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We used variant typing polymerase chain reaction to describe the evolution of severe acute respiratory syndrome coronavirus 2 Omicron sublineages between December 2021 and mid-March 2022. The selective advantage of the BA.2 variant over BA.1 is not due to greater nasopharyngeal viral loads.

Fast, accurate sequencing methods are needed to identify new variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome. Single-molecule real-time (SMRT) Pacific Biosciences (PacBio) provides long, highly accurate sequences by circular consensus reads. This study compares the performance of a target capture SMRT PacBio protocol for whole-genome sequencing (WGS) of SARS-CoV-2 to that of an amplicon PacBio SMRT sequencing protocol. The median genome coverage was higher (p 0.05) with the target capture protocol (99.3% [interquartile range, IQR: 96.3-99.5]) than with the amplicon protocol (99.3% [IQR: 69.9-99.3]). The clades of 65 samples determined with both protocols were 100% concordant. After adjusting for C

Objectives To evaluate the routine use of the Sentosa ultra-deep sequencing (UDS) system for HIV-1 polymerase resistance genotyping in treatment-naïve individuals and to analyse the virological response (VR) to first-line antiretroviral treatment. Methods HIV drug resistance was determined on 237 consecutive samples from treatment-naïve individuals using the Sentosa UDS platform with two mutation detection thresholds (3% and 20%). VR was defined as a plasma HIV-1 virus load Results Resistance to at least one antiretroviral drug with a mutation threshold of 3% was identified in 29% and 16% of samples according to ANRS and Stanford algorithms, respectively. The ANRS algorithm also revealed reduced susceptibility to at least one protease inhibitor (PI) in 14.3% of samples, to one reverse transcriptase inhibitor in 12.7%, and to one integrase inhibitor (INSTI) in 5.1%. For a mutation threshold of 20%, resistance was identified in 24% and 13% of samples according to ANRS and Stanford algorithms, respectively. The 6 months VR was 87% and was similar in the 58% of patients given INSTI-based treatment, in the 16% given PI-based treatment and in the 9% given NNRTI-based treatment. Multivariate analysis indicated that the VR was correlated with the baseline HIV virus load and resistance to at least one PI at both 3% and 20% mutation detection thresholds (ANRS algorithm). Conclusions The Vela UDS platform is appropriate for determining antiretroviral resistance in patients on a first-line antiretroviral treatment. Further studies are needed on the use of UDS for therapeutic management.

Background The increasing use of monoclonal antibodies (mAbs) to treat coronavirus disease 2019 raises questions about their impact on the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mAb-resistant variants. We assessed the impact of Casirivimab-Imdevimab on SARS-CoV-2 mutations associated with reduced mAb activity in treated patients. Methods We measured the nasopharyngeal (NP) viral load and sequenced the haplotypes of spike gene of 50 patients infected with the SARS-CoV-2 delta variant and treated with Casirivimab-Imdevimab using single-molecule real-time sequencing. Results The NP SARS-CoV-2 viral load of patients treated with Casirivimab-Imdevimab decreased from 8.13 (interquartile range [IQR], 7.06–8.59) log10 copies/mL pretreatment to 3.67 (IQR, 3.07–5.15) log10 copies/mL 7 days later (P Conclusions Casirivimab-Imdevimab is an effective treatment for patients infected with the SARS-CoV-2 delta variant. Despite selective pressure on SARS-CoV-2 Spike quasispecies, we detected no key mutations that reduced mAb activity in our patients.

International audience; Objectives: To evaluate the impact of neutralizing monoclonal antibody (mAb) treatment and to determine whether the selective pressure of mAbs could facilitate the proliferation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with spike protein mutations that might attenuate mAb effectiveness.Patients and methods: We evaluated the impact of mAbs on the nasopharyngeal (NP) viral load and virus quasispecies of mAb-treated patients using single-molecule real-time sequencing. The mAbs used were: Bamlanivimab alone (four patients), Bamlanivimab/Etesevimab (23 patients) and Casirivimab/Imdevimab (five patients).Results: The NP SARS-CoV-2 viral load of mAb-treated patients decreased from 8.2 log10 copies/mL before administration to 4.3 log10 copies/mL 7 days after administration. Five immunocompromised patients given Bamlanivimab/Etesevimab were found to have mAb activity-reducing spike mutations. Two patients harboured SARS-CoV-2 variants with a Q493R spike mutation 7 days after administration, as did a third patient 14 days after administration. The fourth patient harboured a variant with a Q493K spike mutation 7 days post-treatment, and the fifth patient had a variant with a E484K spike mutation on day 21. The emergence of the spike mutation was accompanied by stabilization or rebound of the NP viral load in three of five patients.Conclusion: Two-mAb therapy can drive the selection of resistant SARS-CoV-2 variants in immunocompromised patients. Patients given mAbs should be closely monitored and measures to limit virus spread should be reinforced.

International audience; The SARS-CoV-2 B.1.1.529 (Omicron) variant, divided in five lineages (BA.1/BA.2/BA.3/BA.4/BA.5), harbors numerous spike protein mutations particularly in the receptor-binding domain (RBD). Recent studies showed that the Omicron variant resists the majority of RBD-targeting monoclonal antibodies (mAb). Sotrovimab, a pan-sarbecovirus neutralizing mAb recently authorized, seems to remain efficient to neutralize the omicron variant. However, as it targets a single epitope, the risk of developing resistance mutations is not negligible. We determined the evolution of the virus load, the development of spike mutations, and the evolution of the virus complexity in nasopharyngeal (NP) swabs from sotrovimab-treated ambulatory Omicron-infected patients.

To study the long-term evolution of the transmitted CXCR4-using viruses. CCR5-using viruses (R5 viruses) predominate during primary HIV-1 infections (PHI) while CXCR4-using viruses are isolated in less than 10% of PHI.Six patients infected with an R5X4 virus, detected by a sensitive phenotypic assay during PHI, were matched with six patients infected with a pure R5 virus for sex, Fiebig stage, time of antiretroviral initiation and duration of follow-up.We used MiSeq ultra-deep sequencing to determine the composition of the virus quasispecies during PHI and at the end of follow-up (median time of follow-up: 12.5 years).X4 viruses were detected by genetic analysis in three of six samples from the R5X4 group, accounting for 1.3-100% of the virus quasispecies, during PHI, and in four of six samples (accounting for 6.7-100%) at the end of follow-up. No X4 virus was detected in the R5 group during PHI and in only one patient (accounting for 1.2%) at the end of follow-up. The complexity of the virus quasispecies at the stage of PHI was higher in the R5X4 group than in the R5 group. Complexity increased from PHI to the end of follow-up in the R5 group but remained stable in the R5X4 group.CXCR4-using viruses persisted in the peripheral blood mononuclear cells of several patients on suppressive antiretroviral therapy for a median duration of 12.5 years after PHI. The genetic complexity of HIV-1 evolved differently post-PHI in patients infected with R5X4 viruses from those infected with R5 viruses.

Motivation The circulating recombinant form of HIV-1 CRF02-AG is the most frequent non-B subtype in Europe. Anti-HIV therapy and pathophysiological studies on the impact of HIV-1 tropism require genotypic determination of HIV-1 tropism for non-B subtypes. But genotypic approaches based on analysis of the V3 envelope region perform poorly when used to determine the tropism of CRF02-AG. We, therefore, designed an algorithm based on information from the gp120 and gp41 ectodomain that better predicts the tropism of HIV-1 subtype CRF02-AG. Results We used a bio-statistical method to identify the genotypic determinants of CRF02-AG coreceptor use. Toulouse HIV Extended Tropism Algorithm (THETA), based on a Least Absolute Shrinkage and Selection Operator method, uses HIV envelope sequence from phenotypically characterized clones. Prediction of R5X4/X4 viruses was 86% sensitive and that of R5 viruses was 89% specific with our model. The overall accuracy of THETA was 88%, making it sufficiently reliable for predicting the tropism of subtype CRF02-AG sequences. Availability and implementation Binaries are freely available for download at https://github.com/viro-tls/THETA. It was implemented in Matlab and supported on MS Windows platform. The sequence data used in this work are available from GenBank under the accession numbers MK618182-MK618417.

International audience; The spread of SARS-CoV-2 and the resulting disease COVID-19 has killed over 2.6 million people as of 18 March 2021. We have used a modified susceptible, infected, recovered (SIR) epidemiological model to predict how the spread of the virus in regions of France will vary depending on the proportions of variants and on the public health strategies adopted, including anti-COVID-19 vaccination. The proportion of SARS-CoV-2 variant B.1.1.7, which was not detected in early January, increased to become 60% of the forms of SARS-CoV-2 circulating in the Toulouse urban area at the beginning of February 2021, but there was no increase in positive nucleic acid tests. Our prediction model indicates that maintaining public health measures and accelerating vaccination are efficient strategies for the sustained control of SARS-CoV-2.

The present study compares the performances of an in-house sequencing protocol developed on MiSeq, the Sanger method, and the 454 GS-FLX for detecting and quantifying drug-resistant mutations (DRMs) in the human immunodeficiency virus polymerase gene (reverse transcriptase [RT] and protease [PR]). MiSeq sequencing identified all the resistance mutations detected by bulk sequencing (n = 84). Both the MiSeq and 454 GS-FLX platforms identified 67 DRMs in the RT and PR regions, but a further 25 DRMs were identified by only one or other of them. Pearson's analysis showed good concordance between the percentage of drug-resistant variants determined by MiSeq and 454 GS-FLX sequencing (ρ = .77, P < .0001). The MiSeq platform is as accurate as the 454 GS-FLX Roche system for determining RT and PR DRMs and could be used for monitoring human immunodeficiency virus type 1 drug resistance.

Objectives To evaluate the diagnostic performance of the Vela next-generation sequencing (NGS) system in conjunction with the Sentosa SQ HIV Genotyping Assay for genotyping HIV-1. Methods Plasma RNA was extracted and templates prepared with the Sentosa SX instrument before sequencing the HIV-1 polymerase on the Sentosa SQ301 Sequencer (PGM IonTorrent). The Vela NGS System was compared with direct sequencing and the 454 GS-FLX (Roche) and MiSeq (Illumina) systems for genotypic resistance testing on clinical samples. Results The Vela NGS system detected majority resistance mutations in subtype B and CRF02-AG samples at 500 copies/mL and minority variants with a sensitivity of 5% at 100 000 copies/mL. The Vela NGS system and direct sequencing identified resistance mutations with 97% concordance in 46 clinical samples. Vela identified 1/20 of the 1%-5% mutations identified by 454, 5/12 of the 5%-20% mutations and 60/61 of the >20% mutations. Vela identified 3/14 of the 1%-5% mutations identified by MiSeq, 0/2 of the 5%-20% mutations and 47/47 of the >20% mutations. The resistance mutation quantifications by Vela and 454 were concordant (bias: 2.31%), as were those by Vela and MiSeq (bias: 1.06%). Conclusions The Vela NGS system provides automated nucleic acid extraction, PCR reagent distribution, library preparation and bioinformatics analysis. The analytical performance was very good when compared with direct sequencing, but was less sensitive than two other NGS platforms for detecting minority variants.

Hepatitis E virus (HEV) can lead to chronic infection in solid-organ transplant patients. Ribavirin is efficient for treatment of chronically infected patients. Recently, the1634R mutation in the HEV polymerase has been associated with treatment failure. However, it is unclear if this mutation can be used as a prognostic marker of treatment outcome. We studied the prevalence of the 1634R mutation in the HEV polymerase of patients starting ribavirin therapy, the influence of the 1634R variants on the viral response, the frequency of the 1634R mutation in patients whose treatment failed, and its impact on ribavirin retreatment. We analyzed pretreatment samples from 63 solid-organ transplant patients with chronic hepatitis E using deep sequencing; 42 patients had a sustained virologic response (SVR), and 21 were non-SVR patients. We detected the 1634R variant by deep sequencing in 36.5% (23/63) of the patients (proportions, 1.3 to 100%). The 1634R variant was detected in 31.0% (13/42) of baseline plasma samples from patients with SVR and in 47.6% (10/21) in the other patients ( P = 0.2). The presence of this mutation did not influence the initial decrease in viral RNA. Lastly, a second prolonged ribavirin treatment led to SVR in 70% of the patients who initially did not have SVR, despite the presence of the 1634R variant. We conclude that the presence of the 1634R variant at ribavirin initiation does not lead to absolute ribavirin resistance. Although its proportion increased in patients whose treatment failed, the presence of the 1634R variant did not compromise the response to a second ribavirin treatment.

Author(s): Dimeglio, Chloe; Raymond, Stephanie; Nicot, Florence; Jeanne, Nicolas; Carcenac, Romain; Lefebvre, Caroline; Izopet, Jacques; French National Agency for Research on AIDS and Viral Hepatitis (ANRS) AC11 Resistance Study Group | Abstract: ObjectivesTo determine how the load of rilpivirine-resistant variants (mutational load) influences the virological response (VR) of HIV-1-infected patients to a rilpivirine-based first-line regimen.Patients and methodsFour hundred and eighty-nine patients infected with HIV-1 whose reverse transcriptase gene had been successfully resistance genotyped using next-generation sequencing were given a first-line regimen containing rilpivirine. Variables associated with the VR at 12 months were identified using a logistic model. The results were used to build a multivariate model for each mutational load threshold and the R2 variations were analysed to identify the mutational load threshold that best predicted the VR.ResultsThe mutational load at baseline was the only variable linked to the VR at 12 months (P l 0.01). The VR at 12 months decreased from 96.9% to 83.4% when the mutational load was g1700 copies/mL and to 50% when the mutational load was g 9000 copies/mL. The threshold of 9000 copies/mL was associated with the VR at 12 months with an OR of 36.7 (95% CI 4.7-285.1). The threshold of 1700 copies/mL was associated with the VR at 12 months with an OR of 7.2 (95% CI 1.4-36.8).ConclusionsThere is quantifiable evidence that determining a mutational load threshold can be used to identify those patients on a first-line regimen containing rilpivirine who are at risk of virological failure. The clinical management of HIV-infected patients can be improved by evaluating the frequency of mutant variants at a threshold of l 20% together with the plasma HIV-1 viral load at the time of resistance genotyping.

International audience; Hepatitis E virus genotype 3 (HEV-3) can lead to chronic infection in immunocompromised patients, and ribavirin is the treatment of choice. Recently, mutations in the polymerase gene have been associated with ribavirin failure but their frequency before treatment according to HEV-3 subtypes has not been studied on a large data set. We used single-molecule real-time sequencing technology to sequence 115 new complete genomes of HEV-3 infecting French patients. We analyzed phylogenetic relationships, the length of the polyproline region, and mutations in the HEV polymerase gene. Eighty-five (74%) were in the clade HEV-3efg, 28 (24%) in HEV-3chi clade, and 2 (2%) in HEV-3ra clade. Using automated partitioning of maximum likelihood phylogenetic trees, complete genomes were classified into subtypes. Polyproline region length differs within HEV-3 clades (from 189 to 315 nt). Investigating mutations in the polymerase gene, distinct polymorphisms between HEV-3 subtypes were found (G1634R in 95% of HEV-3e, G1634K in 56% of HEV-3ra, and V1479I in all HEV-3efg, clade HEV-3ra, and HEV-3k strains). Subtype-specific polymorphisms in the HEV-3 polymerase have been identified. Our study provides new complete genome sequences of HEV-3 that could be useful for comparing strains circulating in humans and the animal reservoir.

The present study compares the performances of an in-house sequencing protocol developed on MiSeq, the Sanger method, and the 454 GS-FLX for detecting and quantifying drug-resistant mutations (DRMs) in the human immunodeficiency virus polymerase gene (reverse transcriptase [RT] and protease [PR]). MiSeq sequencing identified all the resistance mutations detected by bulk sequencing (n = 84). Both the MiSeq and 454 GS-FLX platforms identified 67 DRMs in the RT and PR regions, but a further 25 DRMs were identified by only one or other of them. Pearson's analysis showed good concordance between the percentage of drug-resistant variants determined by MiSeq and 454 GS-FLX sequencing (ρ = .77, P .0001). The MiSeq platform is as accurate as the 454 GS-FLX Roche system for determining RT and PR DRMs and could be used for monitoring human immunodeficiency virus type 1 drug resistance.

The coreceptor used by HIV-1 must be determined before a CCR5 antagonist, part of the arsenal of antiretroviral drugs, is prescribed because viruses that enter cells using the CXCR4 coreceptor are responsible for treatment failure. HIV-1 tropism is also correlated with disease progression and so must be determined for virological studies. Tropism can be determined by next-generation sequencing (NGS), but not all of these new technologies have been fully validated for use in clinical practice. The Illumina NGS technology is used in many laboratories but its ability to predict HIV-1 tropism has not been evaluated while the 454 GS-Junior (Roche) is used for routine diagnosis. The genotypic prediction of HIV-1 tropism is based on sequencing the V3 region and interpreting the results with an appropriate algorithm. We compared the performances of the MiSeq (Illumina) and 454 GS-Junior (Roche) systems with a reference phenotypic assay. We used clinical samples for the NGS tropism predictions and assessed their ability to quantify CXCR4-using variants. The data show that the Illumina platform can be used to detect minor CXCR4-using variants in clinical practice but technical optimization are needed to improve quantification.

Resume L'objectif de cette etude est de preciser la nature juridique et de mieux definir les contours du regime juridique de ce nouveau mode de traitement des infractions detache du declenchement de l'action publique que constitue la transaction penale par officier de police judiciaire (TOPJ), au regard, non seulement de la loi n˚ 2014-896 du 15 aout 2014 relative a l'individualisation des peines et renforcant l'efficacite des sanctions penales et des precisions apportees par le decret n˚ 2015-1272 du 13 octobre 2015, pris pour l'application des articles 41-1-1 du code de procedure penale et L. 132-10-1 du code de la securite interieure, mais aussi des dispositions regissant les autres modes de traitement des infractions.

We used ultradeep sequencing to investigate the evolution of the frequency of CXCR4-using viruses in the peripheral blood mononuclear cells of 22 patients infected with both CCR5 and CXCR4-using viruses treated with the CCR5 antagonist maraviroc for 24 weeks and a stable antiviral therapy. The mean CXCR4-using virus frequency in peripheral blood mononuclear cells was 59% before maraviroc intensification and 52% after 24 weeks of effective treatment, indicating no selection by maraviroc.

Sotos syndrome is an overgrowth syndrome characterized by distinctive facial features, learning difficulties, and macrocephaly with frequent pre- and postnatal overgrowth with advanced bone age. Here, we report on our experience in the molecular diagnostic of Sotos syndrome on 116 patients. Using direct sequencing and a quantitative multiplex PCR of short fluorescent fragments (QMPSF)-based assay allowing accurate detection of both total and partial NSD1 deletions, we identified NSD1 abnormalities in 104 patients corresponding to 102 Sotos families (90%). NSD1 point mutations were detected in 80% of the index cases, large deletions removing the NSD1 gene entirely in 14%, and intragenic NSD1 rearrangements in 6%. Among the 69 detected distinct point mutations, 48 were novel. The QMPSF assay detected an exonic duplication and a mosaic partial deletion. QMPSF mapping of the 15 large deletions revealed the heterogeneity of the deletions, which vary in size from 1 to 4.5 Mb. Clinical features of NSD1-positive Sotos patients revealed that the phenotype in patients with nontruncating mutations was less severe that in patients with truncating mutations. This study confirms the heterogeneity of NSD1 alterations in Sotos syndrome and therefore the need to complete sequencing analysis by screening for partial deletions and duplications to ensure an accurate molecular diagnosis of this syndrome.

HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds.

Received 4 February 2014; returned 25 February 2014; revised 4 April 2014; accepted 4 April 2014Objectives:To trackchanges in the V3 env region of HIV-1 quasispecies and determine virus coreceptor use in cellreservoirs of patients on long-term suppressive antiretroviral therapy (ART).Patients and methods: Tenpatientswhoseplasmaviraemiahadbeensuppressedforamedianof5.5yearswerefollowed for 5 years. TheV3 envregionsofviruses inperipheral blood mononuclearcells were analysed byultra-deepsequencing (UDS). HIV-1 tropism was predicted using the geno2pheno 5.75 algorithm and a phenotypic assay.Results: The UDS and phenotypic assay data were concordant for predicting HIV-1 tropism. CXCR4-using virusesdetected by UDS accounted for 14.7%–76.5% of the virus populations in samples from five patients at enrolment.FivepatientsharbouredpureR5viruspopulationsandnoX4virusesemergedduringthe5 years.Theselectionpres-sures estimated by the dN/dS ratio were acting on the V3 region to produce diversification of the quasispecies inCXCR4-infected patients and purification of the quasispecies in R5-infected patients on effective ART.Conclusions: UDS showed that the virus quasispecies in cell reservoirs of patients on long-term suppressive ARTcontinued to evolve. CXCR4-using variants became more diversified. Analysis of the selection pressures on thevirus quasispecies could provide a clearer picture of virus persistence in patients on effective ART.Keywords: V3 envelope region, ultra-deep sequencing, selection pressure

Geostatistics meets a growing interest for the remediation forecast of potentially contaminated sites, by providing adapted methods to perform both chemical and radiological pollution mapping, to estimate contaminated volumes, potentially integrating auxiliary information, and to set up adaptive sampling strategies. As part of demonstration studies carried out for GeoSiPol (Geostatistics for Polluted Sites), geostatistics has been applied for the detailed diagnosis of a former oil depot in France. The ability within the geostatistical framework to generate pessimistic / probable / optimistic scenarios for the contaminated volumes allows a quantification of the risks associated to the remediation process: e.g. the financial risk to excavate clean soils, the sanitary risk to leave contaminated soils in place. After a first mapping, an iterative approach leads to collect additional samples in areas previously identified as highly uncertain. Estimated volumes are then updated and compared to the volumes actually excavated. This benchmarking therefore provides a practical feedback on the performance of the geostatistical methodology.Copyright © 2011 by ASME

International audience