分枝杆菌散在分布重复单位分型法在分辨北京株结核分枝杆菌基因分型中型中的初步研究

随机按比例抽取武汉市结核病防治所2005年7-12月肺结核病患者的菌株,为评估分枝杆菌散在分布重复单位(MIRU)方法对北京家族株的分辨能力进行分型分析.     

Genotype analysis of Mycobacterium tuberculosis isolates from a sentinel surveillance population

As part of the National Tuberculosis and Genotyping Surveillance Network, isolates obtained from all new cases of tuberculosis occurring in seven geographically separate surveillance sites from 1996 through 2000 were genotyped. A total of 10883 isolates were fingerprinted by the IS6110-restriction fragment length polymorphism method, yielding 6128 distinct patterns. Low-copy isolates (those with six or fewer bands) were also spoligotyped. The distribution of specific genotype clusters was examined. Databases were also examined for families of related genotypes. Analysis of IS6110 patterns showed 497 patterns related to the W-Beijing family; these patterns represent 946 (9%) of all isolates in the study. Six new sets of related fingerprint patterns were also proposed for isolates containing 6-15 copies of IS6110. These fingerprint sets contain up to 251 patterns and 414 isolates; together, they contain 21% of isolates in this copy number range. These sets of fingerprints may represent endemic strains distributed across the United States.     

Mycobacterium tuberculosis Beijing genotype

Molecular epidemiologic studies of strains of Mycobacterium tuberculosis are currently conducted worldwide. The genetically distinct Beijing family of strains has been associated with large outbreaks of tuberculosis, increased virulence, and multidrug resistance. However, in this first population-based search for Beijing strains in the Danish DNA fingerprint database, analysis of 97% of all culture-positive tuberculosis patients in 1992 to 2001 showed that 2.5% of 3,844 patients, 1.0% of Danish-born patients, and 3.6% of immigrants (from 85 countries) had Beijing strains. No Beijing strains were found among 201 strains from Danish-born patients sampled in the 1960s, and no evidence of an increase in Beijing strains was found over time. The true prevalence of Beijing strains worldwide is unknown because only a fraction of global strains have been analyzed.     

MIRU-VNTR和Spoligotyping用于重庆地区210株儿童结核分枝杆菌临床分离株的基因分型

目的 评价间隔区寡核苷酸分型(Spoligotyping)和基于结核分枝杆菌散在分布数目可变串联重复序列分析(MIRU-VNTR)方法在重庆地区儿童结核病分子流行病学中的应用.方法 收集重庆地区210株儿童结核分枝杆菌(MTB)临床分离株,应用上述两种分型方法进行比较分析.结果 采用Spoligotyping分型方法,210株菌可分为2个基因群44种基因型,其中最大的1个基因群即北京家族(北京基因型)含有130株菌(61.90%).采用MIRU-VNTR分析发现24个位点的多态性差异较大,不同MIRU位点组合(12、15和24位点)的分辨率指数依次升高,后两个组合的差异是由位点ETR-B引起.各位点和各位点组合在北京家族菌株中的分辨率指数均高于非北京家族菌株.结论 重庆地区儿童MTB具有明显的基因多态性,其主要流行型为北京家族.在结核病原学监测中,可先采用Spoligotyping,再对成簇菌株进行15位点与ETR-B组合二次分型的联合分型策略,可提高分子流行病学调查效果.

Abstract：

Objective To evaluate the application of spacer oligonucleotide typing (Spoligotyping) and mycobaeterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) analysis in the molecular-epidemiological study of tuberculosis and to discuss the characteristics of pediatric Mycobacterium (M.) tuberculosis strains in Chongqing. Methods M. tuberculosis strains isolated and typed by Spoligotyping and MIRU-VNTR respectively, from the children patients in Chongqing and to compare the results from both methods, epidemiologically. Results By means of Spoligotyping, 210 clinical isolates were divided into 2 gene groups, displaying 44 genotypes. Among them, the biggest group was M. tuberculosis Beijing family, including 130 strains (61.90%) ,using the Spoligotyping. From the results of MIRU-VNTR, 24 loci showed different polymorphism and the HGI of different loci set (12 old loci, 15 basic loci and 24-loci set) increased accordingly. The subtle difference in HGI was originated from one locus ETR-B, which was included in the 24-locus system. The diversity of each loci and MIRU-VNTR set for non-Beijing genotype strains was higher than that of the Beijing genotype strains. Conclusion In this study, it was preliminarily confirmed the existence of high polymorphism of M. tuberculosis while the Beijing Family was the main genotype and main prevalent strain in children of Chongqing area. Spoligotyping prior to 15-locus with ETR-B combination seemed more suitable for the massive epidemiological investigation of pediatric tuberculosis patients.     

Transmission of Mycobacterium tuberculosis in a rural community,Arkansas, 1945-2000

A cluster of tuberculosis cases in a rural community in Arkansas persisted from 1991 to 1999. The cluster had 13 members, 11 linked epidemiologically. Old records identified 24 additional patients for 40 linked cases during a 54-year period. Residents of this neighborhood represent a population at high risk who should be considered for tuberculin testing and treatment for latent tuberculosis infection.     

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.     

Molecular differentiation of Mycobacterium tuberculosis strains without IS6110 insertions

By using standard restriction fragment length polymorphism, 6 zero-copy IS6110 Mycobacterium tuberculosis isolates were identified from 1180 Maryland isolates as part of the National Tuberculosis Genotyping and Surveillance Network Project. By using various genotyping methods, we demonstrated that this zero band cluster can be differentiated into six genotypes.     

Mycobacterium tuberculosis Beijing genotype and risk for treatment failure and relapse, Vietnam

Among 2,901 new smear-positive tuberculosis cases in Ho Chi Minh City, Vietnam, 40 cases of treatment failure and 39 relapsing cases were diagnosed. All initial and follow-up Mycobacterium tuberculosis isolates of these case-patients had (nearly) identical restriction fragment length polymorphism patterns, and the Beijing genotype was a significant risk factor for treatment failure and relapse (odds ratio 2.8, 95% confidence interval 1.5 to 5.2).     

Quality assessment of Mycobacterium tuberculosis genotyping in a large laboratory network

Quality assessment exercises were conducted to evaluate the reproducibility of IS6110 DNA fingerprinting performed by eight laboratories in the National Tuberculosis Genotyping and Surveillance Network. Three panels, each with 8 to 16 isolates, were typed at all laboratories, resulting in 280 images. When the pattern obtained by the majority for each isolate was used as the standard, exact matches were obtained for 73% of patterns; 90% and 97% of patterns matched within one- and two-band differences, respectively. A second approach involved retyping of randomly selected isolates at the Centers for Disease Control and Prevention. Retyping was done for 8-19 isolates per laboratory (76 total). Paired images matched exactly for 54% of isolates and within one and two band differences, 78% and 93%, respectively. We evaluated reasons for mismatching. We also evaluated the reproducibility of spoligotyping using a test panel of 13 isolates; a discrepancy of 1 in 91 results was noted.     

344株结核分枝杆菌的基因分型研究

目的研究间隔区寡核苷酸序列基因分型技术、结核分枝杆菌散在分布重复单位(MIRU),以及多位点串联重复序列(VNTR)在河南省结核病分枝杆菌基因分型中的应用,了解河南省局部地区MIRU-VNTR基因型种类与分布,以及北京家族基因型在河南省的分布特征。方法应用2007年在河南省的嵩县、新密县、扶沟县、中牟县等4个县及南阳市的结核病防治机构实验室分离培养的344株结核分枝杆菌,设计引物,应用PCR和琼脂糖凝胶电泳技术及间隔区寡核苷酸分型技术对结核分枝杆菌进行VNTR分型检测分析,基因分型聚类分析采用Bio-Numerics软件。结果对344株结核分枝杆菌临床分离株的间隔区寡核苷酸分型结果显示,有299株为北京家族基因型,占86.9%;12个MIRU位点检测结果显示明显的基因多态性;经基因聚类分析,可分5个基因群(Ⅰ~Ⅴ群),292个基因型,Ⅰ~Ⅴ群分别占5.1%、67.8%、21.9%、1.4%、2.8%。结论河南省结核分枝杆菌MIRUs基因存在明显的多态性,存在≥5个MIRU型,主要流行型为Ⅱ型;北京家族基因型占主导地位,北京家族基因型与耐药无明显相关性。     

Mycobacterium tuberculosis Beijing genotype, the Netherlands

To determine whether the Beijing genotype of Mycobacterium tuberculosis is emerging in the Netherlands, we collected data on 6,829 patients during 1993 to 2000. Six percent had the Beijing genotype. This genotype was associated with diagnosis in recent years, young age, nationality, and multidrug resistance.     

Human exposure following Mycobacterium tuberculosis infection of multiple animal species in a Metropolitan Zoo

From 1997 to 2000, Mycobacterium tuberculosis was diagnosed in two Asian elephants (Elephas maximus), three Rocky Mountain goats (Oreamnos americanus), and one black rhinoceros (Diceros bicornis) in the Los Angeles Zoo. DNA fingerprint patterns suggested recent transmission. An investigation found no active cases of tuberculosis in humans; however, tuberculin skin-test conversions in humans were associated with training elephants and attending an elephant necropsy.     

Diagnosis of pulmonary tuberculosis in a microbiological laboratory

The positive diagnosis of pulmonary tuberculosis still relies on the direct detection of Mycobacterium tuberculosis complex strains after isolation, culture, and identification, or on the detection of specific DNA sequences. Clinical specimens for the direct diagnosis include respiratory tract and stool specimens, which can all be collected with a “tuberculosis kit” to simplify and standardize clinical and laboratory tasks. Gastric juice and other specimens are no longer useful. The association of new decontamination technics, new liquid and solid Middlebrook blood-enriched culture media allow for the isolation of tuberculous mycobacteria in 10 days. The microscopic-observation drug susceptibility and thin layer agar assays allow for rapid detection of M. tuberculosis including drug-resistant tuberculosis. “Closed molecular tests” adapted to “point-of-care” use allow for the detection of tuberculous mycobacteria in Ziehl-positive samples and the detection of rifampicin resistance in three hours. Rapid identification can be achieved with protein spectrum analysis by mass spectrometry. Finally, genotyping by various technics of profile analysis (spoligotyping, VNTR-MIRU) or by sequence analysis (multispacer sequence typing) allows for the identification of isolates among the six Mycobacterium tuberculosis complex species, and for the detection of laboratory cross-contaminations. It also allows diagnosing cross-transmission between patients including nosocomial cases, discriminating between reactivation and new infection, and determining the geographical origin of strains (geotyping). The ongoing search for new culture media is a priority to improve the speed of direct diagnosis of pulmonary tuberculosis.     

Use of DNA fingerprinting to investigate a multiyear,multistate tuberculosis outbreak

In 1998-1999, the Baltimore TB control program detected a cluster of 21 tuberculosis (TB) cases. Patients reported frequent travel to various East Coast cities. An investigation was conducted to determine whether transmission of the same Mycobacterium tuberculosis strain was occurring in these other localities. A collaborative investigation among federal, state, and local TB controllers included TB record reviews, interviews of patients, and restriction fragment length polymorphism (RFLP) analysis of selected M. tuberculosis isolates from diagnosed TB patients in several cities in 1996-2001. A national TB genotyping database was searched for RFLP patterns that matched the outbreak pattern. Eighteen additional outbreak-related cases were detected outside of Baltimore-the earliest diagnosed in New Jersey in 1996, and the most recent in New York City in late 2001. The outbreak demonstrates the need for strategies to detect links among patients diagnosed with TB across multiple TB control jurisdictions.     

Predominant tuberculosis spoligotypes, Delhi, India

One hundred five Mycobacterium tuberculosis clinical isolates from the Delhi area were typed by spoligotyping; 45 patterns were identified. Comparison with an international spoligotype database showed type 26, Delhi type (22%), type 54 (12%), and type 1, Beijing type (8%), as the most common. Eighteen spoligotypes did not match any existing database pattern.     

Mycobacterium tuberculosis infection of domesticated Asian elephants, Thailand

Four Asian elephants were confirmed to be infected with Mycobacterium tuberculosis by bacterial culture, other diagnostic procedures, and sequencing of 16S-23S rDNA internal transcribed spacer region, 16S rRNA, and gyrase B gene sequences. Genotyping showed that the infectious agents originated from 4 sources in Thailand. To identify infections, a combination of diagnostic assays is essential.     

Mycobacterium tuberculosis transmission between cluster members with similar fingerprint patterns

Molecular epidemiologic studies provide evidence of transmission of Mycobacterium tuberculosis within clusters of patients whose isolates share identical IS6110-DNA fingerprint patterns. However, M. tuberculosis transmission among patients whose isolates have similar but not identical DNA fingerprint patterns (i.e., differing by a single band) has not been well documented. We used DNA fingerprinting, combined with conventional epidemiology, to show unsuspected patterns of tuberculosis transmission associated with three public bars in the same city. Among clustered TB cases, DNA fingerprinting analysis of isolates with similar and identical fingerprints helped us discover epidemiologic links missed during routine tuberculosis contact investigations.     

DNA fingerprinting of Mycobacterium tuberculosis isolates from epidemiologically linked case pairs

DNA fingerprinting was used to evaluate epidemiologically linked case pairs found during routine tuberculosis (TB) contact investigations in seven sentinel sites from 1996 to 2000. Transmission was confirmed when the DNA fingerprints of source and secondary cases matched. Of 538 case pairs identified, 156 (29%) did not have matching fingerprints. Case pairs from the same household were no more likely to have confirmed transmission than those linked elsewhere. Case pairs with unconfirmed transmission were more likely to include a smear-negative source case (odds ratio [OR] 2.0) or a foreign-born secondary case (OR 3.4) and less likely to include a secondary case <15 years old (OR 0.3). Our study suggests that contact investigations should focus not only on the household but also on all settings frequented by an index case. Foreign-born persons with TB may have been infected previously in high-prevalence countries; screening and preventive measures recommended by the Institute of Medicine could prevent TB reactivation in these cases.