宏基因组测序技术在感染性疾病诊断中的应用

感染性疾病仍是危害人类健康和全球公共卫生安全的重要问题。感染性疾病病原体种类繁杂,传统的检测方法检出率低、且仅可针对有限的病原体种类,难以满足临床诊断的高要求。因此,急需快速、广谱、准确的鉴定方法,满足临床感染性疾病的诊断需求。近年来,宏基因组测序技术得到了快速发展,具有通量高、速度快、信息全等特点,逐渐应用于临床检测。本文对宏基因组测序技术在感染性疾病病原体检测中的应用进行综述。     

慢性阻塞性肺疾病患者肺泡灌洗液宏基因组测序分析北大核心CSCD

目的评价PMseq病原微生物宏基因组测序检测用于慢性阻塞性肺疾病(COPD)合并肺部感染患者诊断的价值。方法以2019年11月至2022年5月在呼吸科确诊的67例稳定期COPD患者(合并肺部感染47例)为研究对象,同时采用肺泡灌洗液培养和PMseq病原微生物宏基因组测序,对培养及测序鉴定出的病原体进行描述性分析,并对检测结果进行比较分析。结果67例稳定期COPD患者中,宏基因组测序阳性65例、肺泡灌洗液培养阳性28例,差异有统计学意义(χ^(2)=48.11,P<0.01)。其中,宏基因组测序和肺泡灌洗液培养均阳性27例。该27例患者中,宏基因组测序和肺泡灌洗液培养检测结果完全一致8例、部分一致12例、不一致7例(Kappa值=0.46)。28例肺泡灌洗液培养阳性患者中以细菌感染多见,其中链球菌属及奈瑟菌感染分别为19例和17例,肠球菌感染5例,金黄色葡萄球菌、铜绿假单胞菌、肺炎克雷伯菌各1例;真菌感染以念珠菌多见(5例),土曲霉菌感染1例。65例高通量测序阳性COPD患者细菌感染以链球菌属为主,真菌感染以曲霉菌为主(5例)。高通量测序患者中,46例为2种及以上病原体感染、21例为单一病原体感染。结论COPD合并肺部感染患者病原体谱广,并存在混合感染。PMseq病原微生物宏基因组测序法敏感、特异,可用于COPD患者感染的病原体精准诊断,值得临床推广应用。     

高通量测序在鸟类肠道微生物中的研究进展

鸟类作为分布广泛、种群数量庞大和高度多样化的物种之一,在病原体传播中发挥了重要作用。本文综述了基于高通量测序技术的3种测序策略(16S rDNA测序、宏基因组测序和宏转录组测序)的特点及其在鸟类肠道菌群多样性、抗生素抗性基因和病原微生物发现中的应用,并对未来发展趋势进行了展望。     

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...     

内脏利什曼病1例

利什曼病是由利什曼原虫引起的人畜共患病, 在中国发病率较低。因其临床表现无特异性, 医师对此病认识不足, 易漏诊。现报道1例长期间断发热, 伴全血细胞减少、脾大等表现的患者, 排除血液系统等非感染性疾病, 多次经验性抗感染治疗无效, 传统方法未检测到病原体, 仍考虑感染性发热, 遂行外周血宏基因组学二代测序检测出利什曼原虫, 给予两性霉素B脂质体小剂量爬坡治疗后病情好转。     

糖尿病足感染微生物分布特点的研究进展

糖尿病足感染(DFI)致残、致死率高,是导致糖尿病患者住院和截肢率升高的主要原因。治疗DFI最重要的环节是选用合适的抗生素,准确选用抗生素的基础是明确致病菌。本文就DFI微生物分布特点进行综述,分析使用16S r RNA及宏基因组测序等高通量测序技术鉴定细菌的利弊,为临床准确选用抗生素治疗DFI提供依据。     

宏基因组病原学测序在下呼吸道感染的诊断价值和应用进展

下呼吸道感染(lower respiratory tract infection, LRTI)迄今仍是全球关注的重大卫生问题,及时准确地检出感染病原体是该病对因治疗的关键。现有各类微生物检测方法尚难满足日益发展的临床病原诊断需求。宏基因组下一代测序(metagenomic next-generation sequencing, mNGS)作为高通量、广覆盖、灵敏快速、无需预判的新型检测技术,可极大地提高感染性疾病的病因诊断率。本文综述了mNGS的概念、技术优势、在LRTI病原诊断的临床应用现状、以及mNGS当前面临的部分责任病原体序列数低,或与背景菌区分鉴别困难的挑战,并从技术和临床不同层面提出了应对措施和未来展望。     

细菌耐药性及耐药基因检测技术和方法的研究进展

［摘要］　抗生素过度使用加速了细菌耐药性的发生发展。当前,细菌耐药性已成为威胁全球公共卫生健康的重要因素之一。细菌耐药性检测技术包括以经典方法为主的肉汤稀释法、琼脂稀释法、纸片扩散法和浓度梯度法等。随后出现了一系列物理、化学方法和分子生物学检测技术,包括微流控图像检测、飞行时间质谱等。随着高通量测序技术的发展,各类细菌耐药基因数据库的开发和完善,全基因组测序、宏基因组测序和微生物单细胞转录组测序等新技术已逐步应用于细菌耐药性检测领域。该文将细菌耐药性检测方法分为传统经典方法、分子生物学技术和高通量测序技术三大类,阐述各类技术方法的原理、优点和局限性。回顾和反思当前细菌耐药性检测的技术和方法,对于规范抗生素的使用至关重要,也为临床超级耐药菌的预防和监测提供新的技术支持。     

宏基因组下一代测序技术检测肺部感染病原体研究进展

肺部感染发病率、死亡率高,是最常见和最重要的感染性疾病之一。细菌、病毒、真菌、寄生虫等一种或多种病原体均可导致肺部感染,早期、精准识别引起肺部感染的病原体对于制定治疗方案、改善预后具有重要意义。目前,肺部感染的主要诊断方法包括微生物培养法、血清学方法、抗原/抗体检测以及基于PCR的核酸检测技术等,但这些方法均存在不足。宏基因组下一代测序技术(metagenomic next generation sequencing, mNGS)可用于脑脊液、血浆、呼吸道分泌物、尿液、粪便、组织等任何种类临床样本检测,现已被广泛用于检测感染性病原体,特别适用于识别罕见或新发病原体。本文主要就mNGS用于肺部病原体感染检测研究进展作一综述。     

宏基因组二代测序辅助诊断鹦鹉热衣原体肺炎一例

<正>鹦鹉热衣原体作为一种革兰氏阴性、胞内寄生的病原体,通常在鹦鹉及鸽子中被发现。由于鹦鹉热的低发病率及无特异性的临床表现,其发生易被忽视。而且目前所存在的传统检测方法检出鹦鹉热衣原体病原体较为困难,然而宏基因组二代测序(metagenomic next-generation sequencing, mNGS)作为一种新型宏基因组二代测序方法,具有快速、准确、全面检测的优势。     

儿童重症肺炎的病原学特点及诊治进展

儿童重症肺炎是一种病死率较高的感染性疾病.其病原学及临床特点因患儿的年龄、不同的季节、不同的环境以及合并其他疾病而有着不同的表现.其病原菌耐药形势也愈发严峻.本文即对其病原学的特点及其诊断治疗方面的进展进行综述.     

Nanopore metagenomic sequencing of influenza virus directly from respiratory samples: diagnosis,drug resistance and nosocomial transmission,United Kingdom, 2018/19 influenza season

BackgroundInfluenza virus presents a considerable challenge to public health by causing seasonal epidemics and occasional pandemics. Nanopore metagenomic sequencing has the potential to be deployed for near-patient testing, providing rapid infection diagnosis, rationalising antimicrobial therapy, and supporting infection-control interventions.AimTo evaluate the applicability of this sequencing approach as a routine laboratory test for influenza in clinical settings.MethodsWe conducted Oxford Nanopore Technologies (Oxford, United Kingdom (UK)) metagenomic sequencing for 180 respiratory samples from a UK hospital during the 2018/19 influenza season, and compared results to routine molecular diagnostic standards (Xpert Xpress Flu/RSV assay; BioFire FilmArray Respiratory Panel 2 assay). We investigated drug resistance, genetic diversity, and nosocomial transmission using influenza sequence data.ResultsCompared to standard testing, Nanopore metagenomic sequencing was 83% (75/90) sensitive and 93% (84/90) specific for detecting influenza A viruses. Of 59 samples with haemagglutinin subtype determined, 40 were H1 and 19 H3. We identified an influenza A(H3N2) genome encoding the oseltamivir resistance S331R mutation in neuraminidase, potentially associated with an emerging distinct intra-subtype reassortant. Whole genome phylogeny refuted suspicions of a transmission cluster in a ward, but identified two other clusters that likely reflected nosocomial transmission, associated with a predominant community-circulating strain. We also detected other potentially pathogenic viruses and bacteria from the metagenome.ConclusionNanopore metagenomic sequencing can detect the emergence of novel variants and drug resistance, providing timely insights into antimicrobial stewardship and vaccine design. Full genome generation can help investigate and manage nosocomial outbreaks.     

蜱媒传染病相关病原体混合感染

目前,蜱媒传染病对公共健康产生了严重的威胁,不断有新的致病的蜱媒病原体被报道。由于大多数的蜱媒传染病临床症状缺乏特异性,无法迅速准确的对单一的蜱媒病原体感染与多种蜱媒病原体混合感染进行区分,甚至因为疏忽混合感染的可能性而耽误治疗,造成严重的后果。因此,本文希望通过综述蜱媒传染病相关病原体可能出现的混合感染种类,减少临床工作中的漏诊和误诊,为临床医生的诊断和治疗提供更多的思路。     

Pyopneumothorax with bronchopleural fistula due to pulmonary infection caused by Porphyromonas gingivalis in a patient with periodontitis

Pyopneumothorax with bronchopleural fistula is a rare complication of lung infection. We herein report a case of pyopneumothorax with bronchopleural fistula caused by Porphyromonas gingivalis infection, a common pathogenic pathogen of periodontitis, in a 49-year-old man with periodontitis. The patient was admitted with respiratory failure. Pleural puncture yielded a lot of gas continually and foul-smelling light brown pus, which was found to be caused due to infection with P. gingivalis by the metagenomic next generation sequencing (mNGS) and anaerobic culture.     

纳米孔测序技术鉴定陕西省首例马尔尼菲青霉菌感染病例

目的 对1例严重感染性疾病进行病原学检测,查明感染来源,为临床诊断治疗提供依据。方法 调查了解患者流行病学史、临床症状及体征,开展一般实验室检测。提取患者血样核酸,利用基于纳米孔测序的宏基因组分析技术,查询病原线索;利用真菌ITS通用引物、马尔尼菲青霉菌PCR以及利什曼原虫、组织荚膜胞浆菌PCR引物进行鉴别;对患者血样进行真菌双相培养确诊。结果 患者曾有不洁同性及异性性交史,常去东南亚地区旅行,临床症状及体征符合HIV感染表现,HIV初筛及确证实验为阳性,利什曼原虫抗体阴性。利用纳米孔测序仪MinION实际测序时间3.5 h,共获得84 000条Reads,序列长度均值7 088.7 base,最长为87 471 base,最短为562 base,总数据量为495 Mb。将basecalling所获数据上传EPI2Me,以WIMP workflow分析,有63 617条为人类基因组数据,13条马尔尼菲青霉菌序列;真菌ITS扩增测序结果鉴定为马尔尼菲青霉菌,马尔尼菲青霉菌PCR结果阳性,组织荚膜胞浆菌PCR阴性;真菌双相培养在25 ℃为霉菌样菌落,37 ℃为酵母样菌落,经鉴定均为马尔尼菲青霉菌。结论 该患者为中国陕西省首次报道的HIV合并马尔尼菲青霉菌感染病例,基于纳米孔测序的宏基因组分析技术可做为临床疑难感染性疾病快速诊断的一种指示方法。     

Perspective on the clone library method for infectious diseases

Recently, culture-independent molecular methods, such as DNA sequencing techniques targeting the 16S-ribosomal RNA (rRNA) gene and/or other housekeeping genes with Sanger method-based technologies, next generation sequencing (NGS), and metagenomic analysis, have been developed for detecting microorganisms in the human body; these can provide information on microbiomes of samples from individuals with or without infectious diseases. Determining the bacterial species is crucial in identifying causative bacteria of upper and lower respiratory tract infections, especially for Streptococcus species, but NGS analysis is often not precise enough to identify bacteria at the species level. This review briefly introduces previous observations of the microbiome of samples from various respiratory and other infections assessed using the clone library method with Sanger sequencing of the 16S-rRNA gene. On analysis of 16S-rRNA gene-sequence data of bronchoalveolar lavage fluid obtained from pneumonia lesions in patients with bacterial pneumonia and lung abscess, anaerobes are often detected in non-elderly patients with pneumonia, and the detection rate of Staphylococcus aureus in patients with hospital-acquired pneumonia is lower than that previously reported. Analysis of pleural effusion samples from patients with pleurisy indicated a more important role of anaerobes than previous believed. The other topics reviewed include microbiomes of nontuberculous mycobacteriosis and lower respiratory tract infections in children with permanent tracheostomy due to neuromuscular disorders, in nasal discharge, in bacterial vaginosis, in the intracystic fluid of postoperative maxillary cyst, and in bacterial conjunctivitis; urine microbiota in urethritis; fecal microbiota; and newly detected infectious organisms in the human respiratory tract.     

DsRNA Sequencing for RNA Virus Surveillance Using Human Clinical Samples

Although viruses infect various organs and are associated with diseases, there may be many unidentified pathogenic viruses. The recent development of next-generation sequencing technologies has facilitated the establishment of an environmental viral metagenomic approach targeting the intracellular viral genome. However, an efficient method for the detection of a viral genome derived from an RNA virus in animal or human samples has not been established. Here, we established a method for the efficient detection of RNA viruses in human clinical samples. We then tested the efficiency of the method compared to other conventional methods by using tissue samples collected from 57 recipients of living donor liver transplantations performed between June 2017 and February 2019 at Kyushu University Hospital. The viral read ratio in human clinical samples was higher by the new method than by the other conventional methods. In addition, the new method correctly identified viral RNA from liver tissues infected with hepatitis C virus. This new technique will be an effective tool for intracellular RNA virus surveillance in human clinical samples and may be useful for the detection of new RNA viruses associated with diseases.     

尿蛋白质组分析及其在病原生物学领域的应用

近年来，新发传染病不断出现，过去认为已得到有效控制的传染病死灰复燃，病原生物对人类健康的威胁日益增加。传统的病原生物学诊断方法的研发周期较长，许多疾病尚无可靠的诊断试剂和可用的预防性疫苗，病原生物学研究急需新的技术方法来解决目前所面临的严峻问题。尿蛋白质组学研究的技术方法多样，高通量、高灵敏度，能够对尿液中存在的蛋白质或多肽进行微量分析并且能够得到结构信息，具有潜在的快速准确获得患者尿液中病原生物标志物的特点，在疾病的快速诊断、病程监测以及药物靶点分析中具有重要的意义和应用前景。本文重点综述了尿蛋白质组学研究的策略、方法及其在病原生物学研究领域的应用，提出了建议和展望。     

叙利亚仓鼠应用于病毒感染性疾病动物模型的研究进展

全球传染病致死的主要病因是病毒感染.建立一种类似临床人类病毒感染性疾病的动物模型,对病毒感染的致病机制、预防与治疗研究有重要意义.近年来叙利亚仓鼠多用于病毒感染性疾病的研究,其作为动物模型的价值和优势突显出来.本文从叙利亚仓鼠的生理学和免疫学等方面阐述其主要生物学特征,并对叙利亚仓鼠在冠状病毒、副粘病毒、汉坦病毒和西尼...