Detection and Typing of Human Enteroviruses from Clinical Samples by Entire-Capsid Next Generation Sequencing

There are increasing concerns of infections by enteroviruses (EVs) causing severe disease in humans. EV diagnostic laboratory methods show differences in sensitivity and specificity as well as the level of genetic information provided. We examined a detection method for EVs based on next generation sequencing (NGS) analysis of amplicons covering the entire capsid coding region directly synthesized from clinical samples. One hundred and twelve clinical samples from England; previously shown to be positive for EVs, were analyzed. There was high concordance between the results obtained by the new NGS approach and those from the conventional Sanger method used originally with agreement in the serotypes identified in the 83 samples that were typed by both methods. The sensitivity and specificity of the NGS method compared to those of the conventional Sanger sequencing typing assay were 94.74% (95% confidence interval, 73.97% to 99.87%) and 97.85% (92.45% to 99.74%) for Enterovirus A, 93.75% (82.80% to 98.69%) and 89.06% (78.75% to 95.49%) for Enterovirus B, 100% (59.04% to 100%) and 98.10% (93.29% to 99.77%) for Enterovirus C, and 100% (75.29% to 100%) and 100% (96.34% to 100%) for Enterovirus D. The NGS method identified five EVs in previously untyped samples as well as additional viruses in some samples, indicating co-infection. This method can be easily expanded to generate whole-genome EV sequences as we show here for EV-D68. Information from capsid and whole-genome sequences is critical to help identifying the genetic basis for changes in viral properties and establishing accurate spatial-temporal associations between EV strains of public health relevance.     

Detection of Enterovirus D68 in Wastewater Samples from the UK between July and November 2021

Infection with enterovirus D68 (EV-D68) has been linked with severe neurological disease such as acute flaccid myelitis (AFM) in recent years. However, active surveillance for EV-D68 is lacking, which makes full assessment of this association difficult. Although a high number of EV-D68 infections were expected in 2020 based on the EV-D68′s known biannual circulation patterns, no apparent increase in EV-D68 detections or AFM cases was observed during 2020. We describe an upsurge of EV-D68 detections in wastewater samples from the United Kingdom between July and November 2021 mirroring the recently reported rise in EV-D68 detections in clinical samples from various European countries. We provide the first publicly available 2021 EV-D68 sequences showing co-circulation of EV-D68 strains from genetic clade D and sub-clade B3 as in previous years. Our results show the value of environmental surveillance (ES) for the early detection of circulating and clinically relevant human viruses. The use of a next-generation sequencing (NGS) approach helped us to estimate the prevalence of EV-D68 viruses among EV strains from other EV serotypes and to detect EV-D68 minor variants. The utility of ES at reducing gaps in virus surveillance for EV-D68 and the possible impact of nonpharmaceutical interventions introduced to control the COVID-19 pandemic on EV-D68 transmission dynamics are discussed.     

质粒介导的印第安纳沙门氏菌耐药机制及分子溯源研究

目的 通过全基因测序方-法(NGS)分析印第安纳沙门氏菌耐药克隆的质粒结构特征并与肠杆菌科质粒BLAST分子溯源,通过质粒消除研究其耐药表型和基因型的变化,探索印第安纳沙门氏菌耐药克隆成因及起源。方法 通过对2株(编号222、15)泛耐药印第安纳沙门菌全基因测序结-果尤其是其质粒序列耐药I类整合子、耐药基因、移动元件的分析,并与NCBI中28株肠杆菌科菌的质粒BLAST比对溯源。 对3株印第安纳沙门氏菌(编号15、38、222),1株肠炎沙门氏菌(编号17)、1株鼠伤寒沙门氏菌(编号32)、1株德尔卑沙门氏菌(编号163)进行高温-SDS质粒消除试验并比较质粒消除后菌株耐药表型及基因型的变化。结果 全基因测序结-果显示5株菌株拥有1个带有1个以上的耐药I类整合子及多个转座酶(Tns)和插入序列(ISs)等多种移动元件的结构相似、携带大量耐药基因且同源性大于99%的质粒(>210 000 bp)。经质粒消除实验后菌株15、38对四环素、庆大霉素、环丙沙星、阿奇霉素、阿米卡星等抗生素恢复敏感,菌株222对头孢噻圬、庆大霉素、环丙沙星、萘啶酸、卡那霉素等抗生素恢复敏感。菌株15不再携带TEM-1、OXA-1、sul1、aacC4、dfrA17、floR、qnrB等7种耐药基因,菌株38不再携带TEM-1、OXA-1、CTX-M、sul1、aacC4、dfrA17、floR,菌株222不再携带TEM-1、OXA-1、sul1、aacC4、aac6-1b。BLAST溯源结-果表明印第安纳携带的质粒与大肠杆菌质粒高度相似,同源性大于99%。结论 印第安纳沙门氏菌拥有含耐药I类整合子、多种移动元件(Tns)、插入序列(ISs)及大量耐药基因的耐药质粒是印第安纳沙门氏菌泛耐药克隆形成的主要原因。该质粒可能是其进化过程中从大肠杆菌类菌中获得。     

Full-Genome Sequences and Phylogenetic Analysis of Archived Danish European Bat Lyssavirus 1 (EBLV-1) Emphasize a Higher Genetic Resolution and Spatial Segregation for Sublineage 1a

European bat lyssavirus type 1 (EBLV-1) is the causative agent for almost all reported rabies cases found in European bats. In recent years, increasing numbers of available EBLV-1 full genomes and their phylogenetic analyses helped to further elucidate the distribution and genetic characteristics of EBLV-1 and its two subtypes, namely EBLV-1a and EBLV-1b. Nonetheless, the absence of full-genome sequences from regions with known detections of EBLV-1 still limit the understanding of the phylogeographic relations between viruses from different European regions. In this study, a set of 21 archived Danish EBLV-1 samples from the years 1985 to 2009 was processed for the acquisition of full-genome sequences using a high-throughput sequencing approach. Subsequent phylogenetic analysis encompassing all available EBLV-1 full genomes from databases revealed the Danish sequences belong to the EBLV-1a subtype and further highlighted the distinct, close phylogenetic relationship of Danish, Dutch and German isolates in this region. In addition, the formation of five putative groups nearly exclusively formed by Danish isolates and the overall increased resolution of the EBLV-1a branch indicate a higher genetic diversity and spatial segregation for this sublineage than was previously known. These results emphasize the importance of phylogenetic analyses of full-genome sequences of lyssaviruses for genetic geography.     

Multiplex PCR-Based Nanopore Sequencing and Epidemiological Surveillance of Hantaan orthohantavirus in Apodemus agrarius,Republic of Korea

Whole-genome sequencing of infectious agents enables the identification and characterization of emerging viruses. The MinION device is a portable sequencer that allows real-time sequencing in fields or hospitals. Hantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius, causes hemorrhagic fever with renal syndrome (HFRS) and poses a critical public health threat worldwide. In this study, we aimed to evaluate the feasibility of using nanopore sequencing for whole-genome sequencing of HTNV from samples having different viral copy numbers. Amplicon-based next-generation sequencing was performed in A. agrarius lung tissues collected from the Republic of Korea. Genomic sequences of HTNV were analyzed based on the viral RNA copy numbers. Amplicon-based nanopore sequencing provided nearly full-length genomic sequences of HTNV and showed sufficient read depth for phylogenetic analysis after 8 h of sequencing. The average identity of the HTNV genome sequences for the nanopore sequencer compared to those of generated from Illumina MiSeq revealed 99.8% (L and M segments) and 99.7% (S segment) identities, respectively. This study highlights the potential of the portable nanopore sequencer for rapid generation of accurate genomic sequences of HTNV for quicker decision making in point-of-care testing of HFRS patients during a hantavirus outbreak.     

Changes in the Cervical Microbiota of Women with Different High-Risk Human Papillomavirus Loads

The cervical microbiota is essential in female sexual health, and its altered states seem to have a central role in the dynamic of high-risk papillomavirus (hrHPV) infections. This study aimed to evaluate the variation in bacterial communities’ compositions according to hrHPV. We collected two samples per woman, with a difference of 12 ± 1 months between them, and performed a follow-up on 66 of these women. The viral load (VL) of the hrHPV was estimated by quantitative PCR (qPCR), then it was normalized (using the HMBS gene as reference) and transformed to the Log₁₀ scale to facilitate the interpretation. The VL was categorized as Negative, without hrHPV copies; Low, less than 10⁰ hrHPV copies; Medium, between 10⁰ to 10² hrHPV copies; and High, >10² hrHPV copies. The microbiota composition was described through the Illumina Novaseq PE250 platform. The diversity analyses revealed changes regarding the hrHPV VL, where women with low VL (<10⁰ hrHPV copies) presented high diversity. The community state type (CST) IV was the most common. However, in women with high VL, a lower association with Lactobacillus depletion was found. Lactobacillus gallinarum and L. iners were the most abundant species in women with high VL, whereas women with low VL had a 6.06 greater probability of exhibiting Lactobacillus dominance. We identified conspicuous differences in the abundance of 78 bacterial genera between women with low and high VL, where 26 were depleted (e.g., Gardnerella) and 52 increased (e.g., Mycoplasma). A multilevel mixed-effects linear regression showed changes in the diversity due to the interaction between the measurement time and the VL, with a decrease in diversity in the second follow-up in women with low VL (Coeff. = 0.47), whereas the women with medium VL displayed an increase in diversity (Coeff. = 0.58). Here, we report for the first time that the cervical microbiota is influenced by the number of copies of hrHPV, where a decrease in the abundance of Lactobacillus, greater diversity, and enrichment of bacterial taxa is relevant in women with low VL.     

宏基因组技术在感染性疾病中的应用

宏基因组测序(mNGS)正越来越多地应用于临床实验室的诊断.该文探讨了这项诊断传染病的新技术的目前实施情况,并讨论将mNGS转变为常规诊断技术的可行性.大量研究表明,mNGS在直接从临床样本中识别常规、罕见、协同感染病原体并对解决抗生素耐药性问题,提供了新的诊断证据,可用于指导治疗方案和改进抗生素管理.mNGS是解决临...     

Cell-free DNA for detection of clonal B cells in diffuse large B cell lymphoma by sequencing

Introduction

Diffuse large B cell lymphoma (DLBCL) is the most common lymphoma in the western world. It is highly heterogeneous with a variable clinical course, but curable with chemo-immunotherapy in up to 70% of all cases. The lymphoma presents in lymph nodes and/or extranodal lymphoid tissue, and the diagnosis is based on invasive procedures for histopathologic evaluation.

Methods

In this technical study, we evaluated cell-free DNA (cfDNA) from blood plasma to detect clonal B cells in patients with DLBCL using rearranged immunoglobulin heavy chain gene as targets by next-generation sequencing. Clonal B cell sequences and frequencies were determined from blood plasma cfDNA and cellular DNA from matched excised lymphoma tissues and mononuclear cells isolated from diagnostic bone marrow and blood samples from 15 patients.

Results

We showed that identical clonal rearrangements could be detected in blood plasma and excised lymphoma tissue and that plasma cfDNA was superior in detecting clonal rearrangements compared to blood or bone marrow-derived cellular DNA.

Conclusion

These findings consolidate the role of blood plasma as a reliable and easily accessible source for detecting neoplastic cells in DLBCL.     

The presence of genomic imbalances is associated with poor outcome in patients with burkitt lymphoma treated with dose‐intensive chemotherapy including rituximab

The introduction of Rituximab has improved the outcome and survival rates of Burkitt lymphoma (BL). However, early relapse and refractoriness are current limitations of BL treatment and new biological factors affecting the outcome of these patients have not been explored. This study aimed to identify the presence of genomic changes that could predict the response to new therapies in BL. Forty adolescent and adult BL patients treated with the Dose‐Intensive Chemotherapy Including Rituximab (Burkimab) protocol (Spanish Programme for the Study and Treatment of Haematological Malignancies; PETHEMA) were analysed using array‐based comparative genomic hybridization (CGH). In addition, the presence of TP53, TCF3 (E2A), ID3 and GNA13 mutations was assessed by next‐generation sequencing (NGS). Ninety‐seven per cent of the patients harboured genomic imbalances. Losses on 11q, 13q, 15q or 17p were associated with a poor response to Burkimab therapy (P = 0·038), shorter progression‐free survival (PFS; P = 0·007) and overall survival (OS; P = 0·009). The integrative analysis of array‐CGH and NGS showed that 26·3% (5/19) and 36·8% (7/19) of patients carried alterations in the TP53 and TCF3 genes, respectively. TP53 alterations were associated with shorter PFS (P = 0·011) while TCF3 alterations were associated with shorter OS (P = 0·032). Genetic studies could be used for risk stratification of BL patients treated with the Burkimab protocol.