Identification of two novel subtypes of hepatitis C virus genotype 8 and a potential new genotype successfully treated with direct acting antivirals.

BACKGROUND: Hepatitis C virus (HCV) has a high genetic diversity and is classified into 8 genotypes and over 90 subtypes with some endemic to specific world regions. This could compromise direct-acting antiviral (DAA) efficacy and global HCV elimination. METHODS: We characterised HCV subtypes 'rare' to the UK (non-1a/1b/2b/3a/4d) by whole genome sequencing via a national surveillance programme. Genetic analyses to determine the genotype of samples with unresolved genotypes were undertaken by comparison with ICTV HCV reference sequences. RESULTS: Two HCV variants were characterised as being closely related to the recently identified genotype 8 (GT8), with >85% pairwise genetic distance similarity to GT8 sequences and within the typical inter-subtype genetic distance range. The individuals infected by the variants were UK residents originally from Pakistan and India. In contrast, a third variant was only confidently identified to be more similar to GT6 compared to other genotypes across 6% of the genome and was isolated from a UK resident originally from Guyana. All three were cured with pangenotypic DAAs (Sofosbuvir + Velpatasvir or Glecaprevir + Pibrentasvir) despite the presence of resistance polymorphisms in NS3 (80 K/168E), NS5A (28 V/30S/62L/92S/93S) and NS5B (159F). CONCLUSIONS: This study expands our knowledge of HCV diversity by identifying two new GT8 subtypes and potentially a new genotype.

High Performance of a Dominant/X-Linked Gene Panel in Patients with Neurodevelopmental Disorders.

Neurodevelopmental disorders (NDDs) affect 2-5% of the population and approximately 50% of cases are due to genetic factors. Since de novo pathogenic variants account for the majority of cases, a gene panel including 460 dominant and X-linked genes was designed and applied to 398 patients affected by intellectual disability (ID)/global developmental delay (GDD) and/or autism (ASD). Pathogenic variants were identified in 83 different genes showing the high genetic heterogeneity of NDDs. A molecular diagnosis was established in 28.6% of patients after high-depth sequencing and stringent variant filtering. Compared to other available gene panel solutions for NDD molecular diagnosis, our panel has a higher diagnostic yield for both ID/GDD and ASD. As reported previously, a significantly higher diagnostic yield was observed: (i) in patients affected by ID/GDD compared to those affected only by ASD, and (ii) in females despite the higher proportion of males among our patients. No differences in diagnostic rates were found between patients affected by different levels of ID severity. Interestingly, patients harboring pathogenic variants presented different phenotypic features, suggesting that deep phenotypic profiling may help in predicting the presence of a pathogenic variant. Despite the high performance of our panel, whole exome-sequencing (WES) approaches may represent a more robust solution. For this reason, we propose the list of genes included in our customized gene panel and the variant filtering procedure presented here as a first-tier approach for the molecular diagnosis of NDDs in WES studies.

Clinical evaluation of a Hepatitis C Virus whole-genome sequencing pipeline for genotyping and resistance testing.

OBJECTIVES: We sought to evaluate clinically a hepatitis C virus (HCV) whole-genome, next-generation sequencing (NGS) pipeline that is agnostic to viral genotype. METHODS: Performance of the NGS pipeline was assessed through comparison of results with Sanger sequencing (SS) of partial HCV genomes. RESULTS: There was 98.7% (376/381) concordance for viral subtype between SS and NGS. The positive and negative per cent agreements for determination of resistance-associated substitutions were 97.8% (95% CI 92.5-99.4%) and 99.9% (95% CI 99.5-100.0%), respectively. The NGS pipeline was also able to detect novel subtypes, mixtures, recombinants, transiently occurring resistance mutations and distinguish re-infection with the same subtype from relapse. DISCUSSION: Particular scenarios where NGS may be used include settings without universal access to pan-genotypic antiviral regimens, those infected with a 'rare' subtype or who have been failed by first-line therapy, and in cases of suspected re-infection.

Real world SOF/VEL/VOX retreatment outcomes and viral resistance analysis for HCV patients with prior failure to DAA therapy.

Sustained viral response (SVR) rates for direct-acting antiviral (DAA) therapy for hepatitis C virus (HCV) infection routinely exceed 95%. However, a small number of patients require retreatment. Sofosbuvir, velpatasvir and voxilaprevir (SOF/VEL/VOX) is a potent DAA combination primarily used for the retreatment of patients who failed by DAA therapies. Here we evaluate retreatment outcomes and the effects of resistance-associated substitutions (RAS) in a real-world cohort, including a large number of genotype (GT)3 infected patients. 144 patients from the UK were retreated with SOF/VEL/VOX following virologic failure with first-line DAA treatment regimens. Full-length HCV genome sequencing was performed prior to retreatment with SOF/VEL/VOX. HCV subtypes were assigned and RAS relevant to each genotype were identified. GT1a and GT3a each made up 38% (GT1a n = 55, GT3a n = 54) of the cohort. 40% (n = 58) of patients had liver cirrhosis of whom 7% (n = 4) were decompensated, 10% (n = 14) had hepatocellular carcinoma (HCC) and 8% (n = 12) had received a liver transplant prior to retreatment. The overall retreatment SVR12 rate was 90% (129/144). On univariate analysis, GT3 infection (50/62; SVR = 81%, p = .009), cirrhosis (47/58; SVR = 81%, p = .01) and prior treatment with SOF/VEL (12/17; SVR = 71%, p = .02) or SOF+DCV (14/19; SVR = 74%, p = .012) were significantly associated with retreatment failure, but existence of pre-retreatment RAS was not when viral genotype was taken into account. Retreatment with SOF/VEL/VOX is very successful for non-GT3-infected patients. However, for GT3-infected patients, particularly those with cirrhosis and failed by initial SOF/VEL treatment, SVR rates were significantly lower and alternative retreatment regimens should be considered.

Technical Validation of a Hepatitis C Virus Whole Genome Sequencing Assay for Detection of Genotype and Antiviral Resistance in the Clinical Pathway.

Choice of direct acting antiviral (DAA) therapy for Hepatitis C Virus (HCV) in the United Kingdom and similar settings usually requires knowledge of the genotype and, in some cases, antiviral resistance (AVR) profile of the infecting virus. To determine these, most laboratories currently use Sanger technology, but next-generation sequencing (NGS) offers potential advantages in throughput and accuracy. However, NGS poses unique technical challenges, which require idiosyncratic development and technical validation approaches. This applies particularly to virology, where sequence diversity is high and the amount of starting genetic material is low, making it difficult to distinguish real data from artifacts. We describe the development and technical validation of a sequence capture-based HCV whole genome sequencing (WGS) assay to determine viral genotype and AVR profile. We use clinical samples of known subtypes and viral loads, and simulated FASTQ datasets to validate the analytical performances of both the wet laboratory and bioinformatic pipeline procedures. We show high concordance of the WGS assay compared to current "gold standard" Sanger assays. Specificity was 92.3 and 96.1% for AVR and genotyping, respectively. Discordances were due to the inability of Sanger assays to assign the correct subtype or accurately call mixed drug-resistant variants. We show high repeatability and reproducibility with >99.8% sequence similarity between sequence runs as well as high precision for variant frequency detection at >98.8% in the 95th percentile. Post-sequencing bioinformatics quality control workflows allow the accurate distinction between mixed infections, cross-contaminants and recombinant viruses at a threshold of >5% for the minority population. The sequence capture-based HCV WGS assay is more accurate than legacy AVR and genotyping assays. The assay has now been implemented in the clinical pathway of England's National Health Service HCV treatment programs, representing the first validated HCV WGS pipeline in clinical service. The data generated will additionally provide granular national-level genomic information for public health policy making and support the WHO HCV elimination strategy.

Enhanced Detection of DNA Viruses in the Cerebrospinal Fluid of Encephalitis Patients Using Metagenomic Next-Generation Sequencing.

The long and expanding list of viral pathogens associated with causing encephalitis confounds current diagnostic procedures, and in up to 50% of cases, the etiology remains undetermined. Sequence-agnostic metagenomic next-generation sequencing (mNGS) obviates the need to specify targets in advance and thus has great potential in encephalitis diagnostics. However, the low relative abundance of viral nucleic acids in clinical specimens poses a significant challenge. Our protocol employs two novel techniques to selectively remove human material at two stages, significantly increasing the representation of viral material. Our bioinformatic workflow using open source protein- and nucleotide sequence-matching software balances sensitivity and specificity in diagnosing and characterizing any DNA viruses present. A panel of 12 cerebrospinal fluid (CSFs) from encephalitis cases was retrospectively interrogated by mNGS, with concordant results in seven of nine samples with a definitive DNA virus diagnosis, and a different herpesvirus was identified in the other two. In two samples with an inconclusive diagnosis, DNA viruses were detected and in a virus-negative sample, no viruses were detected. This assay has the potential to detect DNA virus infections in cases of encephalitis of unknown etiology and to improve the current screening tests by identifying new and emerging agents.

Surveillance of HIV-1 transmitted integrase strand transfer inhibitor resistance in the UK.

BACKGROUND: HIV treatment guidelines have traditionally recommended that all HIV-positive individuals are tested for evidence of drug resistance prior to starting ART. Testing for resistance to reverse transcriptase inhibitors and PIs is well established in routine care. However, testing for integrase strand transfer inhibitor (InSTI) resistance is less consistent. OBJECTIVES: To inform treatment guidelines by determining the prevalence of InSTI resistance in a national cohort of recently infected individuals. PATIENTS AND METHODS: Recent (within 4 months) HIV-1 infections were identified using a Recent Infection Testing Algorithm of new HIV-1 diagnoses in the UK. Resistance-associated mutations (RAMs) in integrase, protease and reverse transcriptase were detected by ultradeep sequencing, which allows for the sensitive estimation of the frequency of each resistant variant in a sample. RESULTS: The analysis included 655 randomly selected individuals (median age = 33 years, 95% male, 83% MSM, 78% white) sampled in the period 2014 to 2016 and determined to have a recent infection. These comprised 320, 138 and 197 samples from 2014, 2015 and 2016, respectively. None of the samples had major InSTI RAMs occurring at high variant frequency (≥20%). A subset (25/640, 3.9%) had major InSTI RAMs occurring only as low-frequency variants (2%-20%). In contrast, 47/588 (8.0%) had major reverse transcriptase inhibitor and PI RAMs at high frequency. CONCLUSIONS: Between 2014 and 2016, major InSTI RAMs were uncommon in adults with recent HIV-1 infection, only occurring as low-frequency variants of doubtful clinical significance. Continued surveillance of newly diagnosed patients for evidence of transmitted InSTI resistance is recommended to inform clinical practice.

Interpreting Viral Deep Sequencing Data with GLUE.

Using deep sequencing technologies such as Illumina's platform, it is possible to obtain reads from the viral RNA population revealing the viral genome diversity within a single host. A range of software tools and pipelines can transform raw deep sequencing reads into Sequence Alignment Mapping (SAM) files. We propose that interpretation tools should process these SAM files, directly translating individual reads to amino acids in order to extract statistics of interest such as the proportion of different amino acid residues at specific sites. This preserves per-read linkage between nucleotide variants at different positions within a codon location. The samReporter is a subsystem of the GLUE software toolkit which follows this direct read translation approach in its processing of SAM files. We test samReporter on a deep sequencing dataset obtained from a cohort of 241 UK HCV patients for whom prior treatment with direct-acting antivirals has failed; deep sequencing and resistance testing have been suggested to be of clinical use in this context. We compared the polymorphism interpretation results of the samReporter against an approach that does not preserve per-read linkage. We found that the samReporter was able to properly interpret the sequence data at resistance-associated locations in nine patients where the alternative approach was equivocal. In three cases, the samReporter confirmed that resistance or an atypical substitution was present at NS5A position 30. In three further cases, it confirmed that the sofosbuvir-resistant NS5B substitution S282T was absent. This suggests the direct read translation approach implemented is of value for interpreting viral deep sequencing data.

Efficient and unbiased metagenomic recovery of RNA virus genomes from human plasma samples.

RNA viruses cause significant human pathology and are responsible for the majority of emerging zoonoses. Mainstream diagnostic assays are challenged by their intrinsic diversity, leading to false negatives and incomplete characterisation. New sequencing techniques are expanding our ability to agnostically interrogate nucleic acids within diverse sample types, but in the clinical setting are limited by overwhelming host material and ultra-low target frequency. Through selective host RNA depletion and compensatory protocol adjustments for ultra-low RNA inputs, we are able to detect three major blood-borne RNA viruses - HIV, HCV and HEV. We recovered complete genomes and up to 43% of the genome from samples with viral loads of 104 and 103 IU/ml respectively. Additionally, we demonstrated the utility of this method in detecting and characterising members of diverse RNA virus families within a human plasma background, some present at very low levels. By applying this method to a patient sample series, we have simultaneously determined the full genome of both a novel subtype of HCV genotype 6, and a co-infecting human pegivirus. This method builds upon earlier RNA metagenomic techniques and can play an important role in the surveillance and diagnostics of blood-borne viruses.

The Effect of Nucleic Acid Extraction Platforms and Sample Storage on the Integrity of Viral RNA for Use in Whole Genome Sequencing.

Extraction of viral RNA and the storage of sample material are extremely important factors in the detection and whole genome sequencing (WGS) of viral pathogens. Although PCR-based detection methods focus on small amplicons, viral WGS applications require RNA of high quality and integrity for adequate sequence coverage and depth. This study examined the fitness of one manual and four automated RNA extraction platforms commonly used in diagnostic laboratories for use in metagenomic sequencing, how the practice of storing sample material in Qiagen buffer AVL before extraction affected the integrity of viral RNA and its suitability for use in amplicon-based WGS methods, and how the addition of Triton X-100 to buffer AVL affected the capability of the extraction platforms and the integrity of viral RNA in stored samples. This study found that the EZ1 platform gave the best performance of the automated platforms and gave comparable results to the frequently used manual Qiagen extraction protocol when extracted viral RNA was used in metagenomics sequencing. To maintain high levels of viral RNA integrity suitable for amplicon-based WGS, nucleic acid should be extracted from samples immediately, because even short storage periods in buffer AVL have a severe effect on integrity, and the addition of Triton X-100 had little effect on the quality of viral material for WGS.

Efficiency of hepatitis A virus removal in six sewage treatment plants from central Tunisia.

The efficiency of six Tunisian sewage treatment plants (STP) for the removal of hepatitis A virus (HAV) from wastewater was analysed in order to evaluate the potential risk for human health linked to reuse or discharge of treated wastewater into the environment. The STP utilize different biological wastewater treatments including primary treatment, which involves the physical removal of organic and inorganic solids, and secondary treatment that involves different processes, such as activated sludge or lagoon. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) and conventional RT-PCR were used for the analysis of the 325 wastewater samples (163 raw and 162 treated) obtained. Results revealed highest contamination in west-central of Tunisia in raw wastewater with 62.96 % of samples positive for HAV and predominance during winter and autumn, whereas east-central region showed 50.62 % of positive samples with high prevalence from winter through summer. The quantitative analysis revealed a range between 4.29 × 10(1) and 1.24 × 10(5) RNA copies/mL in treated wastewater, showing clearly the inefficiency for total removal of HAV regardless of the treatment method used. The vast majority of HAV sequences belonged to the sub-genotype IA, except one that was assigned to sub-genotype IB.

Role of norovirus in acute gastroenteritis in the Northwest of Spain during 2010-2011.

A total of 2,643 samples from patients with gastroenteritis in Galicia (NW Spain) were tested for the presence of Norovirus (NoV). NoV genogroup GI was detected in 416 (15.7%) samples, while NoV genogroup GII was detected in 278 (10.5%) samples. Mixed infections of NoV GI and GII were observed in 53 (2%) samples. Total prevalence of NoV in the analyzed samples was 28.3%. Besides NoV diagnosis assay, all the specimens were also submitted to routine clinical bacteriology tests. Cryptosporidium spp. as well as adenovirus (AdV) and rotavirus (RV) were determined on some samples after specific request by hospital units. The results obtained allowed to determine the disease etiology in 14.4% of the patients. Taking into account all the microorganisms studied, the etiological agent was determined for 39.5% of the cases. The results indicated that NoVs are the leading cause of acute gastroenteritis in all age-groups in Northwestern Spain, and that the lack of routine NoV diagnosis contributes to the underestimation of the importance of this virus, not only in outbreaks, but also in sporadic cases of acute gastroenteritis.

Detection and characterization of hepatitis A virus and norovirus in mussels from Galicia (NW Spain).

Shellfish are recognized as a potential vehicle of viral disease and despite the control measures for shellfish safety there is periodic emergence of viral outbreaks associated with shellfish consumption. In this study a total of 81 mussel samples from Ría do Burgo, A Coruña (NW Spain) were analysed. Samples were collected in seven different harvesting areas with the aim to establish a correlation between the prevalence of norovirus (NoV) and hepatitis A virus (HAV) in mussel samples and the water quality. In addition, the genogroup of the detected HAV and NoV strains was also determined. The HAV presence was detected in 18.5 % of the samples. Contamination levels for this virus ranged from 1.1 × 10² to 4.1 × 106 RNA copies/g digestive tissue. NoV were detected in 49.4 % of the cases reaching contamination levels from 5.9 × 10³ to 1.6 × 109 RNA copies/g digestive tissue for NoV GI and from 6.1 × 10³ to 5.4 × 106 RNA copies/g digestive tissue for NoV GII. The χ²-test showed no statistical correlation between the number of positive samples and the classification of molluscan harvesting area based on the E. coli number. All the detected HAV strains belong to genogroup IB. NoV strains were assigned to genotype I.4, II.4 and II.6.

Norovirus, hepatitis A virus and enterovirus presence in shellfish from high quality harvesting areas in Portugal.

This is the first report on the screening of shellfish from Portugal for the presence of human enteropathogenic viruses. Approximately 2000 shellfish (Curbicula fluminea, Ruditapes decussatus, Tellina crassa, Spisula solida, Dosinia exoleta, Ensis spp., Mytilus spp., Ostrea edulis and Cerastoderma edule), organized in 49 batches, were collected between March 2008 and February 2009. They were tested for norovirus (NoV), hepatitis A virus (HAV) and enterovirus (EV) by RT-PCR followed by nucleotide sequencing. Bacterial contamination was also evaluated by Escherichia coli counts. Viral contamination was detected throughout the year in all shellfish species and in all collection areas, independently of their harvesting areas classification. Overall, 67% of all analyzed batches were contaminated by at least one of the studied viruses while the simultaneous presence of two and three viruses was detected in 22% and 6% batches, respectively. Of the three viruses, NoV was detected in 37% of the batches, followed by EV in 35%, and HAV in 33%. Nucleotide sequencing of the NoV and HAV RT-PCR products demonstrated that all strains belonged to NoV genotype GII.4 and HAV subgenotype 1B. The presence of NoV and HAV in shellfish from "A class" harvesting areas of Portugal can represent a potential health risk.

[Intravenous antibiotic treatment in a hospital based home care unit. Predictors of hospital readmission]. / Antibioterapia intravenosa domiciliaria en una unidad de hospitalización a domicilio: factores pronósticos de reingreso hospitalario.

BACKGROUND AND OBJECTIVE: Intravenous antibiotic therapy at home has showed its efficacy as an alternative to hospitalization care in many infectious pathologies. The objectives of this study are: a) to expose our experience, as hospital at home unit (HHU) integrated within a service of internal medicine, in the antibiotic treatment, and b) to define those parameters that can predict hospital readmissions. PATIENTS AND METHOD: This study included all patients with infectious pathology and intravenous antibiotic therapy who were admitted in our HHU from March 2006 to March 2007. RESULTS: 145 patients were included in this study. Successful treatment was observed in 92% of patients. Eleven patients were re-admitted at hospital during the episode by infectious disease, and only 2 of them showed adverse effects to treatment. Twenty-two patients were re-admitted at hospital 3 months after due to chronic pathology. CONCLUSIONS: Intravenous antibiotic therapy at home is a good alternative in many infectious pathologies. Infectious pathology and baseline state can be predictors of hospital readmissions.

Antibioterapia intravenosa domiciliaria en una unidad de hospitalización a domicilio: factores pronósticos de reingreso hospitalario / Intravenous antibiotic treatment in a hospital based home care unit. Predictors of hospital readmission

FUNDAMENTO Y OBJETIVO: La antibioterapia domiciliariaes una modalidad asistencial alternativa a lahospitalización a domicilio que ha demostrado suseguridad en un gran número de enfermedades infecciosas.Los objetivos principales de este artículoson: a) exponer nuestra experiencia, como unidadde hospitalización a domicilio (UHD)integrada dentro del servicio de medicina interna,en el tratamiento antibiótico intravenoso, y b) definirlos parámetros que pueden predecir un reingresohospitalario.PACIENTES Y MÉTODO: Se incluyó en el estudio a todoslos pacientes ingresados en nuestra UHD porenfermedad infecciosa subsidiaria de antibioterapiaintravenosa desde marzo de 2006 hasta marzode 2007.RESULTADOS: Se incluyó en el estudio a 145 pacientes.El 92% de los casos recibió el alta porbuena evolución clínica. Once pacientes ingresaronen el hospital durante el episodio clínico comoconsecuencia de la propia infección, y sólo 2 presentaronalgún tipo de reacción adversa a causadel tratamiento. Veintidós pacientes ingresaron enel hospital 3 meses después del alta de la UHD,fundamentalmente por enfermedad crónica.CONCLUSIONES: La antibioterapia intravenosa domiciliariaes una buena opción asistencial en unagran variedad de enfermedades infecciosas. A pesarde ello, la propia infección y el estado basaldel paciente influyen activamente en la probabilidadde reingresos hospitalarios

BACKGROUND AND OBJECTIVE: Intravenous antibiotictherapy at home has showed its efficacy as an alternativeto hospitalization care in many infectiouspathologies. The objectives of this study are: a) toexpose our experience, as hospital at home unit(HHU) integrated within a service of internal medicine,in the antibiotic treatment, and b) to definethose parameters that can predict hospital readmissions.PATIENTS AND METHOD: This study included all patientswith infectious pathology and intravenousantibiotic therapy who were admitted in our HHUfrom March 2006 to March 2007.RESULTS: 145 patients were included in this study.Successful treatment was observed in 92% of patients.Eleven patients were re-admitted at hospitalduring the episode by infectious disease, andonly 2 of them showed adverse effects to treatment.Twenty-two patients were re-admitted athospital 3 months after due to chronic pathology.CONCLUSIONS: Intravenous antibiotic therapy athome is a good alternative in many infectious pathologies.Infectious pathology and baseline statecan be predictors of hospital readmissions