HPV genotyping in clinical samples using long-read single-molecule real-time sequencing.

Human papillomavirus (HPV) genotyping is widely used, particularly in combination with high-risk (HR) HPV tests for cervical cancer screening. We developed a genotyping method using sequences of approximately 800 bp in the E6/E7 region obtained by PacBio single molecule real-time sequencing (SMRT) and evaluated its performance against MY09-11 L1 sequencing and after the APTIMA HPV genotyping assay. The levels of concordance of PacBio E6/E7 SMRT sequencing with MY09-11 L1 sequencing and APTIMA HPV genotyping were 100% and 90.8%, respectively. The sensitivity of PacBio E6/EA7 SMRT was slightly greater than that of L1 sequencing and, as expected, lower than that of HR-HPV tests. In the context of cervical cancer screening, PacBio E6/E7 SMRT is then best used after a positive HPV test. PacBio E6/E7 SMRT genotyping is an attractive alternative for HR and LR-HPV genotyping of clinical samples. PacBio SMRT sequencing provides unbiased genotyping and can detect multiple HPV infections and haplotypes within a genotype.

Acute myocardial damage after electrical injury assessed by MRI.

Electrical injuries are not uncommon, and electrical shock-induced cardiac damage can be life-threatening. We present the case of a young patient who suffered from acute myocardial damage due to an occupational electric shock. Myocardial damage was assessed by very early (day 4) and repeated (up to month 18) MRI. Clinical management and patient risk estimation in such a setting are challenging because data on similar non-lethal cases and practice recommendations are scarce in the literature.

SARS-CoV-2 Co-Infections and Recombinations Identified by Long-Read Single-Molecule Real-Time Sequencing.

Co-infection with at least 2 strains of virus is the prerequisite for recombination, one of the means of genetic diversification. Little is known about the prevalence of these events in SARS-CoV-2, partly because it is difficult to detect them. We used long-read PacBio single-molecule real-time (SMRT) sequencing technology to sequence whole genomes and targeted regions for haplotyping. We identified 17 co-infections with SARS-CoV-2 strains belonging to different clades in 6829 samples sequenced between January and October, 2022 (prevalence 0.25%). There were 3 Delta/Omicron co-infections and 14 Omicron/Omicron co-infections (4 cases of 21K/21L, 1 case of 21L/22A, 2 cases of 21L/22B, 4 cases of 22A/22B, 2 cases of 22B/22C and 1 case of 22B/22E). Four of these patients (24%) also harbored recombinant minor haplotypes, including one with a recombinant virus that was selected in the viral quasispecies over the course of his chronic infection. While co-infections remain rare among SARS-CoV-2-infected individuals, long-read SMRT sequencing is a useful tool for detecting them as well as recombinant events, providing the basis for assessing their clinical impact, and a precise indicator of epidemic evolution. IMPORTANCE SARS-CoV-2 variants have been responsible for the successive waves of infection over the 3 years of pandemic. While co-infection followed by recombination is one driver of virus evolution, there have been few reports of co-infections, mainly between Delta and Omicron variants or between the first 2 Omicron variants 21K_BA.1 and 21L_BA.2. The 17 co-infections we detected during 2022 included cases with the recent clades of Omicron 22A, 22B, 22C, and 22E; 24% harbored recombinant variants. This study shows that long-read SMRT sequencing is well suited to SARS-CoV-2 genomic surveillance.

Whole-genome single molecule real-time sequencing of SARS-CoV-2 Omicron.

New variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome can only be identified using accurate sequencing methods. Single molecule real-time (SMRT) sequencing has been used to characterize Alpha and Delta variants, but not Omicron variants harboring numerous mutations in the SARS-CoV-2 genome. This study assesses the performance of a target capture SMRT sequencing protocol for whole genome sequencing (WGS) of SARS-CoV-2 Omicron variants and compared it to that of an amplicon SMRT sequencing protocol optimized for Omicron variants. The failure rate of the target capture protocol (6%) was lower than that of the amplicon protocol (34%, p < 0.001) on our data set, and the median genome coverage with the target capture protocol (98.6% [interquartile range (IQR): 86-99.4]) was greater than that with the amplicon protocol (76.6% [IQR: 66-89.6], [p < 0.001]). The percentages of samples with >95% whole genome coverage were 64% with the target capture protocol and 19% with the amplicon protocol (p < 0.05). The clades of 96 samples determined with both protocols were 93% concordant and the lineages of 59 samples were 100% concordant. Thus, target capture SMRT sequencing appears to be an efficient method for WGS, genotyping and detecting mutations of SARS-CoV-2 Omicron variants.

HIV-1 resistance genotyping by ultra-deep sequencing and 6-month virological response to first-line treatment.

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 <50 copies/mL after 6 months of treatment. 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.

Whole-genome sequencing of SARS-CoV-2: Comparison of target capture and amplicon single molecule real-time sequencing protocols.

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 Ct values, S gene coverage was higher with the target capture protocol than with the amplicon protocol. After stratification on Ct values, higher S gene coverage with the target capture protocol was observed only for samples with Ct > 17 (p < 0.01). PacBio SMRT sequencing protocols appear to be suitable for WGS, genotyping, and detecting mutations of SARS-CoV-2.

Understanding why patients request euthanasia when it is illegal: a qualitative study in palliative care units on the personal and practical impact of euthanasia requests.

CONTEXT: Some patients in palliative care units request euthanasia regardless of legislation. Although studies have explored the reasons for these requests, little is known about the subjective, relational, and contextual repercussions for the patient. OBJECTIVES: The aim of this study is to understand the purpose of euthanasia requests from the patient's viewpoint and their personal and practical impact. METHODS: We conducted in-depth interviews with patients requesting euthanasia, their family members, and health care providers in 11 French palliative care units. A thematic analysis of the data was performed. RESULTS: In total, 18 patients were interviewed within 48 h of the request being made; 1 week later, 9 patients were interviewed again. Five main themes emerged: assuming the possibility of transgressing the forbidden, a call for unbearable suffering to be recognized, encouragement to change clinical practice, reclaiming a sense of freedom over medical constraints, and imagining a desirable future for oneself. CONCLUSIONS: A request for euthanasia appears to be a willful means to remove oneself from the impasse of an existence paralyzed by suffering. It creates a space for discussion, which promotes negotiation with patients on care practices and therapeutics, and strengthens patients' sense of autonomy. Investigating the relationship between the evolution of euthanasia requests within the palliative care setting could be beneficial. It is important to encourage health care professionals to adopt a readiness to listen by interacting with patients in a way that is not momentarily action-oriented but rather focused on proactive discussion.

Hepatitis E Virus Quasispecies in Cerebrospinal Fluid with Neurological Manifestations.

Hepatitis E virus (HEV) infection can lead to a variety of neurological disorders. While HEV RNA is known to be present in the central nervous system, HEV quasispecies in serum and cerebrospinal fluid (CSF) have rarely been explored. We studied the virus' quasispecies in the blood and the CSF of five patients at the onset of their neurological symptoms. The samples of three patients suffering from meningitis, neuralgic amyotrophy and acute inflammatory polyradiculoneuropathy were taken at the acute phase of the HEV infection. The samples from the other two patients were taken during the chronic phase (5 years after HEV diagnosis) when they presented with clinical signs of encephalitis. We sequenced at least 20 randomly polyproline regions of the selected virus clones. Phylogenetic analysis of the virus variants in the blood and the CSF revealed no virus compartmentalization for the three acute-phase patients but there was clear evidence of HEV quasispecies compartmentalization in the CSF of the two patients during chronic infection. In conclusion, prolonged infection in the immunocompromised condition can lead to independent virus replication in the liver and the tissues, producing viruses in CSF.

Influence of SARS-CoV-2 Variant B.1.1.7, Vaccination, and Public Health Measures on the Spread of SARS-CoV-2.

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.

Use of ADAM-C and CHA2DS2-VASc scores to predict complex aortic atheroma after brain ischemia: A prospective observational study.

BACKGROUND AND AIMS: Complex aortic atheroma (CAA) is a common cause of acute brain ischemia (BI), including ischemic stroke (IS) and transient ischemic attack (TIA), and is associated with recurrence. The CHA2DS2-VASc score is a useful tool for predicting stroke in patients with atrial fibrillation (AF), and can also predict cardiovascular events in other populations, including non-AF populations. The ADAM-C score is a new risk score for predicting the diagnostic yield of transesophageal echocardiography (TEE) after BI. We aimed to evaluate the ability of CHA2DS2-VASc and ADAM-C scores to predict CAA after BI. METHODS: This prospective, multicenter, observational study included 1479 patients aged over 18 years who were hospitalized for BI. CAA was defined as the presence of one or more of the following criteria: thrombus, ulcerated plaque, or plaque thickening ≥ 4 mm. RESULTS: CAA was diagnosed in 216 patients (14.6%). CHA2DS2-VASc and ADAM-C scores were significantly higher in the CAA group versus the non-CAA group (P < .0001 for both). The CHA2DS2-VASc and ADAM-C scores appear to be good predictors of CAA (AUC 0.699 [0.635, 0.761] and 0.759 [0.702, 0.814], respectively). The sensitivity, specificity, predictive positive value (PPV), and negative predictive value (NPV) of the scores for detecting CAA were 94%, 22%, 17%, and 96%, respectively, for a CHA2DS2-VASc score < 2, and 90%, 46%, 22%, and 96%, respectively, for an ADAM-C score < 3 CONCLUSIONS: CHA2DS2-VASc and ADAM-C scores are able to predict CAA after BI. CHA2DS2-VASc < 2 and ADAM-C < 3 both have an interesting NPV of 96%.

Investigation of opposition to diagnostic or therapeutic procedures in older people hospitalized in acute geriatric services: the OPTAH pilot study protocol.

BACKGROUND: Shared decision-making is a process that involves collaborative discussions between a patient and a care team to ensure informed healthcare decisions. This process becomes more complex when the older person's decision-making capacities are affected. In these situations, surrogate decision-making processes are used to define a person-centered care plan. Despite these processes, the implementation of the care plan defined in the best interest of the patient may nevertheless be rejected by the patient, particularly in cases of neurocognitive disorders or delirium. This concept of opposition and/or refusal is frequently used in research. This is not yet well understood in the medical literature, and there is a lack of consensus on its definition. We, therefore, explored this concept by defining opposition to diagnostic or therapeutic proposals. METHOD: Our pilot study protocol is based on a mixed methodology (epidemiological and qualitative research) to quantify this phenomenon, validate the proposed definition, and explore its core elements. Opposition and refusal of care will be quantified, and semi-structured interviews will be conducted with patients, their relatives, and referring carers. Multidisciplinary meetings that will be associated with these situations will also be observed and analyzed. Methodological approaches that can be used to explore opposition and refusal of care in a scientific, reproducible framework are presented. This methodology considers the specificities of the geriatric, polypathological population with neurocognitive disorders. DISCUSSION: Opposition and refusal of care are key concepts in clinical research on ethics, particularly in the geriatric field. These concepts are frequently mentioned in studies involving older patients but have not been specifically defined or studied. This study would undoubtedly lead to greater awareness among professional caregivers and relatives of the significance of such opposition, and more respectful interactions in these complex hospitalization cases. TRIAL REGISTRATION: ClinicalTrial.gov, NCT03373838 . Registered on 14 December 2017.

Update: proposed reference sequences for subtypes of hepatitis E virus (species Orthohepevirus A).

In this recommendation, we update our 2016 table of reference sequences of subtypes of hepatitis E virus (HEV; species Orthohepevirus A, family Hepeviridae) for which complete genome sequences are available (Smith et al., 2016). This takes into account subsequent publications describing novel viruses and additional proposals for subtype names; there are now eight genotypes and 36 subtypes. Although it remains difficult to define strict criteria for distinguishing between virus subtypes, and is not within the remit of the International Committee on Taxonomy of Viruses (ICTV), the use of agreed reference sequences will bring clarity and stability to researchers, epidemiologists and clinicians working with HEV.

Insertions and Duplications in the Polyproline Region of the Hepatitis E Virus.

Recombinant strains of hepatitis E virus (HEV) with insertions of human genomic fragments or HEV sequence duplications in the sequence encoding the polyproline region (PPR) were previously described in chronically infected patients. Such genomic rearrangements confer a replicative advantage in vitro but little is known about their frequency, location, or origin. As the sequences of only a few virus genomes are available, we analyzed the complete genomes of 114 HEV genotype 3 strains from immunocompromised (n = 85) and immunocompetent (n = 29) patients using the single molecular real-time sequencing method to determine the frequency, location, and origin of inserted genomic fragments, plus the proportions of variants with genomic rearrangements in each virus quasispecies. We also examined the amino acid compositions and post-translational modifications conferred by these rearrangements by comparing them to sequences without human gene insertions or HEV gene duplications. We found genomic rearrangements in 7/114 (6.1%) complete genome sequences (4 HEV-3f, 1 HEV-3e, 1 HEV-3 h, and 1 HEV-3chi-new), all from immunocompromised patients, and 3/7 were found at the acute phase of infection. Six of the seven patients harbored virus-host recombinant variants, including one patient with two different recombinant variants. We also detected three recombinant variants with genome duplications of the PPR or PPR + X domains in a single patient. All the genomic rearrangements (seven human fragment insertions of varying origins and three HEV genome duplications) occurred in the PPR. The sequences with genomic rearrangements had specific characteristics: increased net load (p < 0.001) and more ubiquitination (p < 0.001), phosphorylation (p < 0.001), and acetylation (p < 0.001) sites. The human fragment insertions and HEV genome duplications had slightly different characteristics. We believe this is the first description of HEV strains with genomic rearrangements in patients at the acute phase of infection; perhaps these strains are directly transmitted. Clearly, genomic rearrangements produce a greater net load with duplications and insertions having different features. Further studies are needed to clarify the mechanisms by which such modifications influence HEV replication.

Classification of the Zoonotic Hepatitis E Virus Genotype 3 Into Distinct Subgenotypes.

Hepatitis E virus (HEV) genotype 3 is the most common genotype linked to HEV infections in Europe and America. Three major clades (HEV-3.1, HEV-3.2, and HEV-3.3) have been identified but the overlaps between intra-subtype and inter-subtype p-distances make subtype classification inconsistent. Reference sequences have been proposed to facilitate communication between researchers and new putative subtypes have been identified recently. We have used the full or near full-length HEV-3 genome sequences available in the Genbank database (April 2020; n = 503) and distance analyses of clades HEV-3.1 and HEV-3.2 to determine a p-distance cut-off (0.093 nt substitutions/site) in order to define subtypes. This could help to harmonize HEV-3 genotyping, facilitate molecular epidemiology studies and investigations of the biological and clinical differences between HEV-3 subtypes.

THETA: a new genotypic approach for predicting HIV-1 CRF02-AG coreceptor usage.

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.

Performance evaluation of the Vela Dx Sentosa next-generation sequencing system for HIV-1 DNA genotypic resistance.

BACKGROUND: Patients on antiretroviral therapy could benefit from HIV-1 DNA resistance genotyping for exploring virological failure with low viral load or to guide treatment simplification. Few new generation sequencing data are available. OBJECTIVE: To check that the automated deep sequencing Sentosa platform (Vela DX) detected minority resistant variants well enough for HIV DNA genotyping. STUDY DESIGN: We evaluated the Sentosa SQ HIV genotyping assay with automated extraction on 40 DNA longitudinal samples from treatment-experienced patients by comparison with Sanger sequencing. HIV drug resistance was interpreted using the ANRS algorithm (v29) at the threshold of 20 % and 3 %. RESULTS: The Sentosa SQ HIV genotyping assay was 100 % successful to amplify and sequence PR and RT and 86 % to amplify and sequence IN when the HIV DNA load was >2.5 log copies/million cells. The Sentosa and Sanger sequencing were concordant for predicting PR-RT resistance at the threshold of 20 % in 14/18 samples successfully sequenced. A higher level of resistance was predicted by Sentosa in three samples and by Sanger in one sample. The prevalence of resistance was 7 % to PI, 59 % to NRTI, 31 % to NNRTI and 20 % to integrase inhibitors using the Sentosa SQ genotyping assay at the threshold of 3 %. Seven additional mutations <20 % were detected using the Sentosa assay. CONCLUSION: Automated DNA extraction and sequencing using the Sentosa SQ HIV genotyping assay accurately predicted HIV DNA drug resistance by comparison with Sanger. Prospective studies are needed to evaluate the clinical interest of HIV DNA genotyping.

Long-term evolution of transmitted CXCR4-using HIV-1 under effective antiretroviral therapy.

OBJECTIVE: 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. DESIGN: 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. METHODS: 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). RESULTS: 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. CONCLUSION: 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.

Impact of the mutational load on the virological response to a first-line rilpivirine-based regimen.

OBJECTIVES: To 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 METHODS: Four 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. RESULTS: The mutational load at baseline was the only variable linked to the VR at 12 months (P  < 0.01). The VR at 12 months decreased from 96.9% to 83.4% when the mutational load was >1700 copies/mL and to 50% when the mutational load was > 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). CONCLUSIONS: There 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 < 20% together with the plasma HIV-1 viral load at the time of resistance genotyping.

Accurate quantification of CXCR4-using HIV-1 variants by Illumina deep-sequencing.

: Next-generation sequencing is a sensitive method for determining HIV-1 tropism but there is a lack of data on the quantification of X4 variants. We evaluated MiSeq and 454 GS-Junior platforms for determining HIV-1 tropism and for quantifying X4 variants. Both platforms were 93% concordant for determining HIV-1 tropism and correlated well for determining the proportion of X4 variants (Spearman correlation, ρ = 0.748; P < 0.0001). MiSeq Illumina sequencing seems to be well adapted for characterizing X4-containing samples.