We initially screened 1,200 specimens by parallel testing with HIV-1 and HIV-2 pepEIA (Figure ). One sample (FEH0045) was non-reactive to the gp41 and gp36 derived peptide. Further testing with the Vironostika HIV Uni-Form II plus O and the HIV-1 Western blot was also negative. Consistently with this serological finding, a PCR reaction set to amplify the reverse trasncriptase and protease regions from HIV-1 and HIV-2 was negative.. This sample was removed from analysis.
Results of the pepEIA screening test for HIV types 1 and 2 in 1,200 HIV infected patients followed-up at the Alto Maé outpatient clinic. Rx = pepEIA reactive and NRx = pepEIA non-reactive.
A total of 1,167 specimens were reactive only on the HIV-1 gp41 pepEIA after the initial screening. Thirty samples were simultaneously reactive to gp41 and gp36 peptides and after re-testing these specimens with a 1:10 dilution, 17 became reactive to HIV-1 peptide only. The remaining 13 samples remained reactive to both HIV-1 and HIV-2 peptides (Table ). Finally, two samples were reactive with the gp36 peptide only after the initial screening. For these last two samples, HIV-2 reactivity persisted after 1:10 specimen dilution.
Serological and genomic tests results of 32 patients reactive on the pepEIA HIV-2 screening assay
The 30 initially dually reactive samples and the two HIV-2 reactive samples were further tested on the INNO-LIA immunoblot assay (Table ). All 30 pepEIA dually reactive samples reacted against all five HIV-1 specific proteins present on the INNO-LIA (gp120, gp41, p31, p24 and p17), except for sample FEH1196 that was negative for p17. The analysis of reactivity against HIV-2 proteins (gp105 and gp36) revealed that among the 17 samples that became HIV-2 negative after re-testing with 1:10 sample dilution in the pepEIA assay, gp36 alone was present on six samples, gp105 alone was present on two samples and both bands were absent for nine samples. Of the 13 samples that remained HIV-2 positive after pepEIA re-testing with 1:10 sample dilution, 10 did not react to gp36 or gp105 while two reacted against gp36 only. Solely one sample (FEH0089) reacted to both gp36 and gp105 HIV-2 proteins. The two samples reactive on gp36 pepEIA only, reacted to both HIV-2 gp36 and gp105 proteins. One of these specimens (FEH0726) did not react to any HIV-1 proteins present on the INNO-LIA assay while the other (FEH0924) showed weak reactivity to the HIV-1 p24 protein (Table ).
In order to explore the discriminatory power of HIV-2 Western blot we have analyzed the capacity of 9 HIV-1/HIV-2 dually reactive samples in recognizing HIV-2 proteins (Figure ). The data indicated no discriminatory pattern and, on the basis of the manufacturer's WB interpretation criteria, all samples could be considered as HIV-2 positive. Likewise, the reactive on the HIV-1 Western blot from the 2 solely HIV-2 pepEIA reactive samples were evaluated. Sample FEH0726 did not react to any HIV-1 proteins; however, sample FEH0924 showed a strong reactivity to gp120 and weaker reactivity to p24, p31, p51 and p66 (data not shown).
Figure 2 Evaluation of HIV-2 Western blot profile (New Lav Blot II). The first two strips are negative (CTR Neg) and positive (CTR Pos) controls. The other lanes show samples from patients infected with HIV-2 (FEH 00726 and 0924), dually infected (FEH 0089) and (more ...)
All 32 samples showing reactivity to the HIV-2 gp36 peptide on the pepEIA were submitted to HIV-2 specific PCR. Only three samples were positive by PCR: one double- reactive on the pepEIA (FEH0089) and the two samples that reacted with the gp36 peptide only (FEH0726 and FEH0924). With the exception of these last two samples, all other 30 samples were also amplified using HIV-1 specific primers and the majority of those analyzed grouped with subtype C based on Stanford HIV Database subtyping tool (Table ). Interestingly, the three HIV-2 PCR positive samples were the only samples reactive to both gp36 and gp105 on the INNO-LIA immunoblot. All HIV-2 positive samples (FEH0089, FEH0726 and FEH0924) pol gene (1507 bp fragment) were analyzed and sequences clustered with HIV-2 subtype A (Table ) sequences after phylogenetic analysis.
In order to explore the cross-reactivity between HIV-1 and HIV-2 antibodies, a sub-group of HIV-1 positive samples (n = 22), some displaying cross-reactivity to HIV-2 (n = 16), were sequenced at the gp41 gene which included the 14 amino acid present at the gp41 immunodominant peptide used in the pepEIA. The gp41/36 IDR aa alignment showed that consensus sequence of 5 HIV-1 samples displaying no cross-reactivity to HIV-2 were not drastically different from 16 samples displaying cross-reactivity to HIV-2 IDR peptide. Of note, we found samples like FEH0871 and FEH0281 from both groups showing some similar aa signatures in IDR peptide to HIV-2 consensus such as A27 and R29 (Table ).
Analysis of amino acid sequence of the variable region of gp41 IDR peptide at positions 21 to 34 from 22 samples showing mono and dually reaction to HIV-1/HIV-2 gp41 IDR peptide