ABSTRACT
Recent reports have documented that certain HIV-1 antibody tests may not reliably detect divergent variants of HIV-1; provisionally classified as HIV-1 group O. Although most known cases of group O infections are found in Cameroon; the prevalence and worldwide distribution of these unusual variants is unclear. In Uganda; where serveral HIV-1 subtypes have been identified; approximately 15of confirmed HIV-1-positive specimens tested negative using an in-house peptide enzyme immunoassay (PLEA) based on the immuno-dominant region of gp41 (QQLLGIWGCSGKLICTT) of the prototypic HIV-1 strain LAI. This result prompted us to further investigate these specimens using PEIA's based on the representative V3-loops of each known serotype; including group O (ANT70 and MVP5180) sequences; and other immuno-dominant petides representing the genetic groups M (LAI and ELI) and O (MVP518 and ANT70). No group O infections were found among these samples. This was confirmed by genetic analysis of the C2V3 region of the envelope protein (gp 20) and/or the immuno-dominant region pf the gp41. Our analysis showed that a single amino-acid substitution (leuu to His) in position607 of gp41; was associated with the diminished sero-reactivity with the immuno-dominant gp41 peptide baserd on LAI. Since synthetic peptides with similar sequences are being used in many commercial third-generation assays for HIV-1; our findings indicate the need to evaluate the performance of these assays before they are used in Uganda. The results of these evaluations may be useful in developing improved diagnostic assays for HIV-1 in Uganda and other African countries
Subject(s)
AIDS Serodiagnosis , HIV Infections/diagnosisABSTRACT
A strategy was developed for a long-term; large scale study of HIV-1 genetic variation in Uganda. Our approach was based on the premisethat DNA sequencing is costly and time consiming with respect to screening largenumbers of specimens. We adopted a dot-blod hybridization method using oligonucleotide probes specific for HIV-1 subtypes A and D in the envC2V3 region to screen for HIV-1 subtypes in Uganda. Specicimens which could not be subtyped in this way were sequenced directly. Genomic DNA from 72 HIV-1 seropositive subjects were amplified by PCR in the envC2V3 region. The results of dot-blot hybridization indicated that 40 were subtype A and 29 were subtype D. The remaining three specimens could not be typed due to either non-binding to probes of both subtypes or non specific binding. Hybdridization results were compared with results obtained by sequence phylogenetic analysis. Sequences were generated from 50 DNA specimens and analysis in the C2V3 region showed that 29 were subtype A and 21 were subtype D. Sequences were also generated from the immuno-dominant env gp41 region (384bp fragment) from specimens for which C2V3 sequences were not available. Phylogenetic analysis of these sequences indicated that 8 were subtype A and 8 were subtype D. Regardless of the region of the HIV genome under study sequence data fully supports the subtype assignment derived from hybridization data. Our findings suggest that probe hybridization using A and D subtype specific probes will be effective for large scale screening og the HIV-infected populationin Uganda. Application of this method should lead to significant savings in cost and time for large; population-based investigations
ABSTRACT
Recent reports have documented that certain HIV-1 antibody tests may not reliably detect divergent variants of HIV-1; provisionally classified as HIV-1 group O. Although most known cases of group O infections are found in Cameroon; the prevalence and worldwide distribution of these unusual variants is unclear. In Uganda; where serveral HIV-1 subtypes have been identified; approaximately 15of confirmed HIV-1-positive specimens tested negative using an in-house peptide enzyme immunoassay (PLEA) based on the immuno-dominant region of gp41 (QQLLGIWGCSGKLICTT) of the prototypic HIV-1 strain LAI. This result prompted us to further investigate these specimens using PEIA's based on the representative V3-loops of each known serotype; including group O (ANT70 and MVP5180) sequences; and other immuno-dominant petides representing the genetic groups M (LAI and ELI) and O (MVP518 and ANT70). No group O infections were found among these samples. This was confirmed by genetic analysis of the C2V3 region of the envelope protein (gp 20) and/or the immuno-dominant region pf the gp41. Ouranalysis showed that a single amino-acid substitution (leuu to His) in position607 of gp41; was associated with the diminished sero-reactivity with the immuno-dominant gp41 peptide baserd on LAI. Since synthetic peptides with similar sequences are being used in many commercial third-generation assays for HIV-1; our findings indicate the need to evaluate the performance of these assays before they are used in Uganda. The results of these evaluations may be useful in developing improved diagnostic assays for HIV-1 in Uganda and other African countries
Subject(s)
HIVABSTRACT
A strategy was developed for a long-term; large scale study of HIV-1 genetic variation in Uganda. Our approach was based on the premisethat DNA sequencing is costly and time consiming with respect to screening largenumbers of specimens. We adopted a dot-blod hybridization method using oligonucleotide probes specific for HIV-1 subtypes A and D in the envC2V3 region to screen for HIV-1 subtypes in Uganda. Specicimens which could not be subtyped in this way were sequenced directly. Genomic DNA from 72 HIV-1 seropositive subjects were amplified by PCR in the envC2V3 region. The results of dot-blot hybridization indicated that 40 were subtype A and 29 were subtype D. The remaining three specimens could not be typed due to either non-binding to probes of both subtypes or non specific binding. Hybdridization results were compared with results obtained by sequence phylogenetic analysis. Sequences were generated from 50 DNA specimens and analysis in the C2V3 region showed that 29 were subtype A and 21 were subtype D. Sequences were also generated from the immuno-dominant env gp41 region (384bp fragment) from specimens for which C2V3 sequences were not available. Phylogenetic analysis of these sequences indicated that 8 were subtype A and 8 were subtype D. Regardless of the region of the HIV genome under study sequence data fully supports the subtype assignment derived from hybridization data. Our findings suggest that probe hybridization using A and D subtype specific probes will be effective for large scale screening og the HIV-infected populationin Uganda. Application of this method should lead to significant savings in cost and time for large; population-based investigations