High level of HIV-1 resistance in patients failing long-term first-line antiretroviral therapy in Mali.

OBJECTIVES: In resource-limited settings, few data are available on virological failure after long-term first-line antiretroviral therapy. This study characterized the genotypic resistance patterns at the time of failure after at least 36 months of a first-line regimen in Mali, West Africa. METHODS: Plasma samples from 84 patients who were receiving first-line antiretroviral treatment and with an HIV-1 RNA viral load (VL) >1000 copies/mL were analysed. Genotypic resistance testing was performed and HIV-1 drug resistance was interpreted according to the latest version of the National Agency for HIV and Hepatitis Research algorithm. RESULTS: At the time of resistance testing, patients had been treated for a median of 60 months (IQR 36-132 months) and had a median CD4 cell count of 292 cells/mm(3) (IQR 6-1319 cells/mm(3)), a median HIV-1 RNA level of 28266 copies/mL (IQR 1000-2 93 495 copies/mL) and a median genotypic susceptibility score of 1 (IQR 1-4). The prevalence of nucleoside reverse transcriptase inhibitor (NRTI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations was 78% and 82%, respectively. Viruses were resistant to at least one drug in 92% of cases. Although etravirine and rilpivirine were not used in the first-line regimens, viruses were resistant to etravirine in 34% of cases and to rilpivirine in 49% of cases. The treatment duration, median number of NRTI and NNRTI mutations and some reverse transcriptase mutations (T215Y/F/N, L210W, L74I, M41L and H221Y) were associated with the VL at virological failure. CONCLUSIONS: This study demonstrated a high level of resistance to NRTIs and NNRTIs, compromising second-generation NNRTIs, for patients who stayed on long-term first-line regimens. It is crucial to expand the accessibility of virological testing in resource-limited settings to limit the expansion of resistance and preserve second-line treatment efficacy.

Upgraded Cobas Ampliprep-Cobas TaqMan version 2.0 HIV-1 RNA quantification assay versus first version: correction of underestimations.

The large underestimations of HIV RNA quantification observed in 17 patients with the first version of Cobas TaqMan assay have been successfully corrected in the upgraded version 2.0. In comparison with the Abbott RealTime assay, the mean difference that was 1.18 log(10) copies/ml is now zero. The discrepancies have disappeared.

[Evaluation of the LightCycler 480 real-time PCR system for the measurement of CMV, EBV, HHV-6 and BKV viral loads in whole blood]. / Evaluation de la plate-forme de PCR en temps réel LightCycler 480 pour la mesure des charges virales CMV, EBV, HHV-6 et BKV dans le sang total.

OBJECTIVE: The Roche LightCycler 480 (LC480) system was evaluated for quantitative molecular diagnosis of opportunistic viral infections caused by human cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and BK virus (BKV), in comparison with "in-house" real-time PCR assays. PATIENTS AND METHODS: A total of 253 whole blood specimens obtained from transplant recipients were tested. RESULTS: Both the "in-house" and the LC480 methods were highly correlated (Spearman correlation coefficient Rho> or =0.85; p<0.0001) with an excellent overall qualitative agreement (90.5%) and no significant quantitative difference between both techniques for the four viruses tested. The accuracy of the LC480 protocols were confirmed further by the results obtained with the 44 samples from the Quality Control for Molecular Diagnosis (QCMD) 2008 proficiency panel. CONCLUSION: The LC480 system constitutes a suitable and versatile real-time PCR platform in a routine laboratory setting for the diagnosis and monitoring of opportunistic viral infections in transplant recipients, by measuring HCMV, EBV, HHV-6, and BKV loads in whole blood samples.

Impact of discrepancies between the Abbott realtime and cobas TaqMan assays for quantification of human immunodeficiency virus type 1 group M non-B subtypes.

Viral loads in 249 clinical samples from individual patients infected with human immunodeficiency virus type 1 non-B subtypes were determined with both the Abbott RealTime and Cobas TaqMan assays. The differences exceeded 0.5 log for about 20% of samples and 1 log for 3%, with higher values always from the Abbott assay in the latter cases.

Quantitative analysis of human mitochondrial DNA using a real-time PCR assay.

OBJECTIVES: Known for their ability to inhibit the human DNA polymerase-gamma, nucleoside analogues induce toxic effects on mitochondria ranging from increased serum lactate levels to fatal lactic acidosis. DNA polymerase-gamma ensures the mitochondrial DNA (mtDNA) replication and, thus, its inhibition leads to the decrease of the mtDNA. We describe a real-time PCR assay for mtDNA quantification associating DNA extraction procedures applied on peripheral blood mononuclear cells (PBMCs) and subcutaneous adipose tissues and to study the antiretroviral effect on mitochondria. METHODS: Total DNA was extracted from PBMCs and subcutaneous adipose tissues. Nuclear and mitochondrial genes were amplified to determine the number of copies of mtDNA per cell using a cyt-b recombinant plasmid as standard control. We analysed eight HIV-infected asymptomatic patients never treated, four patients who had been treated for 6 months with highly active antiretroviral therapy (HAART) and six non-infected donors. RESULTS: The mtDNA quantification gave rise to reproducible results as the mean coefficients of variation were 1.09% for replicates of samples undertaken 10 times within the same run, and 5.78% and 3.7% for replicates tested in five different runs at 1:100 and 1:1000 dilutions, respectively. Median levels of mtDNA in PBMCs of healthy donors, naive and treated HIV-infected patients were 2.94, 2.78 and 1.93 log HIV-1 RNA copies/mL, respectively. Whereas DNA from PBMCs was shown to be devoid of inhibitors, subcutaneous adipose tissues needed an extra treatment as they were found to be highly inhibited. CONCLUSIONS: The method generated consistent and reproducible results and was successfully applied to DNAs extracted from PBMCs and subcutaneous adipose tissues with adapted extraction. The mtDNA changes in PBMCs were found to be fast as they fall off after 6 months' therapy, decreasing from 2.78 to 1.93 log copies/mL.

Multiple herpes simplex virus infections with various resistance patterns in a matched unrelated donor transplant recipient.

A 45-year-old matched unrelated BMT recipient had sequential mucocutaneous herpes simplex virus (HSV) type 2 infections. Five months after BMT, a penile lesion occurred and was cured using acyclovir, as expected from in vitro susceptibility results. The same lesion recurred 1 month later but worsened with acyclovir. The HSV isolate was resistant to acyclovir (IC(50) = 105 microM), and a nucleotide (G) was added to the thymidine kinase gene leading to a premature stop codon. The lesion improved markedly with foscarnet. During this treatment a second HSV infection occurred on the buttocks 2 weeks after the first one and healed completely with acyclovir. This course correlated with in vitro results of the buttock HSV isolate which was foscarnet-resistant (IC(50) = 300 microg/ml) and acyclovir-sensitive. Surprisingly, no mutation gene of the foscarnet-resistant isolate was detected in the DNA polymerase gene. This case shows that an HSV acyclovir-resistant infection may be followed by an acyclovir-sensitive one. Determination of antiviral susceptibility is needed to monitor the treatment of various HSV infections in immunocompromised BMT recipients.

Comparison of an amplified enzyme-linked immunosorbent assay with procedures based on molecular biology for assessing human immunodeficiency virus type 1 viral load.

The sensitivity of the enzyme-linked amplified sorbent test (ELAST) was compared with those of other classic enzyme-linked immunosorbent assays (ELISAs), with or without previous acidic immunocomplex dissociation (ICD), in a series of samples at different stages of human immunodeficiency virus type 1 (HIV-1) infection. The limit of viral detection of ELAST was assessed with fresh HIV-1 preparations quantified by reverse transcription-PCR and with the P24 antigen (Ag) Sanofi Pasteur Calibrator containing lyophilized virus. The P24 Ag detection capacity of ELAST was compared with that of NASBA in samples obtained from infected subjects with less than 250 CD4+ cells. The results of the present study show that ELAST was the most sensitive method for detecting P24 Ag compared to classic ELISA and ICD plus ELISA. ELAST was able to detect 0.5 pg of P24 Ag per ml in a whole virus preparation and the equivalent of 330 to 1,000 RNA copies/ml of HIV. The rate of detection of P24 Ag was always higher in subjects with low levels of anti-P24 antibodies. The number of positive results was dramatically enhanced (from 37% to 94% for subjects with <250 CD4+ cells) when the incubation period was prolonged from 1 to 16 h. In a third series of 84 samples (<250 CD4+ cells) tested in parallel, NASBA yielded 83% of the positive results and ELAST yielded 79%. Considering the high sensitivity, low cost, simplicity of equipment (only a plate reader), and possibility for full automation, ELAST appears to be a promising new tool for measuring viral load, especially in areas with few resources, in which the procedures based on molecular biology techniques may be difficult to install.