Three observers read all the WB results independently. In our laboratory, a WB test is interpreted as positive for HIV-1 antibodies according to the American Red Cross (ARC) criteria, requiring the presence of at least three bands, one from each gene product group of gag, pol, and env ( 4). In Murex ELISA, the HIV antigens comprise the highly purified immunodominant antigens of the core and envelope proteins of HIV-1 and an immunodominant peptide of the HIV-2 envelope. In Vironostika ELISA, the HIV antigens are a mixture of HIV-1 p24, HIV-1 gp160, HIV-1 ANT70 peptide, and HIV-2 env peptide (amino acids 592 to 603). In HIVSPOT, the capture HIV proteins are the recombinant protein of HIV-1, corresponding to a region overlapping the junction between the gp120 and gp41 fragment of the env protein, plus a highly purified peptide which corresponds to a region of the envelope transmembrane protein of HIV-1. Only discrepant results were then evaluated by WB (HIV Blot 2.2 Genelabs Diagnostics). Specimens testing reactive were then confirmed by another methodologically different ELISA (HIV 1+2 Murex Biotech Ltd., Dartford, United Kingdom). HIV screening for samples from other studies was initially performed with an ELISA (Vironostika-HIV Uni-Form II plus O). The algorithm for HIV testing is shown in Fig. Specimens giving discordant results or those testing reactive by both rapid test and ELISA were then confirmed by WB (HIV Blot 2.2 Genelabs Diagnostics). HIV screening for samples from ENARP cohorts was performed by a rapid test (HIVSPOT Genelabs Diagnostics, Singapore) and ELISA (Vironostika-HIV Uni-Form II plus O Organon Teknika, Boxtel, The Netherlands). The ARC criteria best met the specified objectives for diagnosis in our setting. In general, there was 97.8% concordance between the ARC and WHO criteria and 85.7% concordance between the ARC and CDC criteria for an indeterminate WB result. In addition, 17 indeterminate samples were negative when assessed by a nucleic acid-based amplification assay for HIV-1 viremia. Using CDC and WHO criteria, 6 (19.4%) and 2 (6.5%), respectively, of these WB assays would have been considered falsely positive. Of 31 WB assays initially indeterminate by the ARC criteria and with follow-up samples, 29 (93.5%) became negative when retested subsequently while 2 (6.5%) remained indeterminate for more than a year and were thus considered negative. Only two samples (2.2%) were reactive to both env glycoproteins gp41 and gp120/160 (positive by the World Health Organization criteria). However, 12 samples (13.2%) displayed reactivity to p24 and gp41 or to p24 and gp120/160 proteins (positive by Centers for Disease Control and Prevention criteria). Most (30.4%) of these indeterminate WB results were due to p24 reactivity. Ninety-one (≈0.8%) gave equivocal results because of discordant results among the various screening assays and indeterminate WB profiles by the American Red Cross (ARC) criteria. Overall, 1,437 (11.9%) were positive for HIV-1 antibody. Between 19, a total of 12,124 specimens were tested for HIV-1 antibodies. Here, we describe the profiles of indeterminate WB reactivity in Ethiopians with discordant screening assays. The profiles of WB reactivity among Ethiopians are hardly known. However, indeterminate WB reactivity to HIV-1 proteins may occur in individuals who do not appear to be infected with HIV. It is demonstrated in this study that PCR can be used not only to resolve the infection status of individuals with indeterminate Western blots but also to distinguish between HTLV-I and HTLV-II.The Western blot (WB) assay is the most widely accepted confirmatory assay for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1). HTLV sequences were not detected in the remaining samples, which suggested that the majority of individuals with indeterminate results on Western blots that used one set of commercially available reagents are not infected with HTLV. HTLV-I sequences were detected in the second Western blot-positive sample. HTLV-II sequences were detected by PCR in one of the Western blot-positive samples, as well as in one Western blot-indeterminate sample that showed reactivity to p24 only. Of the 61 repeatably reactive samples, 2 were positive, 26 were negative, and 33 were interpreted as indeterminate on Western blot. Western blot analyses were performed on the sera and DNA was prepared from the PBMCs and analyzed by the polymerase chain reaction (PCR). Peripheral blood mononuclear cells (PBMCs) were recovered from platelet units of 61 blood donors who were HTLV-I positive and 3 blood donors who were HTLV-I negative on enzyme-linked immunosorbent assay (ELISA).
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