Diagnostic yield of viral multiplex PCR during acute exacerbation of COPD admitted to the intensive care unit: a pilot study.

Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is one of the leading causes of admission to the intensive care unit, often triggered by a respiratory tract infection of bacterial or viral aetiology. Managing antibiotic therapy in this context remains a challenge. Respiratory panel molecular tests allow identifying viral aetiologies of AECOPD. We hypothesized that the systematic use of a respiratory multiplex PCR (mPCR) would help antibiotics saving in severe AECOPD. Our objectives were to describe the spectrum of infectious aetiologies of severe AECOPD, using a diagnostic approach combining conventional diagnostic tests and mPCR, and to measure antibiotics exposure. The study was bicentric, prospective, observational, and included 105 critically ill patients with a severe AECOPD of presumed infectious aetiology, in whom a respiratory mPCR with a viral panel was performed in addition to conventional microbiological tests. Altogether, the microbiological documentation rate was 50%, including bacteria alone (19%), respiratory viruses alone (16%), and mixed viruses and bacterial species (16%). The duration of antibiotic therapy was shorter in patients without documented bacterial infection (5.6 vs. 9 days; P = 0.0006). This pilot study suggests that molecular tests may help for the proper use of anti-infective treatments in critically ill patients with severe AECOPD.

Methyl-qPCR: a new method to investigate Epstein-Barr virus infection in post-transplant lymphoproliferative diseases.

Epstein-Barr virus DNA viral load is used as a surrogate marker to start Rituximab in transplant recipients at risk of developing PTLD. However, an elevated EBV DNAemia does not discriminate lymphoproliferation and replication. We designed a new molecular assay (methyl-qPCR) to distinguish methylated versus unmethylated viral genomes. In blood, viral genomes were highly methylated in EBV primary infections, PTLD and 4/5 transplant recipients with high viral load. The only patient with under-methylated EBV genomes did not respond to rituximab. Methyl-qPCR is a convenient method to discriminate between latent and lytic EBV genomes and could be useful in treatment decisions.

Factors associated with the emergence of integrase resistance mutations in patients failing dual or triple integrase inhibitor-based regimens in a French national survey.

BACKGROUND: Successful 2-drug regimens (2DRs) for HIV were made possible by the availability of drugs combining potency and tolerability with a high genetic barrier to resistance. How these deal with resistance development/re-emergence, compared with 3DRs, is thus of paramount importance. MATERIALS AND METHODS: A national survey including patients who were either naive or experienced with any 2DR or 3DR but failing integrase strand transfer inhibitor (INSTI)-containing regimens [two consecutive plasma viral load (VL) values >50 copies/mL] was conducted between 2014 and 2019. Genotypic resistance tests were interpreted with the v28 ANRS algorithm. RESULTS: Overall, 1104 patients failing any INSTI-containing regimen (2DRs, n = 207; 3DRs, n = 897) were analysed. Five hundred and seventy-seven (52.3%) patients were infected with a B subtype and 527 (47.3%) with non-B subtypes. Overall, 644 (58%) patients showed no known integrase resistance mutations at failure. In multivariate analysis, factors associated with the emergence of at least one integrase mutation were: high VL at failure (OR = 1.24 per 1 log10 copies/mL increase); non-B versus B subtype (OR = 1.75); low genotypic sensitivity score (GSS) (OR = 0.10 for GSS = 2 versus GSS = 0-0.5); and dolutegravir versus raltegravir (OR = 0.46). Although 3DRs versus 2DRs reached statistical significance in univariate analysis (OR = 0.59, P = 0.007), the variable is not retained in the final model. CONCLUSIONS: This study is one of the largest studies characterizing integrase resistance in patients failing any INSTI-containing 2DR or 3DR in routine clinical care and reveals factors associated with emergence of integrase resistance that should be taken into consideration in clinical management. No difference was evidenced between patients receiving a 2DR or a 3DR.

Caution in interpretation of SARS-CoV-2 quantification based on RT-PCR cycle threshold value.

RT-PCR is the reference method for diagnosis of a Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection. During the setting up of 6 SARS-CoV-2 RT-PCR assays in our laboratory, comparative evaluations were systematically undertaken and allowed to evidence major discrepancies on cycle threshold RT-PCR results between techniques. These tendencies were confirmed in routine application when analyzing sequential samples from the same patients. Our aim was to examine the impact of the technique among factors influencing RT-PCR result, a far surrogate of 'viral load' in the heterogeneous environment of respiratory specimens.

Low level of baseline resistance in recently HCV-infected men who have sex with men with high-risk behaviours.

OBJECTIVES: Presence of baseline hepatitis C virus (HCV) resistance-associated substitutions (RASs) can impair treatment outcome of direct-acting antivirals. We investigated the prevalence of pre-treatment HCV resistance among recently HCV-infected men who have sex with men (MSM) with high risk behaviours, either human immunodeficiency virus (HIV) co-infected or at high risk of HIV acquisition and under pre-exposure prophylaxis (PrEP). METHODS: NS5A and NS3 fragments were deep sequenced on pre-treatment samples of 72 subjects using Illumina MiSeq paired-end sequencing technology. Ultra-deep sequencing data were analysed by SmartGene® platform. RASs mentioned in the literature were analysed and interpreted depending on genotype (GT) at 10% cut-off. RESULTS: HCV genotyping showed 36 (50.0%) GT1a, 31 (43.1%) GT4d and 5 (6.9%) GT3a infections. Fifty-five patients (76.4%) were co-infected with HIV and 15 (20.8%) received PrEP. In GT1a viruses, NS3 RASs were found in 4/30 viruses (13.3%; S122 G/N, R155 K and I170 V) and Q80 K polymorphism was present in 14/30 viruses (46.7%). No NS3 RASs were detected in GT4d and GT3a viruses. NS5A RASs were detected in 3/36 GT1a viruses (8.3%; Q30E/R, L31 M and H58 L). NS5A subtype-specific polymorphisms L30R and T58 P were found at high frequencies in 31/31 (100%) and 16/31 (51.6%) GT4d viruses, respectively. One RAS M31 L was also observed along with the polymorphisms L30R and T58 P. No NS5A RASs were detected in GT3a viruses. CONCLUSION: A low level of RASs to NS3 and NS5A inhibitors in pre-treatment samples was detected in the study population. Our findings reassure the clinical management of HCV infection in this high-risk population.

New Kaposi's sarcoma-associated herpesvirus variant in men who have sex with men associated with severe pathologies.

BACKGROUND: Kaposi sarcoma (KS)-associated herpesvirus (KSHV) subtype depends mostly on patient origin. The current study aimed to assess KSHV diversity in a population of men who have sex with men (MSM) living in France. METHODS: The study included 264 patients. In 65 MSM, including 57 human immunodeficiency virus (HIV)-infected men with KS, multicentric Castleman disease, or primary effusion lymphoma and 8 HIV-uninfected men receiving HIV preexposure prophylaxis (PrEP), we performed KSHV typing with K1 open reading frame Sanger and KSHV whole-genome sequencing. In 199 other patients, we performed real-time polymerase chain reaction screening for the new variant. RESULTS: We found that 51% of KSHV-strains were subtype C (85% C3), and 33% were subtype A. Four patients with severe KSHV disease (2 with visceral KS, 1 with multicentric Castleman disease, and 1 with primary effusion lymphoma) and 1 asymptomatic PrEP user had a new variant resembling the Ugandan subtype F, but with different K1 open reading frame and KSHV whole-genome sequences and a different epidemiological context (MSM vs African population). Its prevalence was 4.5% in Caucasian MSM, and it was absent in other epidemiological groups. CONCLUSIONS: Subtype C predominated among MSM living in France. The new F variant was identified in Caucasian MSM and associated with severe KSHV disease, suggesting that subtype F could be split into F1 and F2 variants. Careful screening for this variant may be required in MSM, given the severe clinical presentation of associated diseases.

Pre-emptive rituximab treatment for Epstein-Barr virus reactivation after allogeneic hematopoietic stem cell transplantation is a worthwhile strategy in high-risk recipients: a comparative study for immune recovery and clinical outcomes.

This retrospective study evaluated the impact of a pre-emptive rituximab (RTX) strategy for Epstein-Barr virus (EBV) reactivation on immune recovery and outcomes of 219 high-risk recipients undergoing allogeneic stem cell transplantation (allo-SCT) for hematological malignancies or bone marrow failure. One-hundred and seven patients received pre-emptive RTX for EBV reactivation (RTX group) and 112 did not (control group). The median onset time of EBV reactivation was 49 days (range, 14-561), including five patients who developed post-transplant lymphoproliferative disorder (EBV-PTLD). RTX and control groups were pair-matched to assess the impact of RTX on all endpoints. In RTX patients, CD19 + B cells were significantly decreased until 1-year post-transplant, so were immunoglobulin levels. Twenty-one patients (17%) developed RTX-related neutropenia. There was, in the RTX group, a trend towards a lower cumulative incidence of chronic GvHD (P = 0.059). Overall survival, progression-free survival, non-relapse mortality, relapse incidence, and incidence of overall infections at 2 years following allo-SCT were comparable in the two groups. We conclude that pre-emptive RTX, despite inducing a delayed B-cell reconstitution and a high risk of RTX-related neutropenia, may be considered as a worthwhile treatment, given the absence of negative impact on post allo-SCT outcomes and a low incidence of EBV-PTLD.

Surveillance of HIV-1 primary infections in France from 2014 to 2016: toward stable resistance, but higher diversity, clustering and virulence?

OBJECTIVES: Patients with primary HIV-1 infection (PHI) are a particular population, giving important insight about ongoing evolution of transmitted drug resistance-associated mutation (TDRAM) prevalence, HIV diversity and clustering patterns. We describe these evolutions of PHI patients diagnosed in France from 2014 to 2016. METHODS: A total of 1121 PHI patients were included. TDRAMs were characterized using the 2009 Stanford list and the French ANRS algorithm. Viral subtypes and recent transmission clusters (RTCs) were also determined. RESULTS: Patients were mainly MSM (70%) living in the Paris area (42%). TDRAMs were identified among 10.8% of patients and rose to 18.6% when including etravirine and rilpivirine TDRAMs. Prevalences of PI-, NRTI-, first-generation NNRTI-, second-generation NNRTI- and integrase inhibitor-associated TDRAMs were 2.9%, 5.0%, 4.0%, 9.4% and 5.4%, respectively. In a multivariable analysis, age >40 years and non-R5 tropic viruses were associated with a >2-fold increased risk of TDRAMs. Regarding HIV diversity, subtype B and CRF02_AG (where CRF stands for circulating recombinant form) were the two main lineages (56% and 20%, respectively). CRF02_AG was associated with higher viral load than subtype B (5.83 versus 5.40 log10 copies/mL, P=0.004). We identified 138 RTCs ranging from 2 to 14 patients and including overall 41% from the global population. Patients in RTCs were younger, more frequently born in France and more frequently MSM. CONCLUSIONS: Since 2007, the proportion of TDRAMs has been stable among French PHI patients. Non-B lineages are increasing and may be associated with more virulent CRF02_AG strains. The presence of large RTCs highlights the need for real-time cluster identification to trigger specific prevention action to achieve better control of the epidemic.

Uncommon Detection of Mixed HCV Genotype Infections in Recently Infected Men Who Have Sex with Men.

INTRODUCTION: Mixed hepatitis C virus (HCV) genotype (GT) infections are clinically important as different genotypes have varied sensitivities to direct-acting antivirals (DAAs). A high prevalence of mixed GT infections was observed in individuals who inject drugs and had multiple HCV exposures. The prevalence of mixed HCV GT infections in men who have sex with men (MSM) and high-risk behaviors was investigated by ultra-deep sequencing (UDS). METHODS: NS5B fragment was sequenced from viruses of patients with recent HCV infection: there were 50 HIV-positive and 18 HIV-negative patients, including 13 from the ANRS Pre-Exposure Prophylaxis (PrEP) IPERGAY study. UDS data were analysed using Geneious (version 10.3.2). Phylogenetic trees were constructed using FastTree (version 2.1). RESULTS: HCV sequencing showed GT1a (47.1%), GT4d (41.2%), GT3a (8.8%) and GT2k (2.9%). We detected three (4.4%) mixed GT infections: one between predominant GT4d and minority GT1a, one between predominant GT4d and minority GT1b, and one between predominant GT1a and minority GT4d virus. The rates of minority GT viral populations detected in viruses of the three patients with mixed GT infections were 0.32%, 10.7%, and 1.3%, respectively. The first two patients were HIV co-infected and the third was HIV-negative under PrEP. The anti-HCV treatment was successful in all three patients. CONCLUSION: This work showed uncommon mixed HCV GT infections in MSM at high risk of multiple HCV exposures. The impact of these infections on treatment response has not been established but further studies on more patients are necessary. To prevent treatment failure in this population, regular monitoring of treatment response is needed, particularly when pan-genotypic treatment is not used.

Shared HCV Transmission Networks Among HIV-1-Positive and HIV-1-Negative Men Having Sex With Men by Ultradeep Sequencing.

OBJECTIVE: Several studies reported hepatitis C virus (HCV) transmission networks among men having sex with men (MSM) in Europe and the spread of HCV strains from HIV-HCV coinfected toward HCV monoinfected MSM. We aimed to investigate HCV transmission dynamics among HIV-positive and HIV-negative MSM by ultradeep sequencing (UDS). DESIGN AND METHODS: NS5B fragment (388 bp) was sequenced from virus of 50 HIV-positive and 18 HIV-negative patients diagnosed with recent HCV infection. UDS data were analyzed by Geneious (version 10.3.2). Phylogenetic trees were constructed by FastTree (version 2.1) and submitted to ClusterPicker (version 1.2.3) for transmission chain detection at different thresholds of maximum genetic distance (MGD) (3% for Sanger, 3% and 4.5% for UDS). RESULTS: Ten, 17, and 18 HCV transmission chains were identified by Sanger at 3%, UDS at 3% and at 4.5% of MGD, respectively. Of 68 subjects enrolled, 38 (55.9%), 38 (55.9%), and 43 (65.3%) individuals were involved in transmission networks found by Sanger at 3%, UDS at 3%, and at 4.5% of MGD, respectively. Mixed transmission chains including HIV-positive and HIV-negative subjects were detected for 8/10 chains by Sanger at 3%, for 9/17 by UDS at 3%, and for 10/18 by UDS at 4.5% of MGD. Overall, the number of HIV-negative individuals clustering with HIV-positive ones was 9/18 by Sanger, 9/18 by UDS at 3%, and 10/18 by UDS at 4.5% of MGD. CONCLUSIONS: HIV-positive and HIV-negative MSM shared HCV transmission networks, which emphasizes the need for HCV surveillance and prevention measures in these communities regardless of the HIV status.

Resistance to integrase inhibitors: a national study in HIV-1-infected treatment-naive and -experienced patients.

OBJECTIVES: To describe integrase strand transfer inhibitor (INSTI) resistance profiles and factors associated with resistance in antiretroviral-naive and -experienced patients failing an INSTI-based regimen in clinical practice. METHODS: Data were collected from patients failing an INSTI-containing regimen in a multicentre French study between 2014 and 2017. Failure was defined as two consecutive plasma viral loads (VL) >50 copies/mL. Reverse transcriptase, protease and integrase coding regions were sequenced at baseline and failure. INSTI resistance-associated mutations (RAMs) included in the Agence Nationale de Recherches sur le SIDA genotypic algorithm were investigated. RESULTS: Among the 674 patients, 359 were failing on raltegravir, 154 on elvitegravir and 161 on dolutegravir therapy. Overall, 90% were experienced patients and 389 (58%) patients showed no INSTI RAMs at failure. The strongest factors associated with emergence of at least one INSTI mutation were high VL at failure (OR = 1.2 per 1 log10 copies/mL increase) and low genotypic sensitivity score (GSS) (OR = 0.08 for GSS ≥3 versus GSS = 0-0.5). Patients failing dolutegravir also had significantly fewer INSTI RAMs at failure than patients failing raltegravir (OR = 0.57, P = 0.02) or elvitegravir (OR = 0.45, P = 0.005). Among the 68 patients failing a first-line regimen, 11/41 (27%) patients on raltegravir, 7/18 (39%) on elvitegravir and 0/9 on dolutegravir had viruses with emergent INSTI RAMs at failure. CONCLUSIONS: These results confirmed the robustness of dolutegravir regarding resistance selection in integrase in the case of virological failure in routine clinical care.

Stable prevalence of transmitted drug resistance mutations and increased circulation of non-B subtypes in antiretroviral-naive chronically HIV-infected patients in 2015/2016 in France.

OBJECTIVES: We estimated the prevalence of transmitted-drug-resistance-associated mutations (TDRAMs) in antiretroviral-naive chronically HIV-1-infected patients. PATIENTS AND METHODS: TDRAMs were sought in samples from 660 diagnosed HIV-1-infected individuals in 2015/2016 in 33 HIV clinical centres. Weighted analyses, considering the number of patients followed in each centre, were used to derive representative estimates of the percentage of individuals with TDRAMs. Results were compared with those of the 2010/2011 survey (n = 661) using the same methodology. RESULTS: At inclusion, median CD4 cell counts and plasma HIV-1 RNA were 394 and 350/mm3 (P = 0.056) and 4.6 and 4.6 log10 copies/mL (P = 0.360) in the 2010/2011 survey and the 2015/2016 survey, respectively. The frequency of non-B subtypes increased from 42.9% in 2010/2011 to 54.8% in 2015/2016 (P < 0.001), including 23.4% and 30.6% of CRF02_AG (P = 0.004). The prevalence of virus with protease or reverse-transcriptase TDRAMs was 9.0% (95% CI = 6.8-11.2) in 2010/2011 and 10.8% (95% CI = 8.4-13.2) in 2015/2016 (P = 0.269). No significant increase was observed in integrase inhibitor TDRAMs (6.7% versus 9.2%, P = 0.146). Multivariable analysis showed that men infected with the B subtype were the group with the highest risk of being infected with a resistant virus compared with others (adjusted OR = 2.2, 95% CI = 1.3-3.9). CONCLUSIONS: In France in 2015/2016, the overall prevalence of TDRAMs was 10.8% and stable compared with 9.0% in the 2010/2011 survey. Non-B subtypes dramatically increased after 2010. Men infected with B subtype were the group with the highest risk of being infected with a resistant virus, highlighting the need to re-emphasize safe sex messages.

High clustering of acute HCV infections and high rate of associated STIs among Parisian HIV-positive male patients.

BACKGROUND: Increasing incidence of hepatitis C virus (HCV) infection in human immunodeficiency virus (HIV)-positive men having sex with men (MSM) has been described in recent years. Phylogenetic analyses of acute HCV infections were undertaken to characterize the dynamics during the epidemic in Paris, and associated sexually transmitted infections (STIs) were evaluated. METHODS: Sanger sequencing of polymerase gene was performed. Maximum likelihood phylogenies were reconstructed using FastTree 2.1 under a GTR+CAT model. Transmission chains were defined as clades with a branch probability ≥0.80 and intraclade genetic distances <0.02 nucleotide substitutions per sites. STIs detected ≤1 month before HCV diagnosis were considered. RESULTS: Among the 85 studied patients, at least 81.2% were MSM. Respectively, 47.6%, 39.0%, 11.0% and 2.4% were infected with genotypes 1a, 4d, 3a and 2k. At least 91.8% were co-infected with HIV. HCV re-infection was evidenced for 24.7% of patients and STIs for 20.0% of patients. Twenty-two transmission chains were identified, including 52 acute hepatitis C (11 pairs and 11 clusters from three to seven patients). CONCLUSIONS: These results revealed strong clustering of acute HCV infections. Thus, rapid treatment of both chronic and acute infections is needed among this population to decrease the prevalence of HCV, in combination with preventive behavioural interventions.

Phenotypic analysis of HIV-1 E157Q integrase polymorphism and impact on virological outcome in patients initiating an integrase inhibitor-based regimen.

Objectives: To assess the phenotypic susceptibility of the E157Q polymorphism in HIV-1 integrase (IN) and the virological outcome of patients infected with E157Q-mutated virus initiating an IN inhibitor (INI)-based regimen. Methods: This was a multicentre study assessing IN sequences from INI-naive patients among 17 French HIV clinical centres. E157Q site-directed mutants in pNL4.3 and pCRF02_AG contexts were assessed in a recombinant phenotypic assay. Results: Prevalence of the E157Q polymorphism was 2.7% among 8528 IN sequences from INI-naive patients and its distribution was 1.7%, 5.6% and 2.2% in B, CRF02_AG and various non-B subtypes, respectively. Thirty-nine INI-naive patients with E157Q-mutated virus initiated an INI-based regimen. Among them, 15 had a viral load (VL) <50 copies/mL at initiation and virological suppression was maintained during the first year of follow-up in all but two exhibiting a viral blip. Twenty-four patients had a VL > 50 copies/mL at the time of INI-based regimen initiation. Among them eight were receiving a first-line regimen and the only two patients who did not reach VL < 50 copies/mL at week 24 were receiving elvitegravir. The 16 remaining patients were ART experienced in virological failure with drug-resistant viruses displaying several virological outcomes independently of the genotypic susceptibility score. Phenotypic analyses showed a fold change in EC50 of 0.6, 0.9 and 1.9 for raltegravir, dolutegravir and elvitegravir, respectively, in a subtype B context, and 1.1, 1.9 and 2.4 for raltegravir, dolutegravir and elvitegravir, respectively, in a CRF02_AG context. Conclusions: Assessment of virological response in 39 patients initiating an INI-based regimen with E157Q-mutated virus, in combination with phenotypic analysis, suggests that particular attention should be paid to antiretroviral-naive patients and dolutegravir should be preferentially used in these patients.

Impact of Human Immunodeficiency Virus Type 1 Minority Variants on the Virus Response to a Rilpivirine-Based First-line Regimen.

Background: Minority resistant variants of human immunodeficiency virus type 1 (HIV-1) could influence the virological response to treatment based on nonnucleoside reverse transcriptase inhibitors (NNRTIs). Data on minority rilpivirine-resistant variants are scarce. This study used next-generation sequencing (NGS) to identify patients harboring minority resistant variants to nucleos(t)ide reverse transcriptase inhibitors and NNRTIs and to assess their influence on the virological response (VR). Methods: All the subjects, 541 HIV-1-infected patients started a first-line regimen containing rilpivirine. VR was defined as a HIV-1 RNA load <50 copies/mL at month 6 with continued suppression at month 12. NGS was performed at baseline (retrospectively) on the 454 GS-FLX platform (Roche). Results: NGS revealed resistance-associated mutations accounting for 1% to <5% of variants in 17.2% of samples, for 5%-20% in 5.7% of samples, and for >20% in 29% of samples. We identified 43 (8.8%) and 36 (7.4%) patients who harbored rilpivirine-resistant variants with a 1% sensitivity threshold according to the French National Agency for Research on AIDS and Viral Hepatitis and Stanford algorithms, respectively. The VR was 96.9% at month 12. Detection of minority rilpivirine resistant variants was not associated with virological failure (VF). Multivariate analysis indicated that VF at month 12 was associated with a CD4 count <250 cells/µL at baseline, a slower decrease in viral load at month 3, and rilpivirine resistance at baseline using the Stanford algorithm with a 20% threshold. Conclusions: Minority resistant variants had no impact on the VR of treatment-naive patients to a rilpivirine-based regimen.

Long-term AIDS-related PCNSL outcomes with HD-MTX and combined antiretroviral therapy.

OBJECTIVE: To assess the characteristics and outcomes of patients with AIDS-related primary CNS lymphoma (AR-PCNSL) in the combined antiretroviral therapy (cART) era systematically treated with high-dose methotrexate (HD-MTX). METHODS: We retrospectively analyzed (intention-to-treat analysis) 51 consecutive patients with AR-PCNSL (median age 39 years) who were diagnosed from 1996 to 2014 and treated with a median of 6 (range 1-15) infusions of HD-MTX (3 g/m2) combined with cART. RESULTS: Median all-patients' and survivors' follow-up lasted 23 (range 0-186) and 76 (range 23-186) months, respectively. At PCNSL diagnosis, 83% of the patients were on cART, median plasma HIV load was 175,600 copies/mL, and median CD4+ T-cell count was 24/µL. Median Eastern Cooperative Oncology Group performance status was 2 (range 1-4). Median overall survival (OS) was 5.7 years, with 5- and 10-year rates of 48% and 41%. Median time to progression was not reached (69% at 10 months). PCNSL was the direct cause of 14 deaths, all observed within the 10 months after its diagnosis: 6 patients died before HD-MTX could be administered, 4 had refractory disease, and 4 relapsed. Multivariate analyses retained time interval between AIDS diagnosis and PCNSL diagnosis, age at AR-PCNSL diagnosis, and deep brain structure involvement as independent OS-predictive factors. To restore effective immune function, cART tailored to HIV genotypes was started and combined with HD-MTX; no interactions and no immune reconstitution inflammatory syndrome occurred. No patient died of acute treatment-related toxicity, and 21 of 51 (41%) patients experienced grade 3/4 toxicity. CONCLUSIONS: Combined short-term HD-MTX monochemotherapy and optimal cART simply and effectively treat AR-PCNSL, achieving long-term survival with few relapses. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that short-term HD-MTX monochemotherapy improves long-term survival of patients with AIDS with primary CNS lymphoma receiving cARTs.

Antiretroviral-treated HIV-1 patients can harbour resistant viruses in CSF despite an undetectable viral load in plasma.

Background: HIV therapy reduces the CSF HIV RNA viral load (VL) and prevents disorders related to HIV encephalitis. However, these brain disorders may persist in some cases. A large population of antiretroviral-treated patients who had a VL > 1.7 log 10 copies/mL in CSF with detectable or undetectable VL in plasma associated with cognitive impairment was studied, in order to characterize discriminatory factors of these two patient populations. Methods: Blood and CSF samples were collected at the time of neurological disorders for 227 patients in 22 centres in France and 1 centre in Switzerland. Genotypic HIV resistance tests were performed on CSF. The genotypic susceptibility score was calculated according to the last Agence Nationale de Recherche sur le Sida et les hépatites virales Action Coordonnée 11 (ANRS AC11) genotype interpretation algorithm. Results: Among the 227 studied patients with VL > 1.7 log 10 copies/mL in CSF, 195 had VL detectable in plasma [median (IQR) HIV RNA was 3.7 (2.7-4.7) log 10 copies/mL] and 32 had discordant VL in plasma (VL < 1.7 log 10 copies/mL). The CSF VL was lower (median 2.8 versus 4.0 log 10 copies/mL; P < 0.001) and the CD4 cell count was higher (median 476 versus 214 cells/mm 3 ; P < 0.001) in the group of patients with VL < 1.7 log 10 copies/mL in plasma compared with patients with plasma VL > 1.7 log 10 copies/mL. Resistance to antiretrovirals was observed in CSF for the two groups of patients. Conclusions: Fourteen percent of this population of patients with cognitive impairment and detectable VL in CSF had well controlled VL in plasma. Thus, it is important to explore CSF HIV (VL and genotype) even if the HIV VL is controlled in plasma because HIV resistance may be observed.

Drug resistance and tropism as markers of the dynamics of HIV-1 DNA quasispecies in blood cells of heavily pretreated patients who achieved sustained virological suppression.

OBJECTIVES: The objective of this study was to address the dynamics of archived resistant quasispecies in cell-associated HIV-1 DNA over time in heavily ART-experienced patients with currently suppressed plasma HIV-1 RNA. METHODS: Longitudinal ultra-deep sequencing (UDS) analysis of reverse transcriptase, protease and V3 Env regions was performed on blood-cell-associated HIV-1 DNA samples. Drug-resistance-associated mutations (DRAMs) and tropism were interpreted using the ANRS and Geno2Pheno algorithms. We analysed frozen blood cells from patients enrolled in the INNOVE and ANRS 123 ETOILE studies who achieved sustained viral suppression after salvage optimized ART (SOT). RESULTS: Samples were available at baseline and 6 and ≥12 months after SOT initiation in 10 patients. V3 loop sequences displayed wide intra-individual dynamics over time. Viral variants harbouring DRAMs exhibited three non-exclusive scenarios. First, when SOT exerted the same selective pressure as previous failing regimens, some viral quasispecies still harboured the same DRAMs at the same level as at the time of virological failure. Thus, as DRAMs were mostly associated with the same viral variant, variants with a complete resistance pattern remained archived. Second, some viral variants harbouring DRAMs were no longer detected over time when SOT consisted of new antiretroviral classes or had resistance profiles distinct from those of previous failing regimens. Third, variants with new DRAMs associated with SOT emerged in blood cells during follow-up despite sustained virological control. CONCLUSIONS: Using longitudinal UDS analysis and focusing on DRAMs and tropism as markers, we demonstrated that, despite sustained virological control, archived HIV-1 DNA quasispecies continued to evolve.

Virological failure of patients on maraviroc-based antiretroviral therapy.

OBJECTIVES: Virological failure (VF) in patients on maraviroc-based treatment has been associated with altered HIV tropism and resistance to maraviroc. This multicentre study aimed to characterize VF in patients treated with maraviroc. METHODS: We analysed 27 patients whose treatment failed between 2008 and 2011. They had been screened for HIV tropism before maraviroc initiation using population-based V3 genotyping. HIV-1 tropism and resistance of R5 viruses to maraviroc at VF and at baseline were determined retrospectively using an ultrasensitive recombinant virus assay (RVA). RESULTS: Viruses from 27 patients given maraviroc on the basis of the R5 genotype were characterized at the time of treatment failure. The RVA indicated that 12 patients harboured CXCR4-using viruses and 15 (56%) had pure R5 viruses at failure. One-third of those harbouring CXCR4-using viruses (4/12) were infected with R5X4/X4 viruses according to the RVA before maraviroc initiation. We analysed the phenotypic resistance to maraviroc of four patients harbouring R5 viruses at failure; two harboured viruses whose maximum percentage inhibition was reduced by 65%-90%, while the other two were infected with susceptible viruses. All patients had effective concentrations of drugs. CONCLUSIONS: Half of the maraviroc-treated patients who experienced VF harboured CXCR4-using viruses at failure, one-third of them were detected by a phenotypic method before maraviroc initiation. Phenotypic assessment of R5 virus resistance to CCR5 antagonists at failure could help optimize antiretroviral therapy.