Transfer and evaluation of an automated, low-cost real-time reverse transcription-PCR test for diagnosis and monitoring of human immunodeficiency virus type 1 infection in a West African resource-limited setting

Abstract

There is an urgent need for low-cost human immunodeficiency virus type 1 (HIV-1) viral load (VL) monitoring technologies in resource-limited settings. An automated TaqMan real-time reverse transcription-PCR (RT-PCR) assay was transferred to the laboratory of the Centre de Diagnostic et de Recherches sur le SIDA, Abidjan, Côte d'Ivoire, and assessed for HIV-1 RNA VL testing in 806 plasma samples collected within four ANRS research programs. The detection threshold and reproducibility of the assay were first determined. The quantitative results obtained with this assay were compared with two commercial HIV-1 RNA kits (the Versant version 3.0 and Monitor version 1.5 assays) in specimens harboring mainly the circulating recombinant form 02 strain (CRF02). The clinical evaluation of this test was done in different situations including the early diagnosis of pediatric infection and the monitoring of antiretroviral-treated patients. The quantification limit of our method was 300 copies/ml. The HIV-1 RNA values obtained by real-time PCR assay were highly correlated with those obtained by the Versant kit (r = 0.901; P < 0.001) and the Monitor test (r = 0.856; P < 0.001) and homogeneously distributed according to HIV-1 genotypes. For the early diagnosis of pediatric HIV-1 infection, the sensitivity and specificity of the real-time PCR assay were both 100% (95% confidence intervals of 93.7 to 100.0 and 98.3 to 100.0, respectively), compared to the Versant results. Following initiation of antiretroviral treatment, the kinetics of HIV-1 RNA levels were very comparable, with a similar proportion of adults and children below the detection limit during follow-up with our technique and the Versant assay. The TaqMan real-time PCR (12 dollars per test) is now routinely used to monitor HIV-1 infection in our laboratory. This technology should be further evaluated in limited-resource countries where strains other than CRF02 are prevalent.

FIG. 1.

Standard curve of the TaqMan HIV-1 RNA real-time PCR test, Abidjan, Côte d'Ivoire (West Africa). The cycle threshold (C_T) is the number of cycles before the fluorescence passes a fixed limit (time to positivity). The C_T values were obtained from 25 different runs, with three distinct operators. The solid line connects the median values and 25% and 75% interquartile ranges (box plot) of the C_T. The vertical lines show the ranges of C_T.

FIG. 2.

Correlation between HIV-1 RNA viral load as measured with the TaqMan real-time PCR test and with two commercial RNA kits. (A) Comparison between the real-time PCR test and the Versant assay, version 3.0 (320 comparative measurements including 28 samples from children and 171 samples from mothers in the ANRS 1201/1202 Ditrame plus program, 39 adult samples from the Primo-ci Cohort, 36 samples from children in the ANRS 1244/1278 program, and 46 adult samples from the ANRS 1269 trial). (B) Comparison between the real-time PCR test and the Monitor assay version 1.5 (97 comparative measurements in the ANRS 1220 Primo-ci Cohort). The fitted regressions are represented by solid lines.

FIG. 3.

Impact of HIV-1 genotype on plasma HIV-1 RNA levels measured with the TaqMan real-time PCR test and two commercial RNA kits. (A) Difference between the mean viral loads measured with the real-time PCR test and the Versant assay version 3.0 (85 comparisons). (B) Difference between the mean viral loads measured with real-time PCR test and Monitor assay, version 1.5 (92 comparisons). The mean differences are represented by solid lines; the 95% CIs are shown by dashed lines.

FIG. 4.

Kinetics of plasma HIV-1 RNA decrease on HAART. The graph shows a comparison between the TaqMan real-time PCR test and the Versant assay based on the analysis of 149 samples taken from 36 ARV-treated children enrolled in the ANRS 1244/1278 cohort, Abidjan, Côte d'Ivoire. The solid line connects the median values of the individual data points. The vertical bars represent the 25% and 75% interquartile ranges.