Benoit Visseaux

Prevalence of pre-existing resistance-associated mutations to rilpivirine, emtricitabine and tenofovir in antiretroviral-naive patients infected with B and non-B subtype HIV-1 viruses

OBJECTIVES The prevalence of rilpivirine, emtricitabine and tenofovir resistance-associated mutations (RAMs), described in vitro and in vivo, was determined in antiretroviral-naive patients. PATIENTS AND METHODS From 2008 to 2011, 1729 treatment-naive patients were tested for resistance by bulk sequencing. We studied the primary rilpivirine RAMs (K101E/P, E138A/G/K/Q/R, V179L, Y181C/I/V, H221Y, F227C and M230I/L) and other potential rilpivirine-associated mutations (V90I, L100I, K101T, E138S, V179D/I, Y188L, V189I, G190A/E/S and M230V). We also studied the M184V/I and K65R mutations for emtricitabine and tenofovir, respectively. RESULTS Among 1729 sequences, half of patients had B-subtype viruses and the other half non-B (with 26.7% CRF02, n=461). Primary rilpivirine RAMs were infrequent (4.6%, n=79) and the most prevalent were E138A (3%, n=52), E138K, (0.3%, n=5), H221Y (0.3%, n=5), E138G (0.2%, n=4) and Y181C (0.2%, n=4). The frequency of the primary rilpivirine RAMs was similar between B and non-B subtypes. The other potential rilpivirine-associated mutations that were most prevalent were V179I (8.4%, n=145), V90I (3.8%, n=65) and V189I (2.3%, n=40). The common V179I, V189I and V90I polymorphisms have not been associated with virological failure in Phase 3 clinical studies. By the ANRS algorithm, 4.9% (n=84) of samples were resistant to rilpivirine, 3.7% (n=32) of B-subtype viruses versus 6% (n=52) of non-B-subtype viruses (P=0.02, χ(2) test). The prevalence of K65R and M184I/V was 0.06% (1/1729) and 1% (18/1729), respectively. The prevalence of K103N was 2% (35/1729). CONCLUSIONS The prevalence of rilpivirine, emtricitabine and tenofovir resistance mutations was very low in antiretroviral-naive patients. The prevalence of resistance to rilpivirine (4.9%, n=84) was not statistically different from the prevalence of efavirenz and nevirapine resistance in our population.

Molecular Determinants of HIV-2 R5–X4 Tropism in the V3 Loop: Development of a New Genotypic Tool

OBJECTIVE The use of CCR5 inhibitors requires a tool to predict human immunodeficiency virus type 2 (HIV-2) tropism, as established in HIV-1. The aim of our study was to identify genotypic determinants of HIV-2 tropism located in the gp105 V3 loop. METHODS HIV-2 tropism phenotypic assays were performed on 53 HIV-2 clinical isolates using GFP expressing human osteosarcoma T4 [GHOST(3)] cell lines expressing CD4 and CCR5 or CXCR4 coreceptors. The gp105 V3 loop was sequenced and analyzed. RESULTS Thirty-four HIV-2 isolates were classified as R5, 7 as X4, and 12 as X4/R5 (dual). Substitution at residue 18 was always associated with a dual/X4 tropism (P < .00001). The following determinants were associated with dual/X4 tropism: a global net charge of more than +6 (P < .00001), V19K/R mutation (P < .00001), S22A/F/Y mutation (P < .002), Q23R mutation (P < .00001), and insertions at residue 24 (P < .00001), I25L/Y (P < .0004), R28K (P < .0004), and R30K (P < .014). These mutations were not found in R5 isolates, except R28K and R30K, which were detected in 4 and 5 R5 isolates, respectively. The 4 major genotypic determinants of dual/X4 tropism were mutation at residue 18, V19 K/R mutation, insertions at residue 24, and V3 global net charge. CONCLUSIONS We established a strong association between HIV-2 phenotypic tropism and V3-loop sequences, allowing for the prediction of R5- and/or X4-tropic viruses in HIV-2 infection.

In vitro phenotypic susceptibility of HIV-2 clinical isolates to CCR5 inhibitors

ABSTRACT HIV-2 is naturally resistant to nonnucleoside reverse transcriptase inhibitors, to a fusion inhibitor, and to some of the protease inhibitors. Maraviroc is the first drug of the new anti-CCR5 drug class and is effective only on CCR5-tropic (R5) HIV-1. No previous studies concerning HIV-2 susceptibility to maraviroc have been reported yet. We developed a phenotypic maraviroc susceptibility test using a peripheral blood mononuclear cell (PBMC) model. We analyzed the maraviroc susceptibility of 13 R5 HIV-2, 2 X4R5 (dual) HIV-2, and 2 CXCR4-tropic (X4) HIV-2 clinical isolates. We also tested, with the same protocol, 1 X4 HIV-1 and 4 R5 HIV-1 clinical isolates. For the R5 HIV-2 clinical isolates, the 50% effective concentration (EC50) for maraviroc was 0.80 nM (interquartile range [IQR], 0.48 to 1.39 nM), similar to that observed for the R5 HIV-1 isolates. The median maximum percentage of inhibition in the R5 HIV-2 isolates was 93% (IQR, 84 to 98%), similar to that observed in the R5 HIV-1 isolates. As expected, both X4 HIV-1 and HIV-2 were highly resistant to maraviroc. Our study showed for the first time that maraviroc is active in vitro against R5 HIV-2. The new tools we developed will allow identification of HIV-2-infected patients eligible for CCR5 inhibitor use and management of virological failure when receiving a maraviroc-based regimen.

HIV-2 infects resting CD4+ T cells but not monocyte-derived dendritic cells.

BackgroundHuman Immunodeficiency Virus-type 2 (HIV-2) encodes Vpx that degrades SAMHD1, a cellular restriction factor active in non-dividing cells. HIV-2 replicates in lymphocytes but the susceptibility of monocyte-derived dendritic cells (MDDCs) to in vitro infection remains partly characterized.ResultsHere, we investigated HIV-2 replication in primary CD4+ T lymphocytes, both activated and non-activated, as well as in MDDCs. We focused on the requirement of Vpx for productive HIV-2 infection, using the reference HIV-2 ROD strain, the proviral clone GL-AN, as well as two primary HIV-2 isolates. All HIV-2 strains tested replicated in activated CD4+ T cells. Unstimulated CD4+ T cells were not productively infected by HIV-2, but viral replication was triggered upon lymphocyte activation in a Vpx-dependent manner. In contrast, MDDCs were poorly infected when exposed to HIV-2. HIV-2 particles did not potently fuse with MDDCs and did not lead to efficient viral DNA synthesis, even in the presence of Vpx. Moreover, the HIV-2 strains tested were not efficiently sensed by MDDCs, as evidenced by a lack of MxA induction upon viral exposure. Virion pseudotyping with VSV-G rescued fusion, productive infection and HIV-2 sensing by MDDCs.ConclusionVpx allows the non-productive infection of resting CD4+ T cells, but does not confer HIV-2 with the ability to efficiently infect MDDCs. In these cells, an entry defect prevents viral fusion and reverse transcription independently of SAMHD1. We propose that HIV-2, like HIV-1, does not productively infect MDDCs, possibly to avoid triggering an immune response mediated by these cells.

Evolution of the K65R, K103N and M184V/I reverse transcriptase mutations in HIV-1-infected patients experiencing virological failure between 2005 and 2010

OBJECTIVES To assess the prevalence of the K65R, K103N and M184V/I resistance mutations in the reverse transcriptase (RT) region in HIV-1-infected patients failing antiretroviral-based regimens between the years 2005 and 2010. PATIENTS AND METHODS HIV-1-infected patients experiencing virological failure between 2005 and 2010 with RT genotypic resistance tests available at the time of virological failure were analysed. K65R, K103N and M184V/I mutation frequencies were determined each year. Statistical analyses were performed using Fishers exact test. RESULTS Among 9586 patients failing their antiretroviral-based regimens from 2005 to 2010, the prevalence of K65R tended to decrease (P = 0.054), while K103N and M184V/I mutation frequencies decreased significantly over time (P < 0.001). The increased use of a tenofovir/emtricitabine/efavirenz single-tablet regimen was associated with decreased selection of these mutations. CONCLUSIONS The global prevalence of resistance-associated mutations to tenofovir, lamivudine/emtricitabine and efavirenz decreased over time between 2005 and 2010. Despite a stable rate of efavirenz and protease inhibitor use, this phenomenon can be explained by an increased use of single-tablet regimens, which simplify drug intake and maximize adherence.

Positive Impact of HIV-1 gag Cleavage Site Mutations on the Virological Response to Darunavir Boosted with Ritonavir

ABSTRACT We assessed the roles of baseline gag and gag-pol cleavage site mutations (CSM) on the virological outcome of a darunavir-based regimen in highly antiretroviral-experienced patients. We showed the association, in multivariate analysis, between the A431V gag CSM and the virological response, defined as a reduction in plasma HIV-1 RNA to <50 copies/ml at month 3 (P = 0.028). Our results suggest that a specific gag CSM might have a role on protease inhibitor susceptibility in an inhibitor-specific manner.

Usefulness of multiplex PCR methods and respiratory viruses' distribution in children below 15 years old according to age, seasons and clinical units in France: A 3 years retrospective study.

Background To date, only influenza and RSV testing are recommended for respiratory viruses’ detection in paediatric units. In this study, we described, according to seasons, ages and clinical units, the results obtained in children (<15 years old) by multiplex-PCR (mPCR) tests allowing a quick and wide range detection of all respiratory viruses. These results were also compared with RSV specific detection. Methods All nasopharyngeal mPCR and RSV tests requested by clinicians in our French teaching hospitals group between 2011 and 2014 were retrospectively included. All repeated samples for the same children in the same month were discarded. Results Of the 381 mPCR tests (344 children) performed, 51.4% were positive. Positivity and viral co-infection rates were higher in the 6–36 months old strata (81% and 25%, p<0.0001 and p = 0.04, respectively). Viral distribution showed strong variations across ages. During specific influenza epidemic periods, only 1/39 (2.5%) mPCR tests were positive for influenza and 19/39 (48.7%) for other viruses. During specific RSV epidemic periods, only 8/46 (17.4%) mPCR tests were positive for RSV and 14/46 (30.4%) for other viruses. 477/1529 (31.2%) of RSV immunochromatography-tests were positive. Among the negatives immunochromatography-test also explored by mPCR, 28/62 (31%) were positive for other respiratory viruses. Conclusion This study provides a wide description of respiratory viruses’ distribution among children in hospital settings using mPCR over 3 years. It emphasizes the number of undiagnosed respiratory viruses according to the current diagnosis practice in France and gives a better picture of respiratory viruses identified in hospital settings by mPCR all over the year in France.

Concordance between HIV-2 genotypic coreceptor tropism predictions based on plasma RNA and proviral DNA.

In this study, assessing HIV-2 tropism among 43 paired plasma/peripheral blood mononuclear cell specimens, the concordance between proviral DNA and plasma RNA genotypic tropism prediction was 74%. All the discordances were attributable to the prediction of R5 in RNA and X4/dual-mixed in DNA. HIV-2 genotypic tropism test based on proviral DNA is a suitable tool for tropism determination in HIV-2-infected patients with low or undetectable viral load.

Impact of respiratory viruses in hospital-acquired pneumonia in the intensive care unit: A single-center retrospective study

Abstract Background Data on the frequency and role of respiratory viruses (RVs) in hospital-acquired pneumonia (HAP) are still scarce. Objectives We assessed the proportion of RVs and their impact on the outcome of hospital-acquired pneumonia (HAP) in the intensive care unit (ICU). Study design Cases of HAP were retrospectively selected among patients who underwent screening for RVs by multiplex PCR (mPCR) in the ICU of a French tertiary care hospital from May 2014 to April 2016. ICU length of stay and in-hospital mortality were compared between four groups defined according to the identified pathogens: virus only (V), virus/bacteria (V/B), bacteria only (B) and no pathogen (Neg). When available, previous mPCR was retrieved in order to assess possible chronic viral carriage. Results Overall, 95/999 (10%) ICU patients who underwent mPCR had HAP (V(17,18%), V/B(13,14%), B(60,63%), Neg(5,5%)). Median age was 61 years and 45 (47%) were immunocompromised. Influenza (27%) and rhinovirus (27%) were the most common RVs. V/B group had higher mortality rate than B and V groups (62% vs. 40% and 35%, p=0.3) and a significantly longer length of stay (31days (18–48)) than V group (5days (3–11), p=0.0002)) and B group (14.5days (5.5–25.5), p=0.007)). Among the 15 patients with available mPCR tests before viral HAP, seven were negative and eight were positive corresponding to long-term carriage of community-acquired viruses. Discussion RVs were detected in 32% of HAP patients who underwent mPCR. Two situations were encountered: (i) acute acquired viral infection; (ii) long-term viral carriage (mostly rhinovirus) especially in immunocompromised patients complicated by a virus/bacteria coinfection. The latter was associated with a longer length of stay and a trend toward a higher mortality.

Short communication: Prevalence of HIV-1 transmitted drug resistance in Liberia.

Abstract No data on HIV-transmitted drug resistance (TDR) are available in Liberia in which the HIV prevalence in the general population is estimated at 1.5%. The aim of the study was to assess the prevalence of TDR in HIV-1 from recently diagnosed and untreated patients living in Monrovia, Liberia. The study was performed in the John F. Kennedy Medical Center and in the Redemption Hospital, both located in Monrovia. All newly HIV-1 diagnosed patients attending voluntary counseling testing centers and antiretroviral therapy naive were consecutively included. Protease and reverse transcriptase (RT) regions sequencing was performed using the ANRS procedures (www.hivfrenchresistance.org). Drug resistance mutations (DRM) were identified according to the 2009 updated WHO surveillance DRM list. Among the 116 HIV-1-infected patients enrolled in the study, 85 (73%) were women. Protease and RT sequencing was successful in 109 (94%) and 102 (88%) samples, respectively. Seventy-five (66%) patients were infected with...No data on HIV-transmitted drug resistance (TDR) are available in Liberia in which the HIV prevalence in the general population is estimated at 1.5%. The aim of the study was to assess the prevalence of TDR in HIV-1 from recently diagnosed and untreated patients living in Monrovia, Liberia. The study was performed in the John F. Kennedy Medical Center and in the Redemption Hospital, both located in Monrovia. All newly HIV-1 diagnosed patients attending voluntary counseling testing centers and antiretroviral therapy naive were consecutively included. Protease and reverse transcriptase (RT) regions sequencing was performed using the ANRS procedures (www.hivfrenchresistance.org). Drug resistance mutations (DRM) were identified according to the 2009 updated WHO surveillance DRM list. Among the 116 HIV-1-infected patients enrolled in the study, 85 (73%) were women. Protease and RT sequencing was successful in 109 (94%) and 102 (88%) samples, respectively. Seventy-five (66%) patients were infected with CRF02_AG. One DRM was observed in six samples, leading to a TDR prevalence of 5.9% (CI 95%=1.7-10.1). DRM were observed in two patients (2.0%; CI 95%=0.0-4.7), four patients (3.9%; CI 95%=0.1-7.7), and one patient (0.9%; CI 95%=0.0-2.7) for nucleoside RT inhibitors (NRTI), non-NRTI (NNRTI), and protease inhibitors, respectively. Overall, one patient exhibited dual class-resistant viruses, harboring NRTI and NNRTI resistance mutations (1.0%; CI 95%=0.0-2.9). This first survey study in Liberia reported a TDR prevalence of 5.9%, classified as moderate according to the WHO criteria, indicating that further surveillance is warranted to follow the level and evolution of TDR prevalence in recently HIV-1 diagnosed patients.