Polymerase chain reaction detection and subtyping of human immunodeficiency virus type 1 infections

Barlow, Katrina Louise (1999). Polymerase chain reaction detection and subtyping of human immunodeficiency virus type 1 infections. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0001024e

Abstract

The work described in this thesis comprises an evaluation of the performance of the Amplicor HIV-1 DNA PCR test, an investigation of the reasons for amplification failures, and phylogenetic analyses of the sequence which includes the gag p24 genomic region amplified in the assay. Of particular concern for the use of the Amplicor test in a diagnostic context was that 21 of 377 specimens from patients aged under two, and 22 of 611 specimens from older patients were falsely negative (sensitivities of 75% and 95%; overall sensitivity of 91%). This study sought to discover the cause of these false negative reactions. To achieve this, an in-house nested PCR capable of detecting HIV-1 group M viruses was designed. This in-house PCR spanned the Amplicor primer and probe target regions, and the amplicons were sequenced to reveal the extent of diversity in these sites. As well as primer and/or probe template mismatches, other possible causes of the false negative PCR results were investigated, including inhibitors and low proviral DNA loads. Although no single causal factor was identified in the majority of instances, sequence divergence and low proviral DNA load most likely explained specific cases of failure. Interestingly, heterozygosity for a mutation (A32) in the CCR5 coreceptor gene was strongly associated with PCR false negative results in the in-house assay. Phylogenetic analysis of the sequences obtained allowed assignation of viral clade, and representatives of subtypes A, B, C, D, A/E and G were seen. One dual infection with subtype B and A/ E viruses was observed. The subtypes and sequences found were related to epidemiological data, including the country where the infection was thought to originate and whether the infections were related.

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