2012. 44. Quinn TC, Wawer MJ, Sewankambo N, Serwadda D, Li C, Wabwire-Mangen F et al. Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group. N Engl J Med. 2000;342:921–9. 45. Gray RH, Wawer MJ, Brookmeyer R, Sewankambo NK, Serwadda D, Wabwire-Mangen F et al. Probability of HIV-1 transmission per coital act in monogamous, heterosexual, HIV-1- discordant couples in Rakai, Uganda. Lancet. 2001;357:1149–53. 46. Avettand-Fènoël V, Hocqueloux L, Ghosn J, Cheret A, Frange P, Melard A et al. Total HIV-1 DNA, a marker of viral reservoir dynamics with clinical implications. Clin Microbiol Rev. 2016;29:859–80. 47. Parkin NT. Measurement of HIV-1 viral load for drug resistance surveillance using dried blood spots: literature review and modeling of contribution of DNA and RNA. AIDS Rev. 2014;16:160–71. 48. Lee TH, Stromberg RR, Heitman JW, Sawyer L, Hanson CV, Busch MP. Distribution of HIV type 1 (HIV-1) in blood components: detection and significance of high levels of HIV-1 associated with platelets. Transfusion. 1998;38:580–8. 49. Smit PW, Sollis KA, Fiscus S, Ford N, Vitoria M, Essajee S et al. Systematic review of the use of dried blood spots for monitoring HIV viral load and for early infant diagnosis. PLoS One. 2014;9:e86461. 41Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce 50. Vojnov L, Carmona S, Zeh C, Markby J, Boeras D, Prescott MR et al. The performance of using dried blood spot specimens for HIV-1 viral load testing: a systematic review and meta-analysis. In preparation. 51. Holodniy M, Mole L, Yen-Lieberman B, Margolis D, Starkey C, Carroll R et al. Comparative stabilities of quantitative human immunodeficiency virus RNA in plasma from samples collected in VACUTAINER CPT, VACUTAINER PPT, and standard VACUTAINER tubes. J Clin Microbiol. 1995;33:1562–6. 52. Ginocchio CC, Wang XP, Kaplan MH, Mulligan G, Witt D, Romano JW et al. Effects of specimen collection, processing, and storage conditions on stability of human immunodeficiency virus type 1 RNA levels in plasma. J Clin Microbiol. 1997;35:2886–93. 53. Holodniy M, Rainen L, Herman S, Yen-Lieberman B. Stability of plasma human immunodeficiency virus load in VACUTAINER PPT plasma preparation tubes during overnight shipment. J Clin Microbiol. 2000;38:323–6. 54. Elbeik T, Nassos P, Kipnis P, Haller B, Ng VL. Evaluation of the VACUTAINER PPT Plasma Preparation Tube for use with the Bayer VERSANT assay for quantification of human immunodeficiency virus type 1 RNA. J Clin Microbiol. 2005;43:3769–71. 55. Giordano M, Kelleher T, Colonno RJ, Lazzarin A, Squires K. The effects of the Roche AMPLICOR HIV-1 MONITOR UltraSensitive Test versions 1.0 and 1.5 viral load assays and plasma collection tube type on determination of response to antiretroviral therapy and the inappropriateness of cross-study comparisons. J Clin Virol. 2006;35:420–5. 56. Griffith BP, Mayo DR. Increased levels of HIV RNA detected in samples with viral loads close to the detection limit collected in plasma preparation tubes (PPT). J Clin Virol. 2006;35:197–200. 57. Wan H, Seth A, Rainen L, Fernandes H. Coamplification of HIV-1 proviral DNA and viral RNA in assays used for quantification of HIV-1 RNA. J Clin Microbiol. 2010;48:2186–90. 58. Salimnia H, Moore EC, Crane LR, Macarthur RD, Fairfax MR. Discordance between viral loads determined by Roche COBAS AMPLICOR human immunodeficiency virus type 1 monitor (version 1.5): standard and ultrasensitive assays caused by freezing patient plasma in centrifuged Becton-Dickinson Vacutainer brand plasma preparation tubes. J Clin Microbiol. 2005;43:4635–9. 59. García-Bujalance S, Ladrón de Guevara C, González-García J, Arribas JR, Zamora F, Gutiérrez A. Elevation of viral load by PCR and use of plasma preparation tubes for quantification of human immunodeficiency virus type 1. J Microbiol Methods. 2007;69:384–6. 60. Rebeiro PF, Kheshti A, Bebawy SS, Stinnette SE, Erdem H, Tang YW et al. Increased detectability of plasma HIV-1 RNA after introduction of a new assay and altered specimen-processing procedures. Clin Infect Dis. 2008;47:1354–7. 61. Kran AM, Jonassen TØ, Sannes M, Jakobsen K, Lind A, Maeland A et al. Overestimation of human immunodeficiency virus type 1 load caused by the presence of cells in plasma from plasma preparation tubes. J Clin Microbiol. 2009;47:2170–4. 62. Kraft CS, Binongo JN, Burd EM, Eaton ME, McCloskey CB, Fernandes H et al. Successful use of Plasma Preparation Tubes™ (PPTs) in the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 test. J Clin Virol. 2013;57:77–9. 63. Fernandes H, Morosyuk S, Abravaya K, Ramanathan M, Rainen L. Evaluation of effect of specimen-handling parameters for plasma preparation tubes on viral load measurements obtained by using the Abbott RealTime HIV-1 load assay. J Clin Microbiol. 2010;48:2464–8. 64. Adachi D, Benedet M, Tang JW, Taylor GD. Comparative evaluation of Roche’s COBAS Ampliprep/COBAS TaqMan HIV-1 Test v2.0 and Abbott’s RealTime m2000sp/rt HIV-1 assay on PPTs and EDTA samples. J Clin Virol. 2014;60:78–9. 65. Cloherty G, Swanson P, Lucic D, Dieckhaus K, Anthony P, Cataline P et al. Clinical implications of elevated HIV-1 viral load results obtained from samples stored frozen in vacutainer plasma preparation tubes. J Virol Methods. 2014;204:91–2. 66. Goedhals D, Scott LE, Moretti S, Cooper MA, Opperman WJ, Rossouw I. Evaluation of the use of plasma preparation tubes for HIV viral load testing on the COBAS AmpliPrep/COBAS TaqMan HIV-1 version 2.0. J Virol Methods. 2013;187:248–50. 67. BD Vacutainer® PPT instructions for use. Franklin Lakes (NJ): Becton, Dickinson and Company; 2015. 68. K2E K2EDTA Sep. Kremsmünster: Greiner Bio-One; 2019 (https://shop.gbo.com/en/row/products/preanalytics/venous- blood-collection/vacuette-tube/edta/k2e-k2edta-sep, accessed 8 July 2019). 69. K2EDTA Gel Tube. Negeri Sembilan: TUD: 2019 (http://www. tud.my/product_info_901003-100013.htm, accessed 8 July 2019). 70. Putting HIV and HCV to the test. Geneva: Médecins Sans Frontières; 2017 (https://www.msf.org/sites/msf.org/files/ putting_hiv_and_hcv_to_the_test.pdf, accessed ( July2019). 42 42 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce 71. Sacks JA, Fong Y, Perez Gonzalez M, Andreotti M, Baliga S, Garrett N et al. Performance of Cepheid GeneXpert HIV-1 viral load plasma assay to accurately detect treatment failure: a clinical meta-analysis. AIDS. (in press). 72. Sacks JA, Fong Y, Gonzalez MP, Andreotti M, Baliga S, Garrett N et al. Performance of cepheid GeneXpert HIV-1 viral load plasma assay to accurately detect treatment failure: a clinical meta-analysis. AIDS. doi: 10.1097/QAD.0000000000002303. [Epub ahead of print] 73. Information note: considerations for adoption and use of multidisease testing devices in integrated laboratory networks. Geneva: World Health Organization; 2017 (https://www.who. int/tb/publications/2017/considerations_multidisease_testing_ devices_2017/en, accessed 8 July 2019). 74. WHO recommendations on the diagnosis of HIV infection in infants and children. Geneva: World Health Organization; 2010 (https://apps.who.int/iris/handle/10665/44275, accessed 8 July 2019). 75. Recommendations for the use of antiretroviral drugs in pregnant women with HIV infection and interventions to reduce perinatal HIV transmission in the United States. Washington (DC): United States Department of Health and Human Services; 2018 (https://aidsinfo.nih.gov/guidelines/html/3/perinatal/509/ diagnosis-of-hiv-infection-in-infants-and-children, accessed 8 July 2019). 76. Delamere C, Burgard M, Mayaux MJ, Blanche S, Doussin A, Ivanoff S et al. HIV-1 RNA detection in plasma for the diagnosis of infection in neonates. J Acquir Immune Defic Syndr Hum Retrovirol. 1997;15:121–5. 77. Nesheim S, Palumbo P, Sullivan K, Lee F, Vink P, Abrams E et al. Quantitative RNA testing for diagnosis of HIV-infected infants. J Acquir Immune Defic Syndr Hum Retrovirol. 2003;32:192·5. 78. Simonds RJ, Brown TM, Thea DM, Orloff SL, Steketee RW, Lee FK et al. Sensitivity and specificity of a qualitative RNA detection assay to diagnose HIV infection in young infants. AIDS. 1998;12:1545–9. 79. Young N, Shaffer N, Chaowanachan T, Chotpitayasunondh T, Vanparapar N, Mock PA et al. Early diagnosis of HIV-1 infected infants in Thailand using RNA and DNA PCR assays sensitive to non-B subtypes; J Acquir Immune Defic Syndr Hum Retrovirol. 2000;24:401–7. 80. Vubil A, Nhachigule C, Loquiha O, Meggi B, Mabunda N, Bollinger T et al. Viral load assay performs comparably to early infant diagnosis assay to diagnose infants with HIV. J Init AIDS Soc. In preparation. 81. Kiyaga C, Fong Y, Okiira C, Kushemererwa GE, Kayongo I, Tadeo I et al. Viral load assay performed well when used as a diagnostic for infants. 11th International Workshop on HIV Pediatrics, Mexico City, Mexico, 19–20 July 2019. 82. Global Plan towards the elimination of new HIV infections among children by 2015 and keeping their mothers alive. Geneva: UNAIDS; 2011 (https://www.unaids.org/en/resources/ documents/2011/20110609_JC2137_Global-Plan-Elimination- HIV-Children_en.pdf, accessed 8 July 2019). 83. Public report: Alere™q HIV-1/2 Detect. Geneva: World Health Organization; 2016 (http://www.who.int/diagnostics_laboratory/ evaluations/pq-list/ hiv-vrl/160613PQPublicReport_0226-032- 00AlereHIVDetect_ v2.pdf?ua=1, accessed 8 July 2019). 84. Public report: Cepheid Xpert®HIV-1 Qual. Geneva: World Health Organization; 2016 (http://www.who.int/diagnostics_ laboratory/evaluations/pq-list/ hiv-vrl/160613PQPublicRepo rt_0259-0700-00_XpertQualHIV_ v2.pdf?ua=1, accessed 8 July 2019). 85. Carmona S, Wedderburn C, Macleod W, Hsaio M, Jani I, Kroon M et al. Field performance of point-of-care HIV testing for early infant diagnosis: pooled analysis from six countries for the EID Consortium. ASLM2016, Cape Town, South Africa, 3–8 December 2016 (https://eidconsortium.org/Files/EID%20 Poster%20v5%20Low%20res.pdf, accessed 8 July 2019). 86. Jani IV, Meggi B, Mabunda N, Vubil A, Tobaiwa O, Bollinger T et al. Effect of point-of-care early infant diagnosis on retention of patients and rates of antiretroviral therapy initiation on primary health care clinics: a cluster-randomized trial in Mozambique. 21st International AIDS Conference, Durban, South Africa, 18–22 July 2016. 87. Mwenda R. Impact of point-of-care EID testing into the national EID program: pilot experiences from Malawi. 21st International AIDS Conference, Durban, South Africa, 18–22 July 2016. 88. Technau K, Sherman G, Bhowan K, Murnane P, Coovadia AH, Kuhn L. Comparing point of care to laboratory HIV PCR testing at birth in a hospital setting in Johannesburg, South Africa (Abstract O-14). 8th International Workshop on HIV Pediatrics, Durban, South Africa, 15–16 July 2016. 43Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce 89. Bourne DE, Thompson M, Brody LL, Cotton M, Draper B, Laubscher R et al. Emergence of a peak in early infant mortality due to HIV/AIDS in South Africa. AIDS. 2009;23:101–6. 90. Newell ML, Coovadia H, Cortina-Borja M, Rollins N, Gaillard P, Dabis F. Mortality of infected and uninfected infants born to HIV-infected mothers in Africa: a pooled analysis. Lancet. 2004;364:1236–43. 91. WHO list of prequalified in vitro diagnostic products. Geneva: World Health Organization; 2019 https://www.who.int/ diagnostics_laboratory/evaluations/PQ_list/en, accessed 8 July 2019). 92. Vojnov L, Markby J, Boeke C, Harris L, Ford N, Peter T. POC CD4 testing improves linkage to HIV care and timeliness of ART initiation in a public health approach: a systematic review and meta-analysis. PLoS One. 2016;11:e0155256. 93. Mwenda R, Fong Y, Magombo T, Saka E, Midiani D, Mwase C et al. Significant patient impact observed upon implementation of point-of-care early infant diagnosis technologies in an observational study in Malawi. Clin Infect Dis. 2018;67:701–7. 94. Jani IV, Meggi B, Loquiha O, Tobaiwa O, Mudenyanga C, Zitha A et al. Effect of point-of-care early infant diagnosis on antiretroviral therapy initiation and retention of patients. AIDS. 2018;32:1453–63. 95. Improving the quality of HIV-related point-of-care testing: ensuring the reliability and accuracy of test results. Geneva: World Health Organization; 2015 (https://www.who.int/hiv/ pub/toolkits/handbook-point-of-care-testing/en, accessed 8 July 2019). 96. Prequalification of in vitro diagnostics. Geneva: World Health Organization; 2019 (https://www.who.int/diagnostics_ laboratory/evaluations/en, accessed 8 July 2019). 97. Procurement of in vitro diagnostics. Geneva: World Health Organization; 2019 (https://www.who.int/diagnostics_ laboratory/procurement/en, accessed 8 July 2019). 98. Post-market surveillance for in vitro diagnostics (IVDs). Geneva: World Health Organization; 2019 (https://www.who.int/ diagnostics_laboratory/postmarket/en, accessed 8 July 2019). 99. Quality assurance of in vitro diagnostics and laboratory technology. Geneva: World Health Organization; 2019 (https:// www.who.int/diagnostics_laboratory/quality/en, accessed 8 July 2019). 44 44 Kit de ferramentas de diagnóstico molecular do vih para melhorar o acesso aos testes de carga viral e diagnóstico pediátrico precoce ANEXO 1. ALGORITMO DO DIAGNÓSTICO PEDIÁTRICO Recém-nascidos (0-2 dias) Considerar NATa,b Negativo Negativo Iniciar imediatamente a TARc Repetir o NAT para confirmar a infecção Bebé/criança com infecção Bebé ou criança (4-6 semanas a 18 meses) expostos ao VIH Fazer NATb (às 4-6 semanas ou na primeira oportunidade thereafter) Positivo Positivo Iniciar imediatamente a TARc Repetir o NAT para confirmar a infecção Infecção pelo VIH não detectada mas se o bebé/criança for amamentado o risco de contrair a infecção pelo VIH permanece, até completa cessação da amamentaçãod Monitorização clínica regular Fazer NATb (aos 9 meses) Teste de anticorpos aos 18 meses de idade ou 3 meses após cessação da amamentação, conforme o que ocorrer mais tardef Bebé/criança com infecção VIH improvável, a menos que ainda amamentadoe Negativo a Com base nas orientações consolidadas da OMS sobre ARV de 2016, pode ser considerada a adição do NAT à nascença ao actual algoritmo de testes. b O NAT nos POC pode ser usado para diagnosticar a infecção pelo VIH, assim como para confirmar os resultados positivos. c CIniciar imediatamente a TAR. Ao mesmo tempo, voltar a fazer o teste para confirmar a infecção. À medida que o tratamento materno é reforçado e as taxas de transmissão vertical diminuem, prevê-se que os resultados falsos-positivos aumentem: por isso, é importante fazer novo teste após um primeiro NAT positivo, para evitar tratamentos desnecessários, particularmente nos contextos com taxas de transmissão inferiores. Se o segundo teste for negativo, deve fazer-se um terceiro NAT, antes de interromper a TAR. d Para as crianças que nunca foram amamentadas, está incluído neste algoritmo um novo teste após um resultado negativo do NAT às 4–6 semanas, para explicar potenciais resultadosfalsos-negativos do NAT. e O risco de transmissão do VIH permanece, enquanto a amamentação continuar. Se o teste aos 9 meses for feito antes dos 3 meses após a cessação da amamentação, a infecção contraída nos últimos dias da amamentação poderá não ser detectada. Para a avaliação final do estado de VIH, devem fazer-se novos testes aos 18 meses ou 3 meses após cessação da amamentação (o que ocorrer mais tarde). f Se a amamentação continuar para lá dos 18 meses, o diagnóstico final do estado de VIH apenas poderá ser avaliado no final da amamentação. Se a amamentação terminar antes dos 18 meses, o diagnóstico final do estado de VIH com testes de anticorpos apenas poderá ser avaliado aos 18 meses. Os testes de anticorpos devem ser feitos, pelo menos, 3 meses após a cessação da amamentação (para permitir o desenvolvimento de anticorpos do VIH). Para os bebes menores de 18 meses, deve fazer-se o NAT para confirmar a infecção. Se o bebé tiver mais de 18 meses, um teste de anticorpos negativo confirma que o bebé não está infectado; um teste de anticorpos positivo confirma que o bebé está infectado. Fonte: Diagnóstico do VIH e uso de ARV em bebés expostos ao VIH: actualização programática (12) .

Para mais informações, contactar: World Health Organization Department of HIV/AIDS 20, avenue Appia 1211 Geneva Switzerland E–mail: hiv-aids@who.int www.who.int/hiv