Evaluation of an amplified Mycobacterium tuberculosis direct test in clinical specimens.

SETTING: An increase in tuberculosis cases has been observed since the last decade, and rapid and specific tests are needed to directly detect Mycobacterium tuberculosis in respiratory specimens. The Amplified Mycobacterium Tuberculosis Direct Test (AMTDT) is a direct specimen assay for the identification of M. tuberculosis. OBJECTIVE: To evaluate the sensitivity and specificity of the AMTDT. METHODS: We performed the test in 426 patients: 58 patients who had culture-proven and/or clinically diagnosed tuberculosis, and 368 patients who were negative for M. tuberculosis by culture and clinical criteria (the control group). The results were compared to culture and clinical diagnosis of tuberculosis. RESULTS: AMTDT was found to be positive in 35 of the 58 patients who had culture-proven and/or clinically diagnosed tuberculosis and 71 of the 368 controls. Seventeen of the tuberculosis group were smear-negative, and AMTDT was positive in 11 of these patients. The sensitivity and specificity of the test were respectively 60% and 80%. CONCLUSION: AMTDT can be used as a complementary technique in tuberculosis patients in the presence of high clinical suspicion.     

四种结核分枝杆菌检测方法临床诊断效能的评价

目的 评估痰涂片抗酸染色镜检(简称“涂片法”)、L-J培养基(Lowenstein-Jenden medium)固体培养(简称“L-J法”)、BACTEC MGIT 960液体培养(简称“MGIT 960法”)及GeneXpert MTB/RIF(简称“GeneXpert法”)等4种结核分枝杆菌检测方法检出结核病病原菌的能力。方法 搜集2018年1月1日至2018年12月31日北京结核病控制研究所451例疑似肺结核患者的痰标本,同时使用涂片法、L-J法、MGIT 960法及GeneXpert法进行检测。对上述4种方法的检测结果以临床诊断情况(总体分为“肺结核患者”与“非肺结核患者”)为参考标准,计算各种检测方法的敏感度、特异度、阳性预测值、阴性预测值、一致率;同时计算4种方法联合检测结果的病原学阳性率(其中任何一种方法检测结果为阳性,即判断为联合检测结果为阳性)。结果 451例疑似肺结核患者中,临床最终诊断依据《WS 288—2017肺结核诊断》标准进行,诊断为肺结核患者227例(肺结核225例、肺结核并发结核性胸膜炎2例),非肺结核患者224例[非结核分枝杆菌(NTM)肺病11例,陈旧性肺结核6例,肺部阴影待查181例,胸腔积液3例,肺纤维化1例,肺炎3例,肺癌1例,密切接触者筛查18例]。以临床诊断结果为参考标准,涂片法、L-J法、GeneXpert法、MGIT 960法检测敏感度分别为22.5%(51/227)、32.2%(73/227)、37.9%(86/227)、43.2%(98/227),特异度分别为96.0%(215/224)、96.4%(216/224)、100.0%(224/224)、95.1%(213/224),一致率分别为59.0%(266/451)、64.1%(289/451)、68.7%(310/451)、69.0%(311/451)。涂片法和L-J法联合检测,以及涂片法、L-J法和GeneXpert法联合检测,4种方法联合检测的敏感度分别为34.8%(79/227)、43.6%(99/227)、49.8%(113/227),特异度分别为96.0%(215/224)、96.0%(215/224)、93.8%(210/224),一致率分别为65.2%(294/451)、69.6%(314/451)、71.6%(323/451)。结论 以临床诊断为参考标准,MGIT 960法检测的敏感度最高;GeneXpert法检测的特异度最高;4种方法联合检测能够显著提高诊断敏感度,与临床诊断的一致率也显著提高。     

Evaluation of urinalysis parameters to predict urinary-tract infection.

We evaluated the performance of automated-flow cytometry, urinalysis dipsticks and microscopic urine sediment analysis as predictors of urinary tract infection. Urine cultures were used as a reference method for comparison. Six-hundred-seventy-five urine samples from hospitalized and not hospitalized patients attended at Hospital M?e de Deus, Porto Alegre, in south Brazil, were included in the study. Among the individual measures analyzed, intense bacteriuria in the microscopic analysis of urinary sediment gave an accuracy of 92.9%. A combination between intense bacteriuria (microscopic analysis) and >20 leukocytes per microL of urine (flow cytometry) gave a higher accuracy (97.3%). We conclude that though it is laborious, microscopic urinalysis is a good analytical tool. Taken together with flow cytometry and dipsticks, we obtained a clinically-acceptable prediction of urinary-tract infection.     

结核分枝杆菌临床株对利福霉素类药物的耐药特点分析

目的比较分析临床标本结核分枝杆菌(Mycobacterium tuberculosis,Mtb)对利福霉素类药物的耐药特点,为含利福霉素类药物的抗结核治疗方案的改进提供细菌学基础。方法用绝对浓度法检测470份临床标本中Mtb的耐药情况。结果 180份培养阳性的标本中,41份对利福霉素类耐药,对利福平和利福喷汀的耐药水平高度一致,对二者的耐药水平显著高于对利福布汀的耐药水平,对低浓度和高浓度利福布汀耐药的数量差异显著。结论临床标本中的Mtb对利福平和利福喷汀的耐药水平表现出高度一致性,提示利福喷汀不适于作为利福平耐药的后备药物,利福布汀可以作为利福平的后备药物,但服用剂量可能影响治疗效果。     

gidB mutations in streptomycin-resistant Mycobacterium tuberculosis clinical

目的　探索gidB基因突变在结核分枝杆菌链霉素耐药中的作用。方法 结核分枝杆菌临床分离株中,60株为链霉素耐药,30株敏感。同时进行链霉素MIC测定并采用DNA测序法分析gidB,rpsL和rrs基因突变。结果 20%的耐药株含有gidB单基因突变,80%的耐药株除gidB基因突变以外,还有rpsL或rrs基因突变。敏感株中也有gidB基因突变。gidB突变呈散在分布,未发现突变热点。结论 gidB基因突变在结核分枝杆菌链霉素耐药中的作用尚不能肯定。     

Cross-contamination of Mycobacterium tuberculosis culture in clinical laboratories

For many years, it has been thought that positive culture of M. tuberculosis is a definitive diagnostic evidence of tuberculosis and cross-contamination of M. tuberculosis culture in clinical laboratories is rare. However recently introduced RFLP analysis has enabled us to identify a strain of M. tuberculosis, and many cases of the cross-contamination in clinical laboratories confirmed by RFLP analysis have been reported. In this report, we present the first case of the cross-contamination confirmed by RFLP in Japan. In our case, 5 patients without any personal link to each other were suspected based on clinical findings to have cross-contaminated results of M. tuberculosis culture. All their specimens were processed on the same day, and were smear negative and culture positive with only a small number of colonies (less than 8 colonies). The sputum from the suspected source of contamination processed on the same day was strongly positive for AFB smear and heavily culture positive. The RFLP patterns of these 6 patients were identical, so it was concluded that the positive cultures of the sputum from the 5 patients who were not expected to be culture positive on clinical findings were caused by the cross-contamination in our hospital laboratory. We review all the charts of patients with M. tuberculosis culture positive results in the same year of this case, but we didn't find no other cases suspected of the cross-contamination. Then we reviewed the literature of M. tuberculosis culture cross-contamination. The patterns of the cross-contamination are divided into two. One is associated with malfunction of a sampling needle in the BACTEC 460 system and the other associated with the initial processing of the specimens, mostly involving reagents such as NaOH solution. Cross-contaminated specimens are usually smear negative with only a few colonies (less than 5), and processed just after the source specimen of the contamination in most reported cases, but not in all. In almost half of them the cross-contamination results had significant influence on the clinical management. The frequency of the cross-contamination is estimated around 1% of the patients with M. tuberculosis culture positive results. For early detection of the cross-contamination, not only clinicians but also laboratory staffs have important role and close cooperation between them is mandatory. To prevent the contamination, it is advisable to process smear positive and probable culture positive specimens separately from others, and not to use a large same container of reagents for processing of different specimens.     

链霉素耐药结核分枝杆菌临床分离株gidB基因突变研究

目的 探索gidB基因突变在结核分枝杆菌链霉素耐药中的作用.方法 结核分枝杆菌临床分离株中,60株为链霉素耐药,30株敏感.同时进行链霉素MIC测定并采用DNA测序法分析gidB,rpsL和rrs基因突变.结果 20%的耐药株含有gidB单基因突变,80%的耐药株除gidB基因突变以外,还有rpsL或rrs基因突变.敏感株中也有gidB基因突变.gidB突变呈散在分布,未发现突变热点.结论 gidB基因突变在结核分枝杆菌链霉素耐药中的作用尚不能肯定.     

Evaluation of biochemical and molecular polymorphism in extended spectrum β-lactamases of Mycobacterium tuberculosis clinical isolates

Background

Tuberculosis (TB) caused 1.8 million deaths worldwide with increased multiple drug resistance (MDR) cases estimated 4.8 lakhs in the year 2015. β-Lactam antibiotics could be a hope for TB treatment. Therefore, in this study, uniformity in the biochemical and molecular nature of β-lactamases was analyzed to evaluate the potential of β-lactam antibiotics as a treatment regimen against Mycobacterium tuberculosis (MTB).

689例住院老年结核的临床研究

目的通过对老年肺结核患者的临床症状、胸片表现、痰检及PPD皮试等、治疗进行分析,探讨老年肺结核的发病特点。方法对2000~2004年临床收治的60岁及以上的689例老年肺结核患者的临床资料进行分析。结果①临床症状不典型:除69例患者无任何表现,体检时被发现诊断为肺结核外,多以咳嗽(78.2%)、咳痰(59.5%)、胸闷、气喘(49.1%)、咯血(38.6%)等呼吸道表现为症状。结核中毒症状除发热(48.2%)外,而盗汗、乏力等症状不甚明显。②合并症多:伴有一种或多种合并症的为576人,占86.3%。③病程迁延漫长,最长35年。④涂阳率高,占47.0%。⑤不正规抗痨治疗者多。结论全社会要加强对老年肺结核的研究,增强对老年肺结核特点的认识,提高其早期诊断和防治效果。     

Development of sandwich-form biosensor to detect Mycobacterium tuberculosis complex in clinical sputum specimens

Mycobacterium tuberculosis, the causing agent of tuberculosis, comes second only after HIV on the list of infectious agents slaughtering many worldwide. Due to the limitations behind the conventional detection methods, it is therefore critical to develop new sensitive sensing systems capable of quick detection of the infectious agent. In the present study, the surface modified cadmium-telluride quantum dots and gold nanoparticles conjunct with two specific oligonucleotides against early secretory antigenic target 6 were used to develop a sandwich-form fluorescence resonance energy transfer-based biosensor to detect M. tuberculosis complex and differentiate M. tuberculosis and M. bovis Bacille Calmette–Guerin simultaneously. The sensitivity and specificity of the newly developed biosensor were 94.2% and 86.6%, respectively, while the sensitivity and specificity of polymerase chain reaction and nested polymerase chain reaction were considerably lower, 74.2%, 73.3% and 82.8%, 80%, respectively. The detection limits of the sandwich-form fluorescence resonance energy transfer-based biosensor were far lower (10 fg) than those of the polymerase chain reaction and nested polymerase chain reaction (100 fg). Although the cost of the developed nanobiosensor was slightly higher than those of the polymerase chain reaction-based techniques, its unique advantages in terms of turnaround time, higher sensitivity and specificity, as well as a 10-fold lower detection limit would clearly recommend this test as a more appropriate and cost-effective tool for large scale operations.     

Genetic diversity in clinical Mycobacterium tuberculosis isolates from Punjab

SETTING: Two hospitals, Sri Guru Ram Das Institute of Medical Sciences and Research and the TB and Chest Hospital, Amritsar, Punjab. OBJECTIVE: To explore genetic diversity among the clinical Mycobacterium tuberculosis isolates prevalent in Punjab. DESIGN: Fifty-six random clinical isolates of M. tuberculosis were cultured from the sputum specimens of pulmonary tuberculosis patients. DNA was extracted from cultured biomass and analysed using the mycobacterial interspersed repetitive units (MIRU) typing method. RESULTS: MIRU typing of 51 isolates revealed 45 different patterns, with a combined Hunter-Gaston discriminatory index (HGDI) of 0.990. Five clinical isolates failed to amplify for one or more MIRUs and were excluded from the analysis. The remaining isolates were categorised in three groups based on the allelic heterogeneity of individual MIRUs. MIRU 10, 16, 26 and 31 were highly discriminant, with an HGDI value >0.6; MIRU 4, 23, 24, 39 and 40 were designated as moderately discriminant (HGDI value 0.6-0.3) and MIRU 2, 20 and 27 were poorly discriminant (HGDI value <0.3). CONCLUSION: MIRU typing and the HGDI values revealed that M. tuberculosis strains from Punjab are genetically quite heterogeneous.     

pncA mutations in clinical Mycobacterium tuberculosis isolates from Korea

Background  

Pyrazinamide (PZA) is among the first-line drugs for the treatment of tuberculosis. In vitro, it kills semidormant mycobacteria only at low pH. The purpose of this study was to compare PZA resistance with pyrazinamidase (PZase) activity and the genotype to better understand the molecular basis of PZA resistance and to expand the profile of pncA mutations worldwide.     

5种结核分枝杆菌特异蛋白质的抗原性分析

目的评价14、16、38kD、mtb81和 ESAT-6等5种结核分枝杆菌特异蛋白质的抗原性。方法利用14、16、38kD、mtb81和 ESAT-6等5种重组蛋白建立ELISA方法,检测120份结核病人、30份非结核呼吸道感染病人和30份健康人血清中相应抗体。结果ELISA显示5种结核杆菌特异性蛋白质的特异性均高于95%;敏感性和准确性则不尽相同,其中38kD的检测敏感性最高,为80.8%;14、16kD、mtb81和 ESAT-6的检测敏感性分别为52.5%、68.3%、35.8%和44.2%。结论5种结核杆菌特异性蛋白质均具有不同程度的抗原性。进一步提高抗原或抗体的检测灵敏度将有利于结核病的诊断。     

82例初治痰涂片阳性老年肺结核患者的近期临床观察

目的 探讨初治痰涂片阳性老年肺结核患者的疗效、对抗结核药物的耐受性及左氧氟沙星的有效性和安全性。方法 将82例初治痰涂片阳性老年肺结核患者按4：3分为治疗组和对照组进行6个月的临床观察（强化期2个月，巩固期4个月）。治疗组选用可乐必妥，按2HLEV／4HLE方案给药；对照组用吡嗪酰胺，按2HLEZ／4HLE给药。结果 治疗组和对照组2个月痰菌阴转率分别为72．7％和63．3％（P＞0．05），治疗6个月痰菌阴转率分别为88．6％和83．3％（P＞0．05），胸部X线病灶吸收分别为86．4％和86．7％（P＞0．05），空洞闭合率分别为30．0％和24．1％（P＞0．05），药物副反应发生率分别为25．5％和51．4％（P＜0．05）。结论 老年人肺结核治愈率较低，药物副反应发生率高，左氧氟沙星对老年人肺结核安全有效。     

Validity of clinical prediction rules for isolating inpatients with suspected tuberculosis

OBJECTIVE: Declining rates of tuberculosis (TB) in the United States has resulted in a low prevalence of the disease among patients placed on respiratory isolation. The purpose of this study is to systematically review decision rules to predict the patient's risk for active pulmonary TB at the time of admission to the hospital. DATA SOURCES: We searched MEDLINE (1975 to 2003) supplemented by reference tracking. We included studies that reported the sensitivity and specificity of clinical variables for predicting pulmonary TB, used Mycobacterium TB culture as the reference standard, and included at least 50 patients. REVIEW METHOD: Two reviewers independently assessed study quality and abstracted data regarding the sensitivity and specificity of the prediction rules. RESULTS: Nine studies met inclusion criteria. These studies included 2,194 participants. Most studies found that the presence of TB risk factors, chronic symptoms, positive tuberculin skin test (TST), fever, and upper lobe abnormalities on chest radiograph were associated with TB. Positive TST and a chest radiograph consistent with TB were the predictors showing the strongest association with TB (odds ratio: 5.7 to 13.2 and 2.9 to 31.7, respectively). The sensitivity of the prediction rules for identifying patients with active pulmonary TB varied from 81% to 100%; specificity ranged from 19% to 84%. CONCLUSIONS: Our analysis suggests that clinicians can use prediction rules to identify patients with very low risk of infection among those suspected for TB on admission to the hospital, and thus reduce isolation of patients without TB.     

基因芯片技术检测结核分枝杆菌利福平和异烟肼耐药性临床应用评价

目的评价基因芯片技术检测结核分枝杆菌利福平和异烟肼耐药性的临床应用效果。方法利用基因芯片技术对涂阳肺结核患者的痰标本和临床分离株进行利福平和异烟肼耐药基因位点检测,比较痰标本与菌株检测结果的差异;并以BACTEC MGIT 960药敏试验结果为对照评价基因芯片的检测性能。另外,通过对999株鉴定为结核分枝杆菌的临床分离菌株进行rpoB、katG和inhA基因耐药相关区域扩增产物测序,以验证基因芯片对核酸序列检测的准确性。结果在涂阳的1 108例标本中有100例经基因芯片菌种鉴定为非结核分枝杆菌。其余的1 008例痰标本基因芯片结果与BACTEC MGIT960培养阳性的菌株基因芯片结果比较仅有9例结果不一致,符合率为99.1%。以BACTEC MGIT960培养法药敏结果为对照,999株菌株基因芯片法检测利福平耐药性的符合率为98.1%,异烟肼的耐药性的符合率为94.5%。与DNA测序法相比,基因芯片法检测利福平和异烟肼耐药性的准确率分别为99.6%和99.8%。结论基因芯片技术能够快速、准确地进行结核分枝杆菌利福平和异烟肼的耐药性检测,并能直接用于痰标本检测。     

rRNA扩增方法对结核分枝杆菌临床诊断作用的评估

目的 评价rRNA扩增方法 在临床应用的效果。 方法 选取到北京胸科医院、山东省胸科医院、河南疾控中心结核病防治所3个机构就诊的肺结核可疑症状者及健康志愿者的痰标本为研究对象,共纳入551例痰标本,对每例痰标本均进行涂片显微镜检查、罗氏培养、rRNA扩增试验以及实时荧光定量PCR。与罗氏培养方法 比较分析rRNA扩增方法 的敏感性、特异性、阳性预测值和阴性预测值以及与实时荧光定量PCR扩增方法 的一致性。 结果rRNA扩增方法 的敏感性为98.5%,特异性为95.0%,阳性预测值为95.0%,阴性预测值为98.5%,rRNA扩增方法 在涂阳标本和涂阴标本间的敏感度和特异度差异均有统计学意义（χ²=9.60,P=0.002;χ²=79.80,P<0.01）。与PCR检测结果的一致性为93.8%,涂阳和涂阴标本中2种方法 的一致性差异有统计学意义（χ²=4.45,P=0.035）。 结论 rRNA扩增方法 的敏感度和特异度均较好,且能明显缩短诊断时间,该方法 是一种较有前景的结核病实验室诊断方法 。     

Molecular genetic approaches to Mycobacterium tuberculosis

Recent progress of molecular genetics has been providing tools for new approaches to disease treatment and diagnosis of Mycobacterium tuberculosis. In 1998, Cole et al. reported the complete genome sequence of Mycobacterium tuberculosis. The new information will provide us the knowledge and understanding of the biology of Mycobacterium tuberculosis. Further, it will provide us new conception of diagnosis and treatment of the disease. Four topics were selected in this symposium. Dr. Iinuma reviewed and prospected the clinical utility of nucleic acid amplification methods of Mycobacterium tuberculosis. Dr. Suzuki reviewed the molecular mechanism of acquired resistance to anti-TB drugs and reported the early detection of genetic mutation by new designed DNA tip method. Dr. Takahashi reviewed the method of molecular epidemiology and genetic elements as a tool for strain differentiation of tuberculosis. Dr. Mizuguchi interpreted the essential feature of mycobacterial genome maps, and genes and their biological activity. He also reviewed the importance and the utility of the complete genome sequence of tuberculosis in association with pathogenecity. These topics were summarized in this report, based on the symposium of "Molecular genetic approaches to Mycobacterium tuberculosis" in the 75th annual meeting of the Japanese Society for Tuberculosis.