Human immunodeficiency virus type 1 (HIV-1) proviral DNA load in purified CD4+ cells by LightCycler(® )Real-time PCR

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) proviral DNA persists in infected cells, even after prolonged successful HAART. In the present study, a relative quantification assay of HIV-1 proviral DNA by LightCycler(® )real-time PCR based on SYBR Green I detection was developed in comparison to the number of purified CD4+ cells as estimated by the quantification of the β-globin gene. METHODS: The ability of the designed gag primers to quantify HIV-1 Group M and the PCR efficiency were assessed on HIV-1 reference isolate subtypes A, B, C and D. The 8E5 cell line containing a single defective copy of HIV-1 proviral DNA was used as a standard for both the HIV-1 target gene and the β-globin reference gene. The assay was applied on thirty consecutive patient samples received for RNA viral load determinations and on retrospective samples from fifteen patients undergoing 2 years of structured treatment interruption (STI). RESULTS: The lower limit of quantification was 50 HIV-1 DNA proviral copies per CD4+ cell sample. The dynamic range was from 50 to 10(6 )HIV-1 DNA copies per CD4+ cell sample with intra- and inter-assay coefficients of variability ranging from 3.1% to 37.1%. The β-globin reference gene was quantified down to a limit of 1.5 pg of DNA/μl (approximately 5 cells) with intra- and interassay coefficients of variability ranging from 1.8% to 21%. DNA proviral load varies widely among HIV-1 infected patients. Proviral load and plasma viral load rebound were high in STI patients who took longer to achieve an undetectable plasma viral load under therapy. A statistically significant correlation was observed between DNA proviral load and RNA steady state viral load in STI patients (p-value = 0,012). CONCLUSION: We have developed a fast, sensitive and specific relative quantification assay of HIV-1 proviral DNA in purified CD4+ cells. The assay enables the monitoring of HIV-1 proviral load, which may be useful to monitor therapy efficacy especially in patients with undetectable plasma RNA viral load, and allows the exploration of viral reservoirs

Characterization of a C-type retrovirus isolated from an HIV infected cell line: complete nucleotide sequence

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Standardisation of primers and an algorithm for HIV-1 diagnostic PCR evaluated in patients harbouring strains of diverse geographical origin

Eight Belgian AIDS Reference Laboratories established a multicentre quality control to evaluate the performance of their diagnostic human immunodeficiency virus type 1 (HIV-1) DNA polymerase chain reaction (PCR). A set of Belgian and African HIV-1 seropositive and seronegative patient samples, collected in Belgium, and the British Medical Research Council (MRC) HIV-1 PCR reference reagent kit, containing plasmid HIV-1 DNA at several dilutions in human carrier DNA with appropriate negative controls, were tested by the laboratories. No false positive results were reported. All laboratories were able to detect one to two copies of HIV-1 DNA. Among the 17 Belgian and African HIV-1 seropositives, some laboratories reported up to four indeterminate results, mainly due to failure of the SK38-39, SK68-69 (Ou et al. (1988) Science 239, 295-297) and/or gag881-882 (Simmonds et al. (1990) J. Virol. 64, 864-872) primers and a poorly performing algorithm. Only the H1POL4235-4538 nested pol primer set, developed by one of the laboratories, correctly identified all the tested HIV-1 positive and negative samples. Consequently, the laboratories decided to evaluate these pol primers as a reference primer set and to standardise the testing algorithm. All laboratories achieved a sensitivity and specificity of 100% on testing 10 additional Belgian and African patient samples, when adapting a standardised algorithm based on three HIV-1 primer sets, one of which is the H1POL4235-4538 primer set.SCOPUS: ar.jinfo:eu-repo/semantics/publishe