SHORT, MEDIUM AND LONGER TERM PRODUCT DEVELOPMENT PRIORITIES IN HIV-RELATED DIAGNOSTICS WHO EXPERT MEETING REPORT ON 6 7 JUNE 2012 GENEVA, SWITZERLAND

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1 HIV/AIDS Programme WHO EXPERT MEETING REPORT ON SHORT, MEDIUM AND LONGER TERM PRODUCT DEVELOPMENT PRIORITIES IN HIV-RELATED DIAGNOSTICS 6 7 JUNE 2012 GENEVA, SWITZERLAND

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3 HIV/AIDS Programme WHO EXPERT MEETING REPORT ON SHORT, MEDIUM AND LONGER TERM PRODUCT DEVELOPMENT PRIORITIES IN HIV-RELATED DIAGNOSTICS 6 7 JUNE 2012 GENEVA, SWITZERLAND

4 WHO Library Cataloguing-in-Publication Data WHO expert meeting report on short, medium and longer term product development priorities in HIV-related diagnostics, 6-7 June 2012, Geneva, Switzerland. 1.HIV infections diagnosis. 2.Anti-HIV agents therapeutic use. 3.Drug resistance. 4.Technology. I.World Health Organization ISBN (NLM classification: WC 503.1) World Health Organization 2012 All rights reserved. Publications of the World Health Organization are available on the WHO web site ( or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: ; fax: ; bookorders@who.int). Requests for permission to reproduce or translate WHO publications whether for sale or for noncommercial distribution should be addressed to WHO Press through the WHO web site ( copyright_form/en/index.html). 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 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. This publication contains the collective views of an international group of experts and does not necessarily represent the decision or the policies of the World Health Organization. Layout L IV Com Sàrl, Villars-sous-Yens, Switzerland. Printed by the WHO Document Production Services, Geneva, Switzerland.

5 Short, medium and longer term product development priorities in HIV-related diagnostics CONTENTS ACRONYMS AND ABBREVIATIONS EXECUTIVE SUMMARY INTRODUCTION Treatment SCOPE OF THE MEETING Process Outcome HIV TESTING AND LOGISTICS Overview Discussion Technical recommendations for HIV testing CD4 TESTING AND LOGISTICS Overview Discussion Technical recommendations for CD4 testing EARLY INFANT DIAGNOSIS AND VL DETERMINATION Overview Discussion Key statements on the roll out of EID and VL technologies Technology recommendations HIV DRUG RESISTANCE Overview Discussion Technical recommendations for HIV drug resistance TB DRUG RESISTANCE Overview Discussion Technical recommendations for TB drug resistance MULTI-ANALYTE TESTING Overview Discussion Technical recommendations for multiple test platforms TEST RESULT READERS AND INFORMATION TECHNOLOGY Overview Discussion Field examples Technical recommendations for IT APPENDIX I: AGENDA APPENDIX II: LIST OF PARTICIPANTS

6 WHO Expert Meeting Report Geneva, Switzerland, 6 7 June 2012 ACRONYMS AND ABBREVIATIONS ACD acid citrate dextrose AFRO (WHO) Regional Office for Africa AIDS acquired immunodeficiency syndrome ART antiretroviral therapy ARV antiretroviral AS-PCR allele-specific polymerase chain reaction ASPE allele-specific primer extension bdna branched DNA testing CD4 cluster of differentiation 4 CLIA clinical laboratory improvement amendments CRI colorimetric redox indicator DBS dried blood spot DNA deoxyribonucleic acid DPS dried plasma spot DR drug resistance DST drug susceptibility testing EDTA ethylene diamine tetra-acetic acid EIA enzyme immunoassay EID early infant diagnosis ELISA enzyme-linked immunosorbent assay EQA external quality assessment FTA-Abs fluorescent treponemal antibody absorption GLI global laboratory initiative GPS global positioning satellite GPRS general packet radio service HBV hepatitis B virus HCV hepatitis C virus HIV human immunodeficiency virus HS heel stick HTC HIV testing and counselling INSTI integrase strand transfer inhibitor IVD in vitro diagnostic IT information technology LCD liquid crystal display LIS laboratory information system LOD limit of detection LPA line probe assay LTBI latent tuberculosis infection 2

7 Short, medium and longer term product development priorities in HIV-related diagnostics LTR maspe MDR-TB MODS MSM MTB MTCT NAT NNRTI NRA NRTI PCR PEPFAR PI PMA PMTCT POC PPT QA QC RDT RIF RNA RPR RT RUO SMS SOP SRL SS+ STI TB TNA TP TPHA UN UNAIDS VDRL VL WB WHO WWW XDR-TB long terminal repeat multiplex allele-specific primer extension multi-drug resistant tuberculosis microscopic observation of drug susceptibility men who have sex with men mycobacterium tuberculosis mother-to-child transmission nucleic acid testing non-nucleoside reverse transcriptase inhibitor nitrate reductase assay nucleoside reverse transcriptase inhibitor polymerase chain reaction President's Emergency Plan for AIDS Relief protease inhibitor point mutation assay prevention of mother-to-child transmission point-of-care plasma preparation tube quality assurance quality control rapid diagnostic test rifampicin ribonucleic acid rapid plasma reagin reverse transcription research use only short message service standard operating procedure supranational TB reference laboratory sputum smear positive sexually transmitted infection tuberculosis threose nucleic acid treponema pallidum treponema pallidum haemaglutination assay United Nations the Joint UN Programme on HIV/AIDS venereal disease research laboratory viral load whole blood the World Health Organization world-wide web extensively drug resistant tuberculosis 3

8 WHO Expert Meeting Report Geneva, Switzerland, 6 7 June 2012 EXECUTIVE SUMMARY The World Health Organization (WHO), with support from the Pangaea Global AIDS Foundation and funding from the Bill & Melinda Gates Foundation, convened a meeting of experts. The aim was to articulate consensus on which technologies, including those to be used at point-of-care (POC), need to be developed for use in HIV programmes and deployed in the short, medium and long term at community, primary care, district, regional and national levels. These recommendations are intended to inform the development of the WHO 2013 consolidated guidelines on the use of antiretroviral drugs (ARVs) for HIV treatment and prevention and to provide initial guidance on product development priorities in HIV-related diagnostics. The meeting was part of WHO s commitment to the broader Treatment 2.0 initiative, coordinated by WHO and the UNAIDS Secretariat. Over-arching principles are that specimen collection should be minimally invasive and all commodities for carrying out a test should be contained within the test kit, which should be simple to use, have a long shelf life, and can function reliably at high and low temperatures. The use of diagnostics at point-of-care needs to be expanded quickly. All existing and pipeline diagnostics must include appropriate quality control measures and be compatible with existing quality assessment procedures. Scale up of access to good quality reliable HIV rapid diagnostic tests (RDT) must be continued. HIV rapid diagnostic tests suitable for oral fluid specimens should be considered for use in resource-limited settings. It is expected that seven new CD4 testing technologies (five for POC) may be commercially available before the end of The choice of CD4 technology should be informed by consideration of workload (daily specimen throughput), infrastructure (including electricity, clean water, climate-controlled testing areas), and available staffing. National regulatory authorities for diagnostics need to be strengthened and a strong post-market surveillance system for diagnostics is needed. WHO will provide concise guidance for the selection and use of CD4 technologies in different health settings and timely prequalification of new technologies. The development of diagnostics for early infant diagnosis (EID) and viral load (VL) measurement at POC should be expedited. A clear framework is needed to assess the technical specifications and performance characteristics of new virological (molecular) technologies. While diagnostics for POC are important for lower levels of the health care system, access to laboratory-based testing for VL and EID is essential at higher level laboratories and in higher throughput facilities, where infrastructure and skilled staffing permits. The use of dried blood spots (DBS) will facilitate access to laboratory-based molecular technologies until the introduction of technologies for use at POC, and offer potential for their EQA, once POC tests are in use. New technology is emerging and the use of DBS has been validated but its use outside a few highly skilled laboratory technicians faces challenges. HIV drug resistance testing is not widely available as the technologies are technically complex and relatively expensive. In resourcelimited settings, HIV drug resistance testing currently is restricted to population-level surveillance. 4

9 Short, medium and longer term product development priorities in HIV-related diagnostics The priority for the management of tuberculosis (TB) is more wide-spread access to rapid and sensitive diagnosis and rapid drug resistance determination. Currently, there are several technologies for diagnosing TB, with new ones being developed. These technologies occupy different places within a tiered health system with differing levels of sophistication, requiring different levels of training and expertise, from basic microscopy to culture. Microscopy remains the primary diagnostic tool in all settings and a strong microscopy network infrastructure must be maintained. Improved technology is reducing diagnosis reporting time and costs, but more progress is needed. Multi-analyte testing brings together screening for HIV and other infections in the same device or using the same platform with multiple sets of reagents. Technological advances have resulted in improved diagnostics for syphilis and viral hepatitis. Some HIV/syphilis rapid diagnostic tests and HIV/HCV, HIV/ HBsAg are under development and soon will come to market. Multi-analyte tests in the development pipeline must be at least as cheap and as easy to use as current technologies with the same intended purpose. There are potential disadvantages that need to be considered when introducing concurrent diagnosis of different infections, including linkages to care and treatment. Decentralization of HIV testing has resulted in an enormous increase in the amount of testing, with more tests being performed at community health centres, but has highlighted problems, especially with quality control, accurate reading and interpretation of test results. Test readers were developed to address these problems. Dissemination of testing-related information requires the continued integration of laboratory and clinical information systems. There are several rapid diagnostic test readers on the market and some have the capability to transmit results directly to a mobile phone, printer or computer. The ideal rapid diagnostic test reader would be able to be used with multiple brands and formats, operate on open technology and be battery powered. There would be a simple test menu, and an integrated LCD touch screen with icons. Future development for rapid diagnostic test readers will enable data communications with software on readers linked to supply chain management software to prevent stock-outs. 5

10 WHO Expert Meeting Report Geneva, Switzerland, 6 7 June 2012 INTRODUCTION In vitro diagnostics (IVDs), hereafter referred to as diagnostics, are used to identify HIV infection, to decide whether and when to treat it, and to monitor antiretroviral therapy (ART). The development and widespread use of HIV rapid diagnostic tests (RDTs) has helped considerably with the scale-up of access to treatment in areas with limited laboratory services, as RDTs can be performed outside of the traditional laboratory setting by trained non-laboratory personnel. This scale-up in treatment access has created an increased demand for technologies to determine treatment eligibility through CD4 cell counts and to monitor response to treatment and adherence through viral load (VL) measurement. WHO, with support from the Pangaea Global AIDS Foundation and funding from the Bill & Melinda Gates Foundation, convened a meeting of experts to identify short, medium and longer term product development priorities for HIV and HIV-related diagnostics in the context of further scale-up of programmes for HIV diagnosis in adults, children and infants, and HIV treatment initiation and monitoring. The secretariat was drawn from the WHO Interdepartmental Working Group on Diagnostics, Departments of HIV and of Essential Medicines and Health Products (Diagnostics and Laboratory Technology team), WHO Regional Office for Africa (WHO/AFRO), and the Pangaea Global AIDS Foundation. Considering the development of diagnostic technologies for use in HIV programmes, two specific objectives were set for this meeting. 1. To articulate the current consensus on technologies including point of care (POC) technologies, which need to be deployed now for use in HIV programmes at various levels of the national laboratory network. 2. To identify technologies including POC technologies that need to be developed in the short, medium and longer term to make HIV programmes more effective. The Agenda for this meeting followed recommendations from an earlier meeting held in Geneva in October 2011 that examined the short-term priorities for HIV and HIV-related diagnostics. The recommendations of this meeting are intended to inform the development of the WHO s normative guidance to countries on how to make best use of existing diagnostic technologies and approaches, as well as to provide initial guidance to assay developers and manufacturers, policy makers, procurers and users on short, medium and longer term product development priorities in HIV-related diagnostics. 6

11 Short, medium and longer term product development priorities in HIV-related diagnostics TREATMENT 2.0 The meeting was part of WHO s commitment to the broader Treatment 2.0 initiative, coordinated by WHO and the UNAIDS Secretariat, which aims to achieve radical simplification of all aspects of HIV treatment. This includes drugs, diagnostics and healthcare delivery systems, as well as reducing costs and mobilizing communities towards greater engagement in programme design and implementation in resource-limited settings. Treatment 2.0 focuses on short- (1 to 3 years), medium- (4 to 6 years) and long- (7 to 10 years) term objectives to achieve and sustain universal access to treatment for all who need it, and to maximize the preventative benefits of HIV treatment. 7

12 WHO Expert Meeting Report Geneva, Switzerland, 6 7 June 2012 SCOPE OF THE MEETING The meeting considered serological testing for HIV diagnosis, technologies for the early infant diagnosis (EID) of HIV infection, CD4 cell count determination, HIV viral load (VL) measurement, HIV drug resistance testing, diagnosis of hepatitis B (HBV) and hepatitis C (HCV), testing platforms that bring together screening for HIV and other infections (such as tuberculosis (TB), syphilis and hepatitis), improvements in logistics (such as information technology (IT), specimen collection) and technologies aiming to improve operational aspects (such as RDT readers). Due to time constraints, the meeting did not discuss toxicity monitoring. The meeting primarily considered priorities for generalized and concentrated epidemic settings although low-level epidemics would benefit from these recommendations as well. In each of these settings, the needs of patient care in service delivery were considered at five levels of health service delivery as illustrated in figure 1 below: Level 0 Community outreach setting Level 1 Primary care setting Level 2 District Level 3 Regional or provincial Level 4 National FIGURE 1. THE TIERED, INTEGRATED LABORATORY NETWORK, WITH EXAMPLES OF STAFF TYPICALLY FOUND AND TESTS THAT COULD BE PERFORMED AT EACH LEVEL NATIONAL Senior laboratory specialists EIA for diagnosis, higher throughput CD4, HIV molecular technologies including HIV VL, quantitative/qualitative EID, HIV resistance testing REGIONAL/PROVINCIAL Laboratory specialists/senior technicians EIA for diagnosis, higher throughput CD4, HIV molecular technologies including HIV VL, quantitative/qualitative EID DISTRICT Laboratory technicians and assistants EIA for diagnosis, low throughput CD4, chemistry, haematology, microbiology PRIMARY CARE First level trained health care workers: nurses, clinical officers HIV RDTs, other POC tests, DBS collection COMMUNITY OUTREACH Community health worker HIV RDTs 8

13 Short, medium and longer term product development priorities in HIV-related diagnostics PROCESS In each area of interest, a brief technical overview on the state of the art and the current development pipeline was presented. The presentations were followed by discussions on: 1. What products need to be recommended for deployment now? 2. What products need to be developed in short, medium and long term? 3. Which development pipelines need to be enriched so as to foster competition? The participants at this meeting were asked to consider the following general principles when making their deliberations. Adopt a forward thinking approach in making recommendations. Advocate for better access to both current and new technologies. Advocate for better and simpler technology, in particularly for use at POC. A provisional definition of POC testing proposed was medical testing within a specified time period (10-30 minutes of specimen collection) and is followed by provision of an appropriate immediate medical decision based on the results (ASLM meeting May 2012). This definition related more to the objective of testing rather than to the technology itself, i.e. not POC test, but rather testing at the POC. Several professionals believe that POC testing should be defined as testing that is decentralized and provides same-day results to patients in settings where there is linkage to clinical care the linkage to same day care being critical. For POC diagnostics, the ASSURED seven minimum criteria were proposed: Affordable (<5 USD), Sensitive (99%), Specific (98%), User-friendly (requiring minimal training), Robust (no cold chain) and rapid (<1 hour: same-day results and same-day care) and require little or no operator calibration, and minimal routine maintenance (e.g. wiping spills, dusting), Equipment-free (battery-operated; few moving parts), Deliverable (commercially available and approved). It was agreed that: Diagnostics that can be used at the POC improve access to testing at all levels. POC technologies are well suited for lower levels of the health care system, but they are unlikely to completely replace laboratory-based testing platforms, which are generally more efficient for higher levels and in higher throughput facilities. Testing platforms available at higher levels are additional to technologies recommended for lower levels as illustrated in Figure 1 above. 9

14 WHO Expert Meeting Report Geneva, Switzerland, 6 7 June 2012 OUTCOME This report articulates a consensus of expert opinion on: 1. The deployment of POC technologies designed for use in HIV programmes. 2. The technologies that need to be developed in the short, medium and longer term. This meeting report does not present guidelines or guidance officially endorsed by WHO. Its purpose is to inform about the 2013 consolidated guidelines on the use of antiretroviral drugs (ARVs) for the treatment and prevention of HIV infection. 10

15 Short, medium and longer term product development priorities in HIV-related diagnostics 1 HIV TESTING AND LOGISTICS 1.1 OVERVIEW Universal access to antiretroviral therapy (ART) requires high coverage HIV testing and counselling (HTC). In 2010, 95 million people aged 15 to 49 received HTC, offered at 131,000 health centres globally. However, less than 40% of people with HIV are aware of their status, and fewer men than women are aware of their status. Often the linkages to care remain poor, both for those who test positive (access to treatment and disclosure support) and those who test negative (access to prevention interventions). New concepts and approaches to HTC are being implemented. HTC is moving from the health centre into the community, to schools, workplaces and to homes, and is being used in door-to-door testing campaigns. HTC campaigns are being widened to include other infectious diseases (such as malaria), safe water, and non-communicable diseases. Newer technologies are being utilized (such as mobile phones) to reach key populations. RDTs are being offered, often not by health care workers but by peers and lay health care workers. Couples testing is a new focus with recent WHO guidance released by WHO. The current spectrum of HIV in vitro diagnostics (IVDs) RDTs (both immunofiltration and immunochromatographic formats). Simple assays (indirect solid-phase enzyme immunoassays and agglutination assays). Enzyme immunoassays (EIA) (96 well microtitre plates). Simple immunoanalysers (low throughput, more hands-on time, as less automated). Random access immunoanalysers (high throughput, more highly automated). Confirmatory assays (Western blot and line-immunoassays). A wide variety of IVDs for HIV diagnosis are available. The choice of which test format to use for initial screening and subsequent confirmatory testing depends on many factors, such as the performance characteristics of the test, the physical infrastructure, ease of use, cost, and the numbers of tests to be performed per day. Table 1 shows some of the operational characteristics that one would consider when choosing between using EIA and RDTs for HIV serological diagnosis. TABLE 1. EIAS VERSUS HIV RDTS EIAs HIV RDTs Time to result is >60 minutes Time to result is 5 30 minutes Large throughput, batch testing Low throughput, single tests Skilled staff required Minimal skill required Requires maintained equipment None to minimal equipment Test kits require refrigeration Most stored between 2 C 30 C Price (WHO) US$ Price (WHO) US$

16 WHO Expert Meeting Report Geneva, Switzerland, 6 7 June DISCUSSION There was consensus that scale-up of access to quality HIV RDTs needs to be continued. Access to care should be provided to any person, with HIV positive status confirmed by a second independent specimen, ideally at the time of testing for treatment eligibility, therefore verifying if specimen mix-up did not occur during the initial testing visit. RDTs need to be highly sensitive and specific with long shelf lives at high temperatures. RDTs must be used within a testing algorithm that reduces the likelihood of false positives and negatives arising between the first- and second- or third-line tests. Currently available HIV RDTs could be improved to make them easier to perform, with fewer steps, shorter time to result, and results easier to read and interpret, i.e. less faint lines, and clear, diagrammatic (graphical) instructions for use. The kits should include test kit controls and procedural quality controls (QC) that can verify that a biological specimen has been added and that the test is functioning correctly. Eliminating the need for a separate buffer addition step is desirable. Adding barcodes to test devices in order to better track results should be considered, especially in the light of newly developed RDT readers. In summary, test kits should include specimen controls (specifically, a specimen addition quality control), double identification stickers to facilitate sample ID on both device and sample worksheet or patient result form (bar code or user ID), and buffer should be incorporated into the test kit (e.g. a squeezable pouch within the device). RDTs should be more robustly packaged with easier disposability, and should include temperature and humidity indicators. The shelf life following manufacture and heat stability needs to be extended. Issues relating to shelf life following delivery are usually dealt with through the negotiation of the procurement contract but these differences should be clearly understood for end users. WHO sets generic testing strategies for high- and low-prevalence settings, with WHO guidance on this topic released in mid A national testing algorithm (with back-up options) must then be validated, whereby a number of well performing tests are placed in a certain configuration with the appropriate testing strategy in a manner that minimizes false positive and false negative testing results. Testing algorithms must always be validated either at national or regional levels. The number of algorithms used in country should be limited, with assurance of back-up options in the case of product failures or stock- outs, and in order to respond quickly to manufacturing changes or recalled products. Quality systems at all stages of the testing process are paramount. In the laboratory, QC should be performed daily following standard operating procedures (SOPs), and the performance of laboratory personnel must be monitored. The challenges for quality systems include turnover of personnel, their competence and proficiency, and assuring consistent and sustainable quality of service. Inclusion in external quality assessment (EQA) programmes (otherwise known as proficiency testing) is recommended and provision for national EQA programmes that reach each testing site, including all POC sites, should be considered as an achievable goal. The dried tube specimen approach may be useful in this regard, and has been piloted in a number of countries with support from the US CDC. Post-market surveillance for diagnostics needs to be strengthened in order to identify problems with test kits that are being used in the field. 12

17 Short, medium and longer term product development priorities in HIV-related diagnostics Challenges with currently available RDTs include cartridge formats that are not harmonized, a lack of robustness, particularly in adverse environmental conditions with non-laboratory personnel performing testing, and variable lot-to-lot quality of RDTs, lack of traceability of the results, insufficient shelf life upon delivery (stability of diagnostics in terms of temperature and humidity), and how to manage sites with low throughput, especially in low prevalence settings. 1.3 TECHNICAL RECOMMENDATIONS FOR HIV TESTING TECHNOLOGIES RECOMMENDED FOR DEPLOYMENT NOW TABLE 2. HIV TESTING TECHNOLOGIES FOR DEPLOYMENT NOW Level 0 (community outreach) Level 1 (health centre) Level 2 (district) Level 3 (region/ province) Level 4 (national reference) HIV RDTs EIA, simple immunoanalyser Automated immunoanalyser TECHNOLOGIES RECOMMENDED FOR DEPLOYMENT IN THE SHORT TERM HIV RDTs suitable for oral fluid specimens. HIV RDTs which are easier to use, more stable in adverse environmental conditions, with clearer instructions for use TECHNOLOGIES RECOMMENDED FOR DEVELOPMENT IN THE MEDIUM AND LONGER TERM Improved ability to support surveillance data capture through the use of RDT readers that electronically transmit results. Multiplexed IVDs including RDTs, see Section 6.0 Multi-analyte testing. 13

18 WHO Expert Meeting Report Geneva, Switzerland, 6 7 June CD4 TESTING AND LOGISTICS 2.1 OVERVIEW Currently available CD4 technologies include traditional and dedicated flow cytometers, POC cytometers and manual methods. One newly developed POC instrument that is capable of CD4 T-cell analysis from a finger stick or venous whole blood (WB) specimen within 20 minutes has been deployed at POC in many countries, including in sub-saharan Africa. TABLE 2. CHARACTERISTICS OF CURRENTLY AVAILABLE CD4+ T-CELL ENUMERATING METHODS Type Classical FC Dedicated FC POC cytometer Manual methods Daily throughput Complexity (infrastructure/ personnel) >100 Require well established labs and trained manpower Require well established labs and trained manpower 1 20 Simple, portable, can be done outside labs 1 10 Require established labs, labour intensive Precision and accuracy High, used as reference method Instrument cost Test cost $ Examples Lab network Comments k 3 7 High 25 30k 3 10 Medium to high low 5 12k 5 6 Calibur Coulter Epic X PLG FACSCount, Cyflow, Guava, Apogee, BlueOcean LG Pima Cyflow CD4 minipoc Coulter beads Dynabeads Regional National District Health Centre/ Dispensaries/ mobile clinics Health Centre Open platform, can be used for extended testing Mainly closed systems, simpler Closed systems Have low precision, labour intensive A. Currently available high-throughput CD4 Systems: 1 BD FACSCalibur System (BD Biosciences) 2 Coulter Epic X, Cytomics FC 500 MCL or Cytomics FC 500 MPL System (Beckman Coulter, Inc.) B. Currently available medium to low throughput CD4 Systems: 1 BD FACSCount System (BD Biosciences) 2 Millipore-Guava Auto CD4/CD4% System (Merck) 3 Apogee Auto40 Flow Cytometer (Apogee Flow Systems) 4 CyFlow Counter (Partec GmbH) 14

19 Short, medium and longer term product development priorities in HIV-related diagnostics C. Currently available POC CD4 testing platforms: 1 Pima Analyser, CD4 absolute count (Alere Inc.) 2 CyFlow CD4 minipoc, CD4 absolute count and percentage (Partec GmbH) CD4 TECHNOLOGIES IN THE PIPELINE 1 DEDICATED CD4 TECHNOLOGIES IN THE PIPEL INE BlueOcean LG 100/250 (Beckman Coulter) Load-and-go approach % and absolute counts Sample preparation station samples/day Late 2012 FACSClearcount (BD Biosciences) % and absolute counts Sample preparation station >200 samples/day Late 2013 POC CD4 TECHNOLOGIES IN THE PIPELINE CD4 POC Technology (BD Biosciences) CD absolute and percentage, haemoglobin Expected in 2013/14 Daktari CD4 counter (Daktari Diagnostics) CD4 absolute count Expected in Most if not all the photo are from Murtagh, MM. UNITAID Technical Report: HIV/AIDS Diagnostic Technology Landscape; 2 nd edition June Geneva: UNITAID, 2012, edition.pdf. 15

20 WHO Expert Meeting Report Geneva, Switzerland, 6 7 June 2012 mbio Diagnostics CD4 System (mbio Diagnostics, Inc.) CD4 absolute count Expected in 2013/14 Zyomyx CD4 test (Zyomyx, Inc.) CD4 absolute count 2013 (clinical trials in 2012) VISTECT CD4 (Omega Diagnostics Ltd & Burnet Institute) Semi quantitative for CD4 protein Expected in 2013/14 Other possible CD4 POC Tests: Palo Alto Research Center (PARC): research stage. Lab-on-a chip, CD4 absolute count It is expected that seven new CD4 technologies, of which five can be used at POC, may be commercially available available before the end of DISCUSSION The choice of CD4 technology is dependent on workload (specimen throughput per facility per day) and the future anticipated use of CD4 for treatment initiation or monitoring. Existing POC CD4 technologies should be scaled-up in areas of need to reach HIV positive individuals who would not otherwise access laboratory services to determine their eligibility for treatment. CD4 technologies should be established in appropriate sites (considering testing volume, data management systems, and linkage to care) where quality assessment (QA) and a strong supply chain can be ensured. Technologies for CD4 absolute 16

21 Short, medium and longer term product development priorities in HIV-related diagnostics count and CD4 percentage (for infants) determinations could be combined with haematology for added value in a clinical setting. Instruments for POC testing ideally should be self-contained, robust, portable, with few steps and automated incubation. The instrument should have built-in QC including QC material/cartridges to run and internal QC checks and software. It should be able to operate in adverse environmental conditions including both high and low temperature ranges, without need for constant electricity, i.e. it should be battery or solar operated. Required reagents should be heat stable and not need cool storage. Ideally, the required specimen should be finger stick whole blood, eliminating the need for phlebotomy. POC testing for CD4 should have a short turnaround time (less than 30 minutes), and be low cost. Instruments for POC testing should have capability for electronic data collection and transmission. The majority of blood specimens are collected by venepuncture into ethylene diamine tetra-acetic acid (EDTA) tubes and must be tested within 24 hours of collection. The pan-leucogating method with CD45 gating allows blood specimens to be stored for up to five days prior to testing but is a relatively complex technical method using a classical flow cytometer. There are several commercially available blood fixatives which may allow CD4 testing to be performed up to seven days after specimen collection. These fixatives have been validated to varying degrees in some existing instruments. Capillary blood collected by finger stick can be used in smaller, simpler, automated instruments for POC testing. However, ensuring specimen homogeneity and an accurate volume without air bubbles remains a challenge. Training of health workers on collection of finger stick specimens will be a constant requirement. In the medium and long term, specimen collection by finger stick for CD4 determination could be considered at Level 0, performed by trained community health workers, considering the time needed to train people. In any one country, both existing (flow and/or dedicated) cytometers plus instruments for POC testing will be needed, their positioning within the health system will depend on the infrastructure (electricity, water), available staff and their skills, and expected specimen throughput per day (low <20 specimens/ day, medium specimens/day, high >50 specimens/day). In the short term, one new CD4 technology expected to be commercially available before the end of 2013 can quantify both absolute CD4 count and percentage in a POC format that is simpler than existing dedicated cytometers. This instrument has high throughput of 100 to 200 specimens per day. In the short to medium term, small, simpler, automated technologies for POC testing with lower specimen throughput that do not require constant electricity, and that are suitable for use at district level (level 2) and primary care level will be available. Ultimately, one possible technological solution may be the lab-on-a-chip (a device that integrates laboratory functions on a single chip using extremely small fluid volumes) adapted for CD4 count, but this is unlikely to be available in the short to medium term. Accurate counting of CD4 cells requires strict adherence to QA practices, including specimen collection and transportation, staff training, instrument and pipette maintenance and calibration, accurate analysis of specimens including gating, and accurate interpretation of results. Currently, QC materials for classical flow and dedicated cytometers have a relatively short shelf life and require cold storage. The introduction of technologies for POC testing will require stronger emphasis on QA as CD4 testing moves outside the traditional laboratory setting to users who may be unfamiliar or inexperienced with QA practices. Very few laboratories in resource-limited settings currently participate in external quality assessment (EQA) schemes. Support (financial and technical) should be considered to strengthen QA programmes, including provision of national EQA schemes. In many resource-limited settings, supply 17

22 WHO Expert Meeting Report Geneva, Switzerland, 6 7 June 2012 chain management systems are too weak to ensure availability of reagents (particularly reagents that require cold storage), and there are challenges for reliable and prompt preventive and corrective maintenance of instruments. Health care workers should be trained in the basic preventive maintenance where possible and ensure that an overarching maintenance schedule for specialized preventative and corrective maintenance is in place. National and regional regulatory authorities for diagnostics need to be strengthened and a strong post market surveillance system will need to be established. WHO will provide concise guidance for the selection and use of CD4 technologies in different health settings and timely pre-qualification of new technologies. WHO will work with partners to minimize the need for multiple in-country validation studies, so that the technologies can be introduced faster. 2.3 TECHNICAL RECOMMENDATIONS FOR CD4 TESTING TECHNOLOGIES RECOMMENDED FOR DEPLOYMENT NOW TABLE 4. CD4 TECHNOLOGIES FOR DEPLOYMENT NOW Level 0 (community outreach) Level 1 (health centre) Level 2 (district) Level 3 (region/ province) Level 4 (national reference) Sample collection Venous collection in EDTA Capillary blood collection by finger stick Preservatives/fixatives (ex. Streck, Cytomark, BD) which can allow testing to be extended up to 7 days Testing technologies * Manual methods (ex. Coulter beads, Dynabeads) POC cytometer (ex. PIMA, Cyflow CD4 minipoc) Dedicated cytometer (e.g. FACSCount, Cyflow, Guava, Apogee) Classical flow cytometer (e.g. FACSCalibur, Epix, PLG) * * Conditional: when and if trained staff and appropriate POC technology is available in health service with outreach settings to ensure linkage to care. 2 The performance of an instrument may be inadequate for HIV clinical management in adults and/or in children depending on external factors or specific equipment related factors. It is important to choose prequalified laboratory technologies or those approved by the national regulatory authority or conduct /refer to an independent evaluation study of new diagnostic technology platforms before deployment. 18

23 Short, medium and longer term product development priorities in HIV-related diagnostics TECHNOLOGIES RECOMMENDED FOR DEVELOPMENT IN THE SHORT TERM Expedite development and bring to the market CD4 technologies in the pipeline in the immediate short term (by end 2012). BlueOcean LG series (Beckman Coulter), FACSClearcount (BD Biosciences). Expedite development and bring to the market CD4 technologies in the pipeline suitable for POC testing in the short term (by end 2014): BD Biosciences, MBio Diagnostics, Daktari Diagnostics, Zyomyx Inc, Omega Diagnostics TECHNOLOGIES RECOMMENDED FOR DEVELOPMENT IN THE MEDIUM AND LONGER TERM Instrument-free technologies for CD4 POC testing at community (level 0) and primary care (level 1 facilities). 19

24 WHO Expert Meeting Report Geneva, Switzerland, 6 7 June EARLY INFANT DIAGNOSIS AND VIRAL LOAD DETERMINATION 3.1 OVERVIEW The current viral load (VL) test market is dominated by four companies: Abbott Molecular, biomérieux, Roche Molecular Diagnostics and Siemens Health Care Diagnostics, Inc. Most testing is performed using sophisticated, high-throughput instruments in centralized laboratory facilities requiring highlytrained technicians. Some VL assays with lower per-test costs are open platform and can accommodate generic test reagents. Although these assays have been shown to be accurate, they may require an increased hands-on time and a validation of the different components necessary to perform the assay given their generic nature, e.g. extraction kit and amplification kit. The cost (per-test) varies greatly with volumes. In the context of Early Infant Diagnosis (EID) and VL, a comprehensive overview of available technologies for the determination of VL and diagnosis using whole blood (WB), plasma or dried blood spots (DBS) was presented. The technology used, the diagnostic markers, the viral sub-types/groups identified, the logistics for sample collection and transport, the time taken per test, the linear range of each test, and the appropriate setting for each test were detailed. Different technologies are appropriate for different levels of the tiered, integrated laboratory network. The commercially available VL and EID technologies are listed below from WHO/DLT, July 2012: Summary of Commercially Available VL and EID Technologies. 20

25 Short, medium and longer term product development priorities in HIV-related diagnostics TABLE 5. OVERVIEW OF COMMERCIALLY AVAILABLE NAT-BASED HIV VL TESTING TECHNOLOGIES Assay Company Technology COBAS AmpliPrep/ COBAS TaqMan (CAP/ Roche RT-PCR, quantitative Diagnostic Markers RNA (gag, LTR) Subtype/ group Group M: A H and Group O Sample volume (ml) Plasma or DBS Linear range (copies/ ml) 1.0 Plasma, DBS (RUO) CTM) HIV-1Test v2.0 Amplicor HIV-1 MONITOR Roche RT-PCR, Gag Group M: A H Plasma million (Ultrasensitive) 6 8 Utrasensitive/standard quantitative ,000(standard) Test, v1.5 COBAS Amplicor HIV-1 Roche RT-PCR, Gag Group M: A H Plasma ,000 (Ultrasensitive) 6 8 MONITOR Utrasensitive/ quantitative 400 1,000,000 (standard) standard Test, v1.5 COBAS TaqMan HIV-1 test, Roche RT-PCR, Gag Group M: A H 0.5 Plasma million 5 6 v2.0 for use with the high quantitative pure system Generic HIV VL (RUO) Biocentric RT-PCR LTR B and non B 0.5 Plasma, DBS million 4 Abbott RealTime HIV-1 Versant HIV-1 RNA 1.0 (kpcr) Versant HIV-1 RNA 3.0 (bdna) NucliSens EasyQ HIV-1 v2.0 Abbott Real-Time PCR Pol/INT Group M: A H Groups N & O Siemens RT-PCR Pol Group M: A H Group O Siemens Branched DNA (bdna) Artus HIV-1 QS RGQ Kit Qiagen RT-PCR Quantitative Artus HIV-1 RG RT-PCR Qiagen RT-PCR Quantitative 0.2, 0.5, 0.6, 1.0 EDTA & ACD Plasma, DBS 0.5 Plasma, serum, DBS Throughput (hr/test) 20 to 10 million to 10 million (0.6 & 1.0 ml) 75 to 10 million (0.5mL) 150 to 10 million (0.2mL) million 5 6 Pol Group M: A G Plasma, , biomérieux NASBA RNA (gag) Group M: A J 0.5 Plasma, DBS million 3 RNA Group M: A H 1.2 EDTA Plasma million 5 6 RNA Group M: A H EDTA Plasma million

26 WHO Expert Meeting Report Geneva, Switzerland, 6 7 June 2012 TABLE 6. OVERVIEW OF COMMERCIALLY AVAILABLE NAT-BASED QUALITATIVE ASSAYS FOR HIV-1 DIAGNOSIS FOR HIV EID Assay Company Technology COBAS AmpliPrep / COBAS TaqMan (CAP/ CTM) HIV-1 Qual Test v1.0 (RUO) AMPLICOR HIV-1 DNA Test v1.5 (RUO) Abbott RealTime Qualitative Roche Real-Time PCR Qualitative Roche PCR, qualitative Abbott Real-Time PCR Diagnostic Markers Subtype/ group gag Group M, subtypes A H Sample volume (ml) Plasma or DBS Linear range (copies/ ml) gag Group M: A H 0.1 (infants 0.5 (adults) Pol Group M: A H Groups N WB DBS WB DBS 0.2 EDTA & Plasma, or DBS Throughput (hr/test) Qualitative test 5 6 Qualitative test 7 8 Qualitative test 5 HIV-1 Generic HIV VL (RUO) Biocentric Real-Time LTR B and non B 0.5 Plasma, DBS million 4 PCR HIV-1 p24 Ultra ELISA Perkin- p24 EIA p24 All 0.01 Plasma upwards 6 (RUO) Elmer antigen ExaVir RT All 1.0 Plasma 200 upwards 48 Load V3 Aptima HIV-1 RNA Qualitative Assay Cavidi Reverse transcriptase assay Gen-Probe TMA RNA A G, N, O 0.5 Plasma, serum

27 Short, medium and longer term product development priorities in HIV-related diagnostics There are no POC testing options currently available for EID or for VL testing, although as figure 2 shows, several are under development. It is anticipated that technologies in the pipeline will have lower instrument and per-test costs, and lower throughput requirements compared to laboratory-based systems. FIGURE 2. TECHNOLOGY PIPELINE POC VL AND EID (ESTIMATED TIMELINE AND SEQUENCE MIGHT CHANGE) Micronics Alere NAT ALL Liat Gene XPert SAMBA VL SAMBA EID NWGHF EID NWGHF VL Lumora WAVE 80 ECOSCAPE Cavidi AMP Biohelix Source: UNITAID- HIV/AIDS Diagnostic Technology Landscape, 2nd Edition,

28 WHO Expert Meeting Report Geneva, Switzerland, 6 7 June 2012 The potential advantages and disadvantages of POC testing Reduce the need for large infrastructure investments in equipment. Reduce the need for service and maintenance. Reduce the per test costs. Yield same day results for prompt clinical decision-making. Improve patient retention. Reduce the need for sample transport network. There are technical issues to be considered with the placement of POC devices, such as reagent stability, remote connectivity, remote QC management, laboratory information system (LIS) and on site waste disposal. TABLE 7. ADVANTAGES AND DISADVANTAGES OF POC TESTING IN EID Advantages Immediate diagnosis Generally no laboratory personnel required Allows for wider coverage of diagnostic service Disadvantages Requires strict monitoring of QA which cannot be attained at lower levels Trained staff to operate Increased workload at facility level Stock-outs Cost There are several EQA programmes for EID and VL diagnostics available. Some of the challenges include limited number of providers, limited capacity with existing providers, and cost. EQA should be part of a comprehensive QA programme. 3.2 DISCUSSION WHO requirements for HIV testing in infants less than 18 months of age include a minimum sensitivity of 95% (but preferably 98%) and a specificity of 99%. Improvements in HIV tests include automation, which reduces technologist time and improves turnaround time, and improved sensitivity and specificity with fewer incorrect or invalid results. They are able to detect lower levels of virus, enabling earlier diagnosis of HIV, including in the presence of antiretrovirals (ARVs) for the prevention of mother-tochild transmission (PMTCT). Among the technologies available for early diagnosis of HIV in infants, DNA PCR is the most widely used and is generally considered to be the standard method. Recent studies have demonstrated that RNA PCR is a reliable and a suitable alternative. HIV RNA/TNA methods are also used for monitoring the response to treatment, potentially reducing the cost of technology investment. 4 4 Both HIV DNA PCR and HIV RNA PCR are recommended by WHO for the early diagnosis of HIV infection in infants. Total nucleic acid (TNA) is an acceptable alternative as DNA and RNA can be amplified separately. HIV RNA is useful only if there is viral replication, i.e. no recent treatment exposure; p24 is also useful only if there is viral replication. Immunoassays are used for the diagnosis of HIV infection in infants and children aged months and 6 weeks or more after the cessation of all breastfeeding (no maternal antibodies present)

29 Short, medium and longer term product development priorities in HIV-related diagnostics Sampling collection methods include EDTA plasma, plasma preparation tube (PPT), DBS, and dried plasma spot (DPS). Plasma is difficult logistically because blood must be kept between 2 C and 25 C and must be processed in the laboratory within six hours of blood draw. DBSs are much easier logistically with storage in an airtight bag, and transport at ambient temperature (even via post). The use of DBS greatly simplifies the transport of samples and provides an enhanced specimen stability and ease of use for health care workers. DBS is cost effective. Studies have demonstrated a good correlation between DBS and plasma specimens for VL monitoring, albeit with slightly lower sensitivity for DBS specimens. Advanced nursing skills are not required, but health care workers need to be trained to collect and process samples correctly. In the laboratory, cutting of DBS leads to increased turnaround time, and increased sample handling creates the possibility of sample contamination. The use of pre-punched cards has reduced these problems, and their use should be promoted. Remaining challenges are obtaining good quality specimens, the choice of paper and the provision of collection supplies in a bundle. In this context (EID), samples preferably should be obtained by heel stick (HS) and collected as WB samples, and transported as DBS (rather than directly to filter paper), to ensure that adequate quantities are collected for processing. A case study was presented to demonstrate the experience in Malawi of quantifying VL for monitoring ART using DBS. This study compared VL measured on 50 μl finger stick DBS compared with plasma sampling. The NucliSENS EasyQ v2 assay was used on 72 patients with suspected treatment failure. TABLE 8. QUANTIFYING VL IN MALAWI Sensitivity Specificity PPV NPV At 5,000 copies/ml 90.8% 100% 100% 98% At 1,000 copies/ml 89.7% 96.8% 88% 98% In another case study, scaling up EID from 2004 to 2010 in South Africa has provided an opportunity to assess diverse technologies and evaluate different sample collection modalities. In 2002, samples collected were predominantly WB. Currently, with more than 1.5 million tests per year performed for EID, more than 75% are collected as DBS. 3.3 KEY STATEMENTS ON THE ROLL OUT OF EID AND VL TECHNOLOGIES A clear framework is needed that allows funders and potential users to assess the technical specifications and performance characteristics of new tests. Technologies for POC are important for lower levels of the health care system but could improve access to testing at all levels. Access to laboratory-based testing is essential at higher level laboratories and the choice of POC or laboratory-based technology should be volume driven and modular based. The development of POC tests for EID and VL should be expedited, with the location of services and devices appropriate to the local epidemic. POC testing should deliver results on the same day, be linked to care and treatment and have a capacity for a minimum of ten tests per day. Capacity requirements will really 25