We compared several antibody-based approaches to identify persons in the early HIV post-seroconversion period. A key finding was that Western blot assays are quite accurate for identifying persons with recent HIV seroconversion. Our data suggest that simple criteria based on the total number of positive bands on the Western blot can have good-to-excellent diagnostic performance for identifying individuals with early HIV infection. Two different Western blot assays both yielded very high specificity for identifying persons within 30 days of seroconversion with the simple criterion of having 3 or fewer bands, and for persons within 90 days of seroconversion with the criterion of having 6 or fewer bands. The significance of these findings is high because Western blot tests are widely used as confirmatory tests for HIV infection. Thus, data that can help identify persons in the early post-seroconversion period is already available to many clinicians. Similar results were recently reported from Switzerland based on an immunoblot assay (INNO-LIA HIV-I/II SCORE, Innogenetics) that is not available in the United States [17
On the 2 standard EIA assays we examined, an SCO value of <4 had high specificity for seroconversion within the past 30 days. This value, although not always reported to clinicians, can be obtained from testing laboratories. The rapid increase in reactivity on these assays, however, results in poor sensitivity. The threshold of an SCO of 4 is exceeded within days to 2 weeks of seroconversion, and nearly half of persons classified as within the first 30 days following seroconversion had already exceeded this cutoff using the OTV EIA. While results were similar between the 2 standard EIAs we tested, the OTV EIA was slightly more sensitive and more specific for the early post-seroconversion period.
In contrast to the Western blots and EIAs, the LS-EIA and BED incidence assays performed best when detecting persons within longer time windows after seroconversion. These assays had the highest sensitivity for identifying persons within 90 days of seroconversion. To achieve reasonable specificity for this time window required using cutoffs that were significantly lower than those used in incidence studies for identifying persons within longer periods (eg, 180 days) since seroconversion. Although the BED was slightly more sensitive than the LS-EIA for detecting persons within 90 days of seroconversion, it suffered from limited specificity. When the pre-test probability of recent infection is low, this can result in substantial false positive results due to misclassification of chronically infected persons as having just seroconverted. While our study was not designed to assess the specificity of these assays for classifying persons within windows beyond 90 days after seroconversion, other studies have noted limitations in the specificity of these assays in certain settings using higher cutoffs for incidence studies, including false positive results in persons with advanced HIV disease [18
]. These assays are research tests that are not FDA approved and are unavailable in many settings, but they are inexpensive and can potentially be performed in laboratories equipped to perform HIV EIA antibody testing.
Besides potential applications in research settings and epidemiologic studies, estimates of duration of HIV infection based on data available from standard HIV diagnostic tests may be useful for prevention purposes. The first few months of HIV infection are a particularly infectious period [10
]. In a macaque model of Simian Immunodeficiency Virus transmission, the viral load needed to transmit infection was >50 times higher when using plasma from chronically infected macaques compared to plasma from acutely infected macaques, and further experiments suggested that lack of neutralizing antibodies may have been a factor in the higher infectivity of plasma from acutely infected macaques [19
]. If these findings apply to HIV infection in humans, having information to identify recently infected persons may be of particular importance in order to focus HIV prevention efforts on those most likely to transmit HIV.
HIV antibody levels can wane in advanced disease, and this can result in erroneous classifications of such individuals as recently infected using tests we assessed. For this reason, the assays we tested should typically not be applied to persons with clinical AIDS or a CD4+ T-cell count <200 cells/μL. Although we did not test persons with chronic HIV infection, there are prior studies that have addressed this issue for the assays we tested. In a study that evaluated 199 subjects with a CD4+ T-cell count <200 cells/μL, 96.5% had at least 6 of 9 Western blot bands present [20
]. In a second study of persons with chronic HIV infection but without clinical AIDS (WHO stage 2 and 3), 701 of 704 patients (99.8%) had 6 or more bands on Western blot testing, and 100% had 5 or more bands [21
]. These findings provide reassurance that the specificity of criteria such as having 6 or fewer bands on Western blot for identifying persons in early HIV infection should be very high when applied to persons with chronic infection. In a review of incidence assays, the median specificity from 11 estimates in longstanding infection was 98% using higher cutoffs than those used in the current study [18
]. Specificity using the much lower cutoffs we employed should be considerably higher.
There are additional limitations to this study. First, by studying only specimens that were close to the time cutoffs being evaluated, it is likely that we underestimated both the specificity and sensitivity of assays for early infection in contexts in which the majority of newly diagnosed persons are infected for longer periods of time, and hence recent and longstanding infections would be easier to distinguish. Second, our results suggest that while results are generally similar across assays such as standard EIA and Western blots, there were differences between specific assays. Thus, caution must be used in extrapolating the present data to other test kits. In addition, interpretation of the Western blot entails some subjective decisions when the bands are faint. It is possible that the Western blot results we obtained from an expert research laboratory might be more carefully interpreted and hence more consistent than the results obtained from standard clinical laboratories. Third, as we did not have daily blood samples, we estimated exact seroconversion dates, and there is some imprecision in the estimates, even if only by a matter of days. Fourth, our patient population consisted primarily of men who have sex with men with sub-type B infection. It is possible that evolution of antibody responses might differ in heterosexual or injection drug use transmission, although we are not aware of data that indicate such a difference. As some of the antibody tests we used were based on sub-type B antigens, results might also differ using these assays with other HIV subtypes. Finally, one of the incidence assays we evaluated (the LS-EIA based on the OTV EIA) is no longer available. New iterations of the LS-EIA test (based on kits manufactured by Avioq) have been developed, validated, and provide similar results, although there may be some differences that could alter diagnostic performance for the applications we describe.
Despite these limitations, our results suggest that if interpreted carefully, information from standard EIA and Western blot assays, and from so-called incidence assays such as the BED and LS-EIA tests, can be useful to identify persons who are in the early post-seroconversion period of HIV infection. The approaches outlined here may be useful for clinical, research, and prevention purposes that benefit from identifying very recently HIV-infected individuals.