Malaria chemoprevention efficacy study protocol
Malaria chemoprevention efficacy study protocol Malaria chemoprevention efficacy study protocol ISBN 978-92-4-005476-9 (electronic version) ISBN 978-92-4-005477-6 (print version) © World Health Organization 2022 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 (http://www.wipo.int/amc/en/ mediation/rules/). Suggested citation. Malaria chemoprevention efficacy study protocol. Geneva: World Health Organization; 2022. 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 https://www.who.int/ copyright. 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. iiiMalaria chemoprevention efficacy study protocol CONTENTS Acknowledgements v Abbreviations vi 1. Introduction 1 2. Studies of chemoprevention efficacy 2 2.1 Definition 3 2.2 Molecular markers of resistance to antimalarial drugs 3 3. CPES study design 4 3.1 Study population 4 3.2 Study site 4 3.3 Sample size 5 3.4 Treatment 6 3.5 Follow-up 6 3.6 Information and samples collected 7 3.7 Diagnosis and molecular analysis 7 3.8 Reporting and analysing study results 7 4. Additions and modifications to the standard methodology 9 4.1 Modification to study new infection only 9 4.2 Conducting CPES as a two-arm study 9 4.3 Monitoring of antimalarial drug resistance 10 References 11 Annex 1. Malaria chemoprevention strategies covered by CPES 13 Annex 2. Molecular markers of resistance 14 Annex 3. Sample size calculations 15 Annex 4. Overview of activities by day 19 Annex 5. Mandatory and optional activities 20 Annex 6. TES definitions of failure 22
vMalaria chemoprevention efficacy study protocol ACKNOWLEDGEMENTS This document was prepared by the Global Malaria Programme of the World Health Organization (WHO) by Charlotte Rasmussen and Pascal Ringwald. WHO gratefully acknowledges the helpful comments and suggestions made by: Michael Alifrangis (Copenhagen University); Pedro Alonso (Hospital Clinic, University of Barcelona); Craig Bonnington (Malaria Consortium); Andrea Bosman (Global Malaria Programme, WHO Headquarters); Matthew Chico (London School of Hygiene and Tropical Medicine); Sian Clarke (London School of Hygiene and Tropical Medicine); Stephan Duparc (Medicine for Malaria Venture); Myriam El Gaaloul (Medicine for Malaria Venture); Roly Gosling (London School of Hygiene and Tropical Medicine); Scott Miller (Gates Medical Research Institute); Abdisalan Noor (Global Malaria Programme, WHO Headquarters); Lucy Okell (Imperial College London); Christopher Plowe (University of Maryland School of Medicine); Stephen Poyer (Population Services International); Michelle Roh (University of California, San Francisco); Cally Roper (London School of Hygiene and Tropical Medicine); David Schellenberg (London School of Hygiene and Tropical Medicine); Jackson Sillah (WHO Regional Office for Africa); Colin Sutherland (London School of Hygiene and Tropical Medicine); André-Marie Tchouatieu (Medicine for Malaria Venture); Ghasem Zamani (WHO Regional Office for the Eastern Mediterranean). Malaria chemoprevention efficacy study protocolvi ABBREVIATIONS ACT artemisinin-based combination therapy ANC antenatal care CI confidence interval CPES chemoprevention efficacy studies DBS dried blood spot IPTi intermittent preventive treatment of malaria in infants IPTp intermittent preventive treatment of malaria in pregnancy IPTsc intermittent preventive treatment of malaria in school-aged children MDA mass drug administration PMC perennial malaria chemoprevention RDT rapid diagnostic test SMC seasonal malaria chemoprevention SP sulfadoxine-pyrimethamine TES therapeutic efficacy studies WHO World Health Organization 1Malaria chemoprevention efficacy study protocol 1. INTRODUCTION Preventive chemotherapy is the use of medicines, either alone or in combination, to prevent malaria infection and its consequences. It requires giving a full treatment course of an antimalarial medicine, often at predefined intervals to individuals who have not been diagnosed with malaria.1 By providing antimalarial medicine to vulnerable populations, existing undiagnosed malaria infections are treated, and the medicine provides a period of protection against new infections. Chemoprevention2 strategies currently recommended by the World Health Organization (WHO) and covered by this guidance are described in Annex 1. These include intermittent preventive treatment of malaria in pregnancy (IPTp), perennial malaria chemoprevention (PMC), seasonal malaria chemoprevention (SMC), and intermittent preventive treatment of malaria in school-aged children (IPTsc) (1).3 Information on the efficacy of the medicines as used in these malaria chemoprevention strategies is critical for ensuring that the strategies remain effective in different settings with different levels of drug resistance. WHO has prepared a standard protocol for therapeutic efficacy studies (TES), and tools for data analysis and monitoring the parasitological and therapeutic responses to treatment (2). TES are prospective evaluations of patients’ clinical and parasitological responses to treatment for uncomplicated malaria. TES are considered the gold standard for assessing antimalarial drug treatment efficacy, and the resulting data are used to inform national malaria treatment policy in malaria- endemic countries. Unfortunately, systematic reviews have demonstrated that even when TES are done at the same time and place, they do not accurately predict the efficacy of chemoprevention strategies (3). In TES, the therapeutic efficacy in clearing asexual blood-stage parasites is assessed in subjects with uncomplicated malaria. In chemoprevention, the drug regimen given can work by clearing both asexual blood- stage parasites and pre-erythrocytic stage parasites in asymptomatic subjects. In both treatment and chemoprevention, many factors other than drug resistance impact the efficacy, including immunity, drug absorption and metabolism. A mutation that can significantly impact the efficacy of a drug in clearing asexual blood-stage parasites may have a different impact on the drug’s efficacy in clearing pre-erythrocytic stage parasites. Additionally, a lower number of asexual blood-stage parasites in an asymptomatic recipient of chemoprevention may be easier for a drug to clear compared to a high number of asexual blood-stage parasites in a symptomatic patient enrolled in TES. Specific protocols are therefore needed for monitoring chemoprevention efficacy. This document is intended as a guide for studies of chemoprevention efficacy and was developed based on reviews of protocols of ongoing studies. The document adapts some of the principles and practices underlying treatment efficacy monitoring to provide standardized approaches for monitoring and evaluating the efficacy of medicines used for malaria chemoprevention. This guide will be updated once additional experience is gained from studies of chemoprevention efficacy. 1 Those identified with malaria at the time of the intervention are treated as per the national treatment guidelines. Only individuals with symptoms are diagnosed by rapid diagnostic test or microscopy. 2 The term chemoprevention is used as equivalent to and interchangeable with the term preventive chemotherapy. 3 Mass drug administration (MDA) and post-discharge malaria chemoprevention (PDMC) are not addressed in this document. Malaria chemoprevention efficacy study protocol2 2. STUDIES OF CHEMOPREVENTION EFFICACY 2.1 Definition Drug efficacy is defined as the capacity of an antimalarial medicine to achieve the therapeutic objective when administered at the recommended dose (4). Chemoprevention efficacy and impact, as understood in this document, are defined in Box 1. Box 1: Definition of chemoprevention efficacy and impact Chemoprevention efficacy: ability of a course of an antimalarial medicine to clear existing parasitaemia and prevent any new infections for a short period (e.g. 28 days). Chemoprevention impact: impact of chemoprevention on uncomplicated malaria, anaemia, low birthweight, severe malaria and death. The WHO-recommended chemoprevention strategies discussed here (IPTp, PMC, SMC and IPTsc) are all intended to clear infections that are present at the time of treatment and to provide a period of post-treatment prophylaxis after the medicine is given. Potential recipients of chemoprevention with symptoms of malaria would be referred for parasitological diagnosis by rapid diagnostic test (RDT) or microscopy and treated as per the national treatment guidelines if malaria diagnosis is confirmed. All malaria chemoprevention strategies are for areas with moderate to high transmission (seasonal or perennial) where a significant proportion of the population may present with asymptomatic parasitaemia. Chemoprevention efficacy studies (CPES) aim to evaluate the ability of antimalarial medicines to clear parasitaemia among asymptomatic individuals and prevent parasitaemia for a predefined period of follow-up. Parasitaemia detected during the follow-up period can be caused either by the failure to clear an infection present at the time of enrolment or by the failure to prevent a new infection. The study methodology can be modified to focus on the efficacy of the medicine given to prevent infection (described in section 4.1). CPES are meant to be routine studies of the efficacy of medicines used for malaria chemoprevention; the purpose is not to evaluate long-term outcomes for the treated individuals. However, studies that link efficacy and longer term outcomes could help to inform policy-makers about the likely public health impact of the chemoprevention strategies and establish relationships between chemoprevention efficacy and specific outcomes. Drug resistance is only one of several factors that may reduce chemoprevention efficacy. Other factors include incorrect dosage, poor patient adherence to treatment, poor drug quality, drug interactions, poor absorption, rapid elimination (e.g. diarrhoea or vomiting) or poor metabolism of prodrugs. Some of these factors can be ruled out in an efficacy study when drug administration is supervised, and the origin and quality of the drugs are verified. The outcome of the study is influenced by a combination of factors, including those related to the human (e.g. immunity) and parasite (e.g. drug resistance); individual variation leading to differences in the availability of the drug (e.g. pharmacokinetics) (5); and the intensity of malaria transmission. 3Malaria chemoprevention efficacy study protocol Although detection of declining chemoprevention efficacy may signal the presence of resistance to an antimalarial drug, confirmation and characterization of parasite resistance require additional tools in addition to TES (e.g. in vitro or ex vivo tests, measurement of drug concentrations in the blood, and analysis of molecular markers). For further information, see the TES protocol and related documents (6–10). 2.2 Molecular markers of resistance to antimalarial drugs Once genetic changes in the parasite are identified and validated as markers of clinically relevant forms of resistance, drug resistance can be monitored using molecular techniques. These analyses can be performed reliably and inexpensively. While the markers have been found to be imperfectly associated with treatment outcomes, their value in predicting the efficacy and usefulness of a medicine for chemoprevention is still unclear. Therefore, it is currently not possible to rely on surveillance of these markers alone to make decisions on chemoprevention efficacy. Molecular markers of Plasmodium falciparum resistance to antimalarial drugs that have been found to be associated with reduced treatment response and are useful in surveillance for resistance include: • Pfcrt (P. falciparum chloroquine resistance transporter) point mutations conferring resistance to chloroquine and piperaquine; • Pfdhfr (P. falciparum dihydrofolate reductase) point mutations conferring resistance to pyrimethamine; • Pfdhps (P. falciparum dihydropteroate synthase) point mutations conferring resistance to sulfadoxine and other sulfa drugs; • Increased copy numbers of Pfmdr1 (P. falciparum multidrug resistance 1 protein) associated with P. falciparum resistance to mefloquine; • Increased copy numbers of Pfpm2–3 (P. falciparum plasmepsin 2–3) and/or point mutations in Pfcrt associated with P. falciparum resistance to piperaquine; • PfKelch13 gene point mutations associated with P. falciparum partial resistance to artemisinins. A more comprehensive summary of molecular markers for drugs currently or potentially being used for chemoprevention is provided in Annex 2. Malaria chemoprevention efficacy study protocol4 3. CPES STUDY DESIGN CPES are single-arm studies4 that aim to evaluate the ability of one or more rounds of chemoprevention to clear any pre-existing parasitaemia and prevent infection for a predefined period of follow-up. 3.1 Study population The population groups targeted for enrolment consist of asymptomatic individuals eligible for the chemoprevention intervention. Those with malaria symptoms must be tested for malaria (by RDT or microscopy). If malaria is confirmed, they should be treated with the recommended treatment as per national treatment guidelines and excluded from the study. The prophylactic drug is expected to be able to clear low parasitaemia at inclusion (< 2000 asexual parasites/µL in moderate to high transmission area), but easy access to treatment must be ensured. Inclusion and exclusion criteria are listed in Table 1. 3.2 Study site CPES should be done in areas where chemoprevention interventions are conducted or are being considered for introduction/implementation. The specific sites selected must be based on: • a sufficient number of individuals targeted for the chemoprevention intervention in the site to make it feasible to reach the target sample size; • up-to-date information on the local epidemiology of malaria; • trained, motivated personnel capable of recruiting and following up study participants, collecting samples and providing malaria treatment as needed; • the availability of facilities to store samples and supplies securely, and stain slides; and • access to a laboratory equipped for blood film examination. The site can consist of one or more facilities or villages, including schools for IPTsc where the intervention is being provided. If supervision of treatment and follow-up is only feasible in a few of the villages covered by a certain health facility, the site can be defined as these villages. The number of sites needed in a country depends on both the size of the area targeted for chemoprevention interventions and the distribution of drug resistance. If data are available showing markedly different levels of drug resistance in different areas, studies should be planned to cover these different settings. 4 The option of having the study as a two-arm study is covered in section 4.2. 5Malaria chemoprevention efficacy study protocol 3.3 Sample size Not having parasitaemia during the follow-up period may be the result of the chemoprevention working as intended or it may be due to the study participant not being exposed to infective bites. This poses a challenge in terms of both the conclusions that can be drawn and the sample size needed. The hypothesis tested is that the proportion of individuals infected at the end of the study will be significantly lower than it would have been if no chemoprevention had been given. In many settings, it will not be possible to have a control group. It would not be ethical to withhold chemoprevention from individuals who are eligible for a recommended intervention with the aim of having a control group. Therefore, having a control group may be possible only in settings where, for instance, the aim of the study is to assess the efficacy of an intervention before it is generally recommended in an area. Some general guidance on sample size calculations is given in Annex 3. Table 1. Inclusion and exclusion criteria Study population Individuals eligible for chemoprevention as per the recommendations with no malaria symptoms Inclusion criteria All CPES Eligible for chemoprevention for SMC, PMC, IPTp and IPTsc as per the current recommendations Able and willing to comply with the study protocol and follow-up schedule Provides informed consent/parent or guardian provides informed consent on behalf of child Additional intervention-specific inclusion criteria IPTp For women ≥ 18 yearsa after the first trimester of pregnancy Exclusion criteria All CPES Symptoms of malaria (axillary fever ≥ 37.5 °C and/or history of fever in the past 48 hours) Known allergy to the medicine provided Sulfadoxine-pyrimethamine (SP) should not be given to individuals receiving a sulfa- based medication for treatment or prophylaxis, including co-trimoxazole (trimethoprim– sulfamethoxazole). This medicine is widely used in HIV-positive individuals (infants and pregnant women) as prophylaxis against opportunistic infections. Individuals receiving azithromycin should be excluded from the study due to the antimalarial activity of azithromycin. Additional intervention-specific exclusion criteria PMC, SMC, IPTsc Presence of severe malnutrition according to WHO’s child growth standards IPTscb Females of menstruation age (> 12 years) unable or unwilling to take pregnancy test due to socio-cultural constrains a Could include adolescents between 12 and 17 years of age but may need specific ethical approval and informed consent from parents/guardian. b Could include adolescents between 12 and 15 years of age but may need specific ethical approval and informed consent from parents/guardian for pregnancy testing. Malaria chemoprevention efficacy study protocol6 3.4 Treatment Those enrolled in the study will receive the recommended treatment course for chemoprevention under direct supervision. How this is organized can vary among study sites, but supervised treatment is needed to ensure that failure is not caused by lack of adherence. If the individual vomits within one hour of receiving the treatment, the treatment should be repeated and vomiting recorded. Study participants identified with asexual parasitaemia, as measured by microscopy between day 4 (in the case of development of clinical symptoms) and day 28 of the follow-up period, will be reported as chemoprevention failure and must be treated with an alternative regimen known to be efficacious – typically the recommended first-line treatment. 3.5 Follow-up Study participants must be followed up with malaria blood films and dried blood spots (DBS) collected weekly for at least four weeks (days 0, 7, 14, 21 and 28), as well as on any day with symptoms. Additionally, day 2 can be added as a day of follow- up and sample collection; this is optional, but as this is the last day of administration for several of the currently recommended chemoprevention medicines, collecting samples on this day may be relatively easy (see Fig. 1). With the currently recommended chemoprevention medicines, a significant level of protection is expected to be maintained for three to four weeks after administration of each course, but will likely decay rapidly thereafter. Where chemoprevention is administered monthly, follow-up for one course of chemoprevention for longer periods (e.g. 42 or 63 days) would require the next course of chemoprevention to be postponed, potentially increasing the risk to study participants. Therefore, this should only be considered when any risk to the study participants can be minimized through follow-up and easy access to care, and when data from a longer follow- up period are considered essential. Fig. 1. Days of follow-up 7Malaria chemoprevention efficacy study protocol 3.6 Information and samples collected On day 0 and all days of follow-up, a thick blood smear should be collected. In addition, in order to minimize discomfort to the patient due to repeated finger pricks, two or three drops of blood should be collected on filter paper each time a blood smear is required. Additionally, the temperature should be recorded (see Annexes 4 and 5 for details). Patients with symptoms should always be tested for malaria; where this cannot be done with microscopy, an RDT can be used. If neither is available, the patient should be referred to a health facility. For slides read retrospectively, asymptomatic study participants found to have parasitaemia from day 7 should be traced and treated as per the national treatment guidelines. 3.7 Diagnosis and molecular analysis The presence of parasitaemia on day 0 and all days of follow-up should be determined primarily by microscopy. Nucleic acid amplification tests for the detection of low-density parasitaemia are considered experimental at this time and are not recommended for classifying study participants as parasitaemic. If parasites are detected by microscopy, the parasitaemia count should be recorded. If parasitaemia is detected on day 0 and any day of follow-up, PCR genotyping is needed to distinguish between reinfection and recrudescence (11). DBS collected on days with parasitaemia (day 0 or during follow-up) should be analysed for relevant molecular markers of antimalarial drug resistance (12). Recruitment will occur in asymptomatic study participants who are either uninfected or have parasitaemia. For study participants with low parasitaemia, genotyping at day 0 might be difficult. 3.8 Reporting and analysing study results CPES aim to evaluate the ability of the provided medicine to clear any existing parasitaemia and/or subsequently prevent parasitaemia for a predefined period of follow-up. Chemoprevention efficacy will be a combination of curative and preventive efficacy. The main study outcome to be reported across interventions is chemoprevention failures based on study participants identified with asexual parasitaemia, as measured by microscopy from day 4 to the end of follow-up (see Table 2). Chemoprevention failure rates can be reported as the cumulative failure rate using Kaplan-Meier analysis or as the proportion using a per protocol analysis. A time- to-event (parasitaemia) analysis can be a helpful additional analysis for interpreting results. Failure can be classified as clinical failure or parasitological failure, as in TES (see Annex 6), based on the presence of symptoms. Early treatment failures are likely to be rare, as the treatment is given to study participants with no parasitaemia, or with low parasitaemia but no symptoms; however, these should be reported separately. For the same reason, failures from days 4 to 7 will be rare, and they will be recorded if study participants seek care outside of scheduled days of follow-up. Malaria chemoprevention efficacy study protocol8 Failure rates where chemoprevention efficacy is less than 100% will be a function of the prevalence of asymptomatic parasitaemia at the time of treatment and the curative efficacy, plus the risk of reinfection and preventive efficacy. Therefore, care needs to be taken in extrapolating conclusions across sites and seasons. Conclusions may be informed by knowledge of the local epidemiology and molecular findings. Table 2. Main study outcomes to be reported Chemoprevention failures Study participants identified with asexual parasitaemia by microscopy during the follow-up period from day 4 to day 28 Additional details when reporting chemoprevention failures Clinical failures: study participants identified with parasitaemia during the follow-up period from day 4 to day 28 with axillary temperature ≥ 37.5°C Parasitological failures: study participants identified with parasitaemia during the follow-up period from day 7 to day 28 with axillary temperature < 37.5°C Study participants identified with a new infection during the follow-up period Study participants identified with recrudescence during the follow-up period Geometric mean and range of asexual parasitaemia in study participants with chemoprevention failures Days to reported failure (median and range) Prevalence of molecular marker(s) Prevalence of molecular marker(s) of resistance to chemoprevention drug(s) at day 0 and in any parasites detected after day 4 (# of infections carrying markers of interest / # of successfully genotyped infections) Note: In the retrospective analysis, children found to have a parasitemia > 2000 asexual parasites/μL blood can be excluded and reported on separately. 9Malaria chemoprevention efficacy study protocol 4. ADDITIONS AND MODIFICATIONS TO THE STANDARD METHODOLOGY 4.1 Modification to study new infection only In some circumstances, there could be an interest in only studying the efficacy of chemoprevention to prevent infection. This could, for instance, be as a follow-up study in areas where a high number of chemoprevention failures have been found and further analysis as to the mechanism of failure is warranted. If the objective of the study is to focus on the efficacy of chemoprevention to prevent infection, it is recommended to treat study participants with a highly efficacious antimalarial with a relatively short half-life 14 days prior to enrolment (see Fig. 2). Currently, the most suitable candidate for the pre-study treatment is artemether- lumefantrine, as lumefantrine has a shorter half-life than the other available partner drugs in artemisinin-based combination therapies (ACTs). Studies with pre-study treatment enable more precise estimates of the efficacy of chemoprevention to prevent new infections. Studies without pre-study treatment report the proportion of participants with new infections during the follow-up period based on a PCR classification of parasitaemia as recrudescence or a new infection. This estimate is likely to be confounded by intra-host competition affecting the risk of new infection when parasites are already present, incorrect PCR classification, and failure of the PCR to pick up more than one infection. Fig. 2. Study modified to focus on new infection Any day of symptoms 4.2 Conducting CPES as a two-arm study In some settings, conducting CPES as a two-arm study can be done to compare groups receiving different drugs or, in an area where the chemoprevention strategy may be introduced, to compare a group receiving chemoprevention with a group not receiving chemoprevention. Steps need to be taken to ensure comparability between groups and to address any ethical concerns. Malaria chemoprevention efficacy study protocol10 4.3 Monitoring of antimalarial drug resistance Preferably, chemoprevention efficacy is monitored at sentinel sites where treatment efficacy and the prevalence of key molecular markers are also monitored. In-depth knowledge of the changing epidemiology at these sites will help inform the design of studies and interpretation of the results. WHO recommends that first- and second-line treatments be monitored through TES at sentinel sites at least every two years. In general, chemoprevention interventions should not employ the same medicines used for treatment. However, if this is done, treatment efficacy needs to be monitored more closely. Changes in the prevalence of relevant molecular markers should continuously be monitored alongside the use of chemoprevention. In areas where chemoprevention has not yet been introduced and in areas where a change of the drugs used for chemoprevention is planned, a molecular marker survey should be done prior to introducing or changing drugs. The purpose of tracking molecular markers is to track changes potentially caused by the use of the chemoprevention, particularly if pre-intervention data are available. This tracking enables analysis of the association between the efficacy of chemoprevention and the general prevalence of relevant molecular markers. It can provide early evidence and serve as an early warning sign of resistance. Unfortunately, molecular markers of drug resistance are available for only some antimalarial medicines (Annex 2). The correlation between molecular markers and the therapeutic efficacy of many antimalarials is imperfect and should be interpreted with caution. The survey of molecular markers can be done by collecting filter paper with DBS from patients seeking treatment in health facilities after confirmed malaria diagnosis and after the patient has consented to be part of the study. 11Malaria chemoprevention efficacy study protocol REFERENCES 1. Preventive chemotherapies. In: Global Malaria Programme [website]. Geneva: World Health Organization (https://www.who.int/teams/global-malaria- programme/prevention/preventive-chemotherapies, accessed 27 May 2021). 2. Tools for monitoring antimalarial drug efficacy. In: Global Malaria Programme [website]. Geneva: World Health Organization (https://www.who.int/teams/ global-malaria-programme/case-management/drug-efficacy-and-resistance/ tools-for-monitoring-antimalarial-drug-efficacy, accessed 27 May 2021). 3. ter Kuile FO, van Eijk AM, Filler SJ. Effect of sulfadoxine-pyrimethamine resistance on the efficacy of intermittent preventive therapy for malaria control during pregnancy: a systematic review. JAMA. 2007;297(23):2603–16. doi:10.1001/ jama.297.23.2603. 4. WHO malaria terminology, 2021 update. Geneva: World Health Organization; 2021 (https://apps.who.int/iris/handle/10665/349442, accessed 27 May 2021). 5. Rogerson SJ, Wijesinghe RS, Meshnick SR. Host immunity as a determinant of treatment outcome in Plasmodium falciparum malaria. Lancet Infect Dis. 2010;10(1):51–9. doi:10.1016/S1473-3099(09)70322-6. 6. Medicines for Malaria Venture, World Health Organization. Methods and techniques for clinical trials on antimalarial drug efficacy: genotyping to identify parasite populations: informal consultation organized by the Medicines for Malaria Venture and cosponsored by the World Health Organization, 29–31 May 2007. Geneva: World Health Organization; 2008 (https://apps.who.int/iris/ handle/10665/43824, accessed 27 May 2021). 7. Methods for surveillance of antimalarial drug efficacy. Geneva: World Health Organization; 2009 (https://apps.who.int/iris/handle/10665/44048, accessed 27 May 2021). 8. Basco LK, World Health Organization, Informal consultation organized by the World Health Organization with the technical support of the worldwide antimalarial resistance network. Methods and techniques for assessing exposure to antimalarial drugs in clinical field studies. Geneva: World Health Organization; 2011 (https://apps.who.int/iris/handle/10665/44653, accessed 27 May 2021). 9. Basco LK, Heseltine E, World Health Organization. Field applications of in vitro assays for the sensitivity of human malaria parasites to antimalarial drugs. Geneva: World Health Organization; 2007 (https://apps.who.int/iris/ handle/10665/43610, accessed 27 May 2021). 10. Malaria surveillance, monitoring & evaluation: a reference manual. Geneva: World Health Organization; 2018 (https://apps.who.int/iris/handle/10665/272284, accessed 27 May 2021). 11. Informal consultation on methodology to distinguish reinfection from recrudescence in high malaria transmission areas: : report of a virtual meeting, 17–18 May 2021. Geneva: World Health Organization; 2021 (https://apps.who.int/ iris/handle/10665/348385, accessed 1 June 2022). Malaria chemoprevention efficacy study protocol12 12. Report on antimalarial drug efficacy, resistance and response: 10 years of surveillance (2010–2019). Geneva: World Health Organization; 2020 (https:// apps.who.int/iris/handle/10665/336692, accessed 1 June 2022). 13. Conrad MD, Rosenthal PJ. Antimalarial drug resistance in Africa: the calm before the storm? Lancet Infect Dis. 2019;19(10):e338-51. doi:10.1016/S1473- 3099(19)30261-0. 13Malaria chemoprevention efficacy study protocol ANNEX 1. MALARIA CHEMOPREVENTION STRATEGIES COVERED BY CPES Current WHO recommendation Intermittent preventive treatment of malaria in pregnancy (IPTp) In malaria-endemic areas, pregnant women of all gravidities should be given antimalarial medicine at predetermined intervals to reduce disease burden in pregnancy and adverse pregnancy and birth outcomes. SP has been widely used for malaria chemoprevention during pregnancy and remains effective in improving key pregnancy outcomes. IPTp-SP should start as early as possible in the second trimester and not before week 13 of pregnancy. Doses should be given at least one month apart, with the objective of ensuring that at least three doses are received. Antenatal care (ANC) contacts remain an important platform for delivering IPTp. Where inequities in ANC service and reach exist, other delivery methods (such as the use of community health workers) may be explored, ensuring that ANC attendance is maintained and underlying inequities in ANC delivery are addressed. Perennial malaria chemoprevention (PMC) – formerly intermittent preventive treatment of malaria in infants (IPTi) In areas of moderate to high perennial malaria transmission, children belonging to age groups at high risk of severe malaria can be given antimalarial medicines at predefined intervals to reduce disease burden. PMC schedules should be informed by the age pattern of severe malaria admissions, the duration of protection of the selected drug, and the feasibility and affordability of delivering each additional PMC course. SP has been widely used for chemoprevention in Africa, including for PMC. ACTs have been effective when used for PMC, but evidence is limited on their safety, efficacy, adherence to multi-day regimens, and cost-effectiveness in the context of PMC. Seasonal malaria chemoprevention (SMC) In areas of seasonal malaria transmission, children belonging to age groups at high risk of severe malaria should be given antimalarial medicines during peak malaria transmission seasons to reduce disease burden. Eligibility for SMC is defined by the seasonality of malaria transmission and age groups at risk of severe malaria. The added value of a seasonally targeted intervention is likely to be greatest where transmission is intensely seasonal. Monthly cycles of SP plus amodiaquine (SP+AQ) have been widely used for SMC in African children under 5 years old and have been shown to be efficacious, safe, well tolerated, available and inexpensive. Intermittent preventive treatment of malaria in school- aged children (IPTsc) School-aged children living in malaria-endemic settings with moderate to high perennial or seasonal transmission can be given a full therapeutic course of antimalarial medicine at predetermined times as chemoprevention to reduce disease burden. IPTsc has been evaluated in children aged 5–15 years. The burden of malaria and benefits of IPTsc may vary across this age range, but evidence is limited. National malaria programmes can consider IPTsc if resources allow for its introduction among school-aged children without compromising chemoprevention interventions for those carrying the highest burden of severe disease, such as children < 5 years old. Schools may provide a low-cost means to deliver chemoprevention to school-aged children. However, seasonal variation in malaria transmission and the timing of school terms, as well as equity concerns, may mean alternative delivery channels are needed to maximize impact. For more information see: WHO recommendations for malaria chemoprevention and elimination. In: Global Malaria Programme [website]. Geneva: World Health Organization (https://www.who.int/news/item/03-06- 2022-updated-who-recommendations-for-malaria-chemoprevention-and-elimination, accessed 3 June 2022). Malaria chemoprevention efficacy study protocol14 ANNEX 2. MOLECULAR MARKERS OF RESISTANCE Drug Molecular markers Gene Mutation 4-aminoquinolines Chloroquine Pfcrt K76T + different sets of mutations at other codons Pfmdr1 (in combination with Pfcrt mutations only) N86Y, Y184F and D1246Y Amodiaquine Yet to be validated Studies show that amodiaquine selects for Pfmdr1 mutations Piperaquine Pfpm2–3 Pfcrt Pfpm2–3 increased copy number Detected in vivo: T93S, H97Y, F145I, I218F and C350R Antifolates Pyrimethamine Pfdhfr N51I, C59R, S108N and I164L Sulfadoxine Pfdhps S436A/F, A437G, K540E, A581G and A613T/S Proguanil Pfdhfr A16V, N51I, C59R, S108N and I164L Amino-alcohols Lumefantrine Yet to be validated Studies show that lumefantrine selects for Pfmdr1 mutations (N86) Mefloquine Pfmdr1 Pfmdr1 increased copy number Quinine Yet to be validated Mannich base Pyronaridine Yet to be validated Naphthoquinone Atovaquone Pfcytochrome b Y268N/S/C Sesquiterpene lactones Artemisinin and its derivatives PfKelch13 List of candidate and validated markers (WHO, 2020) Antibiotics Doxycycline Resistance not documented Clindamycin Resistance not documented 8-aminoquinolines Primaquine Resistance not documented Tafenoquine Resistance not documented For more information see: Report on antimalarial drug efficacy, resistance and response: 10 years of surveillance (2010–2019). Geneva: World Health Organization; 2016 (https://apps.who.int/iris/handle/10665/336692, accessed 3 June 2022). 15Malaria chemoprevention efficacy study protocol ANNEX 3. SAMPLE SIZE CALCULATIONS The hypothesis tested is that the proportion of individuals infected at the end of the study (i.e. 28 days post-intervention) will be significantly lower than it would have been if no chemoprevention had been given. Two different study designs have been considered to test this hypothesis: a single-arm study where all participants receive chemoprevention, and a two-arm study with a chemoprevention group and a control group (no chemoprevention). Single-arm study design In this study design, the focus is on the proportion of participants infected at the end of the follow-up period compared to a theoretical value. This theoretical value can be calculated from a variety of data sources ranging from data generated from previous infection studies in the area of interest to more generalized region-specific data sources. Estimating incidence rate and prevalence in individuals not receiving chemoprevention The theoretical proportion infected at the end of the follow-up period will be a composite of the expected prevalence at the start of the study and the expected number of new infections. Therefore, it is essential to have an estimate of the proportion of individuals who are likely to become infected within 28 days with no chemoprevention or antimalarial treatment (that is, the cumulative incidence), or the incidence rate. There are several approaches to obtain the required baseline data, each with its own strengths and limitations. The incidence can be based on information from a modest study where a small cohort is followed up over a predefined period (e.g. 28 days). Alternatively, the incidence rate can be estimated indirectly from prevalence data using micro-simulation models (1).1 Once the incidence rate has been estimated, the corresponding 95% confidence interval (CI) should be determined. It is suggested that the lower CI be adopted as the indicator of likely new infections. Where the incidence rate is calculated from a baseline study, the lower 95% CI can be calculated as (log incidence rate, or the Rothman/Greenland method): where EF= e 1.96/ √(number of new infections in baseline study) Lower 95% CI = Incidence rate EF 1 Cameron et al. (1) published modelled prevalence (PfPR2-10) vs. incidence curves for infants and young children (0–5 yrs), older children (5–15 yrs) and adults (> 15 yrs) in areas with low and high seasonal malaria transmission. The source data were derived from 13 countries in Africa. Thus, data on site- specific prevalence could be converted to estimated incidence rates. However, it should be noted that estimated incidence rates are given per person-year of observation. This needs to be converted to “per 28-person-days” to predict the number of individuals expected to become infected during the study follow-up period. In areas with little seasonality, the incidence rate can be converted from person-years to 28-person-days by linear scaling. However, in areas with high seasonality, this scaling should take into account the length of the transmission season and the timing of the study relative to the transmission season. To illustrate this point in a highly simplified example, if transmission only occurred in four months of the year with a relatively constant incidence across those four months, and the proposed study had a follow-up period within those four months, then the estimated value of 1 infection/person-year could be scaled to 0.23 infections/28-person-days (calculated as (1 infection/4- person-months)/ 121 days * 28 days) (1). Malaria chemoprevention efficacy study protocol16 Where the incidence rate is estimated based on prevalence, the published 95% lower CI can be used for the corresponding prevalence rate (1). Since these values are a compilation of results from various studies, rather than an estimate from the specific site of interest, there is greater potential for this theoretical value to deviate from reality. If the proportion of individuals infected at the start of the study is relatively high, it is likely that many of the new infections will be additional infections within already infected individuals. When additional infection occurs, the proportion of individuals infected will not increase. The theoretical proportion of individuals infected at the end of the study should be adjusted for this. Sample size calculation Various online calculators are available to calculate required sample sizes. For a single-arm study, the online calculator should test a one-sample proportion; a one- sided test is appropriate, since the hypothesis is that the proportion of individuals infected at the end of the CPES will be significantly lower than it would have been if no chemoprevention had been given. One suitable online calculator can be found at http://powerandsamplesize.com/Calculators/Test-1-Proportion/1-Sample-1- Sided. Sample size calculations are normally performed using 80% power and 5% significance (or Type 1 error). Table A3.1 contains sample calculations of required sample sizes. Table A3.1. Required sample sizes for a single-arm study – calculations performed with 80% power and 5% statistical significance and no loss to follow-up Required sample size Theoretical proportion parasitaemic at end of follow-up period with no chemoprevention 0.01 0.05 0.1 0.15 0.2 0.25 Expected proportion parasitaemic at end of follow- up period after chemoprevention 0.001 447 62 28 17 12 9 0.005 1987 87 34 20 14 10 0.01 123 42 23 16 11 0.02 252 59 30 19 14 0.03 630 83 38 23 16 0.04 2,737 121 47 27 18 0.05 184 60 32 21 0.06 301 77 38 24 0.07 557 101 46 27 0.08 1301 136 55 31 0.09 5,386 191 67 36 0.1 282 83 42 0.12 823 136 58 0.14 7,724 251 84 0.16 584 129 0.18 2,405 219 0.2 440 17Malaria chemoprevention efficacy study protocol Two-arm study design In this study design, the proportions of participants found to be parasite- positive during the follow-up period (from day 4) are compared between the chemoprevention group and the control group (not receiving chemoprevention). This is a more robust study design, as it directly compares the proportions of infected individuals between the two groups, removing the uncertainty around what the anticipated incidence rate would be in the absence of chemoprevention. The sample size calculation can be based on the overall proportion found to be infected at the end of the study. To calculate the required sample size, initial estimates of the prevalence and incidence rate (without chemoprevention) during the follow-up period are required, as is an estimate of the effect size for the chemoprevention. However, these initial estimates are to guide the sample size calculations only and do not feature in the analysis of the data. In contrast to the single-arm study, the estimate of the incidence rate can be used in the calculations, instead of the lower 95% CI. A sample size calculator comparing two proportions using a one-sided test is appropriate to test the hypothesis that the proportion of individuals with new infections in the follow-up period will be significantly lower in the chemoprevention group than in the control group. The lowest overall sample sizes will be obtained when there are equal numbers in the chemoprevention and control groups, but individual study sites may need to move away from this balanced design for a variety of ethical and logistical reasons. Table A3.2 provides estimated sample sizes for a balanced sample design for a range of different anticipated incidence rates. Alternate values for unbalanced designs can be calculated using http://powerandsamplesize.com/Calculators/Compare-2- Proportions/2-Sample-1-Sided, or another similar online calculator. Malaria chemoprevention efficacy study protocol18 Table A3.2. Minimum required sample sizes (in each group) to be able to detect a statistically significant reduction in the proportion of individuals infected following chemoprevention compared to a control group, with 80% power, 5% significance, a balanced design and no loss to follow-up Required sample size in each group Expected proportion parasitaemic at end of follow-up period with no chemoprevention 0.01 0.05 0.1 0.15 0.2 0.25 Expected proportion parasitaemic at end of follow- up period after chemoprevention 0.001 831 125 58 36 26 19 0.005 3,675 161 65 39 27 20 0.01 222 77 44 30 22 0.02 461 106 54 35 25 0.03 1,183 151 68 41 28 0.04 5,305 221 85 48 32 0.05 340 109 57 37 0.06 566 141 69 42 0.07 1,065 186 83 49 0.08 2,526 254 101 56 0.09 10,615 360 124 65 0.1 538 155 77 0.12 1,600 257 108 0.14 15,309 481 158 0.16 1,137 246 0.18 4,749 423 0.2 859 Reference 1. Cameron E, Battle KE, Bhatt S, Weiss DJ, Bisanzio D, Mappin B, et al. Defining the relationship between infection prevalence and clinical incidence of Plasmodium falciparum malaria. Nat Commun. 2015;6:8170. doi:10.1038/ncomms9170. 19Malaria chemoprevention efficacy study protocol ANNEX 4. OVERVIEW OF ACTIVITIES BY DAY Day 0 Screening • Clinical assessment, including measurement of temperature; for those with symptoms of malaria conduct diagnosis by RDT or microscopy • Informed consent and assent Enrolment • Supervised treatment, first dose • Collect thick blood smear • Collect DBS on filter paper • Collect general information (age, weight, height, gender), information on vector control use, and intervention-specific information: o IPTp: gravidity, last menstrual period (to exclude first trimester) previous use of antimalarial medicine in current pregnancy o PMC: previous use of antimalarial medicine in past six weeks, if child is being breastfed o SMC: previous use of antimalarial medicine in current transmission season, if child is being breastfed o IPTsc: previous use of antimalarial medicine in past six weeks Day 1 • Supervised treatment (as needed, depending on medicine given) Day 2 • Supervised treatment (as needed, depending on medicine given) Optional • Record temperature • Collect thick blood smear • Collect DBS on filter paper Day 7 • Record temperature • Collect thick blood smear • Collect DBS on filter paper Optional • Collect blood to test drug blood level Days 14, 21 and 28, and any other day • Record temperature • Collect thick blood smear • Collect DBS on filter paper Malaria chemoprevention efficacy study protocol20 ANNEX 5. MANDATORY AND OPTIONAL ACTIVITIES Activity Mandatory Optional Treatment Pre-study curative treatment Administer pre-study curative treatment, two weeks prior to the chemoprevention regimen Chemoprevention Provide preventive chemotherapy as per recommendations Supervision of treatment All medicine must be given under direct supervision Clinical malaria Study participants identified as having clinical malaria at enrolment must receive first-line treatment as per the national treatment guidelines Patient follow-up Follow-up period 28 days after start of chemoprevention administration with a drug with a short half-life 42 days after start of treatment with a drug with a long half-life 42 days after start of treatment with a drug with a short half-life. Requires any additional chemoprevention doses to be delayed 56 days after start of treatment with a drug with a long half-life Days of patient follow-up Weekly follow-up (days 7, 14, 21, and 28) + any day that study participants have symptoms Additional follow-up on day 2 and days 35, 42, 49 and 56 Malaria parasitaemia Study participant with chemoprevention failure (early failures, malaria parasitaemia with symptoms after day 4 or malaria parasitaemia without symptoms after day 7 – see Annex 6) must receive first-line treatment as per the national treatment guidelines Information and samples collected Information collected at enrolment • Temperature, symptoms of malaria, antimalarial treatment in past, use of vector control + intervention- specific information: • IPTp: last menstrual period, gravidity, previous use of antimalarial medicine in current pregnancy • PMC: previous use of antimalarial medicine in past six weeks and if child is being breastfed • SMC: previous use of antimalarial medicine in current transmission season, and if child is being breastfed • IPTsc: previous use of antimalarial medicine in past six weeks. For females of >12 years ability and willingness to take pregnancy test. Information collected on days of follow-up Temperature, symptoms of malaria 21Malaria chemoprevention efficacy study protocol Samples collected DBS and thick blood smear on day of enrolment and all days of follow-up Venous blood sample on day 7 to measure drug blood level Molecular analysis Reinfection/ recrudescence markers Blood collected in DBS on day 0 and day of failure for analysis of markers of new infection/recrudescence Drug resistance markers Analysis of markers of drug resistance in DBS collected on day 0 for study participants with parasitaemia Analysis of markers of drug resistance in DBS collected for study participants with parasitaemia during follow-up Malaria chemoprevention efficacy study protocol22 ANNEX 6. TES DEFINITIONS OF FAILURE Early treatment failure (ETF) • danger signs or severe malaria on day 1, 2 or 3, in the presence of parasitaemia; • parasitaemia on day 2 higher than on day 0, irrespective of axillary temperature; • parasitaemia on day 3 with axillary temperature ≥37.5 °C; and • parasitaemia on day 3 ≥25% of count on day 0. Late clinical failure (LCF) • danger signs or severe malaria in the presence of parasitaemia on any day between day 4 and day 28 (day 42) in patients who did not previously meet any of the criteria of early treatment failure; and • presence of parasitaemia on any day between day 4 and day 28 (day 42) with axillary temperature ≥37.5 °C in patients who did not previously meet any of the criteria of early treatment failure. Late parasitological failure (LPF) • presence of parasitaemia on any day between day 7 and day 28 (day 42) with axillary temperature <37.5 °C in patients who did not previously meet any of the criteria of early treatment failure or late clinical failure.
Malaria chemoprevention efficacy study protocol24 For further information please contact: Global Malaria Programme World Health Organization 20, avenue Appia CH-1211 Geneva 27 Switzerland Email: GMPinfo@who.int