Microevolution of the direct repeat locus of Mycobacterium tuberculosis in a strain prevalent in San Francisco

We describe a microevolutionary event of a prevalent strain of Mycobacterium tuberculosis that caused two outbreaks in San Francisco. During the second outbreak, a direct variable repeat was lost. We discuss the mechanisms of this change and the implications of analyzing multiple genetic loci in this context.     

Transmission of Mycobacterium orygis (M. tuberculosis Complex Species) from a Tuberculosis Patient to a Dairy Cow in New Zealand

Mycobacterium orygis, previously called the oryx bacillus, is a member of the Mycobacterium tuberculosis complex and has been reported only recently as a cause of human tuberculosis in patients of South Asian origin. We present the first case documenting the transmission of this organism from a human to a cow.     

Microevolution of extensively drug-resistant tuberculosis in Russia

Extensively drug-resistant (XDR) tuberculosis (TB), which is resistant to both first- and second-line antibiotics, is an escalating problem, particularly in the Russian Federation. Molecular fingerprinting of 2348 Mycobacterium tuberculosis isolates collected in Samara Oblast, Russia, revealed that 72% belonged to the Beijing lineage, a genotype associated with enhanced acquisition of drug resistance and increased virulence. Whole-genome sequencing of 34 Samaran isolates, plus 25 isolates representing global M. tuberculosis complex diversity, revealed that Beijing isolates originating in Eastern Europe formed a monophyletic group. Homoplasic polymorphisms within this clade were almost invariably associated with antibiotic resistance, indicating that the evolution of this population is primarily driven by drug therapy. Resistance genotypes showed a strong correlation with drug susceptibility phenotypes. A novel homoplasic mutation in rpoC, found only in isolates carrying a common rpoB rifampicin-resistance mutation, may play a role in fitness compensation. Most multidrug-resistant (MDR) isolates also had mutations in the promoter of a virulence gene, eis, which increase its expression and confer kanamycin resistance. Kanamycin therapy may thus select for mutants with increased virulence, helping preserve bacterial fitness and promoting transmission of drug-resistant TB strains. The East European clade was dominated by two MDR clusters, each disseminated across Samara. Polymorphisms conferring fluoroquinolone resistance were independently acquired multiple times within each cluster, indicating that XDR TB is currently not widely transmitted.     

Extrapulmonary Dissemination of Mycobacterium bovis but Not Mycobacterium tuberculosis in a Bronchoscopic Rabbit Model of Cavitary Tuberculosis

The rabbit model of tuberculosis is attractive because of its pathophysiologic resemblance to the disease in humans. Rabbits are naturally resistant to infection but may manifest cavitary lung lesions. We describe here a novel approach that utilizes presensitization and bronchoscopic inoculation to reliably produce cavities in the rabbit model. With a fixed inoculum of bacilli, we were able to reproducibly generate cavities by using Mycobacterium bovis Ravenel, M. bovis AF2122, M. bovis BCG, M. tuberculosis H37Rv, M. tuberculosis CDC1551, and the M. tuberculosis CDC1551 ΔsigC mutant. M. bovis infections generated cavitary CFU counts of 10⁶ to 10⁹ bacilli, while non-M. bovis species and BCG yielded CFU counts that ranged from 10⁴ to 10⁸ bacilli. Extrapulmonary dissemination was almost exclusively noted among rabbits infected with M. bovis Ravenel and AF2122. Though all of the species yielded secondary lesions at intrapulmonary sites, M. bovis infections led to the most apparent gross pathology. Using the M. tuberculosis icl and dosR gene expression patterns as molecular sentinels, we demonstrated that both the cavity wall and cavity lumen are microenvironments associated with hypoxia, upregulation of the bacterial dormancy program, and the use of host lipids for bacterial catabolism. This unique cavitary model provides a reliable animal model to study cavity pathogenesis and extrapulmonary dissemination.     

Pre-Multidrug-Resistant Mycobacterium tuberculosis Beijing Strain Associated with Disseminated Tuberculosis in a Pet Dog

Resistance to isoniazid, ethambutol, and streptomycin was detected in a Mycobacterium tuberculosis strain, belonging to the Beijing family lineage, isolated from two nodule exudates of a Yorkshire terrier with generalized tuberculosis. This report alerts medical practitioners to the risk of dissemination of pre-multidrug-resistant tuberculosis (preMDR-TB) through exposure to M. tuberculosis-shedding pets.     

Microevolution of the direct repeat region of Mycobacterium tuberculosis: implications for interpretation of spoligotyping data

The direct repeat (DR) region has been determined to be an important chromosomal domain for studying the evolution of Mycobacterium tuberculosis. Despite this, very little is known about microevolutionary events associated with clonal expansion and how such events influence the interpretation of both restriction fragment length polymorphism (RFLP) and spoligotype data. This study examined the structure of the DR region in three independently evolving lineages of M. tuberculosis with a combination of DR-RFLP, spoligotyping, and partial DNA sequencing. The results show that the duplication of direct variable repeat (DVR) sequences and single-nucleotide polymorphisms is rare; conversely, the deletion of DVR sequences and IS6110-mediated mutation is observed frequently. Deletion of either single or contiguous DVR sequences was observed. The deletion of adjacent DVR sequences occurred in a dependent manner rather than as an accumulation of independent events. Insertion of IS6110 into either the direct repeat or spacer sequences influenced the spoligotype pattern, resulting in apparent deletion of DVR sequences. Homologous recombination between adjacent IS6110 elements led to extensive deletion in the DR region, again demonstrating a dependent evolutionary mechanism. Different isolates from the same strain family and isolates from different strain families were observed to converge to the same spoligotype pattern. In conclusion, the binary data of the spoligotype are unable to provide sufficient information to accurately establish genotypic relationships between certain clinical isolates of M. tuberculosis. This has important implications for molecular epidemiologic strain tracking and for the application of spoligotype data to phylogenetic analysis of M. tuberculosis isolates.     

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.     

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.     

Mycobacterium tuberculosis Lineage 7 Strains Are Associated with Prolonged Patient Delay in Seeking Treatment for Pulmonary Tuberculosis in Amhara Region,Ethiopia

Recent genotyping studies of Mycobacterium tuberculosis in Ethiopia have reported the identification of a new phylogenetically distinct M. tuberculosis lineage, lineage 7. We therefore investigated the genetic diversity and association of specific M. tuberculosis lineages with sociodemographic and clinical parameters among pulmonary TB patients in the Amhara Region, Ethiopia. DNA was isolated from M. tuberculosis-positive sputum specimens (n = 240) and analyzed by PCR and 24-locus mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) analysis and spoligotyping. Bioinformatic analysis assigned the M. tuberculosis genotypes to global lineages, and associations between patient characteristics and genotype were evaluated using logistic regression analysis. The study revealed a high diversity of modern and premodern M. tuberculosis lineages, among which approximately 25% were not previously reported. Among the M. tuberculosis strains (n = 138) assigned to seven subgroups, the largest cluster belonged to the lineage Central Asian (CAS) (n = 60; 26.0%), the second largest to lineage 7 (n = 36; 15.6%), and the third largest to the lineage Haarlem (n = 35; 15.2%). Four sublineages were new in the MIRU-VNTRplus database, designated NW-ETH3, NW-ETH1, NW-ETH2, and NW-ETH4, which included 24 (10.4%), 18 (7.8%), 8 (3.5%), and 5 (2.2%) isolates, respectively. Notably, patient delay in seeking treatment was significantly longer among patients infected with lineage 7 strains (Mann-Whitney test, P < 0.008) than in patients infected with CAS strains (adjusted odds ratio [AOR], 4.7; 95% confidence interval [CI], 1.6 to 13.5). Lineage 7 strains also grew more slowly than other M. tuberculosis strains. Cases of Haarlem (OR, 2.8; 95% CI, 1.2 to 6.6) and NW-ETH3 (OR, 2.8; 95% CI, 1.0 to 7.3) infection appeared in defined clusters. Intensified active case finding and contact tracing activities in the study region are needed to expedite diagnosis and treatment of TB.     

肺结核患者痰中结核分枝杆菌检验的临床价值分析

目的探讨分析肺结核患者痰中结核分枝杆菌检验的临床价值遥方法选取该院近期收治的肺结核患者82 例,根据患 病性质将其分为活动组（58 例）与非活动组（24 例）,对比两组患者晨痰痰液中结核分枝杆菌培养阳性率遥结果活动组结核分 枝杆菌培养阳性率（63.79%）较非活动组高（20.83%),两组比对差异存在统计学意义（约0.05）遥结论结核分枝杆菌培养可提升 结核分枝杆菌阳性检测率,对肺结核患者早期检出以及治疗意义重大,值得推广与借鉴遥     

Detection of Mycobacterium tuberculosis Complex in Sputum Specimens by the Automated Roche Cobas Amplicor Mycobacterium Tuberculosis Test

Three hundred twenty-four sputum specimens from 151 patients with suspected active pulmonary tuberculosis were tested for the presence of the Mycobacterium tuberculosis complex with auramine fluorochrome stain and automated PCR assay (Roche Cobas Amplicor Mycobacterium Tuberculosis Test [MTB]). The results were compared with those of the conventional Löwenstein-Jensen tube culture and the BACTEC radiometer liquid culture. A total of 76 specimens from 32 patients were culture positive for M. tuberculosis. In addition, 37 specimens from 15 patients were smear and culture positive for other Mycobacterium species but negative by the present nucleic acid amplification method and thus were not included in the comparison. Compared with culture, the sensitivities, specificities, and positive and negative predictive values for acid-fast smear were 67, 98, 93, and 91% and those for the Cobas Amplicor MTB were 83, 99, 97, and 95%, respectively. When three consecutive sputum specimens per patient could be obtained, the sensitivity of the Cobas Amplicor MTB improved to 91%, whereas the sensitivity of the acid-fast smear remained unchanged.     

Direct detection of Mycobacterium tuberculosis complex in nonrespiratory specimens by Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test.

The Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (AMTDT) was adapted for the detection of Mycobacterium tuberculosis complex in 224 nonrespiratory specimens from 188 patients. The sensitivity and specificity of the AMTDT for such specimens, after resolution of discrepant results, were 85.7 and 100%, respectively. Pretreatment of nonrespiratory specimens with sodium dodecyl (lauryl) sulfate is mandatory to obtain consistent and reproducible AMTDT results. The use of 500 microliters of decontaminated specimen improves the sensitivity of the test. Because the AMTDT detects stable rRNA from noncultivable bacilli, it is not useful for monitoring patients receiving treatment.     

Rapid Diagnosis of Tuberculosis by Real-Time High-Resolution Imaging of Mycobacterium tuberculosis Colonies

Culture remains the cornerstone of diagnosis for pulmonary tuberculosis, but the fastidiousness of Mycobacterium tuberculosis may delay culture-based diagnosis for weeks. We evaluated the performance of real-time high-resolution imaging for the rapid detection of M. tuberculosis colonies growing on a solid medium. A total of 50 clinical specimens, including 42 sputum specimens, 4 stool specimens, 2 bronchoalveolar lavage fluid specimens, and 2 bronchial aspirate fluid specimens were prospectively inoculated into (i) a commercially available Middlebrook broth and evaluated for mycobacterial growth indirectly detected by measuring oxygen consumption (standard protocol) and (ii) a home-made solid medium incubated in an incubator featuring real-time high-resolution imaging of colonies (real-time protocol). Isolates were identified by Ziehl-Neelsen staining and matrix-assisted laser desorption ionization–time of flight mass spectrometry. Use of the standard protocol yielded 14/50 (28%) M. tuberculosis isolates, which is not significantly different from the 13/50 (26%) M. tuberculosis isolates found using the real-time protocol (P = 1.00 by Fisher''s exact test), and the contamination rate of 1/50 (2%) was not significantly different from the contamination rate of 2/50 (4%) using the real-time protocol (P = 1.00). The real-time imaging protocol showed a 4.4-fold reduction in time to detection, 82 ± 54 h versus 360 ± 142 h (P < 0.05). These preliminary data give the proof of concept that real-time high-resolution imaging of M. tuberculosis colonies is a new technology that shortens the time to growth detection and the laboratory diagnosis of pulmonary tuberculosis.     

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.     

Improved Case Detection Using Xpert Mycobacterium tuberculosis/Rifampicin Assay in Skeletal Tuberculosis

Background: In India, musculoskeletal tuberculosis (TB) accounts for 10%–25% of extrapulmonary TB. Data on drug-resistant skeletal TB are lacking. At present, the diagnosis is based mainly on radiological techniques. Laboratory confirmation of skeletal TB is delayed as 6–8 weeks are required for culture results. Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay is a fully automated test which simultaneously detects MTB and RIF resistance within 3 h. Hence, this study was done to compare the yield of case detection using Xpert assay in comparison with culture in specimens received from clinically suspected skeletal TB cases. Methods: Retrospective analysis of microscopy, culture and Xpert assay results was carried out on specimens received in laboratory from skeletal TB cases from January 2016 to December 2017. Results: Of the 201 patients analysed, majority of the specimens were obtained from the spine (55.72%). MTB was detected in 48.68% of tissue and 24% of pus specimens. Xpert assay was detected MTB in 67 (33.33%) specimens of which 53 (47.32%) were from the spine. Culture was detected MTB in 66 (32.83%) specimens. Xpert assay was detected two specimens more than culture. One specimen was positive by only culture. RIF-resistant MTB was detected in 10 (14.92%) specimens by Xpert assay. Conclusion: The spine is the most common site involved. Tissue specimen is better for early diagnosis. High RIF resistance in skeletal TB is an alarming situation. Ability of Xpert MTB/RIF assay for rapid and simultaneous detection of MTB and RIF resistance in comparison with culture makes it a useful diagnostic tool in skeletal TB.     

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.