Search

A major obstacle to using next generation sequencing (NGS) technology in clinical routine practice is reliable data analysis. Thousands of sequences need to be aligned and validated, to exclude sequencing artifacts and generate accurate results. We compared two analysis pipelines for Roche 454 ultradeep pyrosequencing (UDPS) raw data generated from HIV-1 clinical samples: a commercial and fully automated Web-based software NGS HIV-1 Module (SmartGene, Zug, Switzerland) vs. the Amplicon Variant Analyzer software (AVA, 454 Life Sciences; Roche). Results were also compared to those obtained with Sanger sequencing. HIV-1 reverse transcriptase and protease genes from 34 plasma samples were submitted to Sanger sequencing and GS Junior UDPS. Raw UDPS data (sff files) from all samples were analyzed with AVA 2.7 software plus manual review of the alignments and the fully automated SmartGene NGS HIV-1 Module prototype (SMG). Results obtained with both analysis pipelines showed good correlation (85.0%). Divergent results were mainly observed at homopolymer positions, such as K101, where the frame-aware alignment and error corrections of the automated approach were more efficient and more accurate, both in terms of detecting and quantifying drug resistance mutations. Our study shows that NGS data can easily be analyzed via a fully automated analysis pipeline, here the SmartGene NGS HIV-1 Module, thus minimizing the need for manual review of alignments by the user, otherwise essential to ensure accurate results. Such automated analysis pipelines may facilitate the adoption of NGS platforms in the routine clinical laboratory.

The emergence of viral-resistant strains is a major problem for the medical management of HIV-infected individuals. The aim of this study was to characterize viral subtypes and drug-resistance mutations (DRMs) in HIV-1 isolates from patients failing antiretroviral therapy (ART). A total of 45 HIV-1-infected patients failing ART were enrolled. The viral RT and Prot genes were amplified and sequenced to determine subtypes and potential DRMs. The subtype distribution was 74% subtype B, 11% subtype A, 9% CRF02-AG, 4% subtype G, and 2% subtype C. Virus samples from 34% of the patients had no DRM while 53%, 27%, and 2% of samples carried at least one DRM conferring resistance to drugs of one, two, or three classes, respectively. DRMs were observed in 50% of the patients infected with non-B strains. The prevalence of nucleoside transcriptase inhibitor (NRTI) mutations was 48%, M184V being largely predominant. The prevalence of nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations was 13%, with K103N present in 57% of samples from NNRTIs-exposed patients. The prevalence of protease inhibitor (PI) mutations was 22%, with major mutations V82A and M46I seen in 16% and 11% of viruses from PI-exposed individuals, respectively. Our study shows the emergence of DRMs in HIV-1 isolates from Moroccan patients failing ART. Although not surprising, the data plead for longitudinal surveys of the dynamics of emergence of DRMs (with a focus on multidrug resistance) in treated patients and circulation of resistant HIV-1 strains in this country.

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.

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.

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.

We performed HIV-1 drug resistance genotypic analysis of viral isolates from 100 antiretroviral (ARV)-naive, recently HIV-1-infected (between 2002 and 2006) individuals from Abidjan (Côte d'Ivoire). The overall prevalence of HIV-1 variants with resistance mutations to reverse transcriptase, protease, or fusion inhibitors was 6%. The majority of isolates were CRF02_AG. Compared with a previous study carried out by our group in 2001-2002 in a similar population in Abidjan, our findings confirm the circulation and transmission of HIV-1 carrying key ARV drug resistance mutation.

As the HIV-1 pandemic becomes increasingly complex and as new countries acceed to antiretroviral drugs, the molecular characterization of HIV-1 strains circulating has important implications for vaccine research and for the efficacy of treatments. To follow the evolution of HIV-1 diversity in African countries, we have carried out a molecular analysis of HIV-1 strains collected from 150 HIV-1-positive pregnant women recruited in Bangui, Central African Republic (CAR). We have sequenced reverse transcriptase (RT) and protease (PROT) genes to (1) characterize the subtypes and CRFs, (2) describe the polymorphism of RT and PROT, particularly at the positions of drug resistance mutations in subtype B, and (3) observe potential drug resistance mutations and evaluate the prevalence of isolates bearing such mutations in this untreated population. The results showed that there is a very high and increasing diversity of HIV-1 strains circulating in CAR; out of 117 samples sequenced, we have observed 45 CRF11_cpx, 22 subtypes A1, 13 subtypes G, 7 subtypes CRF01_AE, 3 subtypes B, 3 subtypes CRF02_AG, 2 of each subtype F2 and CRF09_cpx, and one of each subtype D, J, C, H, CRF06_cpx, CRF13_cpx, and CRF19_cpx; the remaining 13 strains showed discordant genomic results suggesting multiple recombinations leading to mosaic viruses. The polymorphism of RT and PROT was high compared to subtype B, particularly at some positions that have been involved in antiretroviral resistance in subtype B, but we could not observe any major resistance mutation in this sample of untreated patients. The prevalence of drug resistance mutations in this population was therefore clearly under the WHO 5% threshold.

We have sequenced different genes of HIV-1 strains from infected individuals recruited in various geographic parts of Algeria; phylogenetic trees were constructed yielding molecular characterization of these strains. Subtype B accounts for 56% of the samples studied and is therefore the predominant subtype, particularly in the north part of the country; but there is a high diversity of the virus including CRF02_AG, CRF06_cpx, CRF02/CRF06 interrecombinants, and different other intersubtype and/or inter-CRF recombinants. The prevalence of these non-B viruses increases in the south part of Algeria that borders sub-Saharan African countries. The high diversity of HIV-1 in Algeria has implications for virological follow-up, resistance surveys, and vaccine design.

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.

The HTLV-I envelope plays a major role in the process of target cell infection. It is implied in the recognition of the viral receptor(s), penetration of the viral genetic material, and induction of host immunity to the virus. It is thus important to study the genetic variability of the viral env gene as well as its variation in terms of evolution. In a new approach to these features, we sequenced the entire env gene of 65 HTLV-I isolates originating from Gabon, French Guiana, West Indies, and Iran, such isolates representing all major HTLVI phylums but the Australo-Melanesian one. The sequences obtained and all PTLV-I (HTLV-I and STLV-I) env sequences available in the literature were analyzed. Phylogenetic studies using different algorithms (minimum evolution, neighbor joining, maximum parsimony, and maximum likelihood) gave the same clear-cut results. Newly sequenced HTLV-I isolates described in this report allocated in three well-defined subtypes: Cosmopolitan, Central African, and a new distinct one that we termed "Maroni" subtype (present in the Maroni Basin, French Guiana, and West Indies). Clearly, the most divergent PTLV-I strains present in Asia- Australo-Melanesia as well as African and Asian STLV-I derived from the same node in the phylogenetic tree as isolates of the Central African subtype. In addition, we showed that within each PTLV-I subtype, groups of isolates may be characterized by nonrandom and systematically associated mutations.

The molecular characterization of HIV-1 isolates in drug-naive cases in the early stages of HIV disease was studied in 128 cases from Mumbai (Bombay), India. Subtype C was largely predominant followed by A-C intersubtype recombinants, one subtype A and one CRF01 AE. Compared to subtype B, subtype C exhibited an important polymorphism; the percentages of substitutions could reach more than 90%. Two isolates showed M184V substitution the reverse transcriptase indicating resistance to 3TC.

A total of 107 HIV-1 isolates from untreated adult patients recruited in Abidjan, CMte d'Ivoire, in 2001 and 2002 were sequenced in the env, reverse transcriptase (RT), and protease genes. The results show that CRF02_AG is still predominant in this west African population; key mutations of resistance to antiretroviral drugs (NRTI, NNRTI, and PIs) were detected in 5.6% of the patients. We hypothesize that these resistant mutants have been acquired through horizontal transmission. Compared to a previous study carried out by our group in 1997-2000 in a similar population of Abidjan, it seems that there is a dynamic process of resistance and that a survey will be necessary.

To study the progression of HIV-1 infection and coreceptor usages in Central African Republic, clinical data, plasma viral load, and coreceptor usage of sequential HIV-1 isolates were analyzed in a seroincident prospective cohort (PRIMOCA). Twenty-three HIV-1 infected individuals from the Central African Armed Forces were followed from 1995 to 2000. Viruses were isolated from 17 patients at various time points after seroconversion and their coreceptor usage was examined using GHOST cells expressing CD4 and one of the HIV-1 chemokine coreceptors CCR5, CXCR4, BOB/GPR15, and Bonzo/STRL33/CXCR6. Eleven patients died from AIDS. Eight of them died between 2 and 5 years after seroconversion, after a brief symptomatic stage. Patients who rapidly progressed to AIDS and death displayed the highest viral loads after seroconversion. All isolates obtained soon after seroconversion used CCR5, albeit, in some cases, CXCR4, BOB, or Bonzo were also used. Most isolates remained R5 (59 out of 61 isolates), although viruses using CXCR4 appeared in some cases of progression to AIDS. In several cases, a broad tropism was observed during the course of infection, with a frequent usage of BOB and Bonzo in addition to CCR5. Rapid progression to disease and short survival time among Central African HIV-1 patients appear more frequent than those reported in industrialized countries. Viral coreceptor used was mainly CCR5, but, interestingly, a large part of isolates also used BOB and Bonzo. However, there was no strict correlation between the clinical outcome and extended viral tropism.

More than 50 HIV-1-infected patients, naive of antiretroviral therapy (ART) but eligible for first line ART in JJ Hospital, Mumbai, India were investigated for surveillance drug resistance mutations (SDRMs); all but one virus belonged to subtype C; we could observe SDRMs to nonnucleoside reverse transcriptase inhibitors and protease inhibitors in 9.6% of the patients.

Env C2/V3, gag p17/p24, pol protease, and RT regions of HIV-1 isolates recently obtained from 25 HIV-1 seropositive individuals from Ho Chi Minh City (Vietnam) were studied, and genes subtypes were determined by DNA sequence analyses. Twenty-three isolates out of 25 were identified as belonging to subtype E, now recognized as circulating recombinant form 1 (CRF01_AE). The motif at the top of the V3 loop (generally GPGQ) was then preceded by an isoleucine or a methionine (M) residue; the M residue might be a local signature of Vietnamese E isolates compared to Thai E viruses. Two isolates (8%) were shown to be intersubtype recombinants: one E/B and one CRF02_AG(IBNG)/D. The polymorphism of pol protease was considered only for CRF01_AE isolates and is clearly different from that recorded for B viruses with substitutions at positions 13, 35, 36, 41, 69, and 89.

Relationships were sought between specific anti-HIV cytotoxic T lymphocyte (CTL) responses (against structural and regulatory proteins of the HIV-1 LAI isolate) and plasma and cellular viral loads (VLs) in 17 recently HIV-1-infected patients including 3 displaying asymptomatic primary infection (PI) followed up for 12 months. Plasma VL was correlated directly with CD8 counts and inversely with CD4 counts. Cytotoxic reactions were observed in all patients and directed mainly against structural proteins. The earliest CTL responses were against Gag and Env proteins detected in 87 and 75% of the subjects, respectively, within the first month following PI. Anti-Env and Gag cytotoxic responses were inversely correlated with the plasma VL. Reactions against the pol gene products were thought to be either less involved in or less efficient for the initial decrease of viremia. Responses against regulatory gene products were weak and variable, apart from Nef, which was recognized by half of the subjects. Neutralizing antibodies were not detected before month 3, and were found only in six patients at subsequent times. Two of three patients with asymptomatic PI had a low viral burden and either a delayed response or one limited to a few protein CTL responses, suggesting that the magnitude of the CTL response depends on the initial plasma VL. The third patient displayed viral and CTL parameters identical to those of the patients with symptomatic PI. However, two subjects with symptomatic PI exhibited similarly low plasma VL and moderate CTL responses. Overall, the results suggest that the CTL response may not be the sole factor controlling viremia.

In a background of high genomic HIV-1 variability with a predominance of CRF11_cpx and CRF22_01A1, we have studied the emergence of resistance mutations in isolates from Central African patients at failure of d4T-AZT/3TC/NVP-EFV plus two at failure of a PI-including regimen; the resistance mutations observed are those which are expected on HIV-1 subtype B.