In this study, HIV Combo detected 13 (61.9%) of 21 acute HIV infections in a high-risk population. In all but one case, the VL in the HIV Combo positive acute samples was >125,000 c/ml. One sample with 976 c/ml most likely tested positive in HIV Combo because it contained low-level anti-HIV antibodies in addition to p24 antigen; this sample tested positive in the IgM-sensitive 3A77 EIA, but tested negative in two other EIAs. The higher median VL seen among the HIV Combo positive vs. HIV Combo negative samples is consistent with results of Fiebig et al., where the median VL in individuals with acute HIV infection was approximately 2 log10
higher in subjects with detectable p24 antigen than in those who were HIV RNA positive and p24 antigen negative [2
]. Results obtained with antibody-negative acute specimens from EXPLORE are also consistent with previous estimates for the sensitivity of HIV Combo based on analysis of viral isolate dilutions [14
]. HIV Combo also detected all 11 samples with indeterminate Western blots; the median VL in these samples was lower than the median VL in HIV Combo positive acute samples. In contrast, a previous study found that the median VL was higher in subjects with indeterminate Western blots than in subjects with p24 antigen positive acute HIV infection [2
In the U.S., pooled HIV RNA testing is the most widely used method for identification of individuals with acute HIV infection [4
]. The sensitivity of pooled HIV RNA testing depends on the sensitivity of the HIV RNA assay used, as well as the size of the initial sample pools. For example, if 96 HIV antibody negative samples are pooled and tested with an HIV RNA assay with a lower level of detection of 50 c/ml HIV RNA, the assay would detect a single acute sample with a VL of 4,800 c/ml or more. Fifteen (71.4%) of the 21 acute samples in the EXPLORE cohort had VLs >4,800 c/ml. This is similar to the portion of individuals with acute HIV infection who tested positive with HIV Combo (13/21=61.9%). Moreover, for 5 of the 8 antibody-negative acute samples not detected by HIV Combo, VL values were <4,800 c/ml (range: 724–3984 c/ml). Thus, these samples would likely have been missed by some HIV RNA pooling algorithms. Since samples are tested individually with HIV Combo, sensitivity is not influenced by the number of samples analyzed.
The capacity of HIV Combo to detect acute and chronic infections in a single step offers several advantages over traditional HIV RNA pool testing. These include, time to result, throughput and labor. HIV Combo is run on automated random access instruments. In the present study an i
2000SR instrument was utilized. On this platform, the time to first result is 28 minutes with a throughput of 200 tests/hour. The assay is less labor-intensive than pooled HIV RNA testing, and each sample is analyzed only once. In contrast, pooled HIV RNA testing typically involves 3 or 4 stages of testing using progressively smaller sample pools, to determine which and how many of the samples in a positive initial pool are HIV RNA positive [1
]. These types of algorithms result in delays in reporting results and increase the overall cost of testing. Incorporation of HIV Combo in routine testing paradigms will likely have a significant impact on cost/benefit calculations, even in high incidence settings. It should be recognized that fourth generation assays differ in HIV p24 antigen sensitivity, antibody sensitivity, specificity, and performance on genetically divergent strains of HIV-1 [16
]. Thus, results obtained in this study for the ARCHITECT® HIV Ag/Ab Combo assay, recognized as among the most sensitive in its class [16
], may not be directly applicable to other fourth generation immunoassays.
In many settings, HIV RNA testing algorithms are not feasible or are cost-prohibitive. The relative yield provided by the immunoassays on this panel of recent/acute specimens provides a compelling argument for utilization of the most sensitive immunoassays possible. On this panel of acute samples, although there was no incremental yield between the second to third generation Genetic System EIAs (both detected 0/21), 3A77 (an alternative third generation assay) detected 3/21 (14.3%) samples whereas HIV Combo detected 13/21 (61.9%) of the panel members. Thus, the fourth generation Ag/Ab combination assay provided dramatic improvement in sensitivity of detection. These data are consistent with several previous evaluations of fourth generation assays showing substantial improvement in detection sensitivity and a reduction in the window period relative to antibody only assays [16
]. For samples from acutely-infected individuals, limited data is available on the relationship between HIV-1 viral load and p24 antigen sensitivity [21
]. The present study, the first analysis of fourth generation assay performance on acute HIV-1 infections identified by non-pooled HIV RNA testing, provides additional perspective on the sensitivity of HIV Combo. Studies have demonstrated the benefit of testing fourth generation HIV assays in both high risk and low risk populations [21
]. The recent recommendation in the UK National Guidelines for HIV Testing 2008 [23
] to use fourth generation immunoassays for first-line testing of blood in all healthcare settings is noteworthy. Our results suggest that ARCHITECT® HIV Ag/Ab Combo may be useful for detection of acute HIV infection.