HIV-1 control after transient antiretroviral treatment initiated in primary infection: role of patient characteristics and effect of therapy.

BACKGROUND: The occurrence of viral control after interruption of an antiretroviral treatment (ART) initiated during primary HIV-1 infection (PHI) is rare and the frequency and predictive factors of such a control are unknown. METHODS: Within the French ANRS PRIMO Cohort, 164 patients interrupted ART initiated during PHI. We compared patients whose viral load (VL) remained undetectable (<50 copies/ml) or low (50-500 copies/ml) 1 year after ART interruption to those who evidenced a rapid viral rebound. RESULTS: After ART interruption, VL remained undetectable for a median time of 4.5 years in 14 patients ('post-ART controllers') and low in another 14 patients for a median time of 1.5 years. Post-ART controllers also maintained higher CD4(+) T-cell counts compared to other patients. Female gender, a high CD4(+) T-cell count and low VL during PHI, and a high CD4(+) T-cell count and low HIV DNA levels at interruption, were associated with post-ART HIV control. Treatment characteristics did not differ between controllers and non-controllers. Post-ART controllers had lower specific CD8(+) T-cell frequencies and CD8(+) T-cell activation on ART and after ART interruption than non-controllers. CONCLUSIONS: Few patients maintain very low VL after interruption of treatment initiated during PHI. Early patient characteristics were the main factors of viral control, although early initiation of ART and the effect of ART on reservoir might contribute to control.

Pivotal role of M-DC8⁺ monocytes from viremic HIV-infected patients in TNFα overproduction in response to microbial products.

HIV infects activated CD4⁺ T cells and induces their depletion. Progressive HIV infection leading to AIDS is fueled by chronic immune hyperactivation, mediated by inflammatory cytokines like TNFα. This has been related to intestinal epithelial damage and microbial LPS translocation into the circulation. Using 11-color flow cytometry, cell sorting, and cell culture, we investigated the numbers and TNFα production of fully defined circulating dendritic cell and monocyte populations during HIV-1 infection. In 15 viremic, untreated patients, compared with 8 treated, virologically suppressed patients or to 13 healthy blood donors, circulating CD141 (BDCA-3)⁺ and CD1c (BDCA-1)⁺ dendritic cell counts were reduced. Conversely, CD14⁺ CD16⁺⁺ monocyte counts were increased, particularly those expressing M-DC8, while classical CD14⁺⁺ CD16⁻ M-DC8⁻ monocyte numbers were unchanged. Blood mononuclear cells from viremic patients produced more TNFα in response to LPS than those from virologically suppressed patients. M-DC8⁺ monocytes were mostly responsible for this overproduction. Moreover, M-DC8⁺ monocytes differentiated in vitro from classical monocytes using M-CSF and GM-CSF, which is increased in viremic patient's plasma. This M-DC8⁺ monocyte population, which is involved in the pathogenesis of chronic inflammatory diseases like Crohn disease, might thus be considered as a major actor in the immune hyperactivation fueling HIV infection progression.

Recent HIV-1 infection contributes to the viral diffusion over the French territory with a recent increasing frequency.

OBJECTIVE: To analyse the contribution of primary human immunodeficiency virus type 1 (HIV-1) infection (PHI) to the French viral epidemic. METHODS: HIV-1 pol sequences included 987 PHI from the French ANRS PRIMO cohort between 1999 and 2010 and were analysed using a population-based phylogenetic approach. Clinical features, risk factors, sexual behaviour and drug resistance for clustered and nonclustered transmission events were ascertained. RESULTS: Viruses from 125 (12.7%) of PHI cosegregated into 56 transmission chains, with increasing frequency during the last years (10.2% before 2006 versus 15.2% of clusters in 2006-2010, p = 0.02). The mean number of patients per cluster was 2.44. Compared to unique PHI, clusters involved more often men, infected through homosexual intercourse, of young age, with a high number of casual sexual partnerships and frequent previous HIV serological tests. Resistant strains were found in 16.0% and 11.1% of clusters and unique PHI, respectively (p = 0.11). Overall, 34% (n = 9) clusters included patients followed in French regions far apart, involving 13 clusters with at least one Parisian patient. CONCLUSIONS: PHIs are a significant source of onward transmission, especially in the MSM population. Recently infected people contribute to the spread of the viral epidemic throughout the French territory. Survey of transmitted drug resistance and behavioural characteristics of patients involved into clustered PHI may help to guide prevention and treatment interventions.

X4 tropic multi-drug resistant quasi-species detected at the time of primary HIV-1 infection remain exclusive or at least dominant far from PHI.

Our objective was to analyze the evolution of resistance mutations (RM) and viral tropism of multi-drug-resistant (MDR) strains detected at primary HIV-1 infection (PHI). MDR HIV strain was defined as the presence of genotypic resistance to at least 1 antiretroviral of the 3 classes. Tropism determinations (CCR5 or CXCR4) were performed on baseline plasma HIV-RNA and/or PBMC-HIV-DNA samples, then during follow-up using population-based sequencing of V3 loop and phenotypic tests. Clonal analysis was performed at baseline for env, RT and protease genes, and for HIV-DNA env gene during follow-up. Five patients were eligible. At baseline, RT, protease and env clones from HIV-RNA and HIV-DNA were highly homogenous for each patient; genotypic tropism was R5 in 3 (A,B,C) and X4 in 2 patients (D,E). MDR strains persisted in HIV-DNA throughout follow-up in all patients. For patient A, tropism remained R5 with concordance between phenotypic and genotypic tests. Clonal analysis on Month (M) 78 HIV-DNA evidenced exclusively R5 (21/21) variants. In patient B, clonal analysis at M36 showed exclusively R5 variants (19/19) using both genotypic and phenotypic tests. In patient C, baseline tropism was R5 by genotypic test and R5/X4 by phenotypic test. An expansion of these X4 clones was evidenced by clonal analysis on M72 HIV-DNA (12/14 X4 and 2/14 R5 variants). In patient D, baseline tropism was X4 with concordance between both techniques and HIV-RNA and HIV-DNA remained X4-tropic up to M72, confirmed by the clonal analysis. Patient E harboured highly homogenous X4-using population at baseline; tropism was unchanged at M1 and M18. In all patients, the initial MDR population was highly homogenous initially, supporting the early expansion of a monoclonal population and its long-term persistence. X4-tropic variants present at baseline were still exclusive (patients D and E) or dominant (at least one time point, patient C) far from PHI.

Trends in unsafe sex and influence of viral load among patients followed since primary HIV infection, 2000-2009.

BACKGROUND: In the current context of increasing unsafe sex, HIV incidence may have evolved, depending on HIV prevalence in sexual networks and, among HIV-infected persons who practice unsafe sex, on their infectivity and partners' HIV serostatus. We examined calendar trends in sexual behaviours at risk of HIV-1 transmission (SBR) among 967 adults followed since primary HIV infection (ANRS PRIMO cohort) and relationship with current treatments and viral load. METHODS: Patients completed since 2000 self-administered questionnaires on sexual practices every 6 months. SBR with HIV-negative/unknown partners were analyzed among 155 heterosexual women, 142 heterosexual men and 670 MSM by using logistic generalized estimating equation models (6656 visits). RESULTS: During 2000-2009, the frequency of SBR did not increase significantly among women with steady partners; risk factors were a low education level and alcohol/smoking use. Among heterosexual men with steady partners, the frequency of SBR doubled since 2006; during this period, the only associated factor was combined antiretroviral treatment for at least 6 months or viral load less than 400 copies/ml. Among MSM, SBR increased gradually over time; SBR with steady partners was associated with a low education level and alcohol use. SBR was more frequent among MSM with casual partners; no association with viral load was found. CONCLUSION: In France, recent trends and risk factors in unprotected sex with HIV-negative/unknown partners differ according to sex/sexual preference. The recent increase in SBR among heterosexual men with low viral load may be related to increasing awareness of the 'treatment-as-prevention' concept. The lack of association between SBR and viral load among MSM supports use of treatment-as-prevention as part of diversified prevention strategies.

Undetectable HIV-1 RNA load with the Cobas TaqMan v1.0 in a patient diagnosed at the time of primary HIV-1 infection.

Diagnosis of primary HIV-1 infection is challenging due to the presence of a serological window; thus, HIV-1-RNA quantitation and/or measurement of p24 antigenemia are recommended in such cases. A patient was diagnosed at the time of primary HIV-1 infection, he harbored a CFR02_AG subtype virus; quantitation of plasma HIV-1-RNA yielded an undetectable result according to one commercial assay, while HIV-1-RNA was detectable when measured with three other assays.

Low frequency of CXCR4-using viruses in patients at the time of primary non-subtype-B HIV-1 infection.

We used genotypic and phenotypic assays to estimate the frequency of X4/DM viruses in 131 patients infected with non-subtype-B viruses at the time of primary HIV-1 infection (PHI). All patients were enrolled in the French PRIMO Cohort from 1996 to 2007. Most strains belonged to CRF02_AG (51.1%) and subtype A (14.5%). Sixteen viruses (12.2%) were classified as CXCR4 tropic ("X4 strains") by the combined criteria of amino acids 11 and 25 of the V3 loop (11/25) and net charge rules and/or the SVMgeno2pheno(10%) algorithm: 6 strains by the combined genotypic rule, 7 by the SVMgeno2pheno(10%) algorithm, and 3, clustering in subtype D, by both algorithms. However, only one strain (0.8%), belonging to subtype A, was defined as a dual-tropic (DM) virus by the phenotypic assay. The 67 CRF02_AG strains included 2 classified as X4 strains by the combined genotypic rule (3%) and 2 others classified as X4 strains by SVMgeno2pheno(10%) (3%), but none of these 4 strains was an X4 or DM strain according to the phenotypic assay. These results suggest that the cellular virus reservoir was established with X4 strains in very few non-subtype-B-infected patients at the time of PHI. Genotypic predictions can overestimate the proportion of non-subtype-B X4 viruses at PHI.

Short communication: evidence of HIV type 1 complex and second generation recombinant strains among patients infected in 1997-2007 in France: ANRS CO06 PRIMO Cohort.

Although subtype B strains are still predominant in France, non-B viruses have been isolated from 26% of patients with a primary HIV-1 infection in 2005-2006. The objective of this study was to characterize recombinant-HIV-1 strains by a subtyping based on the phylogenetic analysis of both pol and env sequences. We studied 591 patients who were part of the French PRIMO-Cohort between 1997 and 2007. The RT and V3 regions were sequenced and phylogenetic analyses were performed. Phylogenetic analyses showed concordant subtype results for 91.7% of viruses: 71.6% of the viruses were subtype B and 28.4% belonged to non-B subtypes or circulating recombinant forms (CRFs). Forty-nine strains showed different phylogenies between the two genes (pol and env), indicating that recombinations were observed in 8.3% of the cases. These recombinants were observed in patients from sub-Saharan Africa (28.3%) and in white patients (6.3%). Moreover, among the 49 recombinant viruses, 15 (30.6%) contained a B sequence in the pol or in the env gene compared to 34 (69.4%), which contained non-B or CRF sequences. Twenty-six different recombination patterns involving subtypes, CRFs, or undetermined strains were observed. We have reported the occurrence of new recombinant forms between the two major viral types of strains circulating in France: subtype B and CRF02_AG. Our study confirms a high HIV-1 diversity among patients infected in France within the past 10 years, with a high proportion of non-B strains and the circulation of complex recombinant strains among both sub-Saharan patients and French patients.

Despite being highly diverse, immunovirological status strongly correlates with clinical symptoms during primary HIV-1 infection: a cross-sectional study based on 674 patients enrolled in the ANRS CO 06 PRIMO cohort.

OBJECTIVES: To analyse immunovirological status during primary HIV-1 infection (PHI) according to contemporary clinical status and time since infection. METHODS: Plasma HIV-RNA and peripheral blood mononuclear cell (PBMC) HIV-DNA levels and CD4 cell counts were determined at enrolment in the ANRS PRIMO cohort. Time since infection was estimated based on both the number of antibodies on western blot at enrolment (0-1, 2-4 or > or =5 specific antibodies) and the estimated interval between infection and enrolment based on clinical and epidemiological features. Patients were classified according to the presence or absence of clinical symptoms at enrolment. RESULTS: Between 1996 and 2006, 674 patients were enrolled an estimated median of 47 days after infection. Median marker values were as follows: HIV-RNA 5.10 log(10) copies/mL (range <1.70-8.33); HIV-DNA 3.30 log(10) copies/10(6) PBMCs (<1.84-4.93); and 506 CD4 cells/mm(3) (40-1542). Median HIV-RNA and PBMC HIV-DNA levels were significantly higher in patients with 0 or 1 specific antibody (n = 71) than in patients with 2-4 (n = 228) or > or =5 antibodies (n = 375). Symptomatic patients had significantly higher HIV-RNA and PBMC HIV-DNA levels and lower CD4 cell counts. However, 10% of symptomatic patients recruited shortly after infection had favourable immunovirological status. CONCLUSIONS: Plasma HIV-RNA, PBMC HIV-DNA and CD4 cell count values were highly diverse and correlated strongly with clinical status during PHI. Early diagnosis was not always associated with severe PHI. Combining PBMC HIV-DNA with HIV-RNA, CD4 cell count and clinical symptoms would have allowed identification of 179 patients (26.5%) at high risk of rapid disease progression who did not meet current guidelines for early treatment initiation.

IL-23 and IL-12p70 production by monocytes and dendritic cells in primary HIV-1 infection.

IL-12 enhances protective responses against HIV replication. Its production after in vitro stimulation is defective in chronic HIV infection, but higher responses can be found. IL-23 shares the p40 chain and some properties with IL-12 and enhances Th17 responses, but its role in HIV infection is unknown. The production of IL-12 and IL-23 and the respective contribution of monocytes and myeloid conventional DC (cDCs) during primary HIV infection were determined. Sixteen patients included in the French PRIMO-ANRS Cohort without antiretroviral treatment were followed prospectively and compared with uninfected donors. Intracellular p40 expression by monocytes and cDCs, analyzed by flow cytometry, was transiently increased in monocytes and cDCs in response to LPS and more consistently, in monocytes in response to LPS + IFN-gamma. IL-23 production, measured by ELISA after PBMC stimulation, was induced by LPS in strong correlation with VLs. IL-12p70 production required the addition of IFN-gamma and was transiently increased in patients compared with controls in correlation with VLs, whereas IL-23 was increased sustainedly. Therefore, an apparent domination of IL-23 over IL-12 responses occurred throughout primary HIV infection, and a potential restoration of IL-12 responses might be expected from a treatment mimicking activated T cell signals.

Genomewide association study of a rapid progression cohort identifies new susceptibility alleles for AIDS (ANRS Genomewide Association Study 03).

BACKGROUND: Previous genomewide association studies (GWASs) of AIDS have targeted end points based on the control of viral load and disease nonprogression. The discovery of genetic factors that predispose individuals to rapid progression to AIDS should also reveal new insights into the molecular etiology of the pathology. METHODS: We undertook a case-control GWAS of a unique cohort of 85 human immunodeficiency virus type 1 (HIV-1)-infected patients who experienced rapid disease progression, using Illumina HumanHap300 BeadChips. The case group was compared with a control group of 1352 individuals for the 291,119 autosomal single-nucleotide polymorphisms (SNPs) passing the quality control tests, using the false-discovery rate (FDR) statistical method for multitest correction. RESULTS: Novel associations with rapid progression (FDR, < or = 25%) were identified for PRMT6 (P = 6.1 x 10(-7); odds ratio [OR], 0.24), SOX5 (P = 1.8 x 10(-6); OR, 0.45), RXRG (P = 3.9 x 10(-6); OR, 3.29), and TGFBRAP1 (P = 7 x 10(-6); OR, 0.34). The haplotype analysis identified exonic and promoter SNPs potentially important for PRMT6 and TGFBRAP1 function. CONCLUSIONS: The statistical and biological relevance of these associations and their high ORs underscore the power of extreme phenotypes for GWASs, even with a modest sample size. These genetic results emphasize the role of the transforming growth factor beta pathway in the pathogenesis of HIV-1 disease. Finally, the wealth of information provided by this study should help unravel new diagnostic and therapeutic targets.

Spontaneous control of viral replication during primary HIV infection: when is "HIV controller" status established?

Eight patients in the ANRS PRIMO cohort experienced early spontaneous viral control. Viral control was established a median of 6.2 months after primary human immunodeficiency virus type 1 infection and lasted a median of 4.1 years. Seven of the patients initially had detectable viral replication. For 4 patients, viral control was lost during follow-up.

No evidence of a change in HIV-1 virulence since 1996 in France.

OBJECTIVE: We investigated temporal trends in the CD4 cell count and in plasma HIV RNA and total HIV DNA levels measured at the time of primary HIV infection, as proxies for HIV-1 virulence, taking changes in patient characteristics into account. DESIGN: We studied 903 patients enrolled during primary HIV infection in the French multicenter ANRS PRIMO cohort from 1996 to 2007. METHODS: Associations between the year of primary HIV infection and the values of the three markers were tested with regression models. The year of primary HIV infection was first introduced as a restricted cubic splines function in a regression model in order to explore the shape of the associations, and then as a continuous/categorical variable. The following confounders were considered in multiple regression analysis: time since infection and age (introduced as restricted cubic spline functions), sex, place of birth (Africa vs. others), symptomatic primary HIV infection, smoking, and virus-related factors (subtype B vs. non-B, and drug resistance mutations). RESULTS: Multivariate analysis showed no temporal trends in the CD4 cell count (square-root) or in HIV-1 RNA and DNA levels (log10) measured at the time of primary HIV infection. We observed the well described associations between the prognostic markers and the time since infection, sex, symptomatic primary HIV infection, and smoking. CONCLUSION: The CD4 cell count and HIV RNA and DNA levels measured at the time of primary HIV-1 infection remained stable across 12 consecutive years (1996-2007) in the ANRS PRIMO cohort, suggesting no major change in virulence, after taking into account changes in patient characteristics.

High frequency of X4/DM-tropic viruses in PBMC samples from patients with primary HIV-1 subtype-B infection in 1996-2007: the French ANRS CO06 PRIMO Cohort Study.

OBJECTIVES: To estimate the frequency of viruses with X4 or dual-X4/DM tropism from peripheral blood mononuclear cells (PBMCs) of 390 human immunodeficiency virus type 1 (HIV-1) subtype-B patients diagnosed at the time of primary HIV-1 infection (PHI) between 1996 and 2007 and enrolled in the PRIMO Cohort. METHODS: V3 loop sequences were amplified from HIV-1-DNA and analysed with a combination of five genotypic rules to predict tropism: (i) the '11/25 rule'; (ii) the net charge rule; (iii) the PSSM(X4/DM) algorithm; (iv) the PSSM(SI/NSI) algorithm; and (v) the SVM(Geno2pheno) algorithm. RESULTS: A high proportion (62/390, 15.9%) of patients harboured X4/DM-tropic viruses. This prevalence was stable over time: 18.1% before 2003 versus 14.8% since 2003. No difference according to HIV tropism was noted in HIV-RNA levels, CD4 cell count, time between infection and enrolment, and HIV infection risk factor. The frequency of X4/DM-tropic virus was similar among patients infected with a resistant virus (12/62, 19.4%) compared with patients harbouring wild-type strains (50/328, 15.2%). CONCLUSIONS: This large French epidemiological study evidenced a high proportion of patients (15.9%) harbouring X4/DM-tropic viruses in PBMCs at the time of PHI, suggesting the existence of a cellular X4/DM viral reservoir that could persist for lengthy period of time. Several reports identified that HIV-1 CXCR4 usage was more frequent among patients who developed AIDS and was a powerful predictor of the response to antiretrovirals. Further studies are needed to evaluate the impact of such strains on the outcome of HIV disease, when they are detected at the time of primary infection.

Genomewide association study of an AIDS-nonprogression cohort emphasizes the role played by HLA genes (ANRS Genomewide Association Study 02).

To elucidate the genetic factors predisposing to AIDS progression, we analyzed a unique cohort of 275 human immunodeficiency virus (HIV) type 1-seropositive nonprogressor patients in relation to a control group of 1352 seronegative individuals in a genomewide association study (GWAS). The strongest association was obtained for HCP5 rs2395029 (P=6.79x10(-10); odds ratio, 3.47) and was possibly linked to an effect of sex. Interestingly, this single-nucleotide polymorphism (SNP) was in high linkage disequilibrium with HLA-B, MICB, TNF, and several other HLA locus SNPs and haplotypes. A meta-analysis of our genomic data combined with data from the previously conducted Euro-CHAVI (Center for HIV/AIDS Vaccine Immunology) GWAS confirmed the HCP5 signal (P=3.02x10(-19)) and identified several new associations, all of them involving HLA genes: MICB, TNF, RDBP, BAT1-5, PSORS1C1, and HLA-C. Finally, stratification by HCP5 rs2395029 genotypes emphasized an independent role for ZNRD1, also in the HLA locus, and this finding was confirmed by experimental data. The present study, the first GWAS of HIV-1 nonprogressors, underscores the potential for some HLA genes to control disease progression soon after infection.

Identification of a particular HIV-specific CD8+ T-cell subset with a CD27+ CD45RO-/RA+ phenotype and memory characteristics after initiation of HAART during acute primary HIV infection.

CD8(+) T cells play an important role in controlling viral infections. Defective CD8(+) T-cell responses during HIV infection could contribute to viral persistence. Early initiation of highly active antiretroviral therapy during acute primary HIV infection helps to preserve HIV-specific immune responses. Here, we describe a particular CD27(+) CD45RO(-)/RA(+) HIV-specific CD8(+) T cell in participants treated early during the primary infection. These cells, which were present at a very low frequency during primary HIV infection, increased markedly after early treatment, whereas their frequency remained unchanged in untreated participants and in participants treated later. These nonnaive antigen-experienced cells are in a resting state and have characteristics of long-lived memory cells. They also possess direct effector capabilities, such as cytokine production, and are able to proliferate and to acquire cytotoxic functions on reactivation. Our results suggest that these HIV-specific CD27(+) CD45RO(-)/RA(+) CD8(+) T cells, observed when early viral replication is inhibited, form a pool of resting cells with memory characteristics.

Distinct genetic loci control plasma HIV-RNA and cellular HIV-DNA levels in HIV-1 infection: the ANRS Genome Wide Association 01 study.

Previous studies of the HIV-1 disease have shown that HLA and Chemokine receptor genetic variants influence disease progression and early viral load. We performed a Genome Wide Association study in a cohort of 605 HIV-1-infected seroconverters for detection of novel genetic factors that influence plasma HIV-RNA and cellular HIV-DNA levels. Most of the SNPs strongly associated with HIV-RNA levels were localised in the 6p21 major histocompatibility complex (MHC) region and were in the vicinity of class I and III genes. Moreover, protective alleles for four disease-associated SNPs in the MHC locus (rs2395029, rs13199524, rs12198173 and rs3093662) were strikingly over-represented among forty-five Long Term HIV controllers. Furthermore, we show that the HIV-DNA levels (reflecting the HIV reservoir) are associated with the same four SNPs, but also with two additional SNPs on chromosome 17 (rs6503919; intergenic region flanked by the DDX40 and YPEL2 genes) and chromosome 8 (rs2575735; within the Syndecan 2 gene). Our data provide evidence that the MHC controls both HIV replication and HIV reservoir. They also indicate that two additional genomic loci may influence the HIV reservoir.

Rapid CD4+ cell decrease after transient cART initiated during primary HIV infection (ANRS PRIMO and SEROCO cohorts).

OBJECTIVE: To modelize the rate of CD4 cell count decline and its determinants after cessation of combination antiretroviral therapy (cART) started during primary HIV infection (PHI) and compare it with never-treated patients. METHODS: Kinetics of CD4 counts were analyzed on the square root scale by using a mixed-effects model in 170 patients who received cART during PHI from the Primary Infection (PRIMO) cohort and 123 never-treated patients from the Seroconverters (SEROCO) cohort. RESULTS: After cART interruption in the PRIMO cohort, the CD4 cell count fell rapidly during the first 5 months and more slowly thereafter. The timing of treatment initiation had no influence on the rate of CD4 cell decline. In contrast, a larger increase in CD4 cell counts during cART was associated with a steeper decline and a larger loss of CD4 cells after treatment interruption. The mean CD4 cell loss 3 years postinterruption was 383 cells per microliter. In the SEROCO cohort, the CD4 T-cell decline was less steep (3-year CD4 loss 239 cells/microL). As a result, the mean CD4 cell counts were similar (416 cells/microL) 3 years after cART interruption (PRIMO) or after infection (SEROCO). CONCLUSIONS: These data question the benefit of a limited course of cART even when initiated within 3 months after PHI diagnosis.

New and old complex recombinant HIV-1 strains among patients with primary infection in 1996-2006 in France: the French ANRS CO06 primo cohort study.

BACKGROUND: Prevalence of HIV-1 non-B subtypes has increased overtime in patients diagnosed at the time of primary infection (PHI) in France. Our objective was to characterize in detail non-B strains which could not be genetically classified into the known subtypes/Circulating Recombinant Forms (CRFs). METHODS: Among 744 patients enrolled in the ANRS PRIMO Cohort since 1996, 176 (23.7%) were infected with HIV-1 non-B strains. The subtype/CRF could not be identified in RT for 15 (2%). The V3-V5 env region was sequenced and 3 strains (04FR-KZS, 06FR-CRN, 04FR-AUK) were full-length sequenced. Phylogenetic and bootscan analyses were used to characterize the mosaic structures. RESULTS: Among V3-V5 sequences, 6 were divergent A, 2 distantly related to E or D, 2 C, 1 B and 2 remained unclassified. 04FR-KZS, isolated in a Congolese woman infected in France, clustered with 2 previously described viruses from the Democratic Republic of Congo. They represent CRF27_cpx involving A/E/G/H/J/K/U subtypes. 06FR-CRN, isolated in a homosexual Caucasian patient, was a B/C/U recombinant involving a Brazilian C strain. 04FR-AUK, isolated in a Congolese patient infected in France, was a A/K/CRF09/U recombinant clustering from gag to vif with HIV-1 MAL. Others PHI were further observed in 2006-2007 with 1 KZS and 5 CRN-like viruses, suggesting their spread in France. CONCLUSION: This study illustrates the increasing HIV-1 diversity in France associating new (06FR-CRN) and old (CRF27_cpx and "MAL-like" 04FR-AUK) strains, which are rare in their region of origin but may have a possible founder effect in France. Our results strengthen the French guidelines recommending viro-epidemiological surveillance of HIV-1 diversity.