Each of these approaches continues to be challenged by the variability of the immune response among recent HIV-1-infected persons, by differential performance in populations infected with different HIV-1 subtypes, and by the impact of ART, late-stage AIDS immunosuppression, and ‘elite controller’ status on individual subject misclassification. It remains unclear exactly how, or to what extent each of these factors influences the precision and specificity of available serologic assays in determining recent HIV seroconversion. However, such factors can lead to an overall lack of specificity in identifying persons with recent infection. Moreover, there is mounting evidence that many STARHs assays (and particularly the BED assay) are susceptible to high rates of misclassification of individuals with long-standing infections as recently infected [i.e., high ‘false recent rates’ (FRR)], impairing their performance in many settings.
To address this issue, several groups have recently proposed using test algorithms employing two or more incidence assays in sequence, as part of a Recent Infection Testing Algorithm (RITA) to reduce the FRR and increase the accuracy of incidence estimations . An example of the application of a RITA based on two different incidence assays in combination with additional clinical information on CD4 cell count and antiretroviral treatment is shown in Fig. 2. This approach is reasonable and merits further evaluation, but may increase the complexity and cost of applying tests for recent infection . The inclusion of CD4 cell counting in the testing algorithm requires whole blood samples, which implies that this information would not be available with dried blood spot specimens typically collected in national population-based surveys. In addition to these problems with assay performance, problems persist with standardization and quality control, and with the cost and continued availability of the commercial assays that have been adapted for incidence testing .
A number of leading public health organizations and scientists involved in incidence assay development and application have recently launched a concerted effort to overcome technical barriers, and to develop robust approaches based on immune maturation testing for incidence estimation. To advance this effort, in 2008, WHO formally convened a Technical Working Group on HIV Incidence Assays (http://www.who.int/diagnostics_laboratory/links/hiv_incidence_assay/en/index.html). This group is made up of epidemiologists, laboratory specialists, and public health officials. Several gaps were identified in assay development, validation, and commercialization. For example, there has been a lack of standard terminology and no clear consensus on statistical methods to use for HIV incidence estimation. It was also evident that the assays currently available and employed have not yet been rigorously validated using appropriate, comprehensive sample sets that would allow a standardized comparison of their performance, and a consideration of the benefits of combining assays into RITAs. The Bill & Melinda Gates Foundation (Seattle, Washington, USA), recognizing the pressing need for assays for estimation of HIV incidence to support prevention and intervention trials, has provided funding and support for the Working Group to tackle these limitations and challenges.
In 2009, Family Health International (FHI) conducted a global landscape and market assessment, to estimate demand for HIV incidence assays (http://www.who.int/diagnostics_laboratory/links/assays_to_estimate_hiv_incidence.pdf). Qualitative interviews with key informants, conducted to assess the potential market for HIV incidence assays, revealed that conceptions of acute and recent HIV infection are often conflated, and terms are used inconsistently. The BED assay was the most widely known and the most commonly used incidence assay among interviewees. Although the BED assay was widely known, acceptance and use of this assay were intensely disputed. Although some praised its low cost and convenience, many investigators familiar with its use strongly criticized the BED assay's tendency to overestimate HIV incidence. Interviewees uniformly voiced a desire for assays with improved performance and were willing to accept incremental increases in cost. About half of interviewees expected that assay performance improvements would lead to increased demand for their use. Donor endorsements and expanded surveillance activities were cited as potential influences on demand.
Estimates of potential market size under each of the three scenarios described above are provided in Table 3. The results indicate that an assay that could be used to identify a sample as HIV-seropositive and from a recent infection could result in an increased demand of 6–12 million specimens tested over 5 years. This increased demand would be owing, in part, to the use of such assays to screen specimens of unknown HIV-serostatus, as opposed to use only on HIV-seropositive specimens as for current HIV incidence assays. However, the limitations of the assays that are currently available have been widely noted and reviewed. Normative bodies have not fully endorsed their use, and consequently, current demand is far below potential future demand.
To date, none of the above approaches have been subjected to rigorous evaluation with regard to their application in incidence estimation. For the moment, the WHO Working Group remains primarily focused on the further development and validation of approaches based on serologic assays for recent infection. The group will explore a supplemental role for one or more of these alternative biomarkers, as well as the use of HIV RNA (to identify individuals with very low-level viremia in elite controllers and HAART-treated patients) and CD4 cell count data (to detect advanced AIDS cases), for identification and exclusion of cases of long-standing infection that are misclassified as recent infection (i.e., reduce the FRR of RITAs).
An essential element of the development and evaluation process for the calibration and validation of assays for recent infection is the availability of relevant biological sample sets. An ongoing research program has been launched to identify and catalogue studies that could potentially contribute specimens to panels that could be used to develop, calibrate, and evaluate assays for recent HIV infection. Results of literature search and survey results from this effort are available in a report to be made available at http://www.who.int/diagnostics_laboratory/links/hiv_incidence_assay/en/index.html. We were able to identify scores of studies capable of collecting and storing specimens that would be highly relevant to evaluation of tests for recent infection (e.g., seroconverter cohorts and studies enrolling known nonrecently infected patients, including elite controllers and patients with advanced disease). However, the overwhelming majority of these existing research studies maintain archived plasma specimens in only limited quantities and for limited numbers of individuals.
In recognition of these results, the WHO Working Group has organized an effort to collect large numbers of well pedigreed and large-volume blood specimens, sufficient to allow head-to-head evaluation of currently available assays. The specimen repository that is currently being planned will optimally include some archived specimens, but will also include specimens collected prospectively for this purpose by collaborating cohorts and blood donation programs with ready access to specific types of patients.
In April 2009, a statistical methodology subgroup of the WHO Working Group met to discuss the latest progress in the theoretical underpinnings of RITA-based cross-sectional incidence inference. If the RITA characteristics – now described as the mean RITA duration or ‘RITA interval,’ and the FRR – are known with precision, in principle this allows incidence to be estimated with minimal bias. Two subtly different versions of this method have been described [37,38], and it has been shown that this approach addresses limitations of previous approaches . Crucially, it is now possible to calculate the uncertainty of incidence estimates by systematically taking into account the statistical effects of sampling in the cross-sectional survey, as well as the uncertainty with which the performance characteristics of the RITA are known. Spreadsheets implementing the methods agreed by the WHO Working Group can aid in the calculation of CIs, P-values, sample sizes and power, and are available on the website of the South African Centre for Epidemiological Modeling and Analysis, http://www.sacema.com/page/assay-based-incidence-estimation.
A framework for systematically characterizing a RITA in terms of a mean RITA interval and a FRR also facilitates an improved analysis of the fundamental trade-offs faced by developers of better tests. In particular, it is desirable to have the mean duration of the test-defined recent state to be as long as possible (to enable small survey sample sizes) while having a low FRR (thus reducing the statistical blurring of any systematic adjustment for this effect). These two crucial factors are fundamentally in conflict, and this is the main source of difficulty in identifying suitable biomarkers and thresholds.
The deterioration of statistical power with an increasing FRR is particularly dramatic, from which it follows that imprecise estimates of the FRR can lead to considerable bias in incidence estimation. No useful analysis of incidence can be done without a reasonably precise estimate of a relatively small FRR, of the order of one or two percentage, although statistical tests for trends can at least be performed without precisely estimating the FRR in the tested population.
Necessity frequently breeds invention. In this case, the need for recent infection testing is most clearly felt by the public health organizations, which need better information on HIV incidence. Importantly, investigators and sponsors of intervention and vaccine trials need to identify high incidence subpopulations in which to conduct studies and then accurately measure incidence as important efficacy endpoints. Because of the lack of market incentive for development and evaluation of recent infection assays, it is likely that public institutions and private foundations will need to assume a greater role to advance development and ensure availability and appropriate application of RITAs for incidence estimation. But this effort, now over a decade in evolution, is vital for the control of the HIV pandemic.
1. Mastro TD, Kim A, Hallett T, Rehle T, Welte A, Laeyendecker O, et al. Estimating HIV incidence in populations using tests for recent infection: issues, challenges and the way forward
. J HIV/AIDS Surveil Epidemiol
2010 [Epub ahead of print].
2. Brown T, Salomon JA, Alkema L, Raftery AE, Gouws E. Progress and challenges in modelling country-level HIV
epidemics: the UNAIDS Estimation and Projection Package 2007. Sex Transm Infect 2008; 84(Suppl 1):i5–i10.
3. Hallett TB, Zaba B, Todd J, Lopman B, Mwita W, Biraro S, et al
. Estimating incidence from prevalence in generalised HIV
epidemics: methods and validation. PLoS Med 2008; 5:e80.
4. Rehle TM, Hallett TB, Shisana O, Pillay-van Wyk V, Zuma K, Carrara H, Jooste S. A decline in new HIV
infections in South Africa: estimating HIV
incidence from three national HIV
surveys in 2002, 2005 and 2008. PLoS One 2010; 5:e11094.
5. Petersen LR, Satten GA, Dodd R, Busch M, Kleinman S, Grindon A, Lenes B. Duration of time from onset of human immunodeficiency virus type 1 infectiousness to development of detectable antibody. The HIV
Seroconversion Study Group. Transfusion 1994; 34:283–289.
6. Schreiber GB, Busch MP, Kleinman SH, Korelitz JJ. The risk of transfusion-transmitted viral infections. The Retrovirus Epidemiology Donor Study. N Engl J Med 1996; 334:1685–1690.
7. Busch MP, Lee LL, Satten GA, Henrard DR, Farzadegan H, Nelson KE, et al
. Time course of detection of viral and serologic markers preceding human immunodeficiency virus type 1 seroconversion: implications for screening of blood and tissue donors. Transfusion 1995; 35:91–97.
8. Fiebig EW, Wright DJ, Rawal BD, Garrett PE, Schumacher RT, Peddada L, et al
. Dynamics of HIV
viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV
9. Brookmeyer R, Quinn TC. Estimation of current human immunodeficiency virus incidence rates from a cross-sectional survey using early diagnostic tests. Am J Epidemiol 1995; 141:166–172.
10. Hecht FM, Busch MP, Rawal B, Webb M, Rosenberg E, Swanson M, et al
. Use of laboratory tests and clinical symptoms for identification of primary HIV
11. Pilcher CD, Fiscus SA, Nguyen TQ, Foust E, Wolf L, Williams D, et al
. Detection of acute infections during HIV
testing in North Carolina. N Engl J Med 2005; 352:1873–1883.
12. Zetola NM, Pilcher CD. Diagnosis and management of acute HIV
infection. Infect Dis Clin North Am 2007; 21:19–48, vii.
13. Janssen RS, Satten GA, Stramer SL, Rawal BD, O'Brien TR, Weiblen BJ, et al
. New testing strategy to detect early HIV
-1 infection for use in incidence estimates and for clinical and prevention purposes. JAMA 1998; 280:42–48.
14. Rawal BD, Degula A, Lebedeva L, Janssen RS, Hecht FM, Sheppard HW, Busch MP. Development of a new less-sensitive enzyme immunoassay for detection of early HIV
-1 infection. J Acquir Immune Defic Syndr 2003; 33:349–355.
15. Kothe D, Byers RH, Caudill SP, Satten GA, Janssen RS, Hannon WH, Mei JV. Performance characteristics of a new less sensitive HIV
-1 enzyme immunoassay for use in estimating HIV
seroincidence. J Acquir Immune Defic Syndr 2003; 33:625–634.
16. McDougal JS, Pilcher CD, Parekh BS, Gershy-Damet G, Branson BM, Marsh K, Wiktor SZ. Surveillance for HIV
-1 incidence using tests for recent infection in resource-constrained countries. AIDS
2005; 19(Suppl 2):S25–30.
17. Le Vu S, Pillonel J, Semaille C, Bernillon P, Le Strat Y, Meyer L, Desenclos JC. Principles and uses of HIV incidence estimation
from recent infection testing: a review. Euro Surveill 2008; 13:18969.
18. Hare CB, Pappalardo BL, Busch MP, Karlsson AC, Phelps BH, Alexander SS, et al
. Seroreversion in subjects receiving antiretroviral therapy during acute/early HIV
infection. Clin Infect Dis 2006; 42:700–708.
19. Parekh BS, Kennedy MS, Dobbs T, Pau CP, Byers R, Green T, et al
. Quantitative detection of increasing HIV
type 1 antibodies after seroconversion: a simple assay for detecting recent HIV
infection and estimating incidence. AIDS
Res Hum Retroviruses 2002; 18:295–307.
20. Suligoi B, Galli C, Massi M, Di Sora F, Sciandra M, Pezzotti P, et al
. Precision and accuracy of a procedure for detecting recent human immunodeficiency virus infections by calculating the antibody avidity index by an automated immunoassay-based method. J Clin Microbiol 2002; 40:4015–4020.
21. Suligoi B, Massi M, Galli C, Sciandra M, Di Sora F, Pezzotti P, et al
. Identifying recent HIV
infections using the avidity index and an automated enzyme immunoassay. J Acquir Immune Defic Syndr 2003; 32:424–428.
22. Chawla A, Murphy G, Donnelly C, Booth CL, Johnson M, Parry JV, et al
. Human immunodeficiency virus (HIV
) antibody avidity testing to identify recent infection in newly diagnosed HIV
type 1 (HIV
-1)-seropositive persons infected with diverse HIV
-1 subtypes. J Clin Microbiol 2007; 45:415–420.
23. Barin F, Meyer L, Lancar R, Deveau C, Gharib M, Laporte A, et al
. Development and validation of an immunoassay for identification of recent human immunodeficiency virus type 1 infections and its use on dried serum spots. J Clin Microbiol 2005; 43:4441–4447.
24. Semaille C, Barin F, Cazein F, Pillonel J, Lot F, Brand D, et al
. Monitoring the dynamics of the HIV
epidemic using assays for recent infection and serotyping among new HIV
diagnoses: experience after 2 years in France. J Infect Dis 2007; 196:377–383.
25. Pillonel J, Barin F, Laperche S, Bernillon P, Le Vu S, Brunet S, et al
. Human immunodeficiency virus type 1 incidence among blood donors in France, through 2006: use of an immunoassay to identify recent infections. Transfusion 2008; 48:1567–1575.
26. Wilson KM, Johnson EI, Croom HA, Richards KM, Doughty L, Cunningham PH, et al
. Incidence immunoassay for distinguishing recent from established HIV
-1 infection in therapy-naive populations. AIDS
27. Schupbach J, Gebhardt MD, Tomasik Z, Niederhauser C, Yerly S, Burgisser P, et al
. Assessment of recent HIV
-1 infection by a line immunoassay for HIV
-1/2 confirmation. PLoS Med 2007; 4:e343.
28. Barin F, Nardone A. Monitoring HIV
epidemiology using assays for recent infection: where are we? Euro Surveill 2008; 13:18967.
29. Murphy G, Parry JV. Assays for the detection of recent infections with human immunodeficiency virus type 1. Euro Surveill 2008; 13:18966.
30. Soroka SD, Granade TC, Candal D, Parekh BS. Modification of rapid human immunodeficiency virus (HIV
) antibody assay protocols for detecting recent HIV
seroconversion. Clin Diagn Lab Immunol 2005; 12:918–921.
31. Kshatriya R, Cachafeiro AA, Kerr RJ, Nelson JA, Fiscus SA. Comparison of two rapid human immunodeficiency virus (HIV
) assays, Determine HIV
-1/2 and OraQuick Advance Rapid HIV
-1/2, for detection of recent HIV
seroconversion. J Clin Microbiol 2008; 46:3482–3483.
32. Wei X, Liu X, Dobbs T, Kuehl D, Nkengasong JN, Hu DJ, Parekh BS. Development of two avidity-based assays to detect recent HIV
type 1 seroconversion using a multisubtype gp41 recombinant protein. AIDS
Res Hum Retroviruses 2010; 26:61–71.
33. Laeyendecker O, Rothman RE, Henson C, Horne BJ, Ketlogetswe KS, Kraus CK, et al
. The effect of viral suppression on cross-sectional incidence testing in the johns hopkins hospital emergency department. J Acquir Immune Defic Syndr 2008; 48:211–215.
34. Guy R, Gold J, Calleja JM, Kim AA, Parekh B, Busch M, et al
. Accuracy of serological assays for detection of recent infection with HIV
and estimation of population incidence: a systematic review. Lancet Infect Dis 2009; 9:747–759.
35. Stacey AR, Norris PJ, Qin L, Haygreen EA, Taylor E, Heitman J, et al
. Induction of a striking systemic cytokine cascade prior to peak viremia in acute human immunodeficiency virus type 1 infection, in contrast to more modest and delayed responses in acute hepatitis B and C virus infections. J Virol 2009; 83:3719–3733.
36. Hughes GJ, Fearnhill E, Dunn D, Lycett SJ, Rambaut A, Leigh Brown AJ. Molecular phylodynamics of the heterosexual HIV epidemic in the United Kingdom
. PLoS Pathog
37. McWalter TA, Welte A. Relating recent infection prevalence to incidence with a sub-population of assay nonprogressors. J Math Biol 2010; 60:687–710.
38. Wang R, Lagakos SW. On the use of adjusted cross-sectional estimators of HIV
incidence. J Acquir Immune Defic Syndr 2009; 52:538–547.
39. McWalter TA, Welte A. A comparison of biomarker based incidence estimators. PLoS One 2009; 4:e7368.