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Objective: To compare performance of testing for human immunodeficiency virus (HIV)-1 DNA and HIV-1 RNA for diagnosis of HIV-1 infection in infants receiving preventive antiretroviral therapy., Study Design: This substudy of the French multicenter prospective cohort of neonates born to HIV-infected mothers, included 1567 infants tested for HIV with polymerase chain reaction (PCR) in a single laboratory, receiving post-natal prophylaxis, not breastfed, and having simultaneous HIV-1 DNA and RNA results before 45 days. The performance of PCR was assessed in reference to the 6-month HIV-1 RNA result., Results: Specificity of both HIV-1 RNA and HIV-1 DNA PCR was 100% at all ages (except 99.8% for DNA at birth); sensitivity was 58% (RNA) and 55% (DNA) at birth, and 89% at 1 month, 100% at 3 months for both, and 100% at 6 months (DNA). Concordance between HIV-1 DNA and RNA results was 0.78 and 0.81 (Kappa) at birth and 1 month and 100% at 3 and 6 months. Type of maternal and neonatal prophylaxis had no effect on sensitivity, but influenced viral load., Conclusion: The performances of testing for HIV-1 DNA and RNA were similar with 100% sensitivity at 3 months. At 1 month during prophylaxis, 11% of infected children had negative PCR results., (Copyright © 2012 Mosby, Inc. All rights reserved.)

Background: Clinical studies support biologically independent roles of cell-free HIV particles and HIV-infected cells in disease progression. The associations between the level of infected cells and immune markers have been poorly studied, particularly in perinatally infected children., Objective: We tested the hypothesis that independent roles of cell-free virus and infected cells in HIV pathogenesis should be revealed by different associations between each of them and specific immune markers., Methods: Levels of HIV RNA and DNA, HIV-specific CD8 T lymphocytes, activated and naive/memory T lymphocytes were determined in 44 untreated HIV-1-infected children. Pearson partial correlation coefficients were used to assess associations between the variables., Results: Here we provide new information, by showing a direct correlation between the percentages of CD4HLA-DR lymphocytes and HIV DNA levels. Furthermore, higher HIV-specific CD8 T-lymphocyte frequencies were associated with lower HIV DNA levels. In contrast, CD838 lymphocytes and memory CD4 lymphocytes were correlated only to the HIV RNA level. All correlations were independent of age and CD4 depletion., Conclusions: Several immune markers were correlated to either the HIV RNA or the HIV DNA level, but never to both of them, supporting the concept that cell-free virus and infected cells play different roles in HIV-1 immunopathogenesis.

The high genetic diversity of HIV-1 has a major impact on the quantification of plasma HIV-1 RNA, representing an increasingly difficult challenge. A total of 898 plasma specimens positive for HIV-1 RNA by commercial assays (Amplicor v1.5; Roche Diagnostic Systems, Alameda, CA or Versant v3.0; Bayer Diagnostics, Emeryville, CA) were tested using the Agence Nationale de Recherches sur le SIDA second-generation (G2) real-time reverse transcriptase polymerase chain reaction (RT-PCR) test: 518 samples containing HIV-1 of known subtype, including 88 from 2 subtype panels and 430 harboring B (n = 266) and non-B (n = 164) group M HIV-1 subtypes from patients followed up in 2002 through 2005 at Necker Hospital (Paris, France), and 380 samples from 10 different countries (Argentina, Cambodia, Cameroon, Central African Republic, France, Ivory Coast, Madagascar, Morocco, Thailand, and Zimbabwe). HIV-1 RNA values obtained by G2 real-time PCR were highly correlated with those obtained by the Amplicor v1.5 for B and non-B subtypes (R = 0.892 and 0.892, respectively) and for samples from diverse countries (R = 0.867 and 0.893 for real-time PCR vs. Amplicor v1.5 and real-time PCR vs. Versant v3.0, respectively). Approximately 30% of specimens harboring non-B subtypes were underquantified by at least -0.51 log10 in Amplicor v1.5 versus 5% underquantified in G2 real-time PCR. Discrepant results were also obtained with subtype B samples (14% underquantified by Amplicor v1.5 vs. 7% by G2 real-time PCR). Similar percentages were observed when comparing results obtained with the G2 real-time PCR assay with those obtained using the Versant assay. Addressing HIV-1 diversity, continual monitoring of HIV-1 RNA assays, together with molecular epidemiology studies, is required to improve the accuracy of all HIV RNA assays.

The impact of HIV-1 genetic diversity on the performance of laboratory testing is an issue that has to be monitored continuously. An "in-house" real-time PCR assay was developed by the Agence Nationale de Recherche sur le SIDA (ANRS) in France for viral load (VL) quantitation based on the amplification of the HIV-1 long terminal repeat (LTR) region. This technology has not been used in Argentina yet and considering the HIV-1 diversity in the country, a comparative analysis of this assay was undertaken versus the Versant HIV-1 RNA 3.0 Assay (b-DNA). The performance was assessed on 30 drug-naïve HIV-1 infected patients who were characterized previously by phylogenetic analysis of the pol and vpu gene. The results showed that there is a significant linear correlation between values of transformed viral load logarithms measured by both, bDNA and real-time PCR assay and that this assay can be used to quantify viral load in samples from BF-infected patients with the same accuracy and reliability as for B subtype samples. The use of "in-house" real-time PCR to measure DNA in PBMCs correlated strongly with the HIV-1 RNA levels in all specimens.

Background: The objective of this work was to assess the role of human immunodeficiency virus (HIV) reservoirs in the risk of disease progression, by studying the relationship between HIV DNA level in peripheral blood mononuclear cells (PBMCs) and progression toward acquired immunodeficiency syndrome (AIDS)., Methods: HIV-1 DNA levels in PBMCs were determined by quantitative polymerase chain reaction for 383 patients enrolled in the SEROCO Cohort Study who had experienced seroconversion and had been followed up for >8 years. We compared the predictive values of HIV DNA level, HIV RNA level, and CD4+ cell count., Results: Between 6 and 24 months after seroconversion, HIV DNA level was a major predictor of progression to AIDS independently of HIV RNA level and CD4+ T cell count (adjusted relative risk [RR] for a 1-log(10) increase, 3.20 [95% confidence interval {CI}, 1.70-6.00]). HIV DNA level was also a major predictor of disease progression during the first 6 months after seroconversion (adjusted RR, 4.16 [95% CI, 1.70-10.21]), when HIV RNA level and CD4+ T cell count were less predictive. Thus, a combination of these 3 markers provides the best estimate of the risk of disease progression for each patient., Conclusions: Our results suggest that HIV DNA level could be a useful additional marker in clinical practice and could aid in helping to define the best time to initiate treatment for each patient.

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.

HIV-specific CD8+ T cells are critical in controlling human immunodeficiency virus (HIV) replication. We present the evaluation of a gamma-interferon (IFN-gamma)-based enzyme linked immunospot (ELISPOT) assay for the quantification of HIV-specific CD8+ T cells from HIV-infected children. We studied 20 HLA-A*0201-positive HIV-infected children. The IFN-gamma production in response to stimulation with two HLA-A*0201-restricted immunodominant CD8 epitopes (SLYNTVATL [SL9] in Gag and ILKEPVHGV [IV9] in Pol) was tested using the ELISPOT assay. The results were compared to labeling with the corresponding tetramers. Among the 20 children, 18 had detectable responses against the SL9 and/or the IV9 epitope using the ELISPOT assay (medians, 351 and 134 spot-forming cells/10(6) peripheral blood mononuclear cells, respectively). Comparison of results from the tetramer and ELISPOT assays suggests that only a fraction of HIV-specific CD8+ T cells were able to produce IFN-gamma. Most importantly, we found that the frequencies of IFN-gamma-producing CD8+ T cells were positively correlated with the viral load whereas the frequencies of tetramer-binding CD8+ T cells were not. The high sensitivity of the ELISPOT assay and the fact that this functional assay provided information different from that of tetramer labeling support its use for measurement of HIV-specific CD8+ T cells. In conclusion, our results show that the ex vivo-activated IFN-gamma-producing HIV-specific CD8+ T-cell subset is dependent upon continuous antigenic stimulation.

The aim of this study was to evaluate residual viral replication by assessing the HIV load of circulating infected cells in patients given an effective antiprotease-containing treatment for 1 year. PBMC HIV RNA and HIV DNA was quantified by techniques standardized and evaluated by interlaboratory quality control testing. Viral markers identified in a multicenter study were validated in a cross-sectional study of 121 patients beginning treatment. A longitudinal study of 3 viral markers was carried out in 18 patients, each of whom had fewer than 200 copies of HIV RNA per milliliter of plasma after 12 months of treatment. The cross-sectional study showed that viral replication in PBMCs was correlated with the number of circulating infected cells (Spearman rank correlation; p = 0.0001, r = 0.35) and the concentration of virus particles in the plasma (Spearman; p = 0.0001, r = 0.54). The longitudinal study showed that the decrease in HIV RNA levels was smaller in PBMCs than in the plasma. The largest decrease in HIV DNA levels after 12 months of treatment was recorded in patients with low levels of intracellular replication (Spearman; p = 0.005, r = 0.69). PBMC HIV RNA and HIV DNA levels were very informative markers, complementary to plasma HIV RNA levels. They should be used in future trials evaluating the long-term efficacy of new associations of highly active antiretroviral treatments.

The predictive value of two methods for measuring HIV RNA concentration in plasma was assessed in relation to CD4 lymphocyte counts during the asymptomatic period of infection. The design was a retrospective longitudinal case-control study for a mean period of 60 months involving 20 asymptomatic patients included in the French National prospective survey. The CD4 counts in these patients during the last 36 months of the study were stable (non-progressors) or declined (progressors). Plasma RNA concentrations were determined in each subject annually using the AMPLICOR and NASBA techniques. Only AMPLICOR gave RNA titers above the cut-off value for all the patients. The techniques agreed satisfactorily, although there was a difference, median 0.4 log10, between the AMPLICOR and NASBA values. The non-progressors had low and stable RNA concentrations. The concentration was higher in the progressors, according to the AMPLICOR technique, from their inclusion in the study, and according to the NASBA technique, from 1 year after inclusion. However, only four of ten individual progressors had stable plasma HIV RNA concentrations significantly above those of the non-progressors before the decline in their CD4 counts. These were all and only the patients with a decline in lymphocyte counts more than 100 CD4/mm3/year. In each of the other progressors, the RNA concentration was not significantly different from those of the non-progressors. Thus, when making decisions about therapy, plasma HIV RNA determinations cannot be used in place of CD4 counts and may provide valuable additional information.

The objective of this study was to assess the frequency of detection of HIV-1 RNA in plasma of neonates born to HIV-1-seropositive mothers and to determine the diagnostic value of this method in the neonatal period. The study involved 96 infants among those enrolled in the French National Prospective Study. HIV-1 RNA was detected in the first 10 days of life by nucleic acid sequence-based amplification (NASBA) in 12 of 48 plasma samples of infected infants and in 39 of 39 of the second samples taken before the age of 3 months. On the same samples, peripheral blood mononuclear cell (PBMC) DNA polymerase chain reaction (PCR) or viral culture that had been routinely performed were found to be positive in 11 of 48 samples taken in the first 10 days of life and 39 of 39 second samples. For the noninfected infants, HIV-1 RNA was never detected in the 48 samples taken in the first 10 days of life and was detected in one of the 48 samples taken before the age of 3 months. HIV-1 RNA detection in plasma by NASBA has sensitivity and specificity equal to those of DNA PCR and culture on PBMC for the diagnosis of infection in infants with the clade B virus. This standardized method gives rapid results on a small volume of plasma and seems well suited for diagnosis on a large scale.