Search

Objective: To define a routine algorithm for the specific diagnosis and complete follow-up of HIV-1 group O (HIV-O) infections in Cameroun., Methods: During 18 months, samples referred to Centre Pasteur du Cameroun for HIV testing or viral monitoring were screened for HIV-O infection with an in-house serotyping assay. HIV-O viral load was quantified by real-time polymerase chain reaction in the LTR gene and resistance genotyping was performed on pol and env sequences., Results: Of the 7030 samples tested, 78 HIV-O infections (1.1%) were identified, including 7 M and O dually seroreactive samples (9%). All treatment-naive patients and 59% of the patients receiving HAART had detectable viral loads. Analysis of pol sequences from 15 treatment-naive patients revealed a high number of polymorphisms in the protease region, with natural residues implicated in genotypic resistance to tipranavir and saquinavir for HIV-1 group M according to the Agence Nationale de Recherches sur le Sida et les Hépatites virales algorithm. Six patients (40%) harbored the 181C mutation conferring natural resistance to nonnucleoside reverse transcriptase inhibitors. Among antiretroviral-treated patients, major resistance mutations described for HIV-1 group M were found., Conclusions: HIV-O prevalence remains relatively low in Cameroun. The cocirculation of groups M and O in this country leads to replicative dual infections. HIV-O-infected patients in this region can now benefit from effective and specific tools for a complete monitoring of infection. However, further studies are needed to understand long-term response to antiretrovirals of these complex variants.

Background: In resource-constrained settings, antiretroviral treatment (ART) is often continued based on clinical and CD4 responses, without virologic monitoring. ART with incomplete viral suppression was assessed in 27 subjects with subtype C HIV-1., Methods: Plasma HIV-1 RNA, drug resistance, viral tropism, and evolution in polymerase (pol) and envelope (env) genes were measured. The association between these viral parameters and CD4 cell change over time was analyzed using linear regression models., Results: Increased area under the curve of HIV-1 RNA replication was a predictor of lower CD4 cell gains (P < 0.007), while less drug resistance measured as a genotypic susceptibility score (GSS) (P = 0.065), and lower rates of evolution in pol and env genes (P = 0.08 and 0.097, respectively) measured as genetic distance were modestly associated with increasing CD4 cell counts. Evolution of pol and env were correlated (R2 = 0.48, P = 0.005), however, greater evolution was identified in env vs. pol (P < 0.05). CXCR4-usage (X4) was detected in 14/27 (52%) but no differences in CD4 cell change or plasma viremia were associated with X4-usage., Discussion: Among subtype C HIV-1 infected patients in Zimbabwe receiving incompletely suppressive ART, higher virus replication and lower CD4 cell gains were associated with drug resistance and evolution of polymerase and envelope.

Objective: We evaluated the feasibility of the oligonucleotide ligation assay (OLA), a specific, sensitive, and economical ligase-based point mutation assay designed to detect HIV-1 drug-resistance mutations at 12 codons of HIV-1 subtype B pol, for potential use in resource-poor settings., Methods: Specimens from HIV-1-infected individuals collected by 7 international laboratories, including subtypes A, B, C, D, F, G, J, and recombinants AE and AG, were tested by the OLA developed for HIV-1 subtype B. Common polymorphisms that interfered with reactivity of the OLA were identified and modified probes designed and evaluated., Results: 92.5% (2,410) of 2,604 codons in specimens from 217 individuals were successfully genotyped by the subtype B OLA. A high rate (range 8.3%-31.2%) of indeterminate results (negative OLA reaction for both mutant and wild type) was observed for 5 codons. Modified probes at reverse transcriptase codons 151 and 184 and protease codon 90 increased the rate of valid OLA to 96.1%., Conclusions: The OLA designed for HIV-1 subtype B genotyped most pol codons in non-B subtypes from Asia and Africa but was improved by addition of several modified probes. International laboratories experienced in molecular techniques were able to perform the OLA.

Background: Sequence variations in HR-1 gp41 env gene region encoding the target for T-20 have previously been reported among patients naive to inhibitory fusion., Objective: To evaluate whether a previous therapeutic history of patients could have an impact on a differential evolution of the gp41 polymorphism., Methods: We assessed the genetic polymorphism within the critical HR-1 gp41 env gene region in HIV-1 variants from 108 T-20-naive patients (Groups I-III) and 12 patients receiving T-20 as part of a salvage regimen (Group IV). T-20-naive patients included 50 patients exhibiting variants harboring resistance mutations to NRTIs, NNRTIs, and PIs (Group I), 24 patients with variants harboring resistance mutations for NRTIs and/or NNRTIs (Group II), and 34 antiretroviral drug-naive patients (Group III)., Results: In T-20-naive patients whose HIV harbored resistance mutations to NRTIs, NNRTIs, and/or PIs, the mean number of synonymous mutations (ds) per patient was decreased and the mean number of nonsynonymous (da) mutations per patient was increased, resulting in a significant decrease in the mean Sigmads/Sigmada ratio as compared with antiretroviral drug-naive patients (Group III; 4.1 vs. 11.6; P < 0.0001). The mean number of polymorphic mutations in HR-1 gp41 per patient was two-fold higher in patients exhibiting antiretroviral drug resistance mutations (Groups I and II) than in antiretroviral drug-naive patients (Group III; 0.41 vs. 0.20; P < 0.05)., Conclusion: Our observations indicate that the HR-1 gp41 T-20 target is subjected to high genetic variability, including intrinsic polymorphism and selection of T-20 resistance mutations under T-20 intake, that is increased by the presence of resistance mutations to NRTIs, NNRTIs, and/or PIs. Our data provide a basis for a potential impact of previous antiretroviral drug history on the therapeutic efficacy of T-20.

Objectives: Published results on primary or transmitted HIV drug resistance may be biased because they have been largely derived from specific cohort studies or higher risk individuals who present symptomatically. Here, we present results from a representative population-based study of newly diagnosed cases of HIV in Canada and compare the prevalence of transmitted drug resistance between recent and established infections., Methods: Available archived sera taken for the purpose of diagnostic HIV testing from all treatment-naive HIV-positive individuals who were newly diagnosed between 2000 and 2001 were tested for recency of infection, HIV-1 subtype, and mutations conferring reduced susceptibility to reverse transcriptase inhibitors and protease inhibitors (PIs). Recent infections were identified using the Organon Teknika Vironostika HIV-1-LS assay. After full-length sequencing of the pol gene, drug resistance mutations were identified using the 2004 International AIDS Society-USA mutations panel. Differences in drug resistance profiles between recent and prevalent infections were examined using the chi test and the Fisher exact test. The variables examined included gender, age at diagnosis, year of diagnosis, exposure category, ethnicity, and HIV-1 subtype., Results: Among the study population, 8.1% had genotypic evidence of transmitted drug resistance: 4.1% against nucleoside reverse transcriptase inhibitors, 1.4% against nonnucleoside reverse transcriptase inhibitors, 1.5% against PIs, and 1% against > or =2 classes of drugs. A higher proportion of recent infections had genotypic evidence of transmitted drug resistance when compared with established infections (12.2% vs. 6.1%, respectively; P = 0.005). Transmitted drug resistance was identified mainly among recently infected Caucasian men who have sex with men but it was not limited to this group. Compared with the year 2000, a higher proportion of recently infected individuals with resistance-conferring mutations were diagnosed during the year 2001 (66.7% vs. 46.6%)., Conclusions: In Canada, transmitted drug resistance is occurring within all 3 drug classes and across different population groups. The results suggest that the prevalence rates may be higher among recent versus established infections. Given the public health implications of transmitting drug-resistant HIV, it is important to continue population-based drug resistance surveillance to guide optimum prevention and treatment of HIV infection.

To analyze superinfection in an HIV-1-infected patient showing high-risk practices, viral quasispecies were analyzed in pol and env genes in several plasma samples. Phylogenetic analysis in the reverse transcriptase fragment in pol gene identified a single virus in the first 3 samples analyzed, but 12 years after primoinfection, 3 different viral strains were detected in the patient quasispecies. This result suggests a superinfection with 2 HIV-1 strains, one of which showed the T215Y + M184V resistance mutations. The analysis in the env gene confirmed the existence of 3 different strains in the viral population, one of them a recombinant. This study illustrates that events of superinfection and recombination contribute to the viral genetic variability observed in HIV-1-infected individuals.

Background: The HIVNET 012 trial in Uganda demonstrated that single-dose nevirapine (NVP) can prevent HIV-1 mother-to-child transmission. NVP resistance (NVPR) mutations were detected in 25% of women 6 to 8 weeks after NVP, with a higher rate of NVPR in women with subtype D than A. This study examined emergence and fading of specific NVPR mutations in women with these subtypes., Methods: Plasma HIV-1 was analyzed with the ViroSeq genotyping system (Celera Diagnostics, Alameda, CA). Genotypes were obtained from paired samples collected 7 days and 6 to 8 weeks after NVP from 140 women, 83 with subtype A and 57 with subtype D., Results: The rate of NVPR was similar in women with subtype A vs. D at 7 days but was higher in subtype D than A at 6 to 8 weeks. The higher rate of NVPR in subtype D was explained by at least 2 factors: Y181C faded from detection at a greater rate in women with subtype A (odds ratio = 3.06; 95% CI, 1.04, 8.90) and K103N accumulated at a greater rate in women with subtype D (odds ratio = 1.74; 95% CI, 0.62, 4.87)., Conclusions: HIV-1 subtype influences selection and fading of HIV-1 variants with specific drug resistance mutations after antiretroviral drug exposure.

Objective: To determine whether HIV-1 replicates locally in the female genital tract during therapy, and to study whether endocervix is the dominant source of virus in cervicovaginal lavage fluid., Design: Sequence analyses of HIV-1 pol were performed from cervicovaginal secretions and blood plasma of HIV-infected women failing antiretroviral therapy with detectable viral load in both compartments, as well as from drug-naive subjects., Methods: Viral RNA was extracted from cervicovaginal lavage fluid, endocervical secretions collected by Sno-strips, and blood plasma. Population sequencing of HIV-1 pol was performed using cycle sequencing. Drug resistance mutations were analyzed. Phylogenies were constructed based on synonymous positions in the sequences., Results: Resistant virus was detected concordantly in blood and genital tract specimens, consistent with drug selection pressure in both compartments. However, drug-selected mutations often differed in each compartment, and phylogenetic analysis showed differences in virus lineage in these compartments, consistent with local replication in female genital tract. Viruses in cervicovaginal lavage and endocervical secretions were genetically distinguishable, suggesting that endocervix is not the only source of virus found in cervicovaginal lavage., Conclusion: These data support the hypothesis that HIV replication is compartmentalized within the female genital tract during antiretroviral therapy, which has implications for pathogenesis and for epidemiologic surveillance of drug-resistant virus.

Phylogenetic analysis of numerous strains of HIV-1 isolated from diverse geographic origins has revealed three distinct groups of HIV-1: groups M, N, and O. Within group M, subtypes, sub-subtypes and circulating recombinant forms (CRFs) exist. Recently, two near-full-length genomes of similar complex mosaic viruses containing fragments of subtypes A, G, I, and J were described in patients from Burkina Faso (BFP-90) and Mali (95ML-84). Here, we report on the characterization of two additional full-length genome sequences with similar mosaic structure in epidemiologically unlinked individuals from Senegal (97SE-1078) and Mali (95ML-127). Phylogenetic and recombinant analysis confirmed that the previously described strains, BFP-90 and 95ML-84, were indeed a new CRF of HIV-1, which we can now designate as CRF06-cpx. This new CRF fits the complex (cpx) designation, because four different subtypes (A, G, K, and J) were involved in the mosaic genome structure. The fragment in the pol gene, which was initially characterized as unknown in the BFP-90 strain and subsequently as subtype I in the 95ML-84 strain, is now, with the recent description of the new K subtype, clearly identified as subtype K. CRF06-cpx circulates in Senegal, Mali, Burkina Faso, Ivory Coast, and Nigeria, although the exact prevalence remains to be determined. Importantly, this new variant has also been documented on other continents (Europe [France] and Australia), showing that these viruses are spreading not only locally but globally.

We report in this study the use of polymerase chain reaction (PCR) for the amplification of the genomic DNA, isolated from thymic tissue, using the primers flanking HTLV-I/II tax-rex genes and the sequence method to analyze the HTLV-I pol sequence of 27 Italian patients with myasthenia gravis. These molecular methods showed that 92.5% of patients tested positive for tax gene and 55% for pol genes; 55.5% samples were positive for both the tax gene of HTLV-I/II, and the pol gene of HTLV-I. Histologic investigation of the thymus showed that 15 samples had thymic hyperplasia, 93% tested positive for the tax gene, and 40% tested positive for both the tax and pol genes of HTLV-I. In contrast, 91.6% of thymoma-positive samples were positive for tax gene I/II and 75% positive for both genes, tax and pol type I. The sequence analysis of PCR product for tax and pol genes confirmed that these amplified products were HTLV-I, with minimal variations. Our date suggested that either HTLV-I or part of the virus genome is involved in the etiopathogenesis of myasthenia gravis.

Objective: We evaluated the prevalence of HIV-1 non-clade B over time in a formerly clade B-restricted area. Protease and reverse transcriptase regions of the pol gene were used for phylogenetic and recombination analysis and for clade assignment to HIV-1 A-D, F-H, J, and K strains of the M group., Methods: The pol gene of 349 HIV-1 patients belonging to the Italian Cohort Naive for Antiretrovirals (ICONA) were genotypically analyzed to study the prevalence of antiretroviral-associated resistance mutations. All HIV-1 pol sequences and 32 HIV reference strains were analyzed, including the reference strains for the major HIV-1 subtypes. The non-clade B sequences according to the HIV-1 Subtyping Tool program were further studied by a bootscan analysis (SimPlot) to investigate the likelihood of recombination between subtypes., Results: Phylogenetic analysis detected 19 of 349 (5.4%) non-clade B subtypes. The proportions of patients carrying non-clade B virus before and after 1997 were 1.9% and 8.4%, respectively (p =.008). Among whites, heterosexual infection and female gender were significantly associated with the presence of non-clade B subtypes (p =.001 and.005, respectively). Non-clade B HIV-1 was harbored by 14.5% of the heterosexuals who were found to be HIV-1 positive after 1997, 60% of whom were women. Bootscan analysis identified four strains as F, two as A, one as C, one as G, and 11 (57.9 %) as non-clade B recombinant subtypes., Conclusion: Detection of HIV-1 subtypes and intersubtype recombinants in a previously clade B-homogeneous area indicates that the HIV-1 epidemic is evolving in Italy and that heterosexuals and women are at increased risk of infection with non-clade B HIV-1 subtypes. Sequences inferred from the pol gene yield to establish the subtype of circulating HIV-1 strains. As a consequence, genotyping of pol gene for testing resistance to antiretrovirals warrants concomitant surveillance of non-clade B subtypes.