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.

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 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.

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.

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.

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%.

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.

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.

Diversity of hepatitis E virus genotype 3.

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.

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.

HIV-1 genotypic resistance testing using the Vela automated next-generation sequencing platform.

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.

Performance comparison of deep sequencing platforms for detecting HIV-1 variants in the pol gene.

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.

Mutation in the Hepatitis E Virus Polymerase and Outcome of Ribavirin Therapy.

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.

HIV-1 subtype B-infected MSM may have driven the spread of transmitted resistant strains in France in 2007-12: impact on susceptibility to first-line strategies.

BACKGROUND: Our study describes the prevalence of transmitted drug resistance (TDR) among 1318 French patients diagnosed at the time of primary HIV-1 infection (PHI) in 2007-12. METHODS: HIV-1 resistance-associated mutations (RAMs) were characterized using both the 2009 WHO list of mutations and the French ANRS algorithm. A genotypic susceptibility score was estimated for each first-line recommended ART combination. RESULTS: Patients were mainly MSM (72.6%). Non-B variants were identified in 33.7% of patients. The proportion of TDR was estimated as 11.7% (95% CI 10.0-13.5). The prevalences of PI-, NRTI-, first-generation NNRTI and etravirine/rilpivirine-associated RAMs were 2.5%, 5.2%, 3.9% and 3.2%, respectively. Single, dual and triple class resistance was found in 9.6%, 1.0% and 1.1% of cases, respectively. Additionally, 5/331 strains isolated in 2010-12 had integrase inhibitor (II)-related RAMs (isolated E157Q mutation in all cases). TDR was more common among MSM than in other groups (12.9% versus 8.6%, P = 0.034), and in case of B versus non-B subtype infections (13.6% versus 7.9%, P = 0.002). The proportions of fully active combinations were ≥99.2%, ≥97.3% and ≥95.3% in cases of PI-, II- and NNRTI-based regimens, respectively. In 2010-12, the proportion of fully active efavirenz-based ART was lower in cases of subtype B versus non-B infection (P = 0.021). CONCLUSIONS: Compared with our previous studies, the proportion of NRTI- and first-generation NNRTI-related TDR has continued to decline in French seroconverters. However, subtype B-infected MSM could drive the spread of resistant HIV strains. Finally, we suggest preferring PI- or II- to NNRTI-based combinations to treat PHI patients.

Cross-resistance to elvitegravir and dolutegravir in 502 patients failing on raltegravir: a French national study of raltegravir-experienced HIV-1-infected patients.

OBJECTIVES: The objectives of this study were to determine the prevalence and patterns of resistance to integrase strand transfer inhibitors (INSTIs) in patients experiencing virological failure on raltegravir-based ART and the impact on susceptibility to INSTIs (raltegravir, elvitegravir and dolutegravir). PATIENTS AND METHODS: Data were collected from 502 treatment-experienced patients failing a raltegravir-containing regimen in a multicentre study. Reverse transcriptase, protease and integrase were sequenced at failure for each patient. INSTI resistance-associated mutations investigated were those included in the last ANRS genotypic algorithm (v23). RESULTS: Among the 502 patients, at failure, median baseline HIV-1 RNA (viral load) was 2.9 log10 copies/mL. Patients had been previously exposed to a median of five NRTIs, one NNRTI and three PIs. Seventy-one percent harboured HIV-1 subtype B and the most frequent non-B subtype was CRF02_AG (13.3%). The most frequent mutations observed were N155H/S (19.1%), Q148G/H/K/R (15.4%) and Y143C/G/H/R/S (6.7%). At failure, viruses were considered as fully susceptible to all INSTIs in 61.0% of cases, whilst 38.6% were considered as resistant to raltegravir, 34.9% to elvitegravir and 13.9% to dolutegravir. In the case of resistance to raltegravir, viruses were considered as susceptible to elvitegravir in 11% and to dolutegravir in 64% of cases. High HIV-1 viral load at failure (P < 0.001) and low genotypic sensitivity score of the associated treatment with raltegravir (P < 0.001) were associated with the presence of raltegravir-associated mutations at failure. Q148 mutations were selected more frequently in B subtypes versus non-B subtypes (P = 0.004). CONCLUSIONS: This study shows that a high proportion of viruses remain susceptible to dolutegravir in the case of failure on a raltegravir-containing regimen.

Is shared decision-making vanishing at the end-of-life? A descriptive and qualitative study of advanced cancer patients' involvement in specific therapies decision-making.

BACKGROUND: Little is known about what is at stake at a subjective level for the oncologists and the advanced cancer patients when they face the question whether to continue, limit or stop specific therapies. We studied (1) the frequency of such questioning, and (2) subjective determinants of the decision-making process from the physicians' and the patients' perspectives. METHODS: (1) All hospitalized patients were screened during 1 week in oncology and/or hematology units of five institutions. We included those with advanced cancer for whom a questioning about the pursuit, the limitation or the withholding of specific therapies (QST) was raised. (2) Qualitative design was based on in-depth interviews. RESULTS: In conventional units, 12.8 % of cancer patients (26 out of 202) were concerned by a QST during the study period. Interviews were conducted with all physicians and 21 advanced cancer patients. The timing of this questioning occurred most frequently as physicians estimated life expectancy between 15 days and 3 months. Faced with the most frequent dilemma (uncertain risk-benefit balance), physicians showed different ways of involving patients. The first two were called the "no choice" models: 1) trying to resolve the dilemma via a technical answer or a "wait-and-see" posture, instead of involving the patients in the questioning and the thinking; and 2), giving a "last minute" choice to the patients, leaving to them the responsibility of the decision. In a third model, they engaged early in shared reflections and dialogue about uncertainties and limits with patients, proxies and care teams. These schematic trends influenced patients' attitudes towards uncertainty and limits, as they were influenced by these ones. Individual and systemic barriers to a shared questioning were pointed out by physicians and patients. CONCLUSIONS: This study indicate to what extent these difficult decisions are related to physicians' and patients' respective and mutually influenced abilities to deal with and share about uncertainties and limits, throughout the disease trajectory. These insights may help physicians, patients and policy makers to enrich their understanding of underestimated and sensitive key issues of the decision-making process.

Using pharmacokinetic and viral kinetic modeling to estimate the antiviral effectiveness of telaprevir, boceprevir, and pegylated interferon during triple therapy in treatment-experienced hepatitis C virus-infected cirrhotic patients.

Triple therapy combining a protease inhibitor (PI) (telaprevir or boceprevir), pegylated interferon (PEG-IFN), and ribavirin (RBV) has dramatically increased the chance of eradicating hepatitis C virus (HCV). However, the efficacy of this treatment remains suboptimal in cirrhotic treatment-experienced patients. Here, we aimed to better understand the origin of this impaired response by estimating the antiviral effectiveness of each drug. Fifteen HCV genotype 1-infected patients with compensated cirrhosis, who were nonresponders to prior PEG-IFN/RBV therapy, were enrolled in a nonrandomized study. HCV RNA and concentrations of PIs, PEG-IFN, and RBV were frequently assessed in the first 12 weeks of treatment and were analyzed using a pharmacokinetic/viral kinetic model. The two PIs achieved similar levels of molar concentrations (P=0.5), but there was a significant difference in the 50% effective concentrations (EC50) (P=0.008), leading to greater effectiveness for telaprevir than for boceprevir in blocking viral production (99.8% versus 99.0%, respectively, P=0.002). In all patients, the antiviral effectiveness of PEG-IFN was modest (43.4%), and there was no significant contribution of RBV exposure to the total antiviral effectiveness. The second phase of viral decline, which is attributed to the loss rate of infected cells, was slow (0.19 day(-1)) and was higher in patients who subsequently eradicated HCV (P=0.03). The two PIs achieved high levels of antiviral effectiveness. However, the suboptimal antiviral effectiveness of PEG-IFN/RBV and the low loss of infected cells suggest that a longer treatment duration might be needed in cirrhotic treatment-experienced patients and that a future IFN-free regimen may be particularly beneficial in these patients.

Characterization of CXCR4-using HIV-1 during primary infection by ultra-deep pyrosequencing.

OBJECTIVES: R5 viruses have long been thought to account for almost all strains present in primary HIV-1 infections (PHIs), but recent studies using sensitive phenotypic assays have revealed that 3%-6.4% of subjects also harbour CXCR4-using viruses. Phenotypic assays provide only qualitative results: the presence or absence of CXCR4-using viruses. We have therefore used ultra-deep pyrosequencing (UDS) to determine the frequency of CXCR4-using viruses among HIV-1 quasispecies. METHODS: We first screened 200 patients for HIV-1 tropism using a sensitive phenotypic assay during PHI and identified 11 infected with an R5X4 dual/mixed (D/M) virus population. We then used UDS of the V3 env region with the geno2pheno algorithm (false positive rate = 5.75) to identify the HIV-1 quasispecies. RESULTS: CXCR4-using viruses were detected in all but 1 of the 11 patients by UDS, and accounted for 0.2%-100% of the virus populations. The frequency of CXCR4-using viruses was <20% in six subjects and 100% in four subjects. Virus populations containing 100% CXCR4-using variants during PHI persisted for at least 1-2 years after the acute phase. The CCR5 Δ32 heterozygous genotype was similarly prevalent in patients infected with D/M (27%) and R5 (15%) viruses. CONCLUSIONS: UDS and the phenotype were concordant for determining HIV-1 coreceptor usage. UDS analysis indicated large differences in the percentage of CXCR4-using viruses in the HIV-1 quasispecies during PHI. Further studies should examine the impact of the proportion of CXCR4-using viruses on disease prognosis.

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.