Microevolution of Mycobacterium tuberculosis in a Tuberculosis Patient

Five Mycobacterium tuberculosis isolates were obtained from three body sites from a Dutch patient. The isolates displayed a single genotype by 24-locus MIRU-VNTR typing (except for a single locus not amplified from one isolate) but were differentiated by small variations in IS6110 fingerprints, spoligotypes, 6 hypervariable MIRU-VNTR loci, and/or DiversiLab profiles, revealing patterns of microevolution in a clonal infection.     

Two-Year Population-Based Molecular Epidemiological Study of Tuberculosis Transmission in the Metropolitan Area of Milan,Italy

A 2-year, population-based, molecular epidemiological study was conducted in Milan, Italy, to determine the proportion of tuberculosis (TB) cases attributable to recent transmission. All strains were typed by restriction fragment length polymorphism (RFLP) analysis; clustering was considered indicative of recent transmission. Of the 581 cases, 239 (41.1%) belonged to clusters that consisted of 2 to 11 patients; 28.1% were attributable to recent transmission (number of clustered patients minus 1). Clustering was associated with multidrug-resistant Mycobacterium tuberculosis strains (74.2% of cases), AIDS (60.2%), and a history of incarceration (67.4%). The frequency of multidrug-resistant Mycobacterium tuberculosis was 5.3% overall (15.4% among AIDS patients). Among AIDS patients, infection with a resistant strain was independently associated with clustering (odds ratio, 1.32; 95% confidence interval, 1.07–1.163), while among non-AIDS patients, three factors were associated with clustering: history of incarceration (odds ratio, 2.03; 95% confidence interval, 1.41–2.92), age <30 years (odds ratio, 1.43; 95% confidence interval, 1.05–1.94), and native-born Italian nationality (odds ratio, 1.44; 95% confidence interval, 1.08–1.92). Of the 118 patients who belonged to either the smallest or the largest cluster, 19 (16.1%) reported an epidemiological link with another study patient. The results of this study highlight the need for control programs that focus on selected high-risk groups consisting primarily of HIV-infected individuals and persons with social and lifestyle risks for TB. These programs should be aimed at reducing the probability of transmission of drug-resistant TB through early identification of cases and provision of effective treatment until the individual is cured. Electronic Publication     

肺结核合并气管支气管结核危险因素及诊断

气管支气管结核是肺结核的一种特殊类型,其临床表现及影像学特征不典型,容易导致漏诊、误诊,晚期可能出现气道狭窄甚至闭塞、毁损肺等严重并发症。近年来多项研究指出女性、症状持续时间大于4周、合并糖尿病、职业为肺结核合并气管支气管结核常见危险因素,本文通过对相关危险因素进行综合分析,并对气管支气管结核诊断现状进行总结,以期控制结核病流行并为气管支气管结核防治措施提供新思路。     

Evidence from Molecular Fingerprinting of Limited Spread of Drug-Resistant Tuberculosis in Texas

To determine the contribution of recent transmission to spread of drug-resistant tuberculosis in Texas, we performed IS6110-based and pTBN12-based restriction fragment length polymorphism (RFLP) analyses on Mycobacterium tuberculosis isolates. Isolates collected from 201 patients in Texas between 1992 and 1994 were studied. The distribution of cases was strikingly focal. All cases were reported from 35 of the 254 counties in Texas, and 74% (148 of 201) were reported from only 9 counties. One hundred sixty-one (80%) of the patients had M. tuberculosis isolates with unique RFLP patterns, and 41 (20%) patients were in 20 clusters, each comprising 2 to 3 patients. The largest number of cases of drug-resistant tuberculosis were reported in counties bordering Mexico, but the percentage of clustered cases was highest in northeast Texas and in counties that included the cities of Dallas, Fort Worth, and Houston. Compared to nonclustered patients, clustered patients were more likely to be African American and to have been born in the United States. Clustered patients were significantly more likely to be from the same geographic area, and clustered patients from the same geographic area were more likely to have isolates with identical drug susceptibility patterns, suggesting that they were linked by recent transmission. In 11 of 20 clusters, clustered patients were from geographically separate regions, and most isolates did not have identical drug susceptibility patterns, suggesting that tuberculosis was contracted from a common source in the remote past. Based on the low percentage of clustered cases and the small cluster size, we conclude that there is no evidence for the extensive transmission of drug-resistant tuberculosis in Texas.     

Mixed-Strain Mycobacterium tuberculosis Infections and the Implications for Tuberculosis Treatment and Control

Summary: Numerous studies have reported that individuals can simultaneously harbor multiple distinct strains of Mycobacterium tuberculosis. To date, there has been limited discussion of the consequences for the individual or the epidemiological importance of mixed infections. Here, we review studies that documented mixed infections, highlight challenges associated with the detection of mixed infections, and discuss possible implications of mixed infections for the diagnosis and treatment of patients and for the community impact of tuberculosis control strategies. We conclude by highlighting questions that should be resolved in order to improve our understanding of the importance of mixed-strain M. tuberculosis infections.     

Molecular epidemiology of Mycobacterium tuberculosis in Norway

The incidence of tuberculosis in Norway is one of the lowest in the world, and approximately half of the cases occur in first- and second-generation immigrants. In the present study, the genetic diversity of 92% of all strains of Mycobacterium tuberculosis isolated in Norway in 1994 to 1998 was assessed using restriction fragment length polymorphism (RFLP) analysis, with the insertion sequence IS6110 and the repetitive element DR as probes, to determine the degree of active transmission between patients. The DR probe was used as a secondary molecular marker to support or rule out clustering of strains with fewer than five copies of IS6110. After exclusion of 20 cultures representing laboratory contamination, 573 different IS6110 patterns were found among the 698 strains analyzed. Of these 573 patterns, 542 were observed only once and 31 were shared by 2 to 14 isolates. Among 81 strains (11.5%) carrying fewer than five copies of IS6110, 56 RFLP patterns were found when the results of both the IS6110 and DR methods were combined. Among the 698 strains, 570 were considered to be independent cases. A total of 14.5% of the native Norwegians and 19.7% of the foreign patients were part of a cluster. Thus, the degree of recent transmission of tuberculosis in Norway is low and the great majority of the cases are due to reactivation of previous disease. Transmission between immigrants and native Norwegians is uncommon. Two outbreaks, one among native Norwegians and one mainly among immigrants, have been ongoing for several years, indicating that, even in a low-incidence country such as Norway, with a good national program for tuberculosis surveillance, certain transmission chains are difficult to break.     

Direct detection of Mycobacterium tuberculosis complex in respiratory specimens by Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test and Roche Amplicor Mycobacterium Tuberculosis Test.

Two hundred and fifty-six sputum, bronchoalveolar lavage, and bronchial and tracheal aspirate specimens from 243 patients were tested for the presence of Mycobacterium tuberculosis complex by auramine fluorochrome staining, rRNA target amplification (Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test [AMTD]), and PCR (Roche Amplicor Mycobacterium Tuberculosis Test [Amplicor PCR]. The results were compared with those of conventional Löwenstein-Jensen tube culture and BACTEC radiometric liquid culture. A total of 26 specimens from 18 patients were culture positive for M. tuberculosis. In addition, seven specimens were positive by staining and by culture for other Mycobacterium species but negative by nucleic acid amplification methods and were not included in the comparison. When compared with that for culture, the sensitivities of the techniques were as follows: for staining, 80.8%; for Gen-Probe AMTD, 84.6%; and for Roche Amplicor PCR, 84.6%. The specificities were 99.1, 98.7, and 99.1%, respectively. After resolution of discrepant results by review of the patients' clinical data, 29 specimens from 21 patients were considered positive, and the overall sensitivities, specificities, and positive and negative predictive values were 89.7, 100, 100, and 98.7% for culture; 75.9, 99.5, 95.7, and 96.9% for staining; 86.2, 100, 100, and 98.2% for Gen-Probe AMTD; and 82.8, 100, 100, and 97.9% for Roche Amplicor PCR, respectively. It is concluded that both nucleic acid amplification methods are rapid, sensitive, and specific methods for the detection of M. tuberculosis in respiratory specimens.     

Antigens of Mycobacterium tuberculosis Recognized by Antibodies during Incipient, Subclinical Tuberculosis

Serum samples obtained from human immunodeficiency virus (HIV)-infected tuberculosis (TB) patients months prior to clinical TB were used to delineate the profile of Mycobacterium tuberculosis culture filtrate proteins recognized during subclinical TB. A subset of ~12 antigens was recognized by antibodies in these serum samples. Antibodies to two of these antigens (81 [88]-kDa malate synthase [GlcB] and MPT51) were present in serum samples obtained during incipient subclinical TB in 19 (~90%) of the 21 HIV-infected TB patients tested. These antigens will be useful for devising diagnostic tests that can identify HIV-positive individuals who are at a high risk for developing clinical TB.     

Molecular mechanisms of isoniazid resistance in Mycobacterium tuberculosis and Mycobacterium bovis.

Genetic and biochemical studies have suggested a link between reduced catalase activity and resistance to isoniazid in Mycobacterium tuberculosis. In this study, we examined the molecular mechanisms of resistance to isoniazid with six in vitro mutants of the M. tuberculosis complex (Mycobacterium bovis and M. tuberculosis). Five of six mutants resistant to isoniazid were negative by catalase assays. Immunoblot analyses using a polyclonal antibody against the katG gene product (catalase-peroxidase) demonstrated that the enzyme is not produced in four of these isoniazid-resistant strains. A complete deletion of the katG gene was detected in only one of these isoniazid-resistant M. tuberculosis complex strains by Southern blot analyses. In two other resistant strains, partial deletions of the katG gene were identified. A point mutation which resulted in the insertion of a termination codon in the katG coding sequence caused a catalase-negative phenotype in a fourth strain. Of the two resistant strains which produce the enzyme, one was shown to be negative by a catalase assay. Single-stranded conformational polymorphism and DNA sequence analyses identified a mutation in the katG gene of this strain which may contribute to reduced enzymatic activity and subsequent isoniazid resistance. These data demonstrate that genetic alterations to the katG gene other than complete deletions are prevalent and may contribute significantly to the number of cases of isoniazid-resistant tuberculosis.     

Molecular Fingerprinting of Mycobacterium tuberculosis Isolates Obtained in Havana, Cuba, by IS6110 Restriction Fragment Length Polymorphism Analysis and by the Double-Repetitive-Element PCR Method

Mycobacterium tuberculosis sputum isolates from 38 patients, obtained in the first 6 months of 1997 in Havana, Cuba, were characterized by IS6110 restriction fragment length polymorphism (RFLP) analysis and the double-repetitive-element PCR (DRE-PCR) method. Among 41 strains from 38 patients, 24 and 25 unique patterns, and 5 and 4 cluster patterns, were found by the RFLP and DRE-PCR methods, respectively. Patients within two of these clusters were found to be epidemiologically related, while no relation was observed in patients in the other clusters. The DRE-PCR method is rapid, and it was as discriminating as IS6110 RFLP analysis in identifying an epidemiological association. Its simplicity makes the technique accessible for subtyping of M. tuberculosis strains in laboratories not equipped to perform RFLP analysis.     

New Assessment of Bovine Tuberculosis Risk Factors in Belgium Based on Nationwide Molecular Epidemiology

This assessment aimed to elaborate a statistical nationwide model for analyzing the space-time dynamics of bovine tuberculosis in search of potential risk factors that could be used to better target surveillance measures. A database comprising Mycobacterium bovis molecular profiles from all isolates obtained from Belgian outbreaks during the 1995-to-2006 period (n = 415) allowed the identification of a predominant spoligotype (SB0162). Various databases compiling 49 parameters to be tested were queried using a multiple stepwise logistic regression to assess bovine tuberculosis risk factors. Two isolate datasets were analyzed: the first included all Mycobacterium bovis isolates, while the second included only data related to the SB0162 type strain. When all Mycobacterium bovis isolates were included in the model, several risk factors were identified: history of bovine tuberculosis in the herd (P < 0.001), proximity of an outbreak (P < 0.001), cattle density (P < 0.001), and annual amplitude of mean middle-infrared temperature (P < 0.001). The approach restricted to the predominant SB0162 type strain additionally highlighted the proportion of movements from an infected area during the current year as a main risk factor (P = 0.009). This study identified several risk factors for bovine tuberculosis in cattle, highlighted the usefulness of molecular typing in the study of bovine tuberculosis epidemiology, and suggests a difference of behavior for the predominant type strain. It also emphasizes the role of animals'' movements in the transmission of the disease and supports the importance of controlling trade movements.Despite significant historical efforts and the implementation of eradication plans, bovine tuberculosis (bTB) remains a preoccupant issue in the European Union, with some member states recently facing a reemergence of the disease (10). Some countries succeeded in biologically eradicating bTB after implementing control measures, while others, declared officially tuberculosis free (OTF), still report outbreaks every year, despite ongoing eradication and control programs (10). Belgium was declared OTF in 2003, yet 5 to 10 outbreaks are reported every year (12). In 2008, an increase in the number of reported outbreaks was noticed (12), as shown in Fig. ​Fig.11.Open in a separate windowFIG. 1.Trends for numbers of bTB outbreaks (N) in Belgium between 1996 and 2009, as reported to the World Animal Health Organization. The situation worsened in 2008 as the number of outbreaks almost tripled compared to that for the year 2007. (Based on data from reference 12.)Numerous risk factors for bTB have been identified in cattle around the world. These risk factors include a variety of parameters in relation to wildlife, cattle contacts, movements, density of animals, etc. (reviewed in reference 20), but a number of studies lack standardization. Furthermore, bTB transmission cycles underlying the failure to eradicate Mycobacterium bovis in cattle in some areas remain poorly understood, and several transmission hypotheses have been formulated: inadequate control measures, agro-environmental factors, latency, wildlife reservoirs, and movements of infected animals (15). Partly because bTB control programs are an economical burden, national animal health authorities are considering downscaling current control measures, e.g., cancelling testing at purchase and reducing herd testing. Nevertheless, animal movements were shown to be a risk factor in other countries, such as the United Kingdom (15, 16). Before these reductive measures are applied, it therefore seems appropriate to investigate the true risk represented by animal movements in the country.A database including all M. bovis isolates grown from outbreaks reported between 1995 and 2006 in Belgium was compiled. This database was instrumental in analyzing bTB dynamics in Belgium during the 1995-to-2006 period. A full literature review for bTB risk factors allowed the identification of several potential risk factors to be tested in Belgium (20). A statistical model initially developed on the basis of data collected in the United Kingdom (15) was then adapted to the Belgian data set in order to test these potential risk factors.In addition, recent studies focusing on M. bovis strains isolated in cattle and badgers from the United Kingdom confirmed the limited number of strains circulating in the United Kingdom, even though the bTB herd prevalence is elevated (14, 38). On the other hand, the situation in Belgium is totally opposite: there is a wide diversity of cocirculating strains, with one predominating, and the herd prevalence is under 0.1% in the cattle population (12). It was thus decided that two approaches would be followed, one including all strains isolated in the country during the period of interest (1995 to 2006) and one focusing on the predominant strain type, in order to possibly highlight a difference in behavior.This molecular epidemiology approach, never carried out so far in Belgium, is valuable for health authorities in reassessing and adapting current control measures applicable for surveillance of bTB and in challenging possible reductions in herd and individual testing.     

Molecular epidemiology of Mycobacterium tuberculosis infection in Israel

Restriction fragment length polymorphism analysis with IS6110 and DR-r probes was used to study 69 Mycobacterium tuberculosis isolates obtained from Israeli patients and new immigrants from the former Soviet Union and Ethiopia. DNA fingerprinting identified unique patterns for almost all isolates, indicating that most patients were infected with a unique strain imported from their country of origin and that their latent infection was reactivated in Israel.     

Molecular epidemiology of Mycobacterium tuberculosis in western Sweden

The genetic diversity of Mycobacterium tuberculosis isolates among patients from Sweden was determined by a combination of two PCR-based techniques (spoligotyping and variable number of tandem repeats analysis). It resulted in a clustering of 23.6% of the isolates and a rate of recent transmission of 14.1%. The clustered isolates mainly belonged to the Haarlem family (23.2%), followed by the Beijing (9.8%), Latin American and Mediterranean (LAM; 8%), and East African-Indian (EAI; 6.2%) families. A comparison of the spoligotypes with those in the international spoligotyping database showed that 62.5% of the clustered isolates and 36.6% of all isolates typed were grouped into six major shared types. A comparison of the spoligotypes with those in databases for Scandinavian countries showed that 33% of the isolates belonged to an ill-defined T family, followed by the EAI (22%), Haarlem (20%), LAM (11%), Central Asian (5%), X (5%), and Beijing (4%) families. Both the highest number of cases and the proportion of clustered cases were observed in patients ages 15 to 39 years. Nearly 10% of the isolates were resistant to one or more drugs (essentially limited to isoniazid monoresistance). However, none of the strains were multidrug resistant. Data on the geographic origins of the patients showed that more than two-thirds of the clustered patients with tuberculosis were foreign-born individuals or refugees. These results are explained on the basis of both the historical links within specific countries and recently imported cases of tuberculosis into Sweden.     

Evaluation of Strategies for Molecular Fingerprinting for Use in the Routine Work of a Mycobacterium Reference Unit

We have investigated the role of two rapid PCR-based typing methods, IS6110-based PCR and spacer-oligonucleotide typing, within a national tuberculosis reference service. The validity of clusters with IS6110 restriction fragment length polymorphism fingerprints with less than 6 bands was also investigated in the context of referred isolates.     

Evaluation of Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test and PCR for direct detection of Mycobacterium tuberculosis in clinical specimens.

The Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (AMTD) is a direct specimen assay for the identification of Mycobacterium tuberculosis from respiratory samples. rRNA is amplified, and the product is detected with a specific chemiluminescent probe. We performed a retrospective evaluation of three separate respiratory specimens from each of 250 patients by using the AMTD and compared the results with those of microscopy, culturing, and a patient chart review. From the latter results, 198 patients (594 specimens) were found negative for M. tuberculosis by culturing and clinical criteria. The overall specificity of the AMTD after discrepancy resolution was 98.5% (585 of 594). There were 52 patients with culture-proven and/or clinically diagnosed tuberculosis. Of these 156 specimens, the organism was cultured from 142 (91%), and acid-fast microscopy was positive for 105 (67.3%). The AMTD was positive for 142 (91%) specimens from these patients. Tuberculosis patient samples were tested by a PCR assay which uses primers for amplification of the IS6110 insertion sequence of the M. tuberculosis complex. The PCR assay detected 144 of the 156 (92.3%) specimens. Overall, when three specimens per patient were examined, the AMTD found all 52 patients positive for tuberculosis, while the PCR assay found 51 patients positive by agarose gel analysis and all 52 patients positive by Southern blot hybridization.     

Use of Mycobacterium tuberculosis Complex-Specific Antigen Cocktails for a Skin Test Specific for Tuberculosis

The tuberculin skin test currently used to diagnose infection with Mycobacterium tuberculosis has poor diagnostic value, especially in geographic areas where the prevalence of tuberculosis is low or where the environmental burden of saprophytic, nontuberculous mycobacteria is high. Inaccuracy of the tuberculin skin test often reflects a low diagnostic specificity due to the presence in tuberculin of antigens shared by many mycobacterial species. Thus, a skin test specific for tuberculosis requires the development of new tuberculins consisting of antigens specific to M. tuberculosis. We have formulated cocktails of two to eight antigens of M. tuberculosis purified from recombinant Escherichia coli. Multiantigen cocktails were evaluated by skin testing guinea pigs sensitized with M. bovis BCG. Reactivity of multiantigen cocktails was greater than that of any single antigen. Cocktail activity increased with the number of antigens in the cocktail even when the same amount of total protein was used for cocktails and for each single antigen. A cocktail of four purified antigens specific for the M. tuberculosis complex elicited skin test responses only in BCG-immunized guinea pigs, not in control animals immunized with M. avium. These findings open the way to designing a multiantigen formulation for a skin test specific for tuberculosis.     

DNA Fingerprinting of Mycobacterium tuberculosis Complex Culture Isolates Collected in Brazil and Spotted onto Filter Paper

The usefulness of filter paper for preservation of bacterial cells was shown by mixed-linker DNA fingerprint analysis of Mycobacterium tuberculosis isolates from 77 Brazilian patients. DNA fingerprints of samples spotted onto filter paper and conventional culture material were identical. Thus, filter paper specimens analyzed by an amplification-based typing method provide a new resource for epidemiological studies of infectious diseases.     

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis isolates from Xi'an, China

The increased prevalence of isoniazid (INH)-resistant tuberculosis (TB) has become a significant challenge for TB control in the last 10 years. It has been reported that INH resistance is most frequently associated with katG 315 and inhA-15 mutations. The relative significance of these mutations in INH-resistant Mycobacterium tuberculosis and their association with the different genotypes of M. tuberculosis in Xi'an, China, was investigated. Eighty-nine INH-resistant isolates were subjected to molecular characterization of hotspot mutations within katG 315 and inhA-15 by Multiplex allele-specific PCR, and results from 10 isolates were confirmed by direct sequencing. Mutations in katG 315 and inhA-15 were detected in 58 and 7 isolates, respectively. These isolates were genotyped by mycobacterial interspersed repetitive unit-variable number of tandem repeats and Spoligotyping. Spoligotyping showed that 79 isolates (89%) belonged to Beijing family. Mycobacterial interspersed repetitive unit-variable number of tandem repeats typing revealed significant differences in clustering level between Beijing and non-Beijing family (56% vs. 9%, p?=?0.002), which indicated the active transmission of Beijing family in INH-resistant isolates. Our results suggested that the katG 315 mutants were predominant in INH-resistant isolates in Xi'an and the hotspot mutations within katG 315 and inhA-15 can be used as genetic markers for detection of INH resistance.