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.
Following initial development of assays for recent HIV infection in the late 1990s, various terms have been used to describe assays and methods to estimate HIV incidence. In order to harmonize terms, WHO convened several meetings to discuss the most appropriate terms to describe assays for recent infection. The results of this meeting are summarized in Table 2 (http://www.who.int/diagnostics_laboratory/links/hiv_incidence_assay/en/index.html).
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.
In contrast to HIV diagnostics that are intended for clinical management, demand for HIV incidence assays is driven almost exclusively by public health and research needs. FHI projected the 5-year demand for three scenarios:
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.
The overwhelming majority of work on testing for recent HIV infection has focused on the use of modified serologic assays for detection of HIV proteins, nucleic acids, and antibodies. Additional approaches, based on alternate biomarkers, have been proposed for measuring incidence that may merit future evaluation:
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.
Although the need for laboratory approaches to measure HIV incidence in populations is apparent, the use of tests for recent infection is greatly hindered by problems affecting test performance, and by the lack of sufficient data to guide the selection, use and interpretation of optimal laboratory-based methods. In order to resolve this critical impasse, the WHO Working Group on HIV incidence assays strongly recommends that a concerted effort be organized to rigorously evaluate the performance of current laboratory assays for recent infection; to develop specific recommendations for assay/algorithm use and interpretation for surveillance and research applications; and to develop working partnerships between private industry, public institutions, private foundations and academia to move the field forward.
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.
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