Background:  The recommended testing strategy for HIV diagnosis is to screen by enzyme immunoassay (EIA) and confirm by Western blot. Advances in EIA screening assays allow for earlier detection; however the Western blot remains relatively insensitive. An alternative strategy has been proposed to use an HIV-1/HIV-2 differentiation immunoassay to verify reactive EIA results and an RNA detection test to resolve discordances. Our laboratory uses these tests and has conducted a retrospective analysis of results to compare the EIA-Western blot strategy to the proposed alternative.

Methods:  We analyzed test results from 38,252 specimens received for diagnostic HIV testing from January 2007 to June 2010. All specimens were tested with a HIV-1/2+O EIA; 1654 repeatedly reactive specimens were tested with the Multispot HIV-1/HIV-2 differentiation test and an HIV-1 Western blot. The Aptima HIV-1 RNA Qualitative Assay was conducted on Western blot indeterminate and negative samples. Based on these results and follow-up specimens, 1579 samples were classified as HIV-1⁺ (EIA⁺/Western blot⁺ or EIA⁺/Aptima HIV-1 RNA Qualitative Assay⁺), 68 as HIV^– (EIA^– or EIA⁺/Aptima HIV-1 RNA Qualitative Assay^–), and 7 as unknown.

Results:  Applying the standard diagnostic strategy, 1547 of 1654 EIA⁺ specimens were Western blot⁺ and would be reported as HIV-1⁺. The remaining 107 had an indeterminate or negative Western blot and would be reported as inconclusive. RNA testing was performed on 93 of the 107; 29 were Aptima HIV-1 RNA Qualitative Assay⁺ and 64 were Aptima HIV-1 RNA Qualitative Assay^–. Of the 14 with no Aptima HIV-1 RNA Qualitative Assay testing, 3 were Western blot⁺ on follow-up. If the alternative strategy had been applied, 1582 (1579 EIA⁺/Multispot HIV-1/HIV-2 differentiation test⁺ and 3 EIA⁺/Multispot HIV-1/HIV-2 differentiation test^–/Aptima HIV-1 RNA Qualitative Assay⁺) would be reported as HIV-1⁺, 63 as HIV^– (EIA⁺/Multispot HIV-1/HIV-2 differentiation test^–/Aptima HIV-1 RNA Qualitative Assay^–), and 9 as inconclusive because no RNA testing was performed. The 1582 positive results include 1579 true positives, 2 false positives, and 1 unknown. One false positive had a strong reactive EIA/ Multispot HIV-1/HIV-2 differentiation test, Western blot indeterminate, and Aptima HIV-1 RNA Qualitative Assay^–; no follow-up specimen was submitted. The other had a weak reactive EIA/ Multispot HIV-1/HIV-2 differentiation test, Western blot indeterminate, and EIA^– on follow-up.

Conclusions:  The alternative strategy was 100% sensitive, confirming all 1579 HIV-1⁺ specimens, compared to the standard strategy that confirmed 98% (1547). It also produced fewer inconclusive results (9 vs 107); consequently, 98 additional patients would receive definitive HIV results without requiring a follow-up specimen. The use of the Multispot HIV-1/HIV-2 differentiation test in this strategy also allows HIV-2 diagnosis without additional testing. Although 2 false positives were generated with the alternative strategy, 1 could be eliminated by requiring Aptima HIV-1 RNA Qualitative Assay testing on weak reactive EIA/Multispot HIV-1/HIV-2 differentiation test results.