全基因组测序技术在结核病流行病学调查中的应用

目的 评估全基因组测序技术在结核病分子流行病学调查中的应用。方法 对2008-2012年在上海市两家结核病定点医院发现9名耐多药患者中分离的结核分枝杆菌具有相同的可变数目串联重复序列,本研究对此进行流行学调查,并对9株结核分枝杆菌进行全基因组测序,分析其传播关系。结果 全基因组序列分析将9株结核分枝杆菌分为两个有传播关系的网络,一个为包括7株结核分枝杆菌（5例和2例患者分别来自不同的医院）的大簇,一个为只有2株结核分枝杆菌的小簇。两个簇之间相差15个单核苷酸多态性（SNP）位点,提示两个簇的遗传距离相对较远,基于菌株SNP差异构建的传播链显示了每个簇内菌株的传播方向和耐药突变积累的过程。结论 基于全基因组测序数据研究耐药结核病的传播网络,能准确判断传播路径和方向,识别传染源和传播缺失环节。     

全基因组测序技术在结核病分子流行病学中的应用进展

结核病给全球带来了严重的疾病负担,防控结核病具有重要意义.结核病分子流行病学将结核分枝杆菌分型技术与流行病学资料相整合,在研究结核病的传播和流行特点中起到了极其重要的作用.全基因组测序能够构建生物体基因组的完整DNA序列,具有高分辨率、高通量、结果精确等优势,在结核病分子流行病学研究中非常有吸引力和发展潜力.比较全基因...     

利用全基因组测序解析耐多药结核病传播路径

目的 应用全基因组测序技术解析耐多药结核分枝杆菌的传播路径。方法 对2009-2012年上海市一起经VNTR分型鉴定的耐多药结核病传播案例中8例簇病例进行全基因组测序,描述耐多药结核分枝杆菌的传播路径。结果 该起传播案例中源病例是1例复发耐多药结核病患者,在3年内传播耐药菌株导致另外至少7例患者发病。结论 全基因组测序技术可以弥补传统流行病学调查与基因分型技术的不足,准确地描述结核病传播的方向与细节,鉴定传染源与缺失病例,有助于准确揭示耐多药结核病的传播路径。     

分子流行病学与人类基因组计划

一个以生命科学为主导的新世纪已经到来。 2 0世纪中叶ＤＮＡ双螺旋结构及遗传机制的阐明 ,蛋白质、核酸人工合成的成功 ,导致基因工程、单克隆抗体、聚合酶链反应 (ＰＣＲ)等技术的应用以及基因芯片的研制成功等 ,促进了医学科技飞速发展。当代科技发展的重大项目是人类基因组计划这一伟大科学工程 ,它不仅改变了人们对疾病的认识 ,而且也改变了各国医学和生物学研究的格局。今天 ,生物医学已经历了从整体水平到器官水平 ,细胞水平到分子水平 ,从个体水平到群体水平 ,到生态水平以至宇宙水平的发展历程。在微观研究不断深入的基础上向宏观…     

全基因测序法分析肺炎克雷伯菌JM45株对喹诺酮类药物耐药基因

目的分析泛耐药肺炎克雷伯菌JM45株携带的喹诺酮类药物耐药基因,研究其对喹诺酮类药物的耐药机制。方法采用Roche454高通量测序技术对肺炎克雷伯菌JM45株做全基因组测序(完成图),分析喹诺酮类耐药基因携带状况,再将gyrA与parC全长基因与其他6株肺炎克雷伯菌做分子进化分析。结果最终得到JM45株一条完整的基因组(染色体)序列及两条质粒序列;基因组(染色体)序列大小为5 273 812bp(GC含量65.8%),质粒1序列大小为317 156bp(GC含量53.0%),质粒2序列大小为12 209bp(GC含量55.3%);在基因组(染色体)序列中携带了gyrA基因(全基因组Feature ID:KPN_1614),parC基因(Feature ID:KPN_0743);与肺炎克雷伯菌喹诺酮类药物敏感株相比较,gyrA基因第83位密码子由TCC→ATC,翻译成氨基酸序列后丝氨酸(Ser)→异亮氨酸(Ile),parC基因第80位密码子由AGC→ATC,翻译成氨基酸序列后丝氨酸(Ser)→异亮氨酸(Ile)。结论肺炎克雷伯菌JM45株喹诺酮类药物耐药是gyrA基因和parC基因喹诺酮耐药决定区(QRDR)突变所致;gyrA与parC全长基因的分子进化分析提示JM45株与肺炎克雷伯菌HS11286关系最为接近(序列相同),与肺炎克雷伯菌342关系最远。     

Cost analysis of whole genome sequencing in German clinical practice

Objectives

Whole genome sequencing (WGS) is an emerging tool in clinical diagnostics. However, little has been said about its procedure costs, owing to a dearth of related cost studies. This study helps fill this research gap by analyzing the execution costs of WGS within the setting of German clinical practice.

Methodology

First, to estimate costs, a sequencing process related to clinical practice was undertaken. Once relevant resources were identified, a quantification and monetary evaluation was conducted using data and information from expert interviews with clinical geneticists, and personnel at private enterprises and hospitals. This study focuses on identifying the costs associated with the standard sequencing process, and the procedure costs for a single WGS were analyzed on the basis of two sequencing platforms—namely, HiSeq 2500 and HiSeq Xten, both by Illumina, Inc. In addition, sensitivity analyses were performed to assess the influence of various uses of sequencing platforms and various coverage values on a fixed-cost degression.

Results

In the base case scenario—which features 80 % utilization and 30-times coverage—the cost of a single WGS analysis with the HiSeq 2500 was estimated at €3858.06. The cost of sequencing materials was estimated at €2848.08; related personnel costs of €396.94 and acquisition/maintenance costs (€607.39) were also found. In comparison, the cost of sequencing that uses the latest technology (i.e., HiSeq Xten) was approximately 63 % cheaper, at €1411.20.

Conclusions

The estimated costs of WGS currently exceed the prediction of a ‘US$1000 per genome’, by more than a factor of 3.8. In particular, the material costs in themselves exceed this predicted cost.

     

Molecular epidemiology of tuberculosis among prisoners

The subject of the investigation is a group of 27 strains of Mycobacterium tuberculosis isolated in 2000 from prisoners with the diagnosis of tuberculosis, 19 Czechs and 8 foreigners (mean age 41 and 35 years resp.). The molecular-epidemiological examination of these strains was made using the RFLP fingerprint technique (Restriction Fragment Length Polymorphism) with evidence of the insertive sequence IS6110 and the technique of spoligotyping, based on detection of hybridization of spacer oligonucleotides. DNA fingerprinting revealed a high polymorphism in the number and molecular weight of sequence IS6110 which is common in Czech and other European strains of M. tuberculosis. All strains with the exception of two had mutually different fingerprint profiles. In the two with identical fingerprints probably a duplicit examination of the same material was involved which occurred by mistake during transport or in the laboratory. The fingerprint method thus did not prove interhuman transmission of tuberculosis between the examined prisoners. The technique of spoligotyping revealed the finding of genotype Beijing M. tuberculosis in two sick prisoners, one Algerian and one Albanese, and in one Czech prisoner. This genotype found in a high percentage of patients in southeastern Asia and in migrants from this area was detected for the first time in the Czech Republic. The findings are evidence of a satisfactory standard of the programme of tuberculosis control in the Czech prison system and at the same time draw attention to the potential possibility of the spread of tuberculosis from migrants coming from areas with a high prevalence.     

Molecular biology in the diagnosis and epidemiology of tuberculosis

Tuberculosis is the predominant infectious cause of mortality today, killing 3 million people annually. The cornerstones of diagnosis rest on microscopy of specimens using auramine and Ziehl-Neelsen stains followed by culture on Lowenstein-Jensen or alternative media. The long generation time of Mycobacterium tuberculosis means 2–8 weeks usually elapse before a result is available to the clinician. This has stimulated research into the use of molecular diagnostic techniques. This article reviews the use and limitations of DNA hybridization, restriction fragment length polymorphism, pulsed-field gel electrophoresis and the polymerase chain reaction in the diagnosis and epidemiology of tuberculosis. The applicability of molecular biology to determine drug resistance is also addressed.     

Phylogenetic understanding of clonal populations in an era of whole genome sequencing

Phylogenetic hypotheses using whole genome sequences have the potential for unprecedented accuracy, yet a failure to understand issues associated with discovery bias, character sampling, and strain sampling can lead to highly erroneous conclusions. For microbial pathogens, phylogenies derived from whole genome sequences are becoming more common, as large numbers of characters distributed across entire genomes can yield extremely accurate phylogenies, particularly for strictly clonal populations. The availability of whole genomes is increasing as new sequencing technologies reduce the cost and time required for genome sequencing. Until entire sample collections can be fully sequenced, harnessing the phylogenetic power from whole genome sequences in more than a small subset of fully sequenced strains requires the integration of whole genome and partial genome genotyping data. Such integration involves discovering evolutionarily stable polymorphic characters by whole genome comparisons, then determining allelic states across a wide panel of isolates using high-throughput genotyping technologies. Here, we demonstrate how such an approach using single nucleotide polymorphisms (SNPs) yields highly accurate, but biased phylogenetic reconstructions and how the accuracy of the resulting tree is compromised by incomplete taxon and character sampling. Despite recent phylogenetic work detailing the strengths and biases of integrating whole genome and partial genome genotype data, these issues are relatively new and remain poorly understood by many researchers. Here, we revisit these biases and provide strategies for maximizing phylogenetic accuracy. Although we write this review with bacterial pathogens in mind, these concepts apply to any clonally reproducing population or indeed to any evolutionarily stable marker that is inherited in a strictly clonal manner. Understanding the ways in which current and emerging technologies can be used to maximize phylogenetic knowledge is advantageous only with a complete understanding of the strengths and weaknesses of these methods.     

Molecular epidemiology of tuberculosis in a sentinel surveillance population

We conducted a population-based study to assess demographic and risk-factor correlates for the most frequently occurring Mycobacterium tuberculosis genotypes from tuberculosis (TB) patients. The study included all incident, culture-positive TB patients from seven sentinel surveillance sites in the United States from 1996 to 2000. M. tuberculosis isolates were genotyped by IS6110-based restriction fragment length polymorphism and spoligotyping. Genotyping was available for 90% of 11923 TB patients. Overall, 48% of cases had isolates that matched those from another patient, including 64% of U.S.-born and 35% of foreign-born patients. By logistic regression analysis, risk factors for clustering of genotypes were being male, U.S.-born, black, homeless, and infected with HIV; having pulmonary disease with cavitations on chest radiograph and a sputum smear with acid-fast bacilli; and excessive drug or alcohol use. Molecular characterization of TB isolates permitted risk correlates for clusters and specific genotypes to be described and provided information regarding cluster dynamics over time.     

High diversity of beta-lactamases in the General Hospital Vienna verified by whole genome sequencing and statistical analysis

The detailed analysis of antibiotic resistance mechanisms is essential for understanding the underlying evolutionary processes, the implementation of appropriate intervention strategies and to guarantee efficient treatment options. In the present study, 110 β-lactam-resistant, clinical isolates of Enterobacteriaceae sampled in 2011 in one of Europe’s largest hospitals, the General Hospital Vienna, were screened for the presence of 31 β-lactamase genes. Twenty of those isolates were selected for whole genome sequencing (WGS). In addition, the number of β-lactamase genes was estimated using biostatistical models.The carbapenemase genes bla_KPC-2, bla_KPC-3, and bla_VIM-4 were identified in carbapenem-resistant and intermediate susceptible isolates, bla_OXA-72 in an extended-spectrum β-lactamase (ESBL)-positive one. Furthermore, the observed high prevalence of the acquired bla_DHA-1 and bla_CMY AmpC β-lactamase genes (70%) in phenotypically AmpC-positive isolates is alarming due to their capability to become carbapenem-resistant upon changes in membrane permeability. The statistical analyses revealed that approximately 55% of all β-lactamase genes present in the General Hospital Vienna were detected by this study. In summary, this work gives a very detailed picture on the disseminated β-lactamases and other resistance genes in one of Europe’s largest hospitals.     

Whole genome sequencing to complement tuberculosis drug resistance surveys in Uganda

Understanding the circulating Mycobacterium tuberculosis resistance mutations is vital for better TB control strategies, especially to inform a new MDR-TB treatment programme. We complemented the phenotypic drug susceptibility testing (DST) based drug resistance surveys (DRSs) conducted in Uganda between 2008 and 2011 with Whole Genome Sequencing (WGS) of 90 Mycobacterium tuberculosis isolates phenotypically resistant to rifampicin and/or isoniazid to better understand the extent of drug resistance.A total of 31 (34.4 %) patients had MDR-TB, 5 (5.6 %) mono-rifampicin resistance and 54 (60.0 %) mono-isoniazid resistance by phenotypic DST. Pyrazinamide resistance mutations were identified in 32.3% of the MDR-TB patients. Resistance to injectable agents was detected in 4/90 (4.4%), and none to fluoroquinolones or novel drugs. Compensatory mutations in rpoC were identified in two patients. The sensitivity and specificity of drug resistance mutations compared to phenotypic DST were for rpoB 88.6% and 98.1%, katG 60.0% and 100%, fabG1 16.5% and 100%, katG and/or fabG1 71.8% and 100%, embCAB 63.0% and 82.5%, rrs 11.4% and 100%, rpsL 20.5% and 95.7% and rrs and/or rpsL 31.8% and 95.7%.Phylogenetic analysis showed dispersed MDR-TB isolate, with only one cluster of three Beijing family from South West Uganda.Among tuberculosis patients in Uganda, resistance beyond first-line drugs as well as compensatory mutations remain low, and MDR-TB isolates did not arise from a dominant clone. Our findings show the potential use of sequencing for complementing DRSs or surveillance in this setting, with good specificity compared to phenotypic DST. The reported high confidence mutations can be included in molecular assays, and population-based studies can track transmission of MDR-TB including the Beijing family strains in the South West of the country.     

Molecular epidemiology of Mycobacterium tuberculosis clinical isolates in Southwest Ireland

Tuberculosis has had significant effects on Ireland over the past two centuries, causing persistently higher morbidity and mortality than in neighbouring countries until the last decade. This study describes the results of genotyping and drug susceptibility testing of 171 strains of Mycobacterium tuberculosis complex isolated between January 2004 and December 2006 in a region of Ireland centred on the city of Cork. Spoligotype comparisons were made with the SpolDB4 database and clustered 130 strains in 23 groups, forty-one strains showed unique Spoligotyping patterns. The commonest spoligotypes detected were ST0137 (X2) (16.9%), and ST0351 (15.8%) (‘U’ clade). The major spoligotype clades were X (26.2%), U (19.3%), T (15.2%), Beijing (5.9%), Haarlem (4.7%), LAM (4.1%), BOVIS (1.75%), with 12.9% unassigned strains. A 24-locus VNTR genotyping produced 15 clusters containing 49 isolates, with high discrimination index (HGDI > 0.99). A combination of Spoligotyping and VNTR reduced the number of clustered isolates to 47 in 15 clusters (27.5%). This study identified ST351 as common among Irish nationals, and found a low rate of drug resistance with little evidence of transmission of drug resistant strains. Strain clustering was significantly associated with age under 55 years and Irish nationality. Only strains of Euro-American lineage formed clusters. Molecular typing did not completely coincide with the results of contact investigations.     

Bioinformatics tools and databases for whole genome sequence analysis of Mycobacterium tuberculosis

Tuberculosis (TB) is an infectious disease of global public health importance caused by Mycobacterium tuberculosis complex (MTC) in which M. tuberculosis (Mtb) is the major causative agent. Recent advancements in genomic technologies such as next generation sequencing have enabled high throughput cost-effective generation of whole genome sequence information from Mtb clinical isolates, providing new insights into the evolution, genomic diversity and transmission of the Mtb bacteria, including molecular mechanisms of antibiotic resistance. The large volume of sequencing data generated however necessitated effective and efficient management, storage, analysis and visualization of the data and results through development of novel and customized bioinformatics software tools and databases. In this review, we aim to provide a comprehensive survey of the current freely available bioinformatics software tools and publicly accessible databases for genomic analysis of Mtb for identifying disease transmission in molecular epidemiology and in rapid determination of the antibiotic profiles of clinical isolates for prompt and optimal patient treatment.     

Whole genome sequencing uses for foodborne contamination and compliance: Discovery of an emerging contamination event in an ice cream facility using whole genome sequencing

We review how FDA surveillance identifies several ways that whole genome sequencing (WGS) improves actionable outcomes for public health and compliance in a case involving Listeria monocytogenes contamination in an ice cream facility. In late August 2017 FDA conducted environmental sampling inside an ice cream facility. These isolates were sequenced and deposited into the GenomeTrakr databases. In September 2018 the Centers for Disease Control and Prevention contacted the Florida Department of Health after finding that the pathogen analyses of three clinical cases of listeriosis (two in 2013, one in 2018) were highly related to the aforementioned L. monocytogenes isolates collected from the ice cream facility. in 2017. FDA returned to the ice cream facility in late September 2018 and conducted further environmental sampling and again recovered L. monocytogenes from environmental subsamples that were genetically related to the clinical cases. A voluntary recall was issued to include all ice cream manufactured from August 2017 to October 2018. Subsequently, FDA suspended this food facility's registration. WGS results for L. monocytogenes found in the facility and from clinical samples clustered together by 0–31 single nucleotide polymorphisms (SNPs). The FDA worked together with the Centers for Disease Control and Prevention, as well as the Florida Department of Health, and the Florida Department of Agriculture and Consumer Services to recall all ice cream products produced by this facility. Our data suggests that when available isolates from food facility inspections are subject to whole genome sequencing and the subsequent sequence data point to linkages between these strains and recent clinical isolates (i.e., <20 nucleotide differences), compliance officials should take regulatory actions early to prevent further potential illness. The utility of WGS for applications related to enforcement of FDA compliance programs in the context of foodborne pathogens is reviewed.     

用全基因组测序追踪临床重要耐药菌院内感染的暴发流行

新一代高通量测序技术的成熟和普及使微生物基因组学获得长足发展,采用全基因组序列分析追踪和溯源临床重要耐药菌院内感染的暴发,已成为近年临床微生物研究领域的热点,并形成了一个新的学科方向——基因组流行病学。在将全基因组序列分析用于临床耐药菌流行溯源时,可利用基因组数据的强大分辨率和丰富信息,在进行菌株充分分型的基础上,更精确地判断耐药菌同源性的远近,分辨出不同的进化路线,推断耐药菌在医院环境中的演化过程,这对临床耐药菌院内感染控制工作意义重大。     

Molecular epidemiology of tuberculosis: achievements and challenges to current knowledge

Over the past 10 years, molecular methods have become available with which to strain-type Mycobacterium tuberculosis. They have allowed researchers to study certain important but previously unresolved issues in the epidemiology of tuberculosis (TB). For example, some unsuspected microepidemics have been revealed and it has been shown that the relative contribution of recently acquired disease to the TB burden in many settings is far greater than had been thought. These findings have led to the strengthening of TB control. Other research has demonstrated the existence and described the frequency of exogenous reinfection in areas of high incidence. Much recent work has focused on the phenotypic variation among strains and has evaluated the relative transmissibility, virulence, and immunogenicity of different lineages of the organism. We summarize the recent achievements in TB epidemiology associated with the introduction of DNA fingerprinting techniques, and consider the implications of this technology for the design and analysis of epidemiological studies.     

Molecular epidemiology of Mycobacterium tuberculosis, Buenos Aires, Argentina

To analyze the molecular epidemiology of Mycobacterium tuberculosis strains at a hospital in Buenos Aires, Argentina, and mutations related to multidrug-resistant and extensively drug-resistant tuberculosis, we conducted a prospective case-control study. Our findings reinforce the value of incorporating already standardized molecular methods for rapidly detecting resistance.