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Emergency guidance: selection and use of Ebola in vitro diagnostic (IVD) assays

June 13, 2015 World Health Organization

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EMERGENCY GUIDANCE

Selection and use of Ebola in vitro diagnostic (IVD) assays June 2015

© World Health Organization 2015. All rights reserved. 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 the World Health Organization 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 the World Health Organization 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 the World Health Organization 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 the World Health Organization be liable for damages arising from its use.

WHO/EVD/HIS/EMP/15.2

Contents

Acknowledgements This emergency guidance has been developed by the WHO-HQ Department of Essential Medicines and Health Products in collaboration with the Department of Pandemic and Epidemic Diseases, WHO Emerging and Dangerous Pathogens Laboratory Network, and the Global Malaria Programme; and WHO Country Offices in Liberia and Sierra Leone based on advice from the WHO Ebola Diagnostics Implementation Group. The Group included experts from UK (Public Health England and University of Liverpool), US (Centers for Disease Control and Prevention), UNICEF, and the Non-Governmental Organizations the Foundation for Innovative New Diagnostics and Médecins Sans Frontières. All non-WHO staff completed Declaration of Interest forms, which were assessed by WHO as showing no conflicts.

Objective To provide interim guidance to Ministries of Health and other organizations on factors to consider in the selection and use of available in vitro diagnostic (IVD) assays for diagnosis of Ebola virus disease (EVD).

Key messages Selection of appropriate diagnostic assays for Ebola requires consideration not only of technical criteria but also the social and medical implications of test results. Given the consequences of a misdiagnosis, WHO recommends that only diagnostics that have undergone independent, comprehensive assessment of quality, safety and performance are used in diagnosing infection with Ebola virus. As incidence and prevalence of Ebola disease continues to decrease, assays with high specificity are required. This is because, in the context of low disease prevalence, widespread use of low specificity assays will generate more false positives than true positives. WHO recommends that nucleic acid testing using technologies such as polymerase chain reaction (PCR) should be the method of choice. A rapid antigen test that has reasonable sensitivity in patients with high concentrations of Ebola virus in the blood may have utility in settings without laboratory infrastructure if the benefits and limitations of the test are understood and appropriately managed.

1. Introduction The role of laboratory diagnosis in response to the Ebola outbreak in West Africa has been critical. Early identification of infected patients assists in the provision of rapid access to health care as well as interruption of the chain of transmission by timely isolation of infected patients. Detection of viral nucleic acid using 1 nucleic acid tests such as PCR technology is the recommended technique for laboratory diagnosis of EVD . PCR methods in common use for this epidemic are highly sensitive and detect only Ebola virus, but are often complex to perform. In contrast, rapid tests for Ebola antigen are much more easily performed, but more often generate false signals. In October 2014, WHO issued a Target Product Profile for the development for 2 rapid, safe and cost effective EVD IVDs . Since then, a number of novel diagnostic assays including those using PCR and also lateral flow (rapid) tests that detect viral antigen have been developed for use in the current outbreak. Few of these have been reviewed by stringent regulatory authorities. Due to the need for enhanced biosafety and biosecurity when handling viruses like Ebola, as well as the social and medical implications of test results, careful selection of appropriate diagnostic assays for use in the African context is vital. Consideration must be given to the design and performance of the diagnostic assays, to ensure testing is safe and effective. The intended use of the diagnostic also influences selection of assay; methods used during an outbreak in which there is widespread transmission may be different to those selected for routine screening as part of a national surveillance programme. As many of the affected countries are now faced with the task of building and strengthening the national public health laboratory network, the selection of diagnostic assays should take into account the need for human resources, training, infrastructure, cost effectiveness, biosafety and infection control and prevention. The following document is designed to help guide decision makers, technical staff and ministries of health to select the most appropriate in vitro diagnostic solutions for procurement and use.

‘Laboratory diagnosis of Ebola virus disease’, Geneva, World Health Organization, September 2014. Available online at http://who.int/csr/resources/publications/ebola/laboratory-guidance/en/. 2 ‘Target Product Profile for Zaïre ebolavirus rapid, simple test to be used in the control of the Ebola outbreak in West Africa ’, Geneva, World Health Organization, October 2014. Available online at http://www.who.int/medicines/publications/target-productprofile.pdf?ua=1.

2. Overview of Ebola IVDs Current routine diagnostic methods for Ebola rely on detection of viral components (proteins (antigens) or nucleic acids) in blood. Molecular testing for Ebola virus nucleic acid (nucleic acid tests/NAT) using a technique known as PCR has become the standard method for Ebola virus detection in outbreaks. Properly designed tests of this type are highly accurate, and may be able to detect very low concentrations of virus. Traditional NAT methods are often well suited to a reference laboratory setting, for diagnostic purposes and for research. Traditional NAT systems can offer high throughput and flexibility to test for many different pathogens. The systems can utilise both commercial assays and those produced in-house (i.e. not for commercial distribution). However they are often complex and can be time-consuming methods to perform (around 3 to 5 hours). Special care needs to be taken to avoid false positive results due to contamination of the work area with by-products produced in the process, and so the different steps in the method often need to be performed in dedicated rooms to avoid this problem. Additionally, it is essential that these tests are performed by experienced laboratory scientists. Recently, several companies have developed automated NAT methods that simplify molecular testing (including PCR and similar assays), increasing reliability, and decreasing the likelihood of false results and the number of ancillary reagents and equipment needed. Several such assays have been developed for Ebola, or are in the process of being developed. These assays may use cartridges pre-loaded with all necessary reagents, some are in a temperature-stable format, and are relatively simple to perform with the accompanying specific equipment. The other advantage of these systems is that the user of the test does not need the same level of understanding of molecular methods as is required for traditional NAT. This provides the opportunity for testing to move out of the reference laboratory to hospital based services. An alternative to nucleic acid testing is direct detection of Ebola proteins (antigens). Antigen-detection tests are generally less sensitive than PCR methods. However, given the large amounts of virus present in most Ebola patients after several days of symptoms, these sensitivity limitations may not be critically important in some situations. Currently, these tests are available in a form similar to an over-the-counter pregnancy test. These rapid antigen detection tests (RDTs) theoretically can be performed anywhere, and without ancillary equipment. However, with these tests, false-positive results are harder to predict and control, and may be more common. This makes clinical study results, as well as an understanding of the probability of true Ebola disease in a given patient or cohort, critical to understand the utility of such tests. A number of these IVDs have recently been developed, and studies are ongoing to compare their performance. In a recent guidance 3 document , WHO recommended that antigen detection RDTs for Ebola have no role in the routine management of Ebola in settings where PCR (molecular) testing is available; however, they may have utility in settings without laboratory infrastructure if their benefits and limitations are understood. Tests detecting Ebola viral antigen in what is known as an ELISA format have been used for many years. These IVDs are sensitive in patients with high levels of virus present. However, most ELISA antigen detection tests are not available commercially. They require comprehensive laboratory infrastructure and skilled laboratory scientists to perform the assays. As such, these assays are generally most useful in a reference laboratory setting. None of these assays have been listed under the WHO EUAL mechanism (see section 3). Serology tests that detect antibodies (IgM or IgG) produced by an infected patient have a role in epidemiologic or vaccine research, but not in routine clinical diagnosis or case management. These tests are most suited to a research setting. None of these assays have been listed under the WHO EUAL mechanism. It is important to remember that all IVDs have the potential for incorrect results. This can be due to the inherent design of the test, how the test is performed, the disease stage, or if they are used in a manner other than stated by the manufacturer (e.g. a different specimen type). A graphic summary of diagnostic assays available in different settings is presented in Figure 1.

‘Interim guidance on the use of rapid Ebola antigen detection tests ’, Geneva, World Health Organization, March 2015. Available online at http://www.who.int/csr/resources/publications/ebola/ebola-antigen-detection/en/

Figure 1. Diagnostic assays available in different settings

3. Description of regulatory approval processes ( e.g. US FDA, WHO EUAL, CE marking) Many EVD IVDs have not had any regulatory assessment of quality, safety or performance. Regulation of IVDs is normally the role of the national regulatory authority (NRA) but few NRAs have assessed EVD IVDs. Additionally, each NRA may have a different approach to IVD regulation, from comprehensive premarket assessment to only requiring a manufacturer to certify that the product meets regulatory requirements and obligations (as is the case of CE marking for EDV IVDs where there is no independent review). As full regulatory review may be lengthy and the need for quality IVDs is urgent, some jurisdictions have established special regulatory processes based on minimal essential requirements during an emergency. An example of this is the United States Food and Drug Administration (US FDA) Emergency Use Authorization (EUA) authority. This risk based authorization is time limited and includes restrictions to the use of the IVD. With some authorizations such as the US FDA EUA, restrictions may extend to who can perform the test and/or where it can be performed and obligations for the manufacturer to report performance problems (see Annex 2). In response to the need for quality assured IVDs for EVD, WHO has also developed an emergency use assessment and listing procedure (EUAL). The EUAL procedure assesses if there is sufficient evidence to demonstrate that the benefits of using the IVD for EVD outweigh the foreseeable risks in the context of the current outbreak. The EUAL adopts many FDA EUA requirements but has further assessment steps, including verifying test sensitivity (LoD) by independent testing, and assessing manufacturing capacity. As for FDA, WHO listing also obliges the manufacturer to report performance issues. Given the consequences of misdiagnosis, WHO highly recommends that only diagnostics that have undergone independent, comprehensive assessment of quality, safety and performance are used in diagnosing infection with Ebola virus (see Annexes 2 and 3).

4. Considerations for the selection and use of Ebola IVDs The following aspects should be considered when choosing appropriate IVDs for EVD. The order in which the characteristics are presented does not represent a prioritization listing since priorities will be contextspecific. General characteristics of the test These may include:  Performance The ability of the assay to correctly identify all infected individuals in different stages of the disease. Where clinical trial data are not available analytical sensitivity (limit of detection) may be used as a general indicator of clinical sensitivity The ability of the assay to correctly identify all non-infected individuals (diagnostic specificity) The spectrum of analytes which can be measured (e.g. if the diagnostic detects only Zaire strains of Ebola, or other Ebola species too) Susceptibility of the assay to genetic changes of the organism over time period (genetic drift) for example whether nucleic acid detection tests target one or two genes Presence of appropriate test procedure controls supplied with the test kits to ensure that each step of the testing process has performed correctly. For lateral flow devices, the presence of a control line and provision of positive and negative controls by the manufacturer, o o o For nucleic acid detection assays, decontamination controls, Internal controls (added to every specimen), provision of positive and negative controls , For automated nucleic acid detection assays, processing and amplification controls, and sample adequacy controls Availability of external Quality Assessment (proficiency) Control specimens (provided by a party other than the manufacturer)

 Targets

 Test procedure controls -

 Transport and shelf-life Clearly defined specimen storage and transportation requirements Shelf-life and storage conditions for all reagents supplied or required for the test The time required (including specimen processing) to obtain results Whether results are subjective and transiently available versus objective and permanent Whether results are qualitative or quantitative Connectivity of the technology including remote communication for data transfer.

 Read-out

Anticipated use When considering the type of assay to be selected for use, consideration should be made of the following:  Setting of intended use, taking into account Whether centralized or decentralized testing is preferred The minimal qualifications of the users and training needs e.g. whether staff with molecular biology expertize are needed to operate the test; for RDTs training in performing the test and training and practical experience in PPE are needed The minimum number of specimens which can be tested per time period (e.g. per day or shift) The need for instrument(s) to conduct the testing; whether such equipment is in place, or whether new instrumentation is needed Clear understanding of the intended use of the tests including whether there is a need to undertake confirmatory testing 7

 Intended use of the assay -

Antigen detection assays in general are less sensitive or specific compared to NAT, although both can be used to establish the laboratory diagnosis of EVD As incidence and prevalence of the disease continues to decrease, assays with higher specificity are required. In the current epidemiological context, widespread use of RDTs will generate more false positives than true positives, (see Annex 1 for an explanation). Widespread use of RDTs is therefore not recommended. Operational research is however encouraged to investigate strategies to address this issue. Need to detect markers for other related infections. How EVD testing will be used in the context of diagnostic testing for fever patients. Except in areas of ongoing EVD transmission, other causes of fever, such as malaria, will be more likely. Because of the overlapping symptomatology between Ebola and malaria, and until the Ebola outbreak is declared officially over, WHO’s current guidance on use of malaria RDTs in Ebola 4 affected areas is to suspend their use UNLESS they are performed with PPE .

Infection prevention and control Based on the fact that Ebola viruses are associated with a high case-fatality-ratio, specific steps must be taken to minimize chance of acquiring the disease during the testing process. This should cover the process of specimen collection, preparation, storage and waste disposal. Aspects to consider include:  Whether manual specimen manipulation is required e.g. centrifugation and plasma separation  Whether testing must occur in a setting of high biosecurity level equipment.  Whether venipuncture is required to collect the specimens (venous whole blood, serum or plasma) or if fingerstick testing may be performed  The need to decontaminate equipment after use  Need for appropriate disposal of laboratory wastes  If pipettes are required, plastic pipettes rather than glass pipettes should be used. Regardless of the setting of use, testing should always be performed under strictly applied universal biosafety precautions by trained and properly equipped personnel. During an Ebola epidemic, health workers should be adequately equipped and trained in both performance and disposal of the test and donning and doffing of personal protective equipment. Infrastructure requirements Infrastructure factors which should be considered may include:  The dedicated space required for the instruments including the need for reinforced benches  Design of laboratories to prevent potential contamination from high titre specimens or amplification products. Conventional PCR methods usually require separated laboratory rooms or space for the different steps of the process (extraction/amplification) Fully automated system may use different compartments inside the instrument, obviating the need for physical separation of the steps. Availability of glove boxes or biosafety cabinets

 Other requirements in this context are additional instruments needed for assay performance (e.g. centrifuges) or need for storage of assay reagents and specimens (e.g. refrigerators).  Availability of electricity, as a number of the EVD diagnostics requires an uninterrupted power supply. Electricity is also required for air conditioning of rooms to prevent overheating of some instruments and for refrigeration of some reagents. Some dedicated technologies and point-of-care technologies can run on direct current such as portable car batteries. Cost Regarding the cost of the test, consideration should include cost of reagents, equipment and accessories that are not supplied with the kits. Other factors include: 4

‘Guidance on temporary malaria control measures in Ebola-affected countries’, Geneva, World Health Organization, November 2014. Available online at http://www.who.int/malaria/publications/atoz/malaria-control-ebola-affected-countries/en/

 the cost of PPE, and waste disposal and management requirements  the cost of maintenance of instrument and the requisite infrastructure,  the cost of servicing and maintenance of the diagnostic  the cost of labour (hands-on times for specimen collection, transport, test performance etc)  Simplicity of supply and procurement, e.g. local support capacity of vendor or representative; number and type of accessories or reagents not provided in the kit. Regulatory status As noted in section 3, it is especially important to know if the claims of quality, safety and performance made by the manufacturer have been verified by a stringent independent assessment. Where regulatory approval exists, it is important to understand what assessment has occurred for the approval to have been given. Conditions may apply, such as restrictions on the use of the diagnostic, including who can perform the test and where it can be performed.

5. Fact sheets of each WHO EUAL-listed IVDs and US FDA EUA-listed IVDs The Fact Sheets are available on the WHO website. This is to facilitate updates as needed, whilst the core document can remain unchanged. Please refer to Annexes 2 and 3 for these documents. It is important to note that there is no priority implied by order of tests listed and also that the list will grow as new tests are developed and evaluated.

6. Criteria to take into account in setting up an EVD IVD testing laboratory network An EVD laboratory test should be used within the context of a laboratory network system. Additional criteria need to be taken into account by decision makers when setting up a diagnostics network ( as distinct from decision-making for the individual laboratory), making it as useful as possible for other diseases , not just Ebola, including technical input to decision-making, and consideration of national/regional surveillance.

Calculate expected workload per site

Review available EVD testing options

Choose a model for the network

Centralized vs de-centralized

Determine requirements in infrastructure, equipment, human resources, funding, and training

Prioritize selection criteria prior to selecting tests (see Section 4)

Implement operational plan including programs for training, supervision, data management, quality management, quality assurance and maintenance Figure 2. Criteria to take into account in setting up an EVD IVD testing laboratory network

Annex 1. Predictive values of Ebola RDTs and implications for decision-makers Based on the currently available evidence rapid antigen detection tests have variable sensitivity and therefore a negative test result, in certain circumstances, cannot exclude Ebola infection. Furthermore, test specificity also varies and therefore a positive test result may be a false positive result. Both false negative and false positive results for Ebola have serious implications for individuals/patients and public health. However, sensitivity and specificity of a test are not the only parameters of importance in evaluating its appropriateness for use. The prevalence (% of true positives) of the disease is important in assessing its positive and negative predictive value (PPV and NPV, respectively) .The predictive value of the same test can differ between different populations in the same country or within a district.

Table A1. PPV and NPV of an antigen-based RDT with sensitivity=92%, specificity=85% Prevalence 100.0% 90.0% 80.0% 70.0% 60.0% 50.0% 40.0% 30.0% 20.0% 10.0% 5.0% 2.5% 1.0% 0.5% 0.2% 0.1% PPV 100.0% 98.2% 96.1% 93.5% 90.2% 86.0% 80.3% 72.4% 60.5% 40.5% 24.4% 13.6% 5.8% 3.0% 1.2% 0.6% NPV 0.0% 54.1% 72.6% 82.0% 87.6% 91.4% 94.1% 96.1% 97.7% 99.0% 99.5% 99.8% 99.9% 100.0% 100.0% 100.0% False Negatives 8.0% 7.2% 6.4% 5.6% 4.8% 4.0% 3.2% 2.4% 1.6% 0.8% 0.4% 0.2% 0.1% 0.0% 0.0% 0.0% False Positives 0.0% 1.5% 3.0% 4.5% 6.0% 7.5% 9.0% 10.5% 12.0% 13.5% 14.3% 14.6% 14.9% 14.9% 15.0% 15.0%

Causes and operational implications of negative and positive RDT results False negative results are most likely linked to the inability of the assay to detect a low viral copy number (assuming test is well performed; other less likely causes could be interfering substances); the false negative status is likely to be resolved by retesting the patient at a later stage (at 24/48/72 hours, if symptoms persist; and preferably refer samples from symptomatic suspect patients with negative RDT for PCR testing). False positive results are most likely linked to cross-reactivity or other non-specific binding of reagents in the test to an non-Ebola antigen in the sample; false positive results are less likely to be resolved over time, as the cause of false positivity will remain. Advice concerning use of RDTs in a low prevalence epidemiological context For an assay with performance characteristics similar to the example given above the number of false positive results will become higher than the number of true positives as the prevalence of infection (% of true positives) falls. There is a risk that use of such a test in this epidemiological context will undermine trust in the testing procedures and in the broader public health response. Therefore widespread use of RDTs that do not meet the WHO Target Product Profile is not recommended. Special settings where use of such RDTs for 1 Ebola may be beneficial are given in other WHO guidance .

Selection and use of Ebola in vitro diagnostic (IVD) assays WHO/EVD/HIS/EMP/15.2/annex

Annex 2. Considerations for the selection of PCRs for the diagnosis of Ebola virus disease Table 1a: IVDs with WHO Emergency Use and Assessment Listing (EUAL) Version: 09 June 2015 The WHO EUAL assessment comprises WHO assessment of manufacturing capacity and quality management systems, documentary evidence of safety and performance, independent verification of analytical sensitivity (LOD) Assay name Manufacturer Kit format Kit size Kit components RealStar® Filovirus RT-PCR Kit 1.0 (CE-IVD) altona Diagnostics GmbH One step RT PCR assay 96 reactions Master A and B mixes Internal control EBOV Positive control MARV Positive control Molecular grade water Zaire ebolavirus (ZEBOV) Bundibugyo ebolavirus (BEBOV) Reston ebolavirus (RESTV) Sudan ebolavirus (SEBOV) Tai Forest ebolavirus (TAFV) Marburg virus (MARV) L Real time PCR platform Specify instruments: - Mx 3005P™ QPCR System (Stratagene) - VERSANT™ kPCR Molecular System AD (Siemens) - ABI Prism® 7500 SDS and 7500 Fast SDS (Applied RealStar® Ebolavirus RT-PCR Kit 1.0 (FDA EUA)* altona Diagnostics GmbH One step RT PCR assay 96 reactions Master A and B mixes Internal control EBOV Positive control Molecular grade water Zaire ebolavirus (ZEBOV) Bundibugyo ebolavirus (BEBOV) Reston ebolavirus (RESTV) Sudan ebolavirus (SEBOV) Tai Forest ebolavirus (TAFV) L Real time PCR instrument - ABI Prism® 7500 SDS and 7500 Fast SDS (Applied Biosystems) - LightCycler® 480 Instrument II (Roche) - CFX96 system/Dx real-time Liferiver™ Ebola virus (EBOV) real time RT-PCR kit Shanghai ZJ Bio-Tech Co., Ltd. One step RT PCR assay 25 reactions EBOV super mix RT PCR enzyme mix Internal control Positive control Molecular grade water Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SUDV), Taï Forest ebolavirus (TAFV), Bundibugyo ebolavirus (BDBV) Xpert® Ebola Assay (Also FDA EUA)** Cepheid Integrated cartridge 10 reactions Cartridge with all reagents on board. Inactivation solution. Swab. Transfer pipettes Ebola sample reagent box Zaire ebolavirus (ZEBOV)

Viruses detected

Gene Target Laboratory instruments required but not provided with kit/assay/

NP Real time PCR platform: Bio-Rad CFX 96; SLAN®-96; ABI Prism®7500; LightCycler®480 Instruments Desktop centrifuge Plate centrifuge Vortex mixer Micropipettes

NP, GP GeneXpert Dx System or GeneXpert Infinity systems Vortex mixer Micropipettes Powder free gloves Biosafety Level 3 laboratory

Selection and use of Ebola in vitro diagnostic (IVD) assays

Assay name

RealStar® Filovirus RT-PCR Kit 1.0 (CE-IVD) Biosystems) - LightCycler® 480 Instrument II (Roche) - Rotor-Gene™ 3000/6000 (Corbett Research) - Rotor-Gene Q5/6 plex Platform (QIAGEN) - CFX96 system/Dx real-time system (BIORAD) Desktop centrifuge Plate centrifuge Vortex mixer Micropipettes Barrier pipette tips Powder free gloves 96 well reaction plates Nuclease-Free Water Biosafety Level 3 laboratory

RealStar® Ebolavirus RT-PCR Kit 1.0 (FDA EUA)* system (BIORAD) Desktop centrifuge Plate centrifuge Vortex mixer Micropipettes Barrier pipette tips Powder free gloves 96 well reaction plates Nuclease-Free Water Biosafety Level 3 laboratory

Liferiver™ Ebola virus (EBOV) real time RT-PCR kit Barrier pipette tips Powder free gloves Biosafety Level 3 laboratory

Xpert® Ebola Assay (Also FDA EUA)**

Other materials required but not provided

QIAamp viral RNA mini Kit (Qiagen) 96 well plates or reaction tubes and suitable optical seal Unidirectional workflow; laboratory area must contain dedicated workspace and equipment for specimen extraction, preparation of mastermix and amplification Yes Recommended maintenance and calibration for real time PCR platforms, micropipettes and standard laboratory equipment Required

RNA Isolation kit (ZJ Biotech), or QIAamp viral RNA mini Kit (Qiagen) 96 well plates or reaction tubes and suitable optical seal Unidirectional workflow; laboratory area must contain dedicated workspace and equipment for specimen extraction, preparation of mastermix and amplification Yes Recommended maintenance and calibration for real time PCR platforms, micropipettes and standard laboratory equipment Required

Chlorine

Laboratory space

Specimen inactivation should be performed under enhanced biosafety conditions at the site of collection Yes Annual calibration self-check

Electricity only Equipment maintenance

Technical support by manufacturer

Required

Selection and use of Ebola in vitro diagnostic (IVD) assays

Assay name Specimen inactivation using enhanced biosafety required by user prior to performing the test Nucleic acid extraction required by user Suitable specimen for testing Storage conditions for reagents Recommended time to process the specimen after collection

RealStar® Filovirus RT-PCR Kit 1.0 (CE-IVD) Specimen inactivated by addition of lysis buffer.

RealStar® Ebolavirus RT-PCR Kit 1.0 (FDA EUA)* Specimen inactivated by addition of lysis buffer.

Liferiver™ Ebola virus (EBOV) real time RT-PCR kit Yes

Xpert® Ebola Assay (Also FDA EUA)** Specimen added to Sample Reagent bottle that contains lysis buffer. No Venous whole blood collected in EDTA 2-8°C (requires cold chain)

Yes Plasma collected in EDTA, cell freebody fluids, swab washes -20°C (requires cold chain)

Yes Plasma collected in EDTA

Yes Venous whole blood or plasma collected in EDTA, serum -20°C (requires cold chain)

-20°C (requires cold chain)

Plasma or serum stored at 2-8°C for up to 6 hours Freezing at -20°C or -80°C in aliquots recommended for long-term storage “The LoD was not assessed by WHO during EUAL assessment of the product. However the manufacturer claim is 1.39 copies/µl specimen eluate as determined by probit analysis using in-vitro transcribed RNA without taking into account the RNA extraction procedure. FDA EUA data shows a sensitivity of 1 PFU Zaire ebolavirus/ml plasma (1 PFU = approx. 3.400 copies).” 4-6 hours for a negative, less for a positive Approximately 50 specimens

Plasma stored at 2-8°C for up to 6 hours Freezing at -20°C or -80°C in aliquots recommended for long-term storage “The LoD was not assessed by WHO during EUAL assessment of the product. However the manufacturer claim is 1 PFU Zaire ebolavirus/ml plasma (1 PFU = approx. 3.400 copies) as determined during the FDA Emergency Use Authorization process.”

Plasma or serum stored at 2-8°C for up to 6 hours Freezing at -20°C or -80°C in aliquots recommended for long-term storage 4.23 10 copies /ml blood 4

Fresh blood specimen should be processed immediately The Reagent-treated blood specimens may be stored for 72hrs at 2-8C, for 48hrs at 8-30C or 24hrs at 28-35C (1.34 – 4.23) 10 copies /ml blood 3

WHO evaluation: Limit of Detection (LOD)

Throughput: Time to results Maximum number of specimens per day/instrument

4-6 hours for a negative, less for a positive Approximately 50 specimens

4-6 hours for a negative, less for a positive Approximately 180 specimens

90 minutes

Depending on version of the instrument

Selection and use of Ebola in vitro diagnostic (IVD) assays

Assay name

RealStar® Filovirus RT-PCR Kit 1.0 (CE-IVD)

RealStar® Ebolavirus RT-PCR Kit 1.0 (FDA EUA)*

Liferiver™ Ebola virus (EBOV) real time RT-PCR kit

Xpert® Ebola Assay (Also FDA EUA)** 24 - 96

Reagents shelf life Minimum specimen volume Limitations of the assay

9 months 140 µL plasma for nucleic acid extraction Presence of RT-PCR inhibitors may cause invalid results. Potential for false positives due to amplicon carry-over Laboratory technicians Biosafety training Donning and doffing PPE Specimen inactivation should be performed under BSL3 conditions Follow national guidelines

12 months 140-200 µL blood, plasma or serum for nucleic acid extraction Presence of RT-PCR inhibitors may cause invalid results. Potential for false positives due to amplicon carry-over Laboratory technicians Biosafety training Donning and doffing PPE Specimen inactivation should be performed under BSL3 conditions Follow national guidelines

12-24 months (expected from accelerated stability testing) 100 µL whole blood Presence of RT-PCR inhibitors may cause invalid results.

Trained manpower

Trained health workers Biosafety training Donning and doffing PPE Specimen inactivation should be performed under BSL3 conditions Follow national guidelines

Biosafety requirements Waste management

*FDA EUA restricts the use of this assay to CLIA High Complexity Laboratories and Similarly Qualified Non-U.S. Laboratories **FDA EUA restricts the use of this assay to CLIA Moderate and High Complexity Laboratories or in similarly qualified non-U.S. laboratories Information on certain characteristics listed in the Table will be updated by WHO as more data become available

Selection and use of Ebola in vitro diagnostic (IVD) assays

Table 1b: IVDs with US FDA Emergency Use Authorization (FDA EUA) Version: 09 June 2015 The FDA EUA assessment comprises FDA assessment of documentary evidence of safety and performance, requirements for reporting of performance issues, and control of end users. Assay name FilmArray Biothreat-E test BioFire, Biomerieux Integrated film pouch Multiplex assay 6 reactions Test pouches Single-use sample buffer ampoules Single-use freeze-dried protease vials Single-use pre-filled hydration injection vials Single-use sample injection vials Transfer pipettes ZEBOV L FilmArray instrument with laptop computer FilmArray Pouch loading station compatible with use of FilmArray injection vials FilmArray NGDS BT-E Assay BioFire Defense, LLC for US Dept. of Defense Integrated film pouch Multiplex assay 30 reactions Test pouches Single-use sample buffer ampoules Single-use freeze-dried protease vials Single-use pre-filled hydration injection vials Single-use sample injection vials Transfer Pipettes ZEBOV NP FilmArray instrument with laptop computer FilmArray Pouch loading station compatible with use of FilmArray injection vials LightMix® Ebola Zaire rRT-PCR Test Roche One step RT PCR assay 96 reactions EBOV primers and probes Amplification control and primers/probes EBOV positive template control EZ1 Real-time RT PCR Assay US Dept. of Defense (in house assay) One step RT PCR assay 40 reactions EZ1 master mix RNAseP master mix RT-Taq polymerase EZ1 Positive Template Control RNAseP (RP) control RP Positive Template Control CDC Ebola Virus NP Real-time RT-PCR Assay US CDC (in house assay) One step RT PCR assay 500 reactions Ebola Virus NP Realtime RT-PCR Primer and Probe Set (includes 2 sets of primers and probes: the NP2 set and the RNase P set) CDC Ebola Virus VP40 Real-time RT-PCR Assay US CDC (in house assay) One step RT PCR assay 500 reactions Ebola Virus VP40 Realtime RT-PCR Primer and Probe Set (includes 2 sets of primers and probes: the VP40 set and the RNase P set)

Manufacturer Kit format Kit size Kit components

Viruses Detected Gene Target Laboratory instruments required but not provided with kit/assay

ZEBOV L PCR platform LightCycler® 480 II Instrument or cobas z 480 Analyzer LightCycler® Software (Version 1.5 or higher) or cobas z 480 Software (Version 1.5 or higher with UDF Version 1.0 or

ZEBOV GP Real time PCR platform - Applied Biosystems 7500 Fast Dx Real-time PCR Systems, Roche LightCycler, or BioFire Defense Joint Biological Agent Identification and Diagnostic System (JBAIDS)

ZEBOV NP Real-time PCR instrument - Applied Biosystems 7500 Fast Dx Real-time PCR Systems or Bio-Rad CFX96 Touch Real-time PCR Detection Extraction instrument Life Technologies Dynal

ZEBOV VP40 Real-time PCR instrument - Applied Biosystems 7500 Fast Dx Real-time PCR Systems or Bio-Rad CFX96 Touch Real-time PCR Detection Extraction instrument Life Technologies Dynal

Selection and use of Ebola in vitro diagnostic (IVD) assays

Assay name

FilmArray Biothreat-E test

FilmArray NGDS BT-E Assay

LightMix® Ebola Zaire rRT-PCR Test higher) Extraction Instrument MagNA Pure 96 Instrument LightCycler® 480 Multiwell Plate 96 white, with seals Barrier pipette tips RNase, DNase freedisposable plasticware Plate centrifuge Vortex mixer Microfuge TriPure Isolation Reagent Microfuge tubes, 1.5-ml

EZ1 Real-time RT PCR Assay Desktop centrifuge Plate centrifuge Vortex mixer Micropipettes Barrier pipette tips Powder free gloves Heat block or water bath Biosafety cabinet C Freezer, -20◦ C Refrigerator, 2-8◦

CDC Ebola Virus NP Real-time RT-PCR Assay BeadRetriever System or MagMAX Express-96 Deep Well Magnetic Particle Processor Powder-free gloves and surgical gowns Aerosol barrier pipette tips Microcentrifuge tubes Vortex mixer Microcentrifuge Micropipettes Multichannel micropipettes Racks for 1.5 mL microcentrifuge tubes 2 x 96-well -20 °C cold blocks PCR reaction Optical Adhesive Film Kit EBOV NP rRT-PCR Assay Positive Control Human Specimen Control SuperScriptTM III Platinum® One-Step qRT-PCR Kit Molecular grade water, nuclease-free Extraction reagents MagMax Pathogen RNA/DNA kit Isopropanol Ethanol DNA Away™

CDC Ebola Virus VP40 Real-time RT-PCR Assay BeadRetriever System or MagMAX Express-96 Deep Well Magnetic Particle Processor Powder-free gloves and surgical gowns Aerosol barrier pipette tips Microcentrifuge tubes Vortex mixer Microcentrifuge Micropipettes Multichannel micropipettes Racks for 1.5 mL microcentrifuge tubes 2 x 96-well -20 °C cold blocks PCR reaction Optical Adhesive Film Kit EBOV VP40 rRT-PCR Assay Positive Control Human Specimen Control SuperScriptTM III Platinum® One-Step qRT-PCR Kit Molecular grade water, nuclease-free Extraction reagents MagMax Pathogen RNA/DNA kit Isopropanol Ethanol DNA Away™

Other materials required but not supplied

Bleach De-ionized water

LightCycler® Multiplex RNA Virus Master MagNA Pure 96 DNA and Viral NA Small Volume Kit MagNA Pure 96 Tips MagNA Pure 96 Processing Cartridges MagNA Pure 96 Output Plate MagNA Pure 96 Sealing Foil MagNA Pure 96 System Fluid

QIAamp viral RNA mini Kit-(Qiagen) 96 well plates or reaction tubes and suitable optical seal TRIzol LS reagent or TRI Reagent LS

Selection and use of Ebola in vitro diagnostic (IVD) assays

Assay name

FilmArray Biothreat-E test

FilmArray NGDS BT-E Assay

LightMix® Ebola Zaire rRT-PCR Test High Pure Viral Nucleic acid extraction kit TriPure Isolation reagent Absolute ethanol Isopropanol

EZ1 Real-time RT PCR Assay

CDC Ebola Virus NP Real-time RT-PCR Assay RNase Away™ 10% bleach

CDC Ebola Virus VP40 Real-time RT-PCR Assay RNase Away™ 10% bleach

Laboratory space

No requirement for multiple rooms. Testing should be performed under the appropriate biosafety conditions in accordance with CDC and WHO guidelines.

Unidirectional workflow; laboratory area must contain dedicated workspace and equipment for specimen extraction, preparation of mastermix and amplification.

Unidirectional workflow; laboratory area must contain dedicated workspace and equipment for specimen extraction, preparation of mastermix and amplification. Specimen extraction must be performed inside biosafety cabinet level 3 if specimens have not been inactivated. Addition of extracted specimens into RT-PCR reactions may not need to be performed inside biosafety cabinet level 3. Yes Recommended maintenance and calibration for real time PCR platforms, micropipettes and standard laboratory equipment Required

Electricity only Equipment maintenance

Yes Per Instrument manual

Yes Recommended maintenance and calibration for real time PCR platforms, micropipettes and standard laboratory equipment Required

Technical support

Required

Selection and use of Ebola in vitro diagnostic (IVD) assays

Assay name

FilmArray Biothreat-E test

FilmArray NGDS BT-E Assay

LightMix® Ebola Zaire rRT-PCR Test

EZ1 Real-time RT PCR Assay

CDC Ebola Virus NP Real-time RT-PCR Assay

CDC Ebola Virus VP40 Real-time RT-PCR Assay

by manufacturer Specimen inactivation using enhanced biosafety required by user prior to performing the test? Nucleic acid extraction required by user Suitable specimen for testing No No Optional Optional Optional Optional

Yes

Venous whole blood, undiluted urine *urine must be tested in conjunction with blood specimen 15-25°C

Venous whole blood, plasma and serum

Whole blood collected in EDTA or TriPureinactivated EDTA whole blood collected in EDTA 4-24°C primers. -15 to -25°C LightCycler® Multiplex RNA Virus Master (requires cold chain) Following extraction the specimen extracts can o be stored at 2-8 C until PCR amplification. If not used immediately freezing at -80°C in aliquots recommended for long-term storage 4,781 PFU/mL with irradiated virus spiked into EDTA-whole blood

Venous whole blood, plasma, Trizolinactivated whole blood, Trizol-inactivated plasma -20°C (requires cold chain)

Venous whole blood, serum, plasma and urine with matched blood specimen -20°C (requires cold chain)

Storage conditions for reagents

15-25°C

Recommended time to process the specimen after collection

No specific specimen stability claims. General specimen storage recommendations for NAT tests are applicable.

Freezing at -20°C in aliquots recommended for long-term storage. Freeze-thaw cycles not to exceed 3

Whole blood can be stored up to 7 days at 28°C prior to extraction. Maintain at -70°C if processing delayed

Limit of detection (LOD) as claimed by manufacturer, not verified by independent assessment

6x10 PFU/mL with irradiated virus spiked into whole blood

1x10 PFU/mL with irradiated virus spiked into whole blood, plasma or serum

5,000 PFU/mL with Trizol inactivated whole blood or plasma. 1,000 PFU/mL with live virus spiked in Trizolinactivated whole blood

30 TCID50/reaction with inactivated virus in whole blood or urine

30 TCID50/reaction with inactivated virus in whole blood or urine.

Selection and use of Ebola in vitro diagnostic (IVD) assays

Assay name

FilmArray Biothreat-E test

FilmArray NGDS BT-E Assay

LightMix® Ebola Zaire rRT-PCR Test

EZ1 Real-time RT PCR Assay or Trizol-inactivated plasma.

CDC Ebola Virus NP Real-time RT-PCR Assay

CDC Ebola Virus VP40 Real-time RT-PCR Assay

Throughput: Time to results Maximum number of specimens per day/instrument Reagents shelf life

75 minutes 8 specimens (1 pouch processed at a time) Not known

4-6 hours Approximately 50 specimens Not known

4-6 hours Approximately 40 specimens Not known

4-6 hours Approximately 40 specimens Rehydrated primers and probes may be stored frozen for up to 12 months 100µL whole blood, serum, plasma or urine for nucleic acid extraction

Minimum specimen volume

200µL blood or urine

200µL blood, plasma, or serum

200µL for nucleic acid extraction

140-200µL whole blood or plasma for nucleic acid extraction (70µL whole blood or plasma for each QIAamp spin column. Presence of RT-PCR inhibitors may cause invalid results. Potential for false positives due to amplicon carry-over Specimens from patients who have received therapeutics or vaccines may exhibit false positive test results

Limitations of the assay

Presence of RT-PCR inhibitors may cause invalid results. Specimens from patients who have received therapeutics or vaccines may exhibit false positive test results

Presence of RT-PCR inhibitors may cause invalid results. Potential for false positives due to amplicon carry-over Performance of assay only validated on EDTA Whole blood and triPure inactive EDTA whole blood This test should not be used to test specimens from asymptomatic individuals Laboratory technicians

Presence of RT-PCR inhibitors may cause invalid results. Potential for false positives due to amplicon carry-over Specimens from patients who have received therapeutics or vaccines may exhibit false positive test results

Trained manpower

Laboratory technicians

Selection and use of Ebola in vitro diagnostic (IVD) assays

Assay name

FilmArray Biothreat-E test Biosafety training Donning and doffing PPE

FilmArray NGDS BT-E Assay Biosafety training Donning and doffing PPE Testing under BSL3 conditions

LightMix® Ebola Zaire rRT-PCR Test Biosafety training Donning and doffing PPE Specimen inactivation should be performed under BSL3 conditions Follow national guidelines CLIA high complexity labs and similar non-US facilities

EZ1 Real-time RT PCR Assay Biosafety training Donning and doffing PPE Specimen inactivation should be performed under BSL3 conditions Follow national guidelines Qualified laboratories designated by the US Dept. of Defense

CDC Ebola Virus NP Real-time RT-PCR Assay Biosafety training Donning and doffing PPE Specimen inactivation or extraction should be performed under BSL3 conditions Follow national guidelines Qualified laboratories designated by the US CDC

CDC Ebola Virus VP40 Real-time RT-PCR Assay Biosafety training Donning and doffing PPE Specimen inactivation or extraction should be performed under BSL3 conditions Follow national guidelines Qualified laboratories designated by the US CDC

Biosafety requirements

Testing under BSL3 conditions

Waste management FDA EUA restrictions regarding sites for use

Follow national guidelines CLIA moderate to high complexity labs and similar non-US facilities

Follow national guidelines US Department of Defense-specified laboratories that currently perform testing with the FilmArray System

Information on certain characteristics listed in the Table will be updated by WHO as more data become available

Selection and use of Ebola in vitro diagnostic (IVD) assays

Annex 3: Considerations for the selection of rapid antigen detection tests for the diagnosis of Ebola virus disease IVDs with WHO Emergency Use and Assessment Listing (EUAL) Version: 09 June 2015 The WHO EUAL assessment comprises WHO assessment of manufacturing capacity and quality management systems, documentary evidence of safety and performance, independent verification of analytical sensitivity (LOD) and clinical performance (sensitivity and specificity) Assay name Manufacturer Kit Components Kit format Kit size Kit components Dipstick - Lateral flow rapid chromatographic immunoassay 50 reactions Test dipsticks Specimen buffer dropper bottles Lyophilized negative control (negative human serum) Lyophilized positive control (recombinant VP40 antigen spiked in negative human serum) Test tubes with caps Disposable test tube rack TM Visual aid - ReEBOV Antigen Rapid Test Results Card Zaire ebolavirus (ZEBOV) VP40 antigen ReEBOV™ Antigen Accessory Kit • 200 Disposable Lancets • 200 Cotton Balls • 200 Alcohol Wipes Precision pipettors capable of delivering between 10 μL and 250 μL, with appropriate tips Deionized water ReEBOV™ Antigen Rapid Test (also US FDA EUA authorized) Corgenix, Inc.

Virus Detected Target Other materials required but not supplied

Infrastructure Laboratory space No requirement except for biosafety requirements

Selection and use of Ebola in vitro diagnostic (IVD) assays

Assay name Electricity only Equipment maintenance Technical support by manufacturer Performance characteristics Specimen inactivation using enhanced biosafety required by user Suitable specimen for testing

ReEBOV™ Antigen Rapid Test (also US FDA EUA authorized) No N/A Not required

Fingerstick (capillary) whole blood, venous whole blood collected in EDTA, or plasma collected in EDTA Store at 2–8°C. Do Not Freeze (requires cold chain) Fresh whole blood specimens should be obtained immediately prior to application to the test dipstick. Plasma can be stored at 2–8°C for up to 1 week, or otherwise store at -20°C

Storage conditions for reagents Recommended time to process the specimen after collection Accurate pipetting of blood critical WHO Performance evaluation

Yes when using plasma. Sensitivity (95% CI) ; 91.8% (84.5, 96.8) Specificity (95% CI); 84.6% (78.8, 89.4) Limit of Detection: 2.11E+08 RNA copies/ml 15-25 minutes If one operator, ~19 per day if run consecutively one at a time (assuming 8 hour day/ 25 min per test) but can stagger testing therefore could probably test 60-100 per day 1 year at 2–8°C 1 full drop of blood from Fingerstick or 30µL venous whole blood or plasma

Throughput: Time to results Maximum number of specimens per day Reagents shelf life Min specimen volume

Selection and use of Ebola in vitro diagnostic (IVD) assays

Assay name Limitations of the assay

ReEBOV™ Antigen Rapid Test (also US FDA EUA authorized) Circulating EBOV VP40 antigen may be absent or undetectable if the patient has progressed to their humoral immune response and anti-EBOV VP40 antibody titers may have developed. Negative results do not preclude Ebola virus infection, particularly within the first 72 hours after appearance of symptoms, and should not be used as the sole basis for patient management decisions. Test is not intended for use for general Ebola infection screening, such as airport screening or contact tracing and should not be used on asymptomatic individuals. Testing patient specimens containing excess hemoglobin may result in false negative readings. Testing patient specimens containing rheumatoid factor may result in false positive readings. Specimens from patients who have received therapeutics or vaccines may exhibit false positive test results or other confounding test results. Trained personnel Biosafety training Donning and doffing PPE Where advanced biocontainment facilities (BSL-4) are not available, the use of all possible universal precautions is highly recommended including safety goggles and/or face shields, masks or respiratory equipment, disposable gowning, boots and gloves. It is highly recommended that health care workers are appropriately trained in the donning and doffing of personal protective equipment. Follow national guidelines Use in laboratories or facilities adequately equipped, trained, and capable of such testing (including treatment centres and public health clinics)

Trained manpower

Biosafety requirements

Waste management FDA EUA restrictions regarding sites for use

Selection and use of Ebola in vitro diagnostic (IVD) assays

Key facts

Organisation World Health Organization (WHO)

Document type Technical Documents

Adoption date June 13, 2015

Source World Health Organization