Nicole Vidal

Unprecedented Degree of Human Immunodeficiency Virus Type 1 (HIV-1) Group M Genetic Diversity in the Democratic Republic of Congo Suggests that the HIV-1 Pandemic Originated in Central Africa

ABSTRACT The purpose of this study was to document the genetic diversity of human immunodeficiency virus type 1 (HIV-1) in the Democratic Republic of Congo (DRC; formerly Zaire). A total of 247 HIV-1-positive samples, collected during an epidemiologic survey conducted in 1997 in three regions (Kinshasa [the capital], Bwamanda [in the north], and Mbuyi-Maya [in the south]), were genetically characterized in theenv V3-V5 region. All known subtypes were found to cocirculate, and for 6% of the samples the subtype could not be identified. Subtype A is predominant, with prevalences decreasing from north to south (69% in the north, 53% in the capital city, and 46% in the south). Subtype C, D, G, and H prevalences range from 7 to 9%, whereas subtype F, J, K, and CRF01-AE strains represent 2 to 4% of the samples; only one subtype B strain was identified. The highest prevalence (25%) of subtype C was in the south, and CRF01-AE was seen mainly in the north. The high intersubtype variability among the V3-V5 sequences is the most probable reason for the low (45%) efficiency of subtype A-specific PCR and HMA (heteroduplex mobility assay). Eighteen (29%) of 62 samples had discordant subtype designations betweenenv and gag. Sequence analysis of the entire envelope from 13 samples confirmed the high degree of diversity and complexity of HIV-1 strains in the DRC; 9 had a complex recombinant structure in gp160, involving fragments of known and unknown subtypes. Interestingly, the unknown fragments from the different strains did not cluster together. Overall, the high number of HIV-1 subtypes cocirculating, the high intrasubtype diversity, and the high numbers of possible recombinant viruses as well as different unclassified strains are all in agreement with an old and mature epidemic in the DRC, suggesting that this region is the epicenter of HIV-1 group M.

Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis

Background Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. Methods and Findings We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05–1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06–1.25), North America (OR = 1.19; 95% CI: 1.12–1.26), Europe (OR = 1.07; 95% CI: 1.01–1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12–1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92–1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. Conclusions Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

Evidence of Two Distinct Subsubtypes within the HIV-1 Subtype A Radiation

Members of HIV-1 group M are responsible for the vast majority of AIDS cases worldwide and have been classified on the basis of their phylogenetic relationships into nine roughly equidistant clades, termed subtypes. Although there are no known phenotypic correlates for these genotypes, the disproportionate spread of certain of these lineages has been taken to indicate that subtype-specific biological differences may exist. The subtype nomenclature thus remains an important molecular epidemiological tool with which to track the course of the group M pandemic. In this study, we have characterized HIV-1 strains described previously as unusual subtype A variants on the basis of partial sequence analysis. Six such strains from Cyprus (CY), South Korea (KR), and the Democratic Republic of Congo (CD) were PCR amplified from infected cell culture or patient PBMC DNA, cloned, and sequences in their entirety (94CY017, 97KR004, 97CDKTB48, and 97CDKP58) or as half genomes (97CDKS10 and 97CDKFE4). Distance and phylogenetic analyses showed that four of these viruses (94CY017, 97CDKTB48, 97CDKFE4, and 97CDKS10) were closely related to each other, but quite divergent from all other HIV-1 strains, except for subtype A viruses, which represented their closest relatives. In phylogenetic trees from gag, pol, env, and nef regions, the four newly characterized HIV-1 strains formed a distinct sister clade to subtype A, which was as closely related to subtype A as subsubtypes F1 and F2 are to each other. According to current nomenclature rules, this defines a subsubtype, which we have tentatively termed A2. The two other viruses, 97KR004 and 97CDKP58, as well as a full-length HIV-1 sequence from the sequence database (ZAM184), were found to represent complex A2/D, A2/G, and A2/C recombinants, respectively. These results indicate that HIV-1 subtype A is composed of two subsubtypes (A1 and A2), both of which appear to have a widespread geographic distribution. The A2 viruses described here represent the first reference reagents for this new group M lineage.

Recombination Confounds the Early Evolutionary History of Human Immunodeficiency Virus Type 1: Subtype G Is a Circulating Recombinant Form

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is classified in nine subtypes (A to D, F, G, H, J, and K), a number of subsubtypes, and several circulating recombinant forms (CRFs). Due to the high level of genetic diversity within HIV-1 and to its worldwide distribution, this classification system is widely used in fields as diverse as vaccine development, evolution, epidemiology, viral fitness, and drug resistance. Here, we demonstrate how the high recombination rates of HIV-1 may confound the study of its evolutionary history and classification. Our data show that subtype G, currently classified as a pure subtype, has in fact a recombinant history, having evolved following recombination between subtypes A and J and a putative subtype G parent. In addition, we find no evidence for recombination within one of the lineages currently classified as a CRF, CRF02_AG. Our analysis indicates that CRF02_AG was the parent of the recombinant subtype G, rather than the two having the opposite evolutionary relationship, as is currently proposed. Our results imply that the current classification of HIV-1 subtypes and CRFs is an artifact of sampling history, rather than reflecting the evolutionary history of the virus. We suggest a reanalysis of all pure subtypes and CRFs in order to better understand how high rates of recombination have influenced HIV-1 evolutionary history.

The predominance of Human Immunodeficiency Virus type 1 (HIV-1) circulating recombinant form 02 (CRF02_AG) in West Central Africa may be related to its replicative fitness

BackgroundCRF02_AG is the predominant HIV strain circulating in West and West Central Africa. The aim of this study was to test whether this predominance is associated with a higher in vitro replicative fitness relative to parental subtype A and G viruses. Primary HIV-1 isolates (10 CRF02_AG, 5 subtype A and 5 subtype G) were obtained from a well-described Cameroonian cohort. Growth competition experiments were carried out at equal multiplicity of infection in activated T cells and monocyte-derived dendritic cells (MO-DC) in parallel.ResultsDual infection/competition experiments in activated T cells clearly indicated that CRF02_AG isolates had a significant replication advantage over the subtype A and subtype G viruses. The higher fitness of CRF02_AG was evident for isolates from patients with CD4+ T cell counts >200 cells/μL (non-AIDS) or CD4+ T cell counts <200 cells/μL (AIDS), and was independent of the co-receptor tropism. In MO-DC cultures, CRF02_AG isolates showed a slightly but not significantly higher replication advantage compared to subtype A or G isolates.ConclusionWe observed a higher ex vivo replicative fitness of CRF02_AG isolates compared to subtype A and G viruses from the same geographic region and showed that this was independent of the co-receptor tropism and irrespective of high or low CD4+ T cell count. This advantage in replicative fitness may contribute to the dominant spread of CRF02_AG over A and G subtypes in West and West Central Africa.

Distribution of HIV-1 variants in the Democratic Republic of Congo suggests increase of subtype C in Kinshasa between 1997 and 2002.

The Democratic Republic of Congo (DRC) is characterized by low and stable HIV prevalences and high HIV-1 genetic diversity and is most probably the epicenter of HIV-1 group M. Our major goal was to study the distribution of HIV-1 variants over a 5-year period against a background of political instability and civil war. A total of 288 HIV-1-positive samples collected in 2002 from sentinel population groups in an HIV serosurveillance study performed in 4 cities (Kinshasa [capital city], Mbuji-Mayi [south], Lubumbashi [southeast], and Kisangani [northeast]) were genetically characterized by sequencing and phylogenetic analysis of the V3-V5 env region. The results were compared with those obtained in 1997. Similarly, as in 1997, an extremely high genetic diversity of HIV-1 strains overall and a heterogeneous geographic distribution were seen in 2002. All subtypes and several circulating recombinant forms were present, high intrasubtype diversity was observed, and 5.6% of the samples could not be classified. In each geographic region of the DRC, the genetic diversity was significantly higher than in neighboring countries. Comparison of subtype distribution in similar population groups in Kinshasa in 1997 and 2002 revealed an overall increase of subtype C in Kinshasa from 2.1% to 9.7% and, more precisely, from 0% to 18.9% in female sex workers (P = 0.013). Genetic characterization of HIV-positive samples from sentinel surveys adds significant additional information on new trends in the HIV epidemic. These changes could have implications regarding the spread of HIV infection in the DRC as well on vaccine and/or treatment strategies.

High prevalence of HIV-1 drug resistance among patients on first-line antiretroviral treatment in Lomé, Togo.

BackgroundWith widespread use of antiretroviral (ARV) drugs in Africa, one of the major potential challenges is the risk of emergence of ARV drug-resistant HIV strains. Our objective is to evaluate the virological failure and genotypic drug-resistance mutations in patients receiving first-line highly active antiretroviral therapy (HAART) in routine clinics that use the World Health Organization public health approach to monitor antiretroviral treatment (ART) in Togo.MethodsPatients on HAART for one year (10-14 months) were enrolled between April and October 2008 at three sites in Lomé, the capital city of Togo. Plasma viral load was measured with the NucliSENS EasyQ HIV-1 assay (Biomérieux, Lyon, France) and/or a Generic viral load assay (Biocentric, Bandol, France). Genotypic drug-resistance testing was performed with an inhouse assay on plasma samples from patients with viral loads of more than 1000 copies/ml. CD4 cell counts and demographic data were also obtained from medical records.ResultsA total of 188 patients receiving first-line antiretroviral treatment were enrolled, and 58 (30.8%) of them experienced virologic failure. Drug-resistance mutations were present in 46 patients, corresponding to 24.5% of all patients enrolled in the study. All 46 patients were resistant to non-nucleoside reverse-transcriptase inhibitors (NNRTIs): of these, 12 were resistant only to NNRTIs, 25 to NNRTIs and lamivudine/emtricitabine, and eight to all three drugs of their ARV regimes. Importantly, eight patients were already predicted to be resistant to etravirine, the new NNRTI, and three patients harboured the K65R mutation, inducing major resistance to tenofovir.ConclusionsIn Togo, efforts to provide access to ARV therapy for infected persons have increased since 2003, and scaling up of ART started in 2007. The high number of resistant strains observed in Togo shows clearly that the emergence of HIV drug resistance is of increasing concern in countries where ART is now widely used, and can compromise the long-term success of first- and second-line ART.

Emergence of complex and diverse CRF02-AG/CRF06-cpx recombinant HIV type 1 strains in Niger, West Africa.

On the basis of partial env and gag subtyping, we documented that the majority of HIV-1 strains circulating in Niger were CRF02-AG (54.3%) or CRF06-cpx (18.1%) and that 9% of the samples were possible recombinants between CRF02 and CRF06. To determine in more detail the precise structure of these viruses we sequenced the full-length genomes for three such strains (97NE-003, 00NE-036, and 00NE-095). From the bootscan and phylogenetic tree analysis it is evident that the new viruses are the result of recombination events between CRF02-AG and CRF06-cpx strains. Importantly, each virus had a different complex recombinant structure with multiple breakpoints, leading to viruses with complex mosaic patterns.

Identification of a Complex env Subtype E HIV Type 1 Virus from the Democratic Republic of Congo, Recombinant with A, G, H, J, K, and Unknown Subtypes

Up to now, all known env subtype E viruses (CRF01-AE) have had the same mosaic structure with subtype A, and no other env subtype E HIV-1 viruses with non-A subtypes in their genomes have been described. In this report we describe the full-length genome sequence of an env subtype E isolate with a recombinant genome different from the prototype CRF01-AE strains. The 97CD-KTB49 strain, obtained from a tuberculosis patient in Kinshasa, has a complex mosaic genome involving subtypes A, E, G, H, J, K, and several unknown fragments. The U sequences formed well-separated clusters together with previously described unknown fragments from CRF04-cpx (subtype I), and from Z321, the oldest intersubtype recombinant isolated in 1976 in the Democratic Republic of Congo. The complex recombinant virus from our study is not an isolated strain; partial sequencing of a second strain, 97CD-KFE45, confirmed the breakpoints observed in the 97CD-KTB49 strain in the regions sequenced. The complexity of these recombinant strains suggests a longstanding presence of subtype E in Central Africa.

Risk of virological failure and drug resistance during first and second-line antiretroviral therapy in a 10-year cohort in Senegal: results from the ANRS 1215 cohort.

Background:In 1998, Senegal launched one of Africas first antiretroviral therapy (ART) programs. Since then, the number of treated patients in Africa has substantially increased thanks to simplification in treatment management. Although good outcomes over the first years of ART have been observed in sub-Saharan Africa, little is known about the long-term (>5 years) risks of virological failure and drug resistance and about second-line treatment response. Methods:Patients from the ANRS-1215 cohort in Senegal, started with either one nonnucleoside reverse transcriptase inhibitor or indinavir, a first-generation nonboosted protease inhibitor, followed for >6 months and having >1 viral load (VL) measurement were included. Virological failure was defined as 2 consecutive VL measurements >1000 copies/mL. Results:Of the 366 patients included, 89% achieved a VL <500 copies/mL. The risk of virological failure at 12, 24, and 60 months was 5%, 16%, and 25%, being higher in younger patients (P = 0.05), those receiving a protease inhibitor–containing regimen (P = 0.05), and those with lower adherence (P = 0.03). The risk of resistance to any drug at 12, 24, and 60 months was 3%, 11%, and 18%. After virological failure, 60% of the patients were switched to second-line treatments. Although 81% of the patients achieved virological success, the risk of virological failure was 27% at 24 months, mostly in patients with multiple resistances. Conclusions:In this cohort, virological outcomes for first-line treatments were good compared with those from high-resource settings. However, the rate of virological failure for second-line treatment was high, probably because of accumulation of resistances.