Analysis of RT sequences of subtype C HIV-type 1 isolates from indian patients at failure of a first-line treatment according to clinical and/or immunological WHO guidelines.

The reverse transcriptase (RT) sequences of HIV-1 subtype C isolates from Indian patients at failure (according to WHO clinical or immunological criteria) of a first-line treatment including d4T/AZT-3TC-NVP/EFV were compared to those of HIV-1 isolates from naive patients and analyzed for drug resistance mutations (DRMs), which were interpreted according to ANRS and Stanford algorithms. All viruses were of subtype C. We have observed a decrease of the polymorphism at positions 36 and 214 of RT while D121Y, V179I, and Q217E could be new DRMs. Numerous crucial DRMs to NRTIs and NNRTIs could be recorded including TAMs of pathway 1 and K65R. According to both algorithms, the accumulation of DRMs may induce resistance to second-line NRTIs including tenofovir.

Pol bootscanning analysis of HIV type 1 can exhibit unexpected recombinations.

In France the recommendation is to sequence the RT gene of HIV-1 isolates prior to initiation of antiretroviral therapy. The data are routinely used for molecular characterization of the viruses yielding the subtype or CRF of the isolates investigated together with the absence or presence of drug resistance mutations. In this study, we performed bootscanning analysis on the whole pol gene, in which in vitro and in vivo intersubtype recombination has been reported to occur frequently. We showed that out of 15 HIV-1 isolates, two exhibited a recombination unexpected by this routine sequencing method.

First observation of HIV type 1 drug resistance mutations in Algeria.

This study demonstrates for the first time HIV-1 resistance mutations to all classes of antiretroviral drugs available in Algeria (NRTIs, NNRTIs, PIs) in treated patients at failure. Moreover, it is shown that mutations to NRTIs and PIs can be observed in untreated patients in this country where there is high HIV-1 diversity.

UL40 human cytomegalovirus variability evolution patterns over time in renal transplant recipients.

BACKGROUND: Human cytomegalovirus (HCMV) is the most common opportunistic pathogen infecting immunocompromised patients after transplantation. Although its immunomodulatory capacities and genomic variability participate in immune system evasion, they are poorly studied in clinical strains without culture amplification. One of HCMV immunomodulatory genes, UL40, confers HCMV-infected cells' protection from natural killer-mediated lysis through its encoded nonapeptide presented in the context of human leukocyte antigen-E. METHODS: In three renal transplant recipients with different HCMV serostatus, we aimed to evidence the co-evolution of mixtures of HCMV variants over time with sequencing and cloning of HCMV UL40 gene. RESULTS: Six months after renal transplantation in patient 1Bx, D+/R+, UL40 phylogenetic and bootscan analysis suggested the emergence of a recombination between two previous viral strains. In patient 8Bx, initially D+/R-, distribution of variants in five samples over 43 months was notably stable, with no visible emerging variants despite two renal engraftments and extended episodes of active infection. In patient 9Bx, also D+/R-, phylogenetic tree of viral variants revealed in the first sample a minor clone, confirmed by a specific polymerase chain reaction, related to the three subsequent dominant clones. CONCLUSIONS: In three HCMV-infected renal transplant recipients, we have evidenced different viral evolutive polymorphisms including point mutations, recombination, and occasionally suggesting the intervention of several HCMV strains or a quasispecies-like distribution. This variability could contribute to viral adaptability in pathogenesis.

Characterization of HIV-1 isolates from antiretroviral drug-naive children in southern India.

Access to antiretroviral therapy has expanded in many developing countries, including India. The standard first-line regimens consist of a combination of two nucleoside reverse transcriptase inhibitors and a nonnucleoside reverse transcriptase inhibitor, in a fixed drug combination. Data regarding resistance to these drugs are scarce, especially in children. We evaluated the pattern of polymorphism and potential drug resistance mutations (DRMs) in HIV-1 isolates from 48 children naive to antiretroviral therapy attending the outpatient clinics of the Tuberculosis Research Center in Chennai. The samples were subjected to genotyping of reverse transcriptase (RT) and protease genes. All the samples showed significant polymorphisms in both RT and protease genes, but none had major DRMs. The currently recommended generic first-line antiretroviral drug combination is an appropriate treatment strategy for HIV-1-infected children in India.

Virological responses to atazanavir-ritonavir-based regimens: resistance-substitutions score and pharmacokinetic parameters (Reyaphar study).

OBJECTIVE: To assess the impact of baseline HIV-1 substitutions, individual pharmacokinetic (PK) parameters (Cmin, Cmax, area under the curve [AUC0-->24 h]) and genotype-inhibitory quotient (GIQ) on virological responses (VR) to atazanavir-ritonavir (300 mg/100 mg)-based highly active antiretroviral therapy (HAART) in 71 antiretroviral-experienced, atazanavir-naive patients in virological failure (VF) on HAART. METHODOLOGY: VR was defined as HIV RNA <1.7 log10 copies/ml at week 12 (W12). A clinically relevant genotype-substitutions score for atazanavir-ritonavir was developed and validated (Reyaphar substitutions score). Previously published substitutions scores were also tested. RESULTS: Patients had a median (Q1; O3) of 6 (3; 8) previous treatment lines during 9 (7; 11) years. Baseline (WO) values were as follows: 262 (187; 435) CD4+/microl, 3.9 (2.6; 4.9) log10 HIV-1 RNA copies/ml, 4 (2; 6) protease substitutions and 3 (1; 4) NRTI-related substitutions. Respective steady-state Cmin, Cmax and AUC0-->24 h were 300 (200; 700) ng/ml, 620 (430; 750) ng/ml and 78,000 (61,000; 94,000) ng.h/ml. At W12, 49% of the patients had VR with a median decrease of -1.2 (-0.5; -2.3) log10 HIV-1 RNA copies/ml. The Reyaphar score included 12 baseline protease substitutions from the International AIDS Society USA list that were associated with poorer VR: L10I/F/R/V, K20I/M/R, L241, M461/L, 154L/M/T/V, L63P, A71I/L/V/T, G73A/C/F/T, V771, V82A/F/S/T, 184V, L90M and the polymorphism substitution Q58E. Comparing <5 versus > or =5 Reyaphar substitutions, the W12-W0 HIV-1 RNA decrease was - 1.4 (-0.7; -2.3) versus -0.5 (-1.2; +0.5) log10 copies/ml (P=0.009) with VR in 63% versus 110% (P<10(-4)), respectively. This score predicted VF at W12 with 46% sensitivity, compared to 33% and 28% for the ANRS 2004 and 2005 scores. PK parameters alone were not associated with VR, but GIQ was associated with virological outcome (P=0.04). 150L, known to be correlated with atazanavir-specific resistance, emerged in 2 (8%) of the 24 failing patients with paired genotypes at WO and VF. CONCLUSIONS: These findings highlight the need to cross-validate genotype-based algorithms to interpret substitution impact on virological outcome using different patient databases before their implementation in routine clinical practice.

Whole blood real-time quantitative PCR for cytomegalovirus infection follow-up in transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) remains a major opportunistic agent among transplant recipients. While detection of CMV pp65-lower matrix protein (pp65Ag) is still widely used for monitoring CMV infection, real-time PCR assays have been recently developed for routine quantitation of CMV DNA. However, correlations are lacking between results of pp65Ag and quantitative PCR assays and there is no consensus yet as to the more appropriate blood compartment (whole blood (WB), leukocytes, plasma) to be tested with PCR assays. OBJECTIVES: The aims of the study were to determine, in a population of transplant recipients: (i) the correlation between pp65Ag and CMV quantitative real-time PCR in our setting and (ii) the utility of plasma CMV DNA quantitation in comparison to WB quantitation. METHODS: In 170 blood samples (from 61 solid organ or bone marrow transplant recipients) with pp65Ag results, CMV quantitation was performed in WB and plasma using an in-house real-time quantitative PCR. RESULTS: Real-time PCR and pp65Ag results in WB were correlated: thresholds of 10 and 50(+) cells/200,000 cells were equivalent to 3.3 log(10)copies/mL (2,000 copies/mL) and 3.8 log(10)copies/mL (6,300 copies/mL), respectively. When WB viral load was >or=3.6 log(10)copies/mL, the risk to have a negative plasma CMV DNA result was

Antiretroviral efficacy and virological profile of a zidovudine/lamivudine/tenofovir disoproxil fumarate combination therapy in antiretroviral-naive patients.

OBJECTIVE: To study the antiviral efficacy and the mutations selected by a triple therapy with zidovudine (AZT), lamivudine (3TC) and tenofovir disoproxil fumarate (TDF). METHODS: Antiretroviral-naive patients received 300 mg AZT/150 mg 3TC twice a day plus 300 mg TDF once a day in an open pilot study. Follow-up was assessed at baseline therapy (MO) and at months 1, 3, 6, 9 and 12. Reverse transcriptase (RT) genotypic resistance analysis and in selected cases, a recombinant drug susceptibility and replication capacity assay were performed from plasma RNA at baseline and in case of virological failure (VF); that is, rebound of viral load >50 copies/microl on therapy. RESULTS: Twenty-four patients were included. At baseline, the median CD4+ T-cell count was 443 cells/microl and the median plasma viral load (VL) was 4.38 log10 copies/ml. RT resistance mutations were observed at MO in 4 patients. At M12, the proportion of patients with a VL <50 copies/ml reached 88% using an on-treatment analysis and 67% with an intent-to-treat analysis. The median increase in CD4+ T cells at M12 was 94 cells/microl. Four patients had a VF on therapy: two with wild-type viruses, one with selection of M184V and thymidine analogue mutations (TAMs) on a background of TAMs, and one with selection of K65R and M184V, with a replication capacity at 2.4%/o. CONCLUSION: The virological response in our study demonstrates the antiviral efficacy of the AZT/3TC/TDF combination therapy, which needs further evaluation. The moderate frequency of selection of K65R could be due to the presence of AZT in the regimen.

Characterization of nevirapine (NVP) resistance mutations and HIV type 1 subtype in women from Abidjan (Côte d'Ivoire) after NVP single-dose prophylaxis of HIV type 1 mother-to-child transmission.

Nevirapine (NVP) single dose is widely used in developing countries to prevent HIV-1 mother-to-child transmission. However, this regimen selects key drug resistance mutations that can impair further HAART efficacy. We studied the HIV-1 reverse transcriptase genotype from 29 Ivoirian women 1 month after an NVP single-dose prophylaxis. NVP resistance mutations were observed in six (20.7%) women. The majority of the isolates were CRF02_AG. These results confirm previous studies and suggest the need for different prophylaxis regimens in this setting.

Can highly active antiretroviral therapy be interrupted in patients with sustained moderate HIV RNA and > 400 CD4+ cells/microl? Impact on immunovirological parameters.

Because the effects of treatment interruption on patients with >400 CD4(+) cells/microl and prolonged moderate HIV loads is unknown, their HIV1 RNA and DNA loads, plasma, and PBMC resistance mutations and CD4+ kinetics were studied during 12 months of treatment interruption. Fifty-seven patients were included: 28 with undetectable (median 40 months) and 29 with moderate (>1 year) HIV1 RNA levels (3.3 log10 copies/ml) at the baseline M0. HIV1 RNA and CD4+ counts were determined monthly. PBMC HIV1 DNA was quantified (real-time PCR) and its reverse transcriptase (RT) and protease (PR) genotypes analyzed (M0, M6, M12). Regardless of HIV1 RNA detectability at the baseline M0, all patients had comparable HIV1 RNA (median 4.6), DNA (median 2.9), CD4+ (median 455) at month 12 (M12). The decisive moment was month 1 (M1), when the HIV1 RNA increase and CD4 decline stabilized, rendering M0 differences non-significant and predicting outcome. The lower the CD4+ nadir was before HAART, the steeper the CD4+ decline at M1. HIV1 DNA shifted to wild-type genotype in 47% (M6) and 79% (M12) of RT; 64% of PR major resistance mutations disappeared (M12). Treatment interruption is possible, regardless of the baseline M0 HIV RNA status, but it must take into consideration the CD4+ nadir, an outcome predictor, to initiate HAART before too severe depletion to preserve the possibility of treatment interruption. HIV1 DNA genotyping helps monitor patients with undetectable HIV RNA at M0.

Epidemiological and phylogenetic evidence for patient-to-patient hepatitis C virus transmission during sclerotherapy of varicose veins.

The aim of this study was to provide evidence for patient-to-patient nosocomial hepatitis C virus (HCV) transmission during sclerotherapy of varicose veins. Forty-three patients who had evidence of current infection by genotype 2 HCV have had sclerotherapy by the same physician. Based on this observation, a detailed epidemiological questionnaire on risk factors for HCV in genotype 2 infected patients was conducted. Seventeen sequences in the hypervariable region 1 (HVR1) of the HCV genome obtained from 17 HCV RNA positive patients with a past history of sclerotherapy, were compared with 17 sequences derived from genotype 2 patients with no past history of sclerotherapy, and with 25 sequences sampled from GenBank. Two hundred seven genotype 2 HCV infected patients were included. The main risk factors for HCV infection were transfusion (n = 76), drug use (n = 6), and sclerotherapy of varicose veins (n = 62 including 43 (20.8%) by the same physician), other or unknown (n = 76). These sclerotherapy sessions were carried out in the 1980s for many years. Five of these 43 patients had jaundice within a few weeks after a sclerotherapy session. Sequence analysis of HVR1 from 17 patients who had sclerotherapy by the same physician revealed that they were all infected with HCV genotype 2c. The phylogenetic tree indicated clustering of the patients with a past history of sclerotherapy. The method by which infection was likely to have been transmitted was probably the use of a single vial for multiple patients. This study provides strong evidence that sclerotherapy of varicose veins is a risk factor for HCV infection.

HIV type 1 isolates from 200 untreated individuals in Ho Chi Minh City (Vietnam): ANRS 1257 Study. Large predominance of CRF01_AE and presence of major resistance mutations to antiretroviral drugs.

HIV-1 isolates from 200 untreated patients recruited in 2001 and 2002 in the south part of Vietnam and particularly in Ho Chi Minh City were sequenced in the RT, protease, and env genes. Out of 200 isolates 198 belonged to CRF01_AE while only one subtype B and one intersubtype (B-CRF01_AE) recombinant could be observed. Of the isolates 6.5% had major resistance mutations to antiretroviral drugs.

Predictive value of provirus load and DNA human immunodeficiency virus genotype for successful abacavir-based simplified therapy.

Of 75 human immunodeficiency virus (HIV) type 1-infected patients successfully responding to 2 nucleoside reverse-transcriptase inhibitors (NRTIs) plus 1 protease inhibitor (PI), 55 started a simplified abacavir (ABC)-based triple NRTI regimen. Influences of DNA load and DNA reverse-transcriptase (RT) mutations on virological responses were assessed at month 6 after initiation of therapy. Baseline heterogeneity was observed: peripheral blood mononuclear cell (PBMC) genotyping showed 31% RT mutations with 1-5 NRTI-related mutations, 78% protease mutations had 1-5 PI-related mutations; and HIV-1-DNA levels were 1.8-3.5 log(10) copies/10(6) PBMC. Outcomes for 49 patients on a regimen of 2 NRTIs plus ABC were as follows: 22 successes, 10 blips ("blip" defined as intermittent plasma HIV-1 RNA levels between 50 and 100 copies/mL and a return to an undetectable level), and 17 failures, whereas, for patients continuing on a regimen of 2 NRTIs plus 1 PI, there were 19 successes and 1 blip. Previous treatment regimens, baseline provirus level, and PBMC genotype predicted virological outcome.