Tuberculosis (TB) remains a major international health problem. Rapid differentiation of Mycobacterium tuberculosis complex (MTB) from non-tuberculous mycobacteria (NTM) is critical for decisions regarding patient management and choice of therapeutic regimen. Recently we developed a 20-compound model to distinguish between MTB and NTM. It is based on thermally assisted hydrolysis and methylation gas chromatography-mass spectrometry and partial least square discriminant analysis. Here we report the validation of this model with two independent sample sets, one consisting of 39 MTB and 17 NTM isolates from the Netherlands, the other comprising 103 isolates (91 MTB and 12 NTM) from Stellenbosch, Cape Town, South Africa. All the MTB strains in the 56 Dutch samples were correctly identified and the model had a sensitivity of 100% and a specificity of 94%. For the South African samples the model had a sensitivity of 88% and specificity of 100%. Based on our model, we have developed a new decision-tree that allows the differentiation of MTB from NTM with 100% accuracy. Encouraged by these findings we will proceed with the development of a simple, rapid, affordable, high-throughput test to identify MTB directly in sputum.
Mycobacterium vaccae is a rapidly growing, nontuberculous Mycobacterium species that is generally not considered a human pathogen and is of major pharmaceutical interest as an immunotherapeutic agent. We report here the annotated genome sequence of the M. vaccae type strain, ATCC 25954.
Despite great effort by health organizations worldwide in fighting tuberculosis (TB), morbidity and mortality are not declining as expected. One of the reasons is related to the evolutionary development of Mycobacterium tuberculosis, in particular the Beijing genotype strains. In a previous study, we showed the association between the Beijing genotype and an increased mutation frequency for rifampin resistance. In this study, we use a Beijing genotype strain and an East-African/Indian genotype strain to investigate with our mouse TB model whether the higher mutation frequency observed in a Beijing genotype strain is associated with treatment failure particularly during noncompliance therapy. Both genotype strains showed high virulence in comparison to that of M. tuberculosis strain H37Rv, resulting in a highly progressive infection with a rapid lethal outcome in untreated mice. Compliance treatment was effective without relapse of TB irrespective of the infecting strain, showing similar decreases in the mycobacterial load in infected organs and similar histopathological changes. Noncompliance treatment, simulated by a reduced duration and dosing frequency, resulted in a relapse of infection. Relapse rates were correlated with the level of noncompliance and were identical for Beijing infection and East African/Indian infection. However, only in Beijing-infected mice, isoniazid-resistant mutants were selected at the highest level of noncompliance. This is in line with the substantial selection of isoniazid-resistant mutants in vitro in a wide isoniazid concentration window observed for the Beijing strain and not for the EAI strain. These results suggest that genotype diversity of M. tuberculosis may be involved in emergence of resistance and indicates that genotype-tailor-made treatment should be investigated.
tuberculosis and other mycobacteria; oryx bacillus; Mycobacterium orygis
Mycobacterium tuberculosis; drug resistance; mutation frequency; rifampin; tuberculosis and other mycobacteria; Beijing type
Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). The accurate identification of the MTC member causing human infection is important because the treatment of TB caused by some MTC members requires an alteration of the standard drug regimen, it can inform whether transmission is human to human or zoonotic, and it enables accurate epidemiology studies that help improve TB control. In this study, an internally controlled two-stage multiplex real-time PCR-based method, SeekTB, was developed for the accurate identification of all members of the MTC. The method was tested against a panel of well-characterized bacterial strains (n = 180) and determined to be 100% specific for members of the MTC. Additionally, 125 Mycobacteria Growth Indicator Tube (MGIT)-positive cultures were blindly tested by using SeekTB, and the results were compared to those of the GenoType MTBC and TBc ID tests. The SeekTB and GenoType MTBC results were 100% concordant, identifying 84 of these isolates as M. tuberculosis isolates and 41 as non-MTC isolates. Nine discordant results between the molecular methods and the TBc ID culture confirmation test were observed; however, nucleotide sequencing confirmed the results obtained with GenoType MTBC and SeekTB. SeekTB is the first-described internally controlled multiplex real-time PCR diagnostic method for the accurate identification of all eight members of the MTC. This method, designed for use on cultured patient samples, is specific, sensitive, and rapid, with a turnaround time to results of approximately 1.5 to 3.5 h, depending on which, if any, member of the MTC is present.
Mycobacterium xenopi is a slow-growing, thermophilic, water-related Mycobacterium species. Like other nontuberculous mycobacteria, M. xenopi more commonly infects humans with altered immune function, such as chronic obstructive pulmonary disease patients. It is considered clinically relevant in a significant proportion of the patients from whom it is isolated. We report here the whole genome sequence of M. xenopi type strain RIVM700367.
Mycobacterium phlei is a rapidly growing nontuberculous Mycobacterium species that is typically nonpathogenic, with few reported cases of human disease. Here we report the whole genome sequence of M. phlei type strain RIVM601174.
Tuberculosis patients may be infected with or have disease caused by more than one Mycobacterium tuberculosis strain, usually referred to as “mixed infections.” These have mainly been observed in settings with a very high tuberculosis incidence and/or high HIV prevalence. We assessed the rate of mixed infections in a population-based study in rural Vietnam, where the prevalences of both HIV and tuberculosis are substantially lower than those in previous studies looking at mixed infections. In total, 1,248 M. tuberculosis isolates from the same number of patients were subjected to IS6110 restriction fragment length polymorphism (RFLP) typing, spoligotyping, and variable-number-tandem-repeat (VNTR) typing. We compared mixed infections identified by the presence of (i) discrepant RFLP and spoligotype patterns in isolates from the same patient and (ii) double alleles at ≥2 loci by VNTR typing and assessed epidemiological characteristics of these infections. RFLP/spoligotyping and VNTR typing identified 39 (3.1%) and 60 (4.8%) mixed infections, respectively (Cohen's kappa statistic, 0.57). The number of loci with double alleles in the VNTR pattern was strongly associated with the proportion of isolates with mixed infections according to RFLP/spoligotyping (P < 0.001). Mixed infections occurred more frequently in newly treated than in previously treated patients, were significantly associated with minor X-ray abnormalities, and were almost significantly associated with lower sputum smear grades. Although the infection pressure in our study area is lower than that in previously studied populations, mixed M. tuberculosis infections do occur in rural South Vietnam in at least 3.1% of cases.
Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future.
Studies have shown that the Mycobacterium tuberculosis Beijing genotype is an emerging pathogen that is frequently associated with drug resistance. This suggests that drug resistant Beijing strains have a relatively high transmission fitness compared to other drug-resistant strains.
Methods and Findings
We studied the relative transmission fitness of the Beijing genotype in relation to anti-tuberculosis drug resistance in a population-based study of smear-positive tuberculosis patients prospectively recruited and studied over a 4-year period in rural Vietnam. Transmission fitness was analyzed by clustering of cases on basis of three DNA typing methods. Of 2531 included patients, 2207 (87%) were eligible for analysis of whom 936 (42%) were in a DNA fingerprint cluster. The clustering rate varied by genotype with 292/786 (37%) for the Beijing genotype, 527/802 (67%) for the East-African Indian (EAI) genotype, and 117/619 (19%) for other genotypes. Clustering was associated with the EAI compared to the Beijing genotype (adjusted odds ratio (ORadj) 3.4: 95% CI 2.8–4.4). Patients infected with streptomycin-resistant strains were less frequently clustered than patients infected with streptomycin-susceptible strains when these were of the EAI genotype (ORadj 0.6, 95% CI 0.4–0.9), while this pattern was reversed for strains of the Beijing genotype (ORadj 1.3, 95% CI 1.0–1.8, p for difference 0.002). The strong association between Beijing and MDR-TB (ORadj 7.2; 95% CI 4.2–12.3) existed only if streptomycin resistance was present.
Beijing genotype strains showed less overall transmissibility than EAI strains, but when comparisons were made within genotypes, Beijing strains showed increased transmission fitness when streptomycin-resistant, while the reverse was observed for EAI strains. The association between MDR-TB and Beijing genotype in this population was strongly dependent on resistance to streptomycin. Streptomycin resistance may provide Beijing strains with a fitness advantage over other genotypes and predispose to multidrug resistance in patients infected with Beijing strains.
Pyrazinamide is important in the treatment of tuberculosis. Unfortunately, the diagnosis of pyrazinamide resistance is hampered by technical difficulties. We hypothesized that mutation analysis combined with the mycobacterial growth indicator tube (MGIT) phenotypic method would be a good predictor of pyrazinamide resistance. We prospectively analyzed 1,650 M. tuberculosis isolates referred to our tuberculosis reference laboratory in 2008 and 2009. In our laboratory, the MGIT 960 system was used for pyrazinamide resistance screening. If a pyrazinamide-resistant strain was detected, we performed a pncA gene mutation analysis. A second MGIT 960 susceptibility assay was performed afterwards to evaluate the accuracy of the pncA mutation analysis to detect true- or false-positive MGIT results. We observed pyrazinamide resistance in 69 samples using the first MGIT 960 analysis. In a second MGIT 960 analysis, 47 of the 69 samples proved susceptible (68% false positivity). Sensitivity of nonsynonymous pncA mutations for detecting resistant isolates was 73% (95% confidence interval [CI], 61% to 73%), and specificity was 100% (95% CI, 95% to 100%). A diagnostic algorithm incorporating phenotypic and molecular methods would have a 100% positive predictive value for detecting pyrazinamide-resistant isolates, indicating that such an algorithm, based on both methods, is a good predictor for pyrazinamide resistance in routine diagnostics.
‘Mycobacterium sherrisii’ is an undescribed species that appears to be emerging, in particular, among HIV-positive patients originating from Africa. To describe ‘M. sherrisii’, to ensure that the species name is validly published and to define its phylogenetic position, we collected 11 of these strains reported in five previous studies, and subjected them to biochemical identification, cell-wall mycolic acid analysis and sequencing of multiple housekeeping genes. The bacteria formed smooth and generally non-chromogenic colonies after 2–3 weeks of subculture at 24–37 °C; photochromogenic and scotochromogenic pigmentation were exhibited by three and two strains, respectively. The strains were positive for the heat-stable catalase test, but negative in tests for hydrolysis of Tween 80, nitrate reduction, β-glucosidase and 3-day arylsulfatase. Mycolic acid patterns, obtained by HPLC, resembled a trimodal profile similar to those of type strains of Mycobacterium simiae, Mycobacterium lentiflavum, Mycobacterium triplex and Mycobacterium genavense. The 16S rRNA gene sequences of the 11 strains differed by 4 bp (99.7 % similarity) from that of the type strain of the closest related species, M. simiae ATCC 25275T. Levels of internal transcribed spacer (ITS) and partial hsp65 and rpoB gene sequence similarity between the two taxa were 95.8 % (271/283 bp), 97.5 % (391/401 bp) and 95.2 % (700/735 bp), respectively. On the basis of these results, we propose the formal recognition of Mycobacterium sherrisii sp. nov. The type strain is 4773T ( = ATCC BAA-832T = DSM 45441T).
To determine differences in the ability of Mycobacterium tuberculosis strains to withstand antituberculosis drug treatment, we compared the activity of antituberculosis drugs against susceptible Beijing and East-African/Indian genotype M. tuberculosis strains. Beijing genotype strains showed high rates of mutation within a wide range of drug concentrations, possibly explaining this genotype’s association with multidrug-resistant tuberculosis.
multidrug-resistant tuberculosis; MDR TB; Beijing genotype strains; antituberculosis drugs; emergence; resistance; antimicrobial resistances; Mycobacterium tuberculosis; tuberculosis and other mycobacteria
The oryx bacilli are Mycobacterium tuberculosis complex organisms for which phylogenetic position and host range are unsettled. We characterized 22 isolates by molecular methods and propose elevation to subspecies status as M. orygis. M. orygis is a causative agent of tuberculosis in animals and humans from Africa and South Asia.
Tuberculosis and other mycobacteria; oryx bacillus; molecular typing; epidemiology; Mycobacterium orygis; bacteria
Mycobacterium cultures, from patients suspected of tuberculosis or nontuberculous mycobacteria (NTM) infection, need to be identified. It is most critical to identify cultures belonging to the Mycobacterium tuberculosis complex, but also important to recognize clinically irrelevant or important NTM to allow appropriate patient management. Identification of M. tuberculosis can be achieved by a simple and cheap lateral flow assay, but identification of other Mycobacterium spp. generally requires more complex molecular methods. Here we demonstrate that a paramagnetic liquid bead array method can be used to capture mycobacterial rRNA in crude lysates of positive cultures and use a robust reader to identify the species in a direct and sensitive manner. We developed an array composed of paramagnetic beads coupled to oligonucleotides to capture 16 rRNA from eight specific Mycobacterium species and a single secondary biotinilated reporter probe to allow the captured rRNA to be detected. A ninth less specific bead and its associated reporter probe, designed to capture 23S rRNA from mycobacteria and related genera, is included as an internal control to confirm the presence of bacterial rRNA from a GC rich Gram variable genera. Using this rRNA capture assay (rCapA) with the array developed we were already able to confirm the presence of members of the M. tuberculosis complex and to discriminate a range of NTM species. This approach is not based on DNA amplification and therefore does not require precautions to avoid amplicon contamination. Moreover, the new generation of stable and cost effective liquid bead readers provides the necessary multiplexing potential to develop a robust and highly discriminatory assay.
The “Harlingen” IS6110 restriction fragment length polymorphism (RFLP) cluster has linked over 100 tuberculosis cases in The Netherlands since 1993. Four Mycobacterium tuberculosis isolates that were epidemiologically linked to this cluster had different spoligotype patterns, as well as slightly divergent IS6110 profiles, compared to the majority of the isolates. Sequencing of the direct repeat (DR) locus revealed sequence polymorphisms at the putative deletion sites. These deletion footprints provided evidence for independent deletions of the central region of the DR locus in three isolates, while the different genotype of the fourth isolate was explained by transmission. Our finding suggests that convergent deletions in the DR locus occur frequently. However, deletion footprints are not suitable to detect convergent deletions in the DR because they seem to be exceptional. Deletion footprints in the DR were not described previously, and we did not observe them in any public M. tuberculosis complex sequences. We conclude that preferential deletions in the DR loci of closely related strains are usually an unnoted event that interferes with clustering of closely related strains.
Early detection, effective treatment, and infection control measures are needed to reduce transmission.
We conducted a case–control study to investigate risk factors for multidrug-resistant tuberculosis (MDR TB) in the People’s Republic of China. Genotyping analysis was used to estimate the percentage of cases from recent transmission among 100 MDR TB case-patients hospitalized during April 2007–July 2009. Molecular subtyping of isolates showed that 41% of MDR TB strains clustered. Beijing genotype was found in 94% of the MDR TB isolates and 79% of the pan-susceptible isolates. In multivariate analysis, MDR TB was independently associated with Beijing genotype, retreatment for TB, symptoms lasting >3 months before first evaluation at the hospital, lack of health insurance, and being a farmer (vs. being a student). MDR TB was associated with Beijing genotype and lower socioeconomic status. A large percentage of MDR TB cases seemed to result from recent transmission. Early detection, effective treatment, and infection control measures for MDR TB are needed to reduce transmission.
Tuberculosis and other mycobacteria; TB; Mycobacterium tuberculosis; M. tuberculosis; drug-resistant tuberculosis; multidrug-resistant tuberculosis; bacteria; MDR tuberculosis; MDR TB; genotype; genetic association studies; genotype-phenotype associations; cluster; clustering; antimicrobial resistance; People’s Republic of China; research
Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC.
We ran a comparative analysis of all patients for whom a positive culture of Mycobacterium tuberculosis complex was available between April 2004 and October 2005 and whose HIV serology results were known, with spoligotyping results (n = 163) split into 49 HIV-positive patients and 114 HIV-negative patients. Spoligotype international type 373 (SIT373) (T1 lineage), which was highly prevalent among the HIV+ patients, was totally absent from the HIV− population, suggesting that we had a specific clone affecting nearly 1/3 of all HIV-tuberculosis (TB)-coinfected patients. Among the LAM10-CAM sublineage strains, we had only a single strain of SIT403 among HIV− patients (0.88%), as opposed to 12.25% of the HIV+ population (χ2 = 10.77; P < 0.01), indicating a strong association between the strain and the HIV+ population. The LAM10-CAM lineage spoligotype SIT61 was prevalent among the 2 subsets (37.72% in HIV− versus 12.24% in HIV+ populations), though, with a significant difference between the 2 groups (χ2 = 10.53; P < 0.01). However, there was no significant difference for SIT53 (T1 lineage) in the 2 subsets: 6.14 versus 8.2% (χ2 = 0.22; P > 0.05). A total of 7/49, or 14.3%, other SITs among HIV+ patients were not found among the HIV− patients. When added to the most prevalent SIT among HIV+ patients (SIT373; n = 16), 23/49, or 47%, isolates among HIV-TB-coinfected patients were unique. We conclude that further studies should be carried out to investigate the evolution of these genotypes and others in the emergence of multidrug resistance and control of tuberculosis in Nigeria.
Using spoligotyping, we identified 13 genotypes and 17 orphan types among 160 Mycobacterium tuberculosis isolates from patients in Valle del Cauca, Colombia. The Beijing genotype represented 15.6% of the isolates and was correlated with multidrug-resistant tuberculosis, female sex of the patients, and residence in Buenaventura and may represent a new public health threat.
tuberculosis and other mycobacteria; bacteria; genotypes; spoligotyping; Beijing; MDR; XDR; multidrug resistance; Colombia; dispatch
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.