Background:  The HIV-1 Western blot can be indeterminate or negative in specimens from persons with HIV-1 infection and, because of cross-reactivity, positive in persons with HIV-2 infection. Proposed laboratory algorithms may use HIV-1/HIV-2 differentiation assays, such as the Multispot HIV-1/HIV-2, in lieu of Western blot as a supplemental test for enzyme immunoassay repeatedly reactive specimens. We compared the current CDC Western blot interpretive criteria (≥2 bands:  p24, gp41, or gp120/160), alternative Western blot criteria (3 bands:  2 envelope plus 1 gag or pol), and Multispot HIV-1/HIV-2 results using specimens sent for diagnostic testing.

Methods:  The New York State Department of Health provided de-identified HIV-1 Western blot and Multispot HIV-1/HIV-2 results from EIA-RR specimens submitted between January 2002 and December 2006. We compared the number of results that were positive, indeterminate, and negative based on CDC and alternative Western blot criteria, and Multispot HIV-1/HIV-2 results (negative, HIV-1⁺, HIV-2⁺, or HIV-1⁺ and -2⁺, but undifferentiated). Follow-up HIV test results (HIV-1 Western blot, HIV-2 enzyme immunoassay, and in-house HIV-2 Western blot, and/or HIV-1 or HIV-2 PCR), when available, were used to resolve HIV-1 and HIV-2 infection status.

Results:  We analyzed results from 2135 enzyme immunoassay repeatedly reactive specimens. Using CDC Western blot criteria, 1790 (83.8%) specimens had HIV-1⁺ results, 96 (4.5%) had HIV-1 indeterminate results, and 249 (11.7%) had HIV-1^– results. Indeterminate results were more frequent using alternative (46, 2.5%) than CDC criteria (31, 1.7%) with Multispot HIV-1/HIV-2, HIV-1⁺ specimens. We confirmed 6 specimens as HIV-2⁺ based on HIV-2 Western blot or follow-up; 4 HIV-2-infected specimens (3 Multispot HIV-1/HIV-2 for HIV-2 only; 1 Multispot HIV-1/HIV-2 for HIV-1/HIV-2) were indeterminate using both Western blot criteria; 1 specimen with a Multispot HIV-1/HIV-2 HIV-1/HIV-2 result was HIV-1⁺ by CDC Western blot criteria and indeterminate by alternative Western blot; the sixth  was HIV-1⁺ by both Western blot criteria. A total of 25 specimens with Multispot HIV-1/HIV-2 undifferentiated results were HIV-1⁺ by both Western blot criteria. We determined 17 specimens to be HIV-1⁺ according to Multispot HIV-1/HIV-2 results and HIV-1⁺ based on follow-up or indeterminate based on HIV-1 PCR (15) or negative (2) by CDC criteria;  2 specimens with negative Multispot HIV-1/HIV-2 results were HIV-1⁺ by CDC criteria.

Conclusions:  Multispot HIV-1/HIV-2 identified 6 cases of HIV-2 infection in 4 years, all of which would have been identified as HIV-1⁺ or indeterminate by CDC Western blot criteria. The alternative Western blot criteria produced more indeterminate results than the CDC criteria, with little reduction in HIV-2 misclassification. Enzyme immunoassay repeatedly reactive specimens with negative or indeterminate Western blot results or with negative or undifferentiated Multispot HIV-1/HIV-2 results require further testing to resolve HIV-1 and HIV-2 infection status.