Rapid, sensitive, and specific detection of Mycobacterium tuberculosis complex by real-time PCR on paraffin-embedded human tissues

The detection of Mycobacterium tuberculosis complex (MTB) in clinical specimens is important for diagnosing and caring for patients in whom tuberculosis is clinically suspected. We collected 129 FFPE specimens, including 56 nontuberculosis cases, 63 MTB cases, and 10 nontuberculous mycobacteria (NTM) cases determined by acid-fast bacilli (AFB) culture. We performed AFB staining; nested MTB PCR, targeting the IS6110 gene; and real-time MTB PCR, targeting the senX3-regX3 intergenic region in the 129 FFPE specimens. The sensitivity and specificity of AFB staining were 37.0% and 98.2%, respectively, using AFB culture results as the reference standard. The sensitivity and specificity of detecting MTB were 68.3% and 98.5%, respectively, by nested PCR; and 74.6% and 98.5% by real-time PCR, respectively. Among the 129 specimens, four were positive by AFB staining but negative by nested or real-time PCR. NTM grew in all four of these cases by AFB culture. AFB density in FFPE tissue sections significantly correlated with MTB DNA load. Thus, real-time PCR is a useful diagnostic tool for rapid and sensitive MTB detection in FFPE specimens, whereas NTM should be included in differential diagnoses of cases positive by AFB staining but negative by PCR.     

Clinicopathologic characteristics of nontuberculous mycobacterial lung disease in Taiwan

Taiwan is an endemic area for tuberculosis (TB), and the incidence of pulmonary infection caused by nontuberculous mycobacteria (NTMs) is also increasing. This study aims to investigate the clinicopathologic characteristics of patients with NTM lung disease during 1998 to 2007 at a medical center in Taiwan. The medical records of patients with confirmed NTM pulmonary infections who underwent open lung surgery in a medical center were reviewed. Twenty-four patients with confirmed NTM pulmonary infections were identified. These patients were histologically classified into 4 types: fibrocavitary/tuberculoid (n = 10), nodular bronchiectatic (n = 4), sarcoidal (n = 6), and other (n = 4). The fibrocavitary/tuberculoid type usually (90%) develops in the upper lobes of old patients with preexisting lung disease. Pulmonary TB (n = 7, 70%) was the major underlying disease before 2003. Nodular bronchiectatic type occurred mainly in the middle lobe of middle-aged women without preexisting lung disease. Sarcoidal type was usually associated with Mycobacterium avium complex infection and develops in middle-aged women. Immunoreactive bacilli were detected in 21 patients (87 %) by immunohistochemical staining using a polyclonal antibody against Mycobacterium tuberculosis and other mycobacterial species (M. avium-intracellulare, Mycobacterium phlei, and Mycobacterium parafortuitum), whereas conventional acid-fast staining was positive in only 21% of patients. In conclusion, TB was the major underlying disease in patients with NTM lung disease in Taiwan. The different histologic types of pulmonary NTM infection suggest each had a distinct pathogenesis.     

2011-2018年四川省自贡市分枝杆菌临床分离株鉴定分析

目的了解自贡市肺部感染分枝杆菌的菌种菌型特征,为临床分枝杆菌病的诊断和治疗及制定有效防控策略提供科学依据。方法收集2011－2018年临床疑似结核病患者分离到的抗酸染色阳性培养物,经对硝基苯甲酸（PNB）和噻吩-2-羧酸肼（TCH）鉴别培养和多位点PCR鉴定为结核分枝杆菌和非结核分枝杆菌（NTM）,再经rpoB和hsp65序列分析对NTM进行种的鉴定。结果2 751株分枝杆菌临床分离株经PNB/TCH鉴别培养基和多位点PCR鉴定,结核分枝杆菌2 647株,占96.22%,非洲分枝杆菌Ⅰ型32株,占1.16%,NTM 72株,占2.62%。 72株NTM鉴定出11个种。结论自贡市分枝杆菌肺部感染主要为结核分枝杆菌所致,感染致病的NTM种类较多,以脓肿分枝杆菌、胞内分枝杆菌、堪萨斯分枝杆菌和鸟分枝杆菌为主。 外来分枝杆菌和奥巴涅分枝杆菌在国内为首次报道。     

Standardization and evaluation of a tetraplex polymerase chain reaction to detect and differentiate Mycobacterium tuberculosis complex and nontuberculous Mycobacteria--a retrospective study on pulmonary TB patients

The clinical presentation of pulmonary tuberculosis by members of Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) cannot be differentiated using the available standard diagnostic procedures. A single-tube tetraplex polymerase chain reaction (T-PCR) was designed to simultaneously amplify 4 well-known DNA targets of MTC. Taguchi's protocol was followed for the optimization of the conditions and was then tested on 288 pulmonary TB patient samples. The analytical sensitivity of the T-PCR was 100 fg of purified mycobacterial DNA, and specificity was found to be 100% in being able to distinguish MTC and NTM in all the cases tested. The results correlated well when validated with hsp65 PCR restriction analysis and sequencing of the 16S-23S internal transcribed spacer region, hsp65, and rpoB. The T-PCR described here is a quick, valuable, and cost-effective tool for determining whether the causative organism is MTC or NTM, and thus is useful for disease surveillance.     

多位点PCR技术用于四川267株分枝杆菌临床分离株的鉴定

目的 初步了解四川省肺结核患者分枝杆菌菌种类型。 方法 收集267株分枝杆菌临床菌株,经PNB/TCH鉴别培养基进行培养鉴定后,采用聚合酶链反应(PCR)对16S rRNA、Rv0577、IS1561、Rv1510、Rv1970、Rv3877/8和Rv3120 基因位点进行扩增,鉴定至种,再经PRA-rpoB、hsp65基因测序进行验证。 结果 267株分枝杆菌临床分离株多位点PCR结果显示结核分枝杆菌262株,非洲分枝杆菌Ⅰ型3株,非结核分枝杆菌2株。PNB/TCH鉴别培养基培养鉴定结果为结核分枝杆菌复合群266株,非结核分枝杆菌1株。2株非结核分枝杆菌分别为鸟分枝杆菌(M. avium)和脓毒分枝杆菌(M. septicum)。多位点PCR结果与rpoB-PRA、hsp65基因测序结果一致。 结论 多位点PCR技术鉴定分枝杆菌菌种结果准确可靠,且具有简便和快速等优点,有较大的分子流行病学应用价值,且对于临床诊断和治疗都具有重要意义。     

海南省肺部感染患者非结核分枝杆菌的菌种鉴定分析

目的 通过对海南省肺部感染患者非结核分枝杆菌（NTM）进行菌种鉴定,探讨海南省NTM肺部感染菌种的类型及人群分布情况。方法 收集2016年7月至2021年6月期间就诊于海南医学院第二附属医院疑似肺结核患者的呼吸道标本进行分枝杆菌培养,对培养阳性标本进行对硝基苯甲酸（PNB）/噻吩-2-羧酸肼（TCH）培养菌型初步鉴定,采用DNA微阵列芯片技术进行分枝杆菌菌种鉴定,无法确定菌种的菌株进一步采用基因测序法鉴定。结果 共收集8 507例疑似肺结核患者的呼吸道标本,剔除重复病例后,有318例经PNB/TCH培养初步鉴定为NTM,315例经DNA微阵列芯片技术和热休克蛋白65(hsp65)基因测序鉴定为NTM。其中308例患者为单一感染模式,6例MTB+NTM和1例2种不同NTM的混合感染模式。快速生长分枝杆菌128株占40.5%,以龟/脓肿分枝杆菌为主（占32.9%）;缓慢生长分枝杆菌188株占59.5%,以胞内分枝杆菌为主（占39.6%）。女性患者多于男性且好发于中老年人,男女性别比为0.79∶1。男女性患者年龄分布差异有统计学意义（Z=2.200,P<0.05）,>40岁的患者...     

中国首例脓毒分枝杆菌感染肺病报告

目的 分析脓毒分枝杆菌感染肺病的临床和细菌学特征,探讨其鉴别诊断方法。 方法 分析脓毒分枝杆菌感染肺病患者的临床表现极其相关实验室检查结果。对其临床分离株采用L-J,TCH/PNB鉴别培养基和多位点PCR初步鉴别,rpoB/hsp65基因PCR-测序鉴定出菌种。 结果 该患者痰涂片和培养检测为阳性。L-J,TCH/PNB鉴别培养基和多位点PCR鉴别为非结核分枝杆菌。rpoB/hsp65基因PCR-测序,经BLAST比对,与脓毒分枝杆菌(ATCC 700731)同源性均为99%。药物敏感性检测证实本病例分离到的菌株对大多数药物(包括四种一线抗结核药物)耐药,而对阿米卡星、环丙沙星、氧氟沙星、左氧氟沙星、卡那霉素和新诺明等药物敏感。 结论 在中国首次鉴定、确诊脓毒分枝杆菌感染肺病。     

FQ-PCR检测肠黏膜结核杆菌DNA对肠结核的诊断价值

目的建立一种实时定量检测结核分枝杆菌（MTB）插入序列IS6110 DNA的方法,探讨其在肠结核（ITB）诊断中的价值。方法采用FQ-PCR技术对36例内镜活检ITB标本（30例石蜡、6例新鲜组织）、36例Crohn’s disease标本（16例石蜡手术标本,15例石蜡内镜活检标本,5例新鲜内镜活检组织）及34例阴性对照标本进行IS6110 DNA的实时定量检测,并比较上述标本抗酸染色结果。结果13例ITB抗酸染色阳性,CD无1例阳性,抗酸染色对ITB诊断的敏感性36.11%。MTBIS6110DNA检测结果：23例ITB（63.89%）阳性,6例CD（16.67%）阳性,另有1例阳性为升结肠腺癌癌旁正常组织。MTBIS6110 DNA在ITB与CD中的阳性率差异有统计学意义（P〈0.05）。FQ-PCR对ITB诊断的敏感性为63.89%,特异性83.33%。FQ-PCR敏感性显著高于抗酸染色（P〈0.05）。MTBIS6110 DNA阳性的ITB标本其定量结果范围为2.44×10^-4-2.26×10^1拷贝/细胞。结论FQ-PCR检测MTBIS6110 DNA是一种快速有效的ITB诊断方法,其定量范围广,检测灵敏度高。对活检组织少、病理改变不典型、抗酸染色阴性组织的诊断和鉴别诊断尤有意义。     

AIDS合并结核与非结核分枝杆菌肺病CT影像表现

目的探讨AIDS合并结核分枝杆菌（Mycobacterium tuberculosis,MTB）和非结核分枝杆菌（non-tuberculous mycobacteria,NTM）肺病CT影像表现的异同。方法回顾性分析32例AIDS合并MTB肺病和13例AIDS合并NTM肺病患者的CT影像资料。结果 2组病变均累及多个肺叶,多种病变同时存在,表现为磨玻璃样或斑片状渗出、大片状实变、沿支气管播散树芽征等;纵隔和/或肺门淋巴结肿大多见;空洞少见;AIDS合并MTB组弥漫粟粒性病变、胸腔积液和/或胸膜增厚比例高于AIDS合并NTM组（P〈0.05）,结节病灶比例低于AIDS合并NTM组（P〈0.05）。结论 AIDS合并MTB和NTM肺病CT均可表现为多肺段分布,多种病变共存,常伴纵隔或肺门淋巴结肿大;如出现两肺弥漫粟粒结节、胸膜增厚或胸腔积液,多提示AIDS合并MTB肺病,而结节病灶多提示AIDS合并NTM肺病。     

Nontubercular mycobacterial pulmonary infection in immunocompetent men

BACKGROUND: Nontubercular mycobacteria (NTM) are increasingly recognized to cause lung disease in immunocompetent patients. We studied the occurrence of pulmonary infection due to NTM in immunocompetent men. METHODS: We retrospectively analyzed all sputum mycobacterial cultures at our institution over a 5-year period. Charts were reviewed to identify patients who met the American Thoracic Society's criteria for mycobacterial pulmonary infection. RESULTS: From the 7,380 sputum mycobacterial cultures obtained, 46 male patients had NTM identified. Forty-two patients were immunocompetent. Five of these patients were found to have NTM--2 with Mycobacterium kansasii, 2 with Mycobacterium avium-intracellulare, and 1 with Mycobacterium gordonae. All 5 patients responded to antimycobacterial therapy. CONCLUSION: Twelve percent of our population of immunocompetent men from whom NTM were isolated from sputum were infected. This study should alert the clinician that NTM cause treatable pulmonary disease in immunocompetent men.     

环介导等温扩增技术快速检测结核分枝杆菌核酸

摘要:目的：建立一种快速准确的检测结核分枝杆菌（MTB）核酸的环介导等温扩增（LAMP）方法。 方法：以重复插入序列IS6110为目的基因,设计LAMP引物,特异检测MTB核酸。用本法与痰涂片抗酸染色镜检法、实时荧光PCR法对100例可疑患者痰标本进行对比检查。 结果：LAMP法特异性强,仅扩增MTB复合群核酸;灵敏度高,检测限达100 fg;而实时荧光PCR检测限为1 pg。对100例疑似结核病患者痰液标本检测,涂片抗酸染色法、LAMP法、实时荧光PCR法的阳性率分别为28%、39%和38%。 结论：本研究建立的LAMP方法检测MTB核酸特异性强、灵敏度高、时间短且操作简便,有望成为临床快速检测MTB的新方法。     

Evaluation of multiple genetic markers for typing drug-resistant Mycobacterium tuberculosis strains from Poland

In the present study, 77 drug-resistant Mycobacterium tuberculosis strains isolated in Poland in 2000 were characterized by the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and our novel method based on PCR amplification of DNA regions between IS6110 and 16-bp GC-rich frequent repeats (designated IS6110-Mtb1/Mtb2 PCR). The results were compared with previous data of the more commonly used methods, IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The discriminatory power of IS6110-Mtb1/Mtb2 method was only slightly lower than that of IS6110 RFLP, whereas MIRU-VNTR typing was the least discriminative among the 4 methods used. Clustering of strains by using results of IS6110-Mtb1/Mtb2 PCR correlated well with RFLP-defined clusters, further confirming epidemiologic relationships among patients. These results indicate that the novel genotyping method could be an attractive alternative for other PCR-based typing procedures, such as spoligotyping and MIRU-VNTR typing. Also, it seems to be a valuable adjunct to the reference IS6110 RFLP method for studying the genetic diversity of drug-resistant M. tuberculosis strains in Poland.     

Diagnosis and treatment of pulmonary diseases caused by Mycobacterium avium complex

Recently, the clinical importance of non-tuberculous mycobacteria(especially, Mycobacterium avium complex [MAC] respiratory infection) has been increasing. In addition, an official ATS/IDSA statement about diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases has been published in February, 2007. In this review article, clinical features and radiological findings of pulmonary MAC diseases mainly i) primarily fibrocavitary disease, and ii) nodular/bronchiectatic disease are described. Primarily fibrocavitary disease is characterized by cavitary lesions in upper lung fields in elderly subjects, smoking patients, or patients with pneumoconiosis. Nodular/bronchiectatic disease is characterized by centrilobular nodules and diffuse bronchiectases in the right middle lobe and the left lingula in middle-aged women. In addition, diagnosis and treatment for pulmonary diseases caused by MAC are also described.     

Nontuberculous mycobacterial infections in cancer patients in a medical center in Taiwan, 2005-2008

Data on the nontuberculous mycobacterial (NTM) species that cause infection and the characteristics of disease caused by these pathogens in cancer patients are limited, so we perform this study to investigate the species distribution of NTM isolates from various clinical specimens and to elucidate the epidemiologic trends in NTM isolates and diseases among cancer patients. From 2005 through 2008, cancer patients with NTM infections as defined by the American Thoracic Society/Infectious Diseases Society of America criteria were identified at the National Taiwan University Hospital. The medical records of all patients were reviewed. During the study period, a total of 219 cancer patients with NTM infections were identified. Among them, 133 (60.7%) patients were older than 65 years, most of whom were men. Lung cancer was the most common type of cancer, followed by hematologic cancer and gastrointestinal tract cancer. Pulmonary NTM infection was the most common type of infection in 205 (93.6%) patients, followed by skin and soft tissue infections (n = 7, 3.2%), disseminated infections (n = 4, 1.8%), and genitourinary tract infection (n = 3, 1.4%). Disseminated infections occurred exclusively in patients with hematologic cancer. Mycobacterium avium complex (MAC) caused the majority of pulmonary NTM infections in cancer patients; in contrast, M. abscessus was the most common causative pathogen of extrapulmonary NTM diseases, followed by MAC. In conclusion, physicians need to be aware of the possibility of co-existing pulmonary NTM infection in patients with lung cancer. In addition, disseminated NTM infection should be considered in patients with hematologic cancer.     

Loop-mediated isothermal amplification (LAMP) in the respiratory specimens for the diagnosis of pediatric pulmonary tuberculosis: A pilot study

BackgroundLoop-mediated isothermal amplification (LAMP) assay is a novel molecular diagnostic technique that can be used for the diagnosis of tuberculosis (TB). It is most suited for developing countries as it is rapid, inexpensive, highly sensitive, requiring minimal infrastructure, training and manpower. Studies in pediatric TB are lacking. We evaluated LAMP in the diagnosis of pediatric pulmonary TB.MethodologyThis was a cross-sectional analytical study conducted at a tertiary care teaching hospital in North India from July 2014 to June 2015 involving 60 children with suspected pulmonary TB. Respiratory specimens (sputum, gastric lavage, bronchoalveolar lavage and/or endotracheal aspirates) were collected and subjected to BACTEC MGIT 960 culture, IS6110 PCR, and LAMP assay targeting IS6110 gene of Mycobacterium tuberculosis.ResultsThirty seven children had confirmed and probable TB according to the composite reference standard (CRS). Among all the 3 tests used for diagnosis of Pulmonary TB, LAMP had highest sensitivity (37.8%) followed by PCR (27%), and culture (21.6%) when compared against the predefined CRS. Culture had maximum specificity of 100%; and PCR, and LAMP had specificity of 95–96%. The sensitivity, specificity, PPV, and NPV of LAMP against culture as reference standard were 75%, 72.4%, 42.9%, and 91.3% respectively. Similarly sensitivity, specificity, PPV, and NPV of PCR against culture as reference standard were 75%, 86.2%, 60%, and 86.2% respectively. On combining LAMP with culture, sensitivity increased to 45.7% (7.8% increase, p = 0.04).ConclusionWe noted that LAMP had highest sensitivity when compared to culture and PCR and comparable specificity.     

实时荧光定量PCR检测血浆结核分枝杆菌DNA的初步临床应用

目的 评价实时荧光定量PCR技术检测血浆（或血清）结核分枝杆菌（Mycobacterium tuberculosis,MTB）DNA的技术。方法 建立实时荧光定期量PCR方法测定血浆MTB DNA,分别检测临床确诊的55例结核病患者血清43份,血浆25份以及非结核肺部疾病患者血清18份,健康体检血清18份和健康体检者血浆11份的MTB DNA含量。结果 18例非结板肺疾病患者血清、18例健康体检者血清以及11例健康体检者血浆MTB DNA全部阴性。55例初诊结核患者中10例（18．2％）治疗前血浆（或血清）MTB DNA阳性。在痰涂片阴性的18例结核患者中,血浆（或血清）MTB DNA阳性检出率为27．8％（5／18）,其特异性达100％。结论 本研究证实了结核患者血浆（或血清）循环MTB DNA的存在,其定量检测对痰涂片阴性及无痰患者的结核病诊断有重要参考价值。     

不同分子生物学方法快速鉴别非结核分枝杆菌的应用评价

目的 评价3种分子生物学方法快速鉴定非结核分枝杆菌的优缺点.方法 收集41株临床分离的非结核分枝杆菌,以16S rRNA基因测序方法为标准,同时以hsp65基因测序方法及PCR-RFLP方法鉴定菌株,与16S rRNA基因测序结果进行比较.结果 41株非结核分枝杆菌16SrRNA基因测序结果:9株龟分枝杆菌复合群,7株偶发分枝杆菌,7株胞内分枝杆菌,3株鸟分枝杆菌,3株堪萨斯分枝杆菌复合群,3株耻垢分枝杆菌,3株土分枝杆菌,2株草分枝杆菌,2株无色分枝杆菌,1株瘰疬分枝杆菌,1株M.arupense.与16S rRNA基因测序相比较,hs65 PCR-RFLP能鉴定9株龟分枝杆菌复合群至亚种脓肿分枝杆菌,3株堪萨斯分枝杆菌复合群鉴定至亚种堪萨斯分枝杆菌;1株偶发分枝杆菌及1株无色分枝杆菌与其不符;其余菌株鉴定结果一致,符合率为95.1%(39/41).hsp65基因测序结果显示,1株爱尔兰分枝杆菌与16S rRNA测序结果不符,其余菌株鉴定结果与其一致,符合率为97.6%(40/41),并且能进一步将9株龟分枝杆菌复合群鉴定至亚种脓肿分枝杆菌,3株堪萨斯分枝杆菌复合群鉴定至亚种堪萨斯分枝杆菌.结论 3种方法均能快速鉴定非结核分枝杆菌.与16S rRNA基因测序相比,hsp65基因测序及hsp65 PCR-RFLP更容易鉴定临床最常见非结核分枝杆菌(如堪萨斯分枝杆菌和脓肿分枝杆菌),可在临床推广使用.     

Molecular identification of nontuberculous mycobacteria isolates in a Brazilian mycobacteria reference laboratory

This study utilized the hsp65 polymerase chain reaction restriction analysis (PRA) method in the identification of nontuberculous mycobacteria (NTMs) isolated in a Brazilian mycobacteria laboratory. NTM isolates from clinical specimens collected from 192 patients were characterized using the hsp65 PRA method and analyzed using both 16S rRNA and hsp65 gene sequencing. Only 30% of the NTM strains were correctly identified through PRA, though the suggested inclusion of an additional restriction enzyme could increase the resolution to roughly 90%. A total of 17 NTM strains were not identified to species level and may represent a new taxonomic entity classified as belonging to the Mycobacterium simiae complex. This study demonstrates the applicability of hsp65 PRA in the identification of several NTM strains in a reference laboratory, though the results suggest that some modifications to the original PRA method could increase its resolution substantially.     

The pathophysiological concept of nontuberculous mycobacterial diseases and pulmonary Mycobacterium avium complex disease

Although nontuberculous mycobacteria (NTM) were initially isolated shortly after Mycobacterium tuberculosis, it has not been confirmed as true human pathogens until the 1930s. Only around 30 NTMs among more than 150 identified can infect to human, and cause pulmonary, lymph node, skin, soft tissue, bone, and disseminated diseases. NTM diseases, especially pulmonary, appear to be increasing all over the world. M. avium complex (MAC) and M. kansasii were the most frequent pulmonary pathogens. There are three forms in MAC respiratory infection; "fibrocavitary", "nodular/bronchiectatic" and "hot tub lung". Fibrocavitary form prefers apical bulla and cavity by pre-existing disease in smoking males. Nodular/bronchiectatic form involves middle lobe or lingula predominantly in non-smoking females. There must be the genetic susceptibility, but details are unknown.     

Blood and urine samples as useful sources for the direct detection of tuberculosis by polymerase chain reaction

The aim of the study was to assess the utility of the polymerase chain reaction (PCR) assay in blood and urine for the diagnosis of tuberculosis (TB). We prospectively evaluated the usefulness of PCR performed in blood and urine samples from patients with proved or probable TB compared with a control group of patients. The PCR technique was performed using IS6110 primers. We included in the study 57 patients (43 with definite TB and 14 with probable TB) and 26 controls. Blood and urine samples were drawn at the time of microbiologic diagnosis and 3, 6, 9, and 12 months later. Cultures were positive in the early period (<1 month after treatment) in 11 of 57 patients (19%) with probable or definite TB, in comparison with 42% of patients (24/57) who yielded a positive PCR (P = 0.02). Urine samples increased the sensitivity of PCR determination in blood samples by 10%. The PCR in blood and/or urine was positive in 41% of patients with pulmonary TB, in 36% of patients with extrapulmonary TB, and in 50% of patients with disseminated TB. Mycobacterium tuberculosis was still detectable by PCR in 5 of 13 patients with cured TB after 1 or more months of antituberculous treatment. The PCR detection of M. tuberculosis in blood and urine samples is useful for the diagnosis of different clinical forms of TB, mostly in those patients in which sample extraction is difficult or requires aggressive techniques. The sensitivity of this technique could be improved studying more than 1 sample in each patient, even after initiating an antituberculous treatment.