Moving towards a reliable HIV incidence test current ......We present the speciﬁc rationale for several of these innovations, and discuss important resources for assay developers

REVIEW ARTICLEMoving towards a reliable HIV incidence test – current status,resources available, future directions and challenges ahead

G. MURPHY1*, C. D. PILCHER2, S. M. KEATING3, R. KASSANJEE4,5,S. N. FACENTE2, A. WELTE4, E. GREBE4, K. MARSON2, M. P. BUSCH3,P. DAILEY6, N. PARKIN6,7, J. OSBORN6, S. ONGARELLO6, K. MARSH8

AND

J. M. GARCIA-CALLEJA 9 on behalf of the Consortium for the Evaluation andPerformance of HIV Incidence Assays (CEPHIA)† Foundation for Innovative New Diagnostics,UNAIDS and the WHO Technical Working Group1Public Health England, London, UK, 2University of California, San Francisco, San Francisco, CA, USA3Blood Systems Research Institute, San Francisco, CA, USA, 4The South African DST/NRF Centre ofExcellence in Epidemiological Modelling and Analysis (SACEMA), Stellenbosch University, Stellenbosch,South Africa, 5Department of Statistical Sciences, University of Cape Town, Cape Town, South Africa,6FIND, Geneva, Switzerland, 7Data First Consulting Inc., Belmont, CA, USA, 8UNAIDS, Geneva, Switzerland,9WHO, Geneva, Switzerland

Received 17 May 2016; Final revision 7 November 2016; Accepted 9 November 2016;first published online 22 December 2016

SUMMARY

In 2011 the Incidence Assay Critical Path Working Group reviewed the current state of HIVincidence assays and helped to determine a critical path to the introduction of an HIV incidenceassay. At that time the Consortium for Evaluation and Performance of HIV Incidence Assays(CEPHIA) was formed to spur progress and raise standards among assay developers, scientists andlaboratories involved in HIV incidence measurement and to structure and conduct a directindependent comparative evaluation of the performance of 10 existing HIV incidence assays, to beconsidered singly and in combinations as recent infection test algorithms. In this paper we report ona new framework for HIV incidence assay evaluation that has emerged from this effort over thepast 5 years, which includes a preliminary target product profile for an incidence assay, a consensusaround key performance metrics along with analytical tools and deployment of a standardizedapproach for incidence assay evaluation. The specimen panels for this evaluation have beencollected in large volumes, characterized using a novel approach for infection dating rules andassembled into panels designed to assess the impact of important sources of measurement error withincidence assays such as viral subtype, elite host control of viraemia and antiretroviral treatment.We present the specific rationale for several of these innovations, and discuss important resourcesfor assay developers and researchers that have recently become available. Finally, we summarize thekey remaining steps on the path to development and implementation of reliable assays formonitoring HIV incidence at a population level.

Key words: HIV/AIDS, incidence.

* Author for correspondence: Dr G. Murphy, Public Health England, 61 Colindale Avenue, London, UK.(Email: [email protected])† CEPHIA collaborators are listed in the Appendix.

Epidemiol. Infect. (2017), 145, 925–941. © Crown Copyright. Published by Cambridge University Press 2016doi:10.1017/S0950268816002910

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INTRODUCTION

Information on the rate of new infections is critical tomonitoring the HIV pandemic and to measuring theimpact of prevention interventions [1, 2]. Over 15years ago, Brookmeyer & Quinn [1] and Janssenet al. [3] introduced the idea that HIV incidencecould be reliably measured by conducting cross-sectional surveys and counting the number of indivi-duals with ‘incident’ infections detected by laboratoryassays. This method was proposed to overcome theexpense and bias associated with longitudinal cohortstudies and the complexities of mathematical model-ling. However, incidence estimation in cross-sectionalsurveys depends on being able to distinguish indivi-duals who have recent infection (i.e. ideally thoseacquiring infection in the last 12 months) from thosewith longstanding infection (>12 months).

However, a host of factors have adversely affectedthe performance of HIV incidence assays, resulting ina substantial number of ‘false recent’ infection resultsamong individuals who actually have longstandingHIV infections; the rate of such results can vary mark-edly between populations and over time. These factorsinclude variability in immune responses at both anindividual and population-level, variability by HIV-1subtype, access to antiretroviral therapy (ART),advanced HIV disease, and other factors that are notwell understood. Notably, the inability to account forthese factors when estimating incidence, especiallyfrom early generation assays, has led to unreliable esti-mates of the number and pattern of new infections insome countries. These limitations, as well as the limitedperiod for which some assays can identify recent infec-tion, present significant barriers to the developmentand application of HIV incidence assays.

In 2011, stakeholders convened as an IncidenceAssay Critical Path Working Group, to review chal-lenges and propose ways to improve assay developmentespecially for use in a surveillance and research context[4]. To help overcome these challenges, the Bill andMelinda Gates Foundation (BMGF) awarded a grantto form the Consortium for the Evaluation andPerformance of HIV Incidence Assays (CEPHIA).CEPHIA was tasked with: providing clear guidelinesfor cross-sectional incidence estimation; fostering scien-tific consensus through defining a target product profilefor an assay designed to assess incidence at a populationlevel; identifying development priorities and perform-ance metrics for incidence assays; and establishing arepository of specimens to assess the most promising

available incidence assays and enable developmentand rigorous evaluations of multi-test algorithms forincidence measurement. Creating this framework hassignificantly reshaped the landscape for developingand evaluating HIV incidence assays.

In this paper, we review these developments anddescribe the remaining challenges to establishing reliabletools, based on HIV recency assays, for cross-sectionalHIV incidence estimation at the population level. Todate, about 20 assays have been developed or adaptedto estimate HIV incidence. These assays work byusing markers of the maturity of immune response toclassify HIV-seropositive specimens as belonging toeither a recently or non-recently infected person. Until2012, only one dedicated incidence assay was commer-cially available (BED) [5]; at that time a second dedi-cated assay, the Sedia™ HIV-1 LAg-Avidity assay,was commercially released [6]. Most of the remainingassays now available are modified commercial HIVdiagnostic assays. Table 1 identifies the major assayscurrently available, as well as historically importantassays.

Multiple strategies are now used to improve inci-dence estimation efforts. For example, a combinationof antibody-based assays and viral load has led toimprovements in the accuracy of HIV incidence estima-tion, reducing the false recency ratio (FRR) of theseassays by mitigating the measurement error resultingfrom patients on ART. Building on this idea is the con-cept of a recent infection testing algorithm (RITA),which uses a series of assays in combination – oftenan HIV screening test, and antibody-based recencyassay, and a viral load assay. RITAs were recentlyused in household surveys in Kenya [7], Botswanaand South Africa [8]. Starting in 2015, the UnitedStates Government has been investing in population-based HIV impact assessment surveys (PHIAs) thatuse RITAs to measure the impact of HIV preventionprogrammes in about 20 sub-Saharan African coun-tries. It is important to have consensus on approachesto estimating incidence using RITAs, and to under-stand the implications of RITA performance on studydesign and analysis, to ensure that accurate and inform-ative incidence estimates are generated.

To promote consistency in estimating HIV incidenceusing RITAs at the population level, the WHOWorking Group on HIV Incidence Assays publishedguidelines in 2010 and have provided technical updatesin 2013 and 2015 [9, 10], with a further technical updateplanned for 2016. These guidelines reflect the most up todate recommendations for using HIV incidence assays

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Table 1. Historically important and currently available HIV incidence assays

HIV incidence assay Market type Principle Format Reference

Limiting antigen assay(LAg) – Sedia/Maxim

Commercial Single-well antibody avidity assay Microtitre plate-based assay Duong et al. [6]

CDC BioRad Avidity Modified commercial Two-well antibody avidity assay Microtitre plate-based assay Masciotra et al. [27]BED EIA - Sedia Commercial Proportion of total antibody that is HIV-specific Microtitre plate-based assay Parekh et al. [5]Ortho Vitros ECi anti-HIV1 + 2

Modified commercial Avidity of anti-HIV antibodies Automated sample processor andchemiluminescent analyser

Chawla et al. [31]

Ortho Vitros ECi anti-HIV1 + 2

Modified commercial Antibody titre – ‘detuned’ – standard assaysensitivity reduced to extend seroconversionwindow

Closed automated sample processor andchemiluminescent analyser

Keating et al. [26]

Architect Avidity, Abbott Modified commercial Avidity of anti-HIV antibodies Closed automated sample processor andchemiluminescent analyser

Suligoi et al. [32]

Geenius, BioRad Modified commercial Comparative reactivity between HIV antigens Line assay Keating et al. [26]Luminex-basedimmunoassay

Home-brew Reactivity of antigens, antibody titre, antibodyavidity

Open luminescent assay platform Curtis et al. [28]

IDE-V3 EIA Home-brew Reactivity with two selected HIV antigens is used topredict likelihood of recent infection

Microtitre plate based assay Barin et al. [33]

Glasgow BioRad Avidity Modified commercial Two-well antibody avidity assay Microtitre plate-based Sheppard et al. [34]IgG3 anti-HIV Home-brew Transient presence of IgG3 isotype antibodies

against HIV p24AgMicrotitre plate-based assay Wilson et al. [35]

InnoLIA HIV Modified commercial Relationship of reactivity with various HIV antigens Line assay Schupbach et al. [36]Particle agglutination(SeroDIA-HIV)

Modified commercial ‘Detuned’ – standard assay sensitivity reduced toextend seroconversion window

Particle agglutination assay Li et al. [37]

Abbott HAVAB (3A11) Modified commercial(withdrawn 2003)

‘Detuned’ – standard assay sensitivity reduced toextend seroconversion window

Microtitre plate-based assay Janssen et al. [3]

bioMérieux VironostikaHIV-1 microELISA

Modified commercial(withdrawn 2008)

‘Detuned’ – standard assay sensitivity reduced toextend seroconversion window

Microtitre plate-based assay Kothe et al. [38]

Abbott AxSYM HIV 1/2 gO Modified commercial(withdrawn 2013)

Avidity of anti-HIV antibodies Automated sample processor andenzyme immunoassy

Suligoi et al. [39]

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for surveillance and epidemic monitoring and have alsobeen used to inform a revision of the 2005 Guidelinesfor Estimating National HIV Prevalence published in2015 by the UNAIDS/WHO HIV Surveillance GlobalWorking Group to address the use of HIV incidenceassays in population surveys.

NEW RESOURCES FOR DEVELOPERSAND USERS OF INCIDENCE ASSAYS

Defining performance metrics, refining statistical tools.

The methods for calculating incidence estimates havebeen refined using laboratory assessment of recentinfection biomarkers [11–19]. The critical constructs,on which this characterization of recency tests rest, are:

. The protocol dependent, estimated date of detectableinfection. The specific assays chosen for use in anyparticular HIV infection screening protocol willhave an impact on when infection becomes detect-able, and, consequently, the calculation of the esti-mation of this date of detectable infection. Allassays have their own median delay of days, frominitial HIV exposure/acquisition until infectionbecomes detectable, which affects this calculation.It is important to be explicit about this largelyneglected ‘front end’ of the case definition of ‘recentinfection’. It is further necessary to account for‘NAAT yield’ (i.e. specimens that test positive forHIV nucleic acid markers, but prior to HIV anti-body seroconversion thus making the antibody-based recency assays unusable) in the case definitionof ‘recent infections’. These NAAT yield positivespecimens offer an increased mean duration ofrecent infection with no significant increase infalse recency, as long as the diagnostic algorithmis highly specific (false positives are likely to be clas-sified as recent on most existing incidence assays).

. Recency cut-off period (T). To determine whether anassay is producing false recent results, a time bound-ary for recency must be defined. Using this pre-defined T, a test result of ‘recent’ obtained from a spe-cimen whose subject was known to have had detect-able infection for a time longer than T can beproperly defined as a false recent result. CurrentWHO recommendations are for T to be set at 2years (http://www.unaids.org/sites/default/files/media_asset/HIVincidenceassayssurveillancemonitoring_en.pdf). Given the lack of a gold standard for ‘recent’infection and the approximate nature of most

estimated dates of infection, it is challenging to decideon an appropriate recency ‘cut-off’ time (T).Ultimately, the period of recency need not bedefined such that assays can be calibrated to shiftfrom ‘recent’ to ‘longstanding’ results at precisely T;rather, the decision regarding the value of T simplyrequires that the period of recency be sufficientlylong lasting so that surveys of feasible size can expectto detect a statistically stable number of recent infec-tions, yet not so long that surveys are reduced toessentially estimating prevalence only. Kassanjeeet al. [13] highlight a consistent method to estimateincidence from cross-sectional surveys using a recencycut-off beyond which a known incorrect ‘recent’ testresult can be reclassified – using this method, cut-offT is variable based on particular situations, and issensibly chosen so that the FRR (defined below) forthe test is very small.

. Mean duration of recent infection (MDRI). This isthe mean time that a group of subjects fit the‘recent’ case definition, after initial detectable infec-tion and within the period T. Note that this does notrequire that progression from recent to non-recent isa once-off transition. Therefore, MDRI calcula-tions consistently account for both inter-subjectvariability in biomarker development and intra-subject fluctuations, both of which are likely.

. False recency ratio (FRR). This is simply the pro-portion of those who are classified as recent bythe test, despite being known to be infected formore than time T. The FRR is context-dependent,because populations of interest have differing pro-portions of elite controllers, antiretroviral-treated vir-ally suppressed persons, and other characteristics lesswell understood that are likely to produce a recentfalse test, such as regional variation in prevalentHIV subtype and host genetic factors. Naturally,an ideal test would have an FRR of 0% in all con-texts, but in practice there will always be residualuncertainty about the FRR. When the FRR isshown to be sufficiently close to zero, biomarker-based incidence estimates will have a greater levelof precision.

Both in assay development and field application, theconcept of estimated date of detectable infection mustbe carefully considered:

. Estimated date of detectable infection (EDDI) of aparticular subject in a study. In practice, an EDDIis a summary measure of uncertain infection time



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(essentially a ‘plausibility window’) derived fromearliest plausible and last plausible dates of detect-able infection, obtained by interpreting diagnostictesting histories (further refinement is possible byincorporating uncertainty in the ‘diagnostic delays’of the relevant tests). A systematic measure ofEDDIs for individual subjects is necessary, as it isalmost inevitable that in a large set of specimensanalysed together, specimens would be obtainedfrom a range of different testing protocols; allthen need to be consistently processed when calcu-lating a single estimate such as MDRI. We proposethat it would be useful to standardize the definitionof ‘detectable infection’ as the date at which a sub-ject would first yield a detectable viral load on ahighly sensitive viral load assay, i.e. with a detectionthreshold of 1 copy per ml.

. Use of an MDRI offset related to the survey testprotocol used. In real-world testing protocols, infec-tions become detectable at different times postexposure depending on the assays used in the par-ticular protocol. The actual screening proceduredetermines the selection of specimens reflexed tothe recency-testing protocol, or meeting the casedefinition of ‘recent’ without further testing. Forthis reason, the MDRI must always be adapted tothe sensitivity of the actual screening procedure. Itwill seldom, if ever, be possible to find a study-specific MDRI in a table of values calculated by atest development or test benchmarking study.

The challenge for a developer of an incidence assay is tomaximise the MDRI (hence having more recent casesto count in a survey, thus improving statistical precisionand power at a given sample size) while keeping theFRR low (reducing the measurement error withinrecency data resulting from misclassified longstandinginfections). By specifying an intended-use context andinterpreting the impact of test properties on the preci-sion of the incidence estimate in that context, this trade-off between MDRI and FRR can be made precise [13] .While there are already recent infection case definitionswith a FRR around 1% in likely real-world contexts,existing tests currently obtain an MDRI of only a fewmonths (Table 4). MDRIs of close to 12 months areneeded to have usefully precise incidence estimates inthe order of 1% or 2% per annum, using samples of afew thousand individuals.

To demonstrate this, Figure 1 displays the impactthat MDRI and FRR have on the real-world applica-tion of these assays, using prevalence and expected

incidence for Botswana as an example. The figureshows the sample sizes required to support a statistic-ally significant comparison between two incidencemeasurements where the second measurement isexpected to be at least 40% lower than the first, plottedas a function of MDRI for several different values ofFRR. In addition to the MDRI and FRR (which arein turn impacted by variables such as HIV-1 subtype,extent of ART use and viral load suppression in thepopulation being surveyed), the sample size is alsodependent on factors related to the nature of the epi-demic (i.e. prevalence and expected incidence), thesurvey methods (and consequent requirement toinclude design effects), and the desired level of preci-sion associated with the incidence estimate. Forexample, in countries with higher incidence thanBotswana, the required sample sizes would be lowersince the expected number of events detected will behigher. Conversely, if the background prevalence ishigher, the required sample size will also be higher,since the proportion of HIV-positive individuals iden-tified in the survey who are recently infected is lower.Given the performance characteristics of currentlyavailable incidence assays, detection of a decrease inincidence of 540% at the national level can be per-formed using a sample size of 420 000 for only twocountries (Lesotho and Swaziland), while assay-basedestimation of incidence at a single point in time with arelative standard error of 30% can be achieved in nearlyall of the 14 countries with prevalence of at least 5% orincidence of at least 0·3% per annum, as recommendedby WHO and UNAIDS [10]. However there is signifi-cant uncertainty regarding the MDRI and FRRassumptions in countries where multiple subtypes areknown to be circulating, such as Kenya, Tanzaniaand Uganda (subtypes A and D) and Cameroon (circu-lating recombinant form CRF02_AG).

In general, to define and estimate the performance of arecent infection test, onemustfirst specify contextual fac-tors (such as incidence, prevalence, treatment coverage)and details of intended use. Then, various conventionalmetrics such as power and precision can be calculatedfrom these inputs, andpotentially be optimised as a func-tion of controllable inputs such as choice of test, or thethreshold applied to the test to define as ‘recent’.

Target product profile (TPP)

Determination of whether or not a particular diagnos-tic test is suitable for a specific application is typically

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achieved by comparing its performance characteristicsto a TPP. In 2011, a TPP for incidence assays wasdescribed to be applied to the estimation of incidencein a population, based on the experience of users ofearly HIV incidence assays, laboratorians, epidemiol-ogists and regulatory agencies [4]. The TPP includesminimal acceptable recommendations for MDRI(at least 120 days) and FRR (<2%). The TPP alsorequires that assay results be reproducible, and thetraining, equipment and sample type requirementsbe practical for the populations to be studied and loca-tion where testing will be performed. Additionally, theTPP addresses practical requirements of a test such asreagent storage conditions, sample testing volume andconditions, training, and infrastructure needs. TPPcriteria related to practicality of use were included toensure that evaluation of an assay takes into accountthe needs of resource-limited settings where complexor costly automated analysers may not be available.

In addition to incidence estimation in a population,recent infection tests could also be used for other pur-poses. Alternative use cases include assessment of theimpact of large-scale HIV prevention interventions,infection staging to guide individual treatment or public

health interventions (e.g. contact tracing), or case-basedsurveillance by central laboratories where additionaltypes of data (e.g. CD4+ T-cell counts and viral loads)maybe available.Thus, additionalTPPsmaybe requiredthat correspond to other use cases (see Table 2 for a sum-mary of potential use cases for HIV incidence assays).

The expansion of use cases and TPPs for HIV inci-dence assays may also impact the market size. In 2010,a market landscape assessment was performed todescribe the projected demand for incidence assaysunder several different scenarios (http://www.who.int/diagnostics_laboratory/links/assays_to_estimate_hiv_incidence_jun_2010.pdf). An updated market land-scape assessment has been commissioned to provideup-to-date information on the potential market forthese assays, taking into account the potential forwider use, including on an individual patient basis.

Defining an HIV incidence assay development criticalpath

The critical path for an incidence assay depends on itsposition on the spectrum ranging from early biomarkerdiscovery to post-marketing performance evaluation.

Fig. 1. Impact of mean duration of recent infection and false recency ratio on sample size requirements to detectreductions in incidence. This figure demonstrates the sample size required to detect a 40% reduction in incidence for thecountry of Botswana (power 0·8, alpha 0·05, design effect 1·3). Calculations come from https://finddx.shinyapps.io/Sample_size_calculator/.



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Figure 2 illustrates some of the distinct challengesfacing developers at each stage of assay discovery anddevelopment; each of these challenges requires specifictypes of resources. Historically, the focus of HIV inci-dence assays was on the maturation of the humoralimmune response. However, it is increasingly clearthat the assays that are likely to be most useful in thenext decade are only now being developed. Newapproaches include rapid tests for incidence andnumerous non-traditional approaches, which arebeing developed with the hope they may not be suscep-tible to measurement error by ART use. In 2011 theNational Institute of Allergy and Infectious Diseases(NIAID) issued a call for studies of viral diversitymeasurement approaches [20–23], followed in 2012 bya call for novel HIV incidence biomarker discoveryprojects from the BMGF [24]. These early-phase dis-covery projects (see Table 3) have resulted in a renewedemphasis on, and increased resources for, development,optimization and validation work.

Repositories of blood and non-blood specimens, andconstruction and distribution of panels for incidenceassay development and evaluation

Previously, a major barrier to development, optimiza-tion and independent evaluation of HIV incidence

assays was the lack of availability of suitable speci-mens but the creation of relatively large specimenrepositories for incidence assay evaluation by theCenters for Disease Control (CDC) and NationalInstitutes of Health (NIH) has enabled rigorousassessment of several assays, leading to criticalinsights on the potential utility of RITAs [2, 4–6, 23,25–28]. However, the specimens collected in theserepositories were available in only limited volumes.

CEPHIA, on the other hand, received funding tobuild a larger repository that could facilitate directcomparative evaluations of assays and biomarker dis-covery. The effort focused on acquiring plasma speci-mens from subjects who had enrolled in prospectiveincidence cohorts from diverse countries of originwhere the dates of detectable infection were knownwith a high level of confidence. Specimens were add-itionally acquired from collaborators who studiedlong-term HIV disease outcomes, response to anti-retroviral treatment, and elite controllers, all ofwhich are populations critical to the establishmentof FRRs applicable to real-world settings. Plasma spe-cimens with large volumes were sought, since theavailability of multiple identical panels of pedigreedsamples would greatly simplify the future task of com-parative assay performance evaluations. Throughactive collaboration with existing clinical HIV

Table 2. Potential uses for HIV incidence assays

Use Description of use

1. Incidence estimate for national surveillance To provide national estimate of incidence; may be part of a broaderdemographic study

2. Incidence estimation for programme, preventionor trial planning

To provide incidence estimate in sub-populations for planning,prioritizing, or other instances when an estimate of incidence isrequired. May often be for only a city or region (e.g. prioritizeprogrammes or investments, or identify sites for intervention trials)

3. Incidence estimate in key or sentinel populations To provide incidence estimates in special sub-population using targetedsampling methods

4. Incidence estimation to assess the impact ofpopulation-level interventions

To assess the impact of a population-level intervention (e.g.community-level intervention) by comparing incidence before and afterthe intervention

5. Incidence estimate from case-based surveillance To provide national or regional incidence estimates via case-basedreporting of newly identified HIV+ individuals

6. Identification of individuals with ‘recent’infections for research purposes

Identification of individuals with ‘recent’ infections for multiplepotential applications (e.g. recruitment of recently infected individualsinto longitudinal cohort studies)

7. Identification of patients with ‘recent’ infectionsfor individual patient management

Identification of patients with ‘recent’ infections for to guide clinicalmanagement and/or public health programmes (e.g. selecting therapy,and/or prioritizing contact tracing)

8. Targeted prevention planning To provide population-level data on recent infections to enable riskfactors analysis or identify hotspots to inform targeted preventionplanning (no incidence estimate is obtained)




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research cohorts and blood banks interested in thedevelopment of HIV incidence assays, the CEPHIArepository now includes more than 9000 highlyselected plasma specimens for which an estimateddate of detectable infection could be calculated usinga standardized approach, or which fell into specificother categories known to result in measurementerror for recent HIV infection (elite controllers,ART-suppressed, etc.). Most samples in the repositoryhave at least 10 ml of plasma available and theyrepresent a broad diversity of subtypes and geograph-ies (see Fig. 3). In the last 3 years, many of theConsortium’s clinical collaborators have begun collect-ing alternative specimen types such as serum, driedblood spots, urine, saliva and stool, which have alsobeen added to the CEPHIA repository to supportevaluation and development of new tests and novelbiomarkers. Relevant clinical and laboratory data,including detailed information on predicate HIV diag-nostic testing, CD4+ T-cell counts, viral load, ARTstatus and co-infections, are ascertained for allsubjects/samples and maintained in a dedicated data-base. Where possible these specimens have been

benchmarked against the currently best-performingincidence assays.

New specimen panels and support for biomarkerdiscovery and assay development

CEPHIA specimen panels have been prepared and areavailable for biomarker discovery work or assayevaluation. Specimen information is accessible andsearchable online, along with instructions for access,on the CEPHIA website (www.incidence-estimation.com/CEPHIA). Applications for repository accessare reviewed to ensure that this valuable resource isused appropriately. Currently supported studiesinclude both focused hypothesis-driven studies (forinstance, how the gut inflammasome and specific sub-classes of HIV antibodies change during the transitionfrom recent to longstanding HIV infection); andnon-hypothesis-driven efforts to identify signatures ofrecent HIV infection, including numerous types ofnovel biomarkers (e.g. searches for antibodies reactiveto peptoids in a large ‘peptoid shape library’; exosomalor extracellular microRNAs; and urinary and blood

Fig. 2. CEPHIA incidence assay critical path. Listed on the right are specific milestones that should be demonstratedbefore an assay moves forward to further development.



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Table 3. Biomarker discovery and new assay development for HIV incidence measurement

Organization Funder Project title Assay format/platformSpecimentypes Development stage

Beijing Genome Instituteand Blood SystemsResearch Institute

BMGF Circulating cellular microRNAs inplasma as biomarkers of HIV-1 infection

Multiplex molecular platform Plasma,PBMCs

Proof of concept,inactive

Duke University BMGF Multiplex antibody markers Multiplex immunoassay platform Plasma,urine, saliva

Proof of concept

Duke University BMGF,Self

Cell associated viral load Molecular platform PBMCs Proof of concept,inactive

University of Pittsburgh BMGF Detection of recent HIV-1 infectionsbased on naturally inspired syntheticoligomers

Multiplex immunoassay platform Plasma Proof of concept,inactive

ISI Global (CRESIB) BMGF Novel GI biomarkers for HIV incidence Multiplex immunoassay platform for antibodiesand cytokines

Plasma,stool,PBMCs

Proof of concept

Metabolistics BMGF Urinary metabolite HIV recencybiomarker profile for incidencemeasurement

NMR spectrum Urine,serum/plasma

Qualification,inactive

Johns Hopkins NIH Laboratory and statistical development ofcross-sectional HIV incidence assays

High-resolution melting (HRM) assay based onincreasing HIV genetic diversity over time sinceinfection

Plasma Qualification/evaluation(non-CEPHIA)

Immunetics NIH Rapid test for recent HIV infection Rapid test format based on CDC LAg assay Plasma Qualification/evaluation(non-CEPHIA)

Sedia Biosciences NIH Development and commercialization ofan innovative rapid HIV-1 incidenceassay

Rapid test format based on CDC LAg assay Plasma Qualification/evaluation

University of SouthernCalifornia

NIH HIV incidence assay via deep sequencingand statistical tests

Viral biomarker based on inter-sequence Hammingdistance via deep sequencing.

Plasma Discovery

Harvard School of PublicHealth

NIH Accurate and efficient measures for HIVincidence

Measure changes over time in the variability of HIVenv gene sequence.

Plasma Discovery

Blood Systems ResearchInstitute

NIH High throughput measurement ofenvelope gene diversity for an HIVincidence assay

Adapting a low-cost assay based on DNAhybridization kinetics (AmpliCot) to measure HIVgene complexity to distinguish recent from chronicinfection.

Plasma Discovery

NCI/NIH NIH HIV-1 genetic variation in infectedindividuals

Using mutations detected by clinical drug resistanceassays to distinguish recent from chronic infection.

Plasma Discovery

Beth Israel DeaconessMedical Center

NIH Measurement of antibody epitopesignatures by peptide microarrays todetermine recency of HIV infection

High throughput antibody-based assay Plasma Discovery

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metabolites). Table 3 summarizes a number of newapproaches being undertaken. If successful, any ofthese new biomarkers will face the challenging transi-tion from research assays to kit-based assays that canbe successfully transferred and implemented in the field.

For assay developers, CEPHIA has developed rela-tively small panels ranging from 100 to 300 specimensin each panel. These panels are available in multiplespecimen types and in multiple replicates.

. A recency biomarker screening panel containing 125specimens from recent and longstanding infections,as well as HIV-negative specimens, is available forearly stage explorations. This panel is available ina wide variety of specimen types.

. For more advanced biomarker candidates, a proof ofconcept panel can be distributed. To determine if acandidate marker has a plausible MDRI (e.g. 4–12months), this panel contains 150 specimens with well-characterized EDDIs from the first 2 years post-infection, and 150 specimens from subjects knownto have longstanding infections ranging from 2 to11 years post-infection. These 300 specimens arebalanced between B and C subtypes. There are anadditional 50 ‘challenge’ specimens, which are 10 vir-ally suppressed specimens from subjects treated within60 days of the last plausible date of detectable infec-tion, and 40 specimens treated later after infectionbut nonetheless virally suppressed.

Specimen panels for independently assessingperformance of HIV incidence assays

For assays where preliminary data are available andan independent evaluation by the CEPHIA is desired,two specific specimen panels have been created:

. A qualification panel (n= 250) is provided under code(blinded) to researchers whose assays have reached anadvanced and well-defined set of criteria, includingthat the assay is available in a kit format. The develo-pers report blinded results that are evaluated by theCEPHIA group to confirm that the assay can reliablydistinguish recent from longstanding infections.

. For assays that are thus qualified, an evaluationpanel (n= 2500) is used by CEPHIA laboratoriesin blinded assay evaluations as part of a processdescribed below. The composition of the evaluationpanel allows determination of the effect of a num-ber of known sources of measurement error forthe performance of the assay (e.g. HIV subtype,T

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antiretroviral treatment, elite controller status andadvanced immunodeficiency) and enables determin-ation of an MDRI and FRR for each assay (orcombinations of assays). Each evaluation panelalso includes multiple blinded aliquots of pedigreedsamples (25 replicates of each) with antibodyreactivity characteristic of recent, intermediate andlongstanding infection to allow evaluation of thereproducibility of each candidate assay.

A process for independent evaluation of incidence assayperformance

The development of TPPs, a specimen repository, andtest panels that are now available represent a newstandard for evaluations of HIV incidence assays.Independent evaluations that are undertaken shouldaim to match the CEPHIA principles of:

(1) Using comprehensive panels of specimens (withspecimen background data blinded to the testoperator) and appropriate specimens to ensurethat statistical analyses of the results can quantifythe impact of known sources of measurementerror for incidence assays.

(2) Evaluations performed independently of the assaydevelopers.

(3) Assay developers/manufacturers provide standardoperating procedures, training, and certification ofproficiency of the laboratory prior to initiatingevaluations.

(4) Laboratories operate within a stringent qualitysystem and with rigorous procedures in place tomonitor and document the evaluations.

(5) All test output data analysed separately to thelaboratory where results can be unblinded, verifiedfor completeness, and compiled for analyses,ensuring all aspects of assay performance evalu-ated in addition to the qualitative output (recent/non-recent designations) from the assay.

To date, CEPHIA has completed 10 assay evaluations(see Table 4). Detailed analyses of individual assayperformance have been presented at various meet-ings and conferences and individual assay perform-ance summaries including assay usability are availableon request (http://www.incidence-estimation.com/page/CEPHIA-assay-evaluations); a report summarizingdata from the first five comparative assay evaluationshas been published separately [29].

These evaluations have highlighted the variabilityin assay performance and provided new insights onfactors to be considered in using tests and designingRITAs. For instance, the effect of viral suppressionon reliability of antibody-based incidence assays wasshown to be highly dependent on the timing of ARTinitiation (i.e. FRRs are substantially higher in sam-ples from persons treated early vs. later in infection).The analyses clearly indicate a need for inclusion ofviral load results in interpretation of results from allantibody maturation-based incidence assays, to miti-gate the impact of ART treatment and elite controllerson FRRs. Going forward, with the increasing uptakeof ART and the availability of laboratory-based dataon ART exposure to monitor progress towards theUNAIDS 90-90-90 targets, the inclusion of ARTdata in RITAs may become part of many nationalpopulation-based survey protocols. In contrast, the

Fig. 3. Clade/geographical breakdown of CEPHIA repository specimens.



http://www.incidence-estimation.com/page/CEPHIA-assay-evaluations)





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Table 4. Main characteristics of assays previously evaluated by CEPHIA

SpecificationSedia™ HIV-1Lag Avidity

BED HIV-1incidence assay

Bio-Rad GSHIV-1/HIV-2PLUS O EIAavidity (CDCmodification)

Ortho Avidity-Vitros ECi

Ortho Less Sensitive(LS)-Vitros ECi

BioRadGeenius™

AbbottArchitect Avidity

Bio-Rad GSHIV-1/HIV-2PLUS O EIAAvidity(Glasgowmodification) ANRS IDE-V3 CDC multiplex

False recencyratio (FRR),%

1·0 7·0 6·0 7·0 9·7 6·0 1·5 1·0 5·2 TBD

Mean durationrecentinfection(MDRI)(days)

188 302 333 285 285 179 128 88 216 TBD

Analyte HIV-1antibodies

HIV-1antibodies

HIV-1antibodies

HIV-1 antibodies HIV-1 antibodies HIV-1antibodies

HIV-1 antibodies HIV-1antibodies

HIV-1antibodies

Multiple HIVantibody andantigen

Sample type Serum orplasma/DBS

Serum orplasma/DBS

Serum orplasma/DBS

Serum or plasma Serum or plasma Serum orplasma

Serum or plasma Serum orplasma

Serum orplasma/DBS

Serum orplasma

Samplevolume

40 µl total 40 µl total 20 µl total 40 µl total 40 µl total 10 µl total 40 µl total 40 µl total 10 µl total 5 µl total

Infrastructurerequirements

Centralizedlab.Commercialassay, generallab.equipment

Centralizedlab.Commercialassay, generallabequipment

Centralizedlab.Modifiedcommercialassay,automatedplatform

Centralized lab.Modifiedcommercial assay,automated platform


Requirescompanysuppliedreader andaccess tosoftware toobtain banddata


Centralizedlab.Modifiedcommercialassay –general labequipment

General labequipment –userresponsiblefor platemanufacturerand qualitycontrol

Specialized labequipment –userresponsiblefor platemanufacturerand qualitycontrol

Storage/shippingconditions

Some reagentsare storedfrozen

Some reagentsare storedfrozen

4–25 °C 2–25 °C 2–25 °C 4–25 °C 4–25 °C 4–25 °C Conjugateshippedfrozen

4 °C; calibratorand controlsshippedfrozen

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Incubation withinautomated platform

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proficientwith 1 week’straining

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same evaluation showed no evidence for a relationshipbetween low CD4+ T-cell count and false-recent mis-classification on the evaluated assays; this finding isimportant because it calls into question the need toperform CD4+ T-cell testing, which adds cost andlogistical challenges in cross-sectional surveys. Inanother example, the benefit of independent evalu-ation, when compared to previous, developer-led stud-ies, was highlighted when conflicting results betweenthe evaluations contributed to the revision of pub-lished recommendations for interpretation of theSedia™ HIV-1 LAg-Avidity assay [30].

A major obstacle now facing the incidence assayfield is the cost and funding of future evaluations.As the funding from the BMGF comes to an end,identifying new support to maintain independentevaluation of future assays is critical. The cost ofcollecting suitable specimens, outside of existingsystems, means any new evaluation may cost up toUS$ 200 000 per evaluation, depending on re-quirements, which may be prohibitive to small com-panies trying to bring new products to market.

REMAINING CHALLENGES TO THEFIELD

Regulatory and policy issues

The potential use of assays to improve individualpatient management and further prevention effortsthrough discriminating recently infected subjects fol-lowing or coincident with initial HIV diagnosiscould substantially increase the market for their use[2]. However, applying the tests to named patientssamples with specific claims for use of results in clin-ical care will increase regulatory hurdles that mightlimit the application of assays in the field. To addressthese considerations, the CEPHIA evaluations wereundertaken using a comprehensive quality assurancesystem to ensure that accurate and complete recordsof the independent evaluation are fully documented.All sample characteristics, test performance documen-tation, and results can be shared with regulatory bod-ies should a company seek a regulatory claim on aparticular assay. The type and scope of informationrequired for regulatory approval of recency assayshas no precedent, and it remains to be seen whetherthe FDA, Council of Europe (CE) or other regulatoryagencies will accept such evaluations as suitable forinclusion in a regulatory claim. Should this not bethe case, then it is difficult to envisage whereT

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companies would be able to access sufficient numbersof appropriately characterized specimens to supportan evaluation.

In many countries, pre- and post-market regulatorycontrol for in-vitro diagnostics is often not adequate toensure that assay use in their markets will meet inter-national standards for safety, quality and perform-ance. In these countries, national authorities mayrely on the WHO for recommendations and advice.For assay developers, prequalification assessment bythe WHO, alongside a performance evaluation byCEPHIA or similar group, may be an alternative toseeking regulatory approval through US FDA orCE marking, which is targeted for the use of assaysin well-resourced laboratories rather than in resource-limited settings. WHO prequalification assessmentconsists of a review of a product dossier that substanti-ates the manufacturer’s claims for safety, quality andperformance alongside an inspection of the site(s) ofmanufacture to review the quality management sys-tem under which the assay is manufactured. Thechoice of pre-market assessment to be undertakenwill depend on where the assay will be supplied,such as in settings of high HIV incidence and in set-tings with greatest need for HIV intervention studies.

Laboratory standards and external quality assurance

During the performance of the CEPHIA evaluations,laboratories often interpreted published methods dif-ferently. These subtle differences may affect the per-formance of the assay and lead to different results.This has highlighted the need for unambiguous stand-ard operating procedures and for effective training inthe performance and interpretation of assays, alongwith the need for an independent quality assessmentsystem using blinded panels of well-characterized spe-cimens to monitor assay performance across labora-tories. Previously, such external quality assurance(EQA) programmes for the BED and Vironostika‘detuned’ assays were supported by the US CDCbut, as the use of these assays reduced, support forthese programmes has been withdrawn. Becausemost incidence assays are either in-house (‘home-brew’) tests, modified versions of commercially avail-able kits, or manufactured by small businesses withrelatively limited QA resources, in-process kit controlsare often unsuitable or insufficient to confirm that theassay is performing as expected in its modified format.Furthermore, it is unclear how sufficient fundingcould be obtained to support production and

management of EQA panels, given the limited marketfor some assays.

In collaborationwithCEPHIAandCDC, theExternalQuality Assurance Program Oversight Laboratory(EQAPOL), based atDukeUniversity, has begun aprofi-ciency testing programme for theLAg-Avidity assay. Thesuccess of this pilot effort offers hope that an independentEQA programme for incidence assays can be developed.However, some challenges remain in this area. The factthat a range of incidence assays are in use worldwidemakes it challenging to predict which other assays needto be included long-term in theEQAscheme, and fundingconstraints to the EQAPOL approach may inhibitwidespread participation of non-US-funded sites.Furthermore, there is an urgent need for EQA panelsthat include dried blood spot specimens aswell as plasma.

Global coordination to advance development andapplication of incidence assays

The WHO Technical HIV Incidence Assay Workinggroup (HIVIWG) has continued to meet on an annualbasis (http://www.who.int/diagnostics_laboratory/links/hiv_incidence_assay/en/) to provide technical guidanceand to advance efforts in this area of work (for example,see http://www.unaids.org/sites/default/files/media_asset/HIVincidenceassayssurveillancemonitoring_en.pdf).The WHO HIVIWG has supported a number of initia-tives, especially related to training of staff in the useand interpretationof data and in the preparationof guid-ance documents. Other groups, including the HIVModelling Consortium, UNAIDS Reference Group onEstimates, Modelling and Projections, and CEPHIAhave met frequently during the 5 years since the 2011Incidence Assay Critical Path meeting. However, itremains unclear how recommendations from groupssuch as CEPHIA will be implemented and endorsed bynormative guidance agencies, or how the regulatoryenvironment for incidence and recent infection assayswill evolve in the post-CEPHIA era. There is uncertaintyregarding funding for purchasing agreements that willguarantee assay supply on a continuing basis in the con-text of limited markets and reliance on small companiesfor themanufacture ofmany of these assays. The CDC’sGlobal Health Initiative provides an example of oneapproach, but one that does not extend to all countries.

CONCLUSIONS

Over the last few years there has been a continued inter-est in the development and application of incidence



http://www.who.int/diagnostics_laboratory/links/hiv_incidence_assay/en/

http://www.who.int/diagnostics_laboratory/links/hiv_incidence_assay/en/




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assays, which have evolved from simple techniquesinvolving the quality or quantity of antibody presentto now include more diverse biomarkers. This willincrease complexity as multiple and novel biomarkersare considered in determining recency, and will poten-tially lead to more specialized equipment beingrequired for biomarker-based incidence estimation.The potential of molecular assays (e.g. viral sequencediversity) for use in HIV incidence estimates, whichseemed considerable a few years ago, has not yet mate-rialized. The use of incidence assays for studies otherthan cross-sectional incidence determination hasincreased, and the desire for the assays to be used onan individual patient basis is gaining momentum.

Further, rapidly changing national guidelines onthe use of ART, resulting in earlier treatment of indi-viduals and increasing use of pre- and post-exposureprophylaxis will render the interpretation of incidenceassays more challenging in the future. This will requireimprovements in the current assays and a betterunderstanding of how to interpret data within a chan-ging and challenging environment.

The landscape for development of incidence assayapproaches has also been changed. A more completeconsensus has formed around a TPP and an assaydevelopment critical path. The formation of theCEPHIA has made more specimens, data, analysistools and technical support available to researchersthan ever before. However, the lack of coordinatedaction on purchasing agreements between funders,governments and developers will likely slow the roll-out of these assays, and uncertainty remains aboutassay regulation and provision of external qualityassurance. Given the considerable funds beinginvested in national surveys, it is disturbing that cur-rently there is only very limited funding available forproper quality control, training and evaluation ofHIV incidence assays. The development of sustainablefunding for this area is critical to ensure the qualityand accuracy of results, if recent and future develop-ments are to be translated into meaningful publichealth interventions.

APPENDIX. CEPHIA Collaborators

The Consortium for the Evaluation and Performanceof HIV Incidence Assays (CEPHIA) is comprised ofthe authors and: Tom Quinn, Oliver Laeyendecker(Johns Hopkins University); David Burns (NationalInstitutes of Health); Anita Sands (World HealthOrganization); Tim Hallett (Imperial College

London); Sherry Michele Owen, Bharat Parekh,Connie Sexton (Centers for Disease Control andPrevention); Anatoli Kamali (International AIDSVaccine Initiative); David Matten, Hilmarié Brand,Trust Chibawara (South African Centre forEpidemiological Modelling and Analysis); ElaineMckinney, Jake Hall (Public Health England); MilaLebedeva, Dylan Hampton (Blood SystemsResearch Institute); Lisa Loeb (The Options Study –

University of California, San Francisco); StevenG. Deeks, Rebecca Hoh (The SCOPE Study –

University of California, San Francisco); ZelindaBartolomei, Natalia Cerqueira (The AMPLIARCohort – University of São Paulo); Breno Santos,Kellin Zabtoski, Rita de Cassia Alves Lira (TheAMPLIAR Cohort – Grupo Hospital Conceição);Rosa Dea Sperhacke, Leonardo R. Motta, MachlinePaganella (The AMPLIAR Cohort – UniversidadeCaxias Do Sul); Helena Tomiyama, ClaudiaTomiyama, Priscilla Costa, Maria A. Nunes, GiseleReis, Mariana M. Sauer, Natalia Cerqueira, ZelindaNakagawa, Lilian Ferrari, Ana P. Amaral, KarineMilani (The São Paulo Cohort – University of SãoPaulo, Brazil); Salim S. Abdool Karim, QuarraishaAbdool Karim, Thumbi Ndungu, Nigel Garret,Nelisile Majola, Natasha Samsunder (CAPRISA,University of Kwazulu-Natal); Denise Naniche (TheGAMA Study – Barcelona Centre for InternationalHealth Research); Inácio Mandomando, Eusebio VMacete (The GAMA Study – Fundacao Manhica);Jorge Sanchez, Javier Lama [SABES Cohort –Asociación Civil Impacta Salud y Educación(IMPACTA)]; Ann Duerr (The Fred HutchinsonCancer Research Center); Maria R. Capobianchi(National Institute for Infectious Diseases ‘L.Spallanzani’, Rome); Barbara Suligoi (IstitutoSuperiore di Sanità, Rome); Susan Stramer(American Red Cross); Phillip Williamson (CreativeTesting Solutions/Blood Systems Research Institute);Marion Vermeulen (South African National BloodService); and Ester Sabino (Hemocentro do SaoPaolo).

DECLARATION OF INTEREST

None.

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Documents

Moving towards a reliable HIV incidence test current ......We present the speciﬁc rationale for several of these innovations, and discuss important resources for assay developers