Indo-Oceanic Mycobacterium tuberculosis strains from Thailand associated with higher mortality.

SETTING: This study was conducted among tuberculosis (TB) patients in a highly endemic Thai province.OBJECTIVE: To evaluate the association between different Mycobacterium tuberculosis lineages and clinical characteristics, especially mortality.DESIGN: We enrolled 1,304 TB patients registered from 2002-2011 with culture isolates whose lineages were identified by specific regions of deletion. Data on mortality within 1 year of follow-up were extracted from the registration system and hospital records. Mortality-associated risk factors, including bacterial lineages, as independent variables were analysed using Cox regression models.RESULTS: Of 1,304 isolates, 521 (40.0%) and 582 (44.6%) belonged to Indo-Oceanic and East-Asian lineages, respectively. Indo-Oceanic strains significantly increased the mortality risk compared with East-Asian strains (adjusted hazard ratio [aHR] 1.42, 95%CI 1.02-1.99) or modern lineages (aHR 1.49, 95%CI 1.08-2.06) in the 172 patients who died within 1 year after TB diagnosis. The former also caused significantly higher mortality than modern lineages among patients who died within 6 months after TB diagnosis (aHR 1.62, 95%CI 1.12-2.35). No significant association was found between drug resistance and death.CONCLUSION: In Thailand, the Indo-Oceanic lineage of M. tuberculosis increased mortality risk compared with modern lineages or the East-Asian lineage, the latter being considered highly virulent in previous studies.

Strain-based HLA association analysis identified HLA-DRB1*09:01 associated with modern strain tuberculosis.

Tuberculosis (TB) occurs as a result of complex interactions between the host immune system and pathogen virulence factors. Human leukocyte antigen (HLA) class II molecules play an important role in the host immune system. However, no study has assessed the association between HLA class II genes and susceptibility to TB caused by specific strains. This study investigated the possible association of HLA class II genes with TB caused by modern and ancient Mycobacterium tuberculosis (MTB). The study included 682 patients with TB and 836 control subjects who were typed for HLA-DRB1 and HLA-DQB1 alleles. MTB strains were classified using a large sequence polymorphism typing method. Association analysis was performed using common HLA alleles and haplotypes in different MTB strains. HLA association analysis of patients infected with modern MTB strains showed significant association for HLA-DRB1*09:01 (odds ratio [OR] = 1.82; P-value = 9.88 × 10-4 ) and HLA-DQB1*03:03 alleles (OR = 1.76; P-value = 1.31 × 10-3 ) with susceptibility to TB. Haplotype analysis confirmed that these alleles were in strong linkage disequilibrium and did not exert an interactive effect. Thus, the results of this study showed an association between HLA class II genes and susceptibility to TB caused by modern MTB strains, suggesting the importance of strain-specific analysis to determine susceptibility genes associated with TB.

Draft Genome Sequence of the Mycobacterium tuberculosis Strain 43-16836, Belonging to the Indo-Oceanic Lineage, Isolated From Tuberculous Meningitis in Thailand.

We present the draft genome sequence of Mycobacterium tuberculosis strain 43-16836, belonging to the Indo-Oceanic lineage, isolated from a tuberculous meningitis patient in Thailand. The genome is 4,381,942 bp long with 4,316 protein-coding genes and contains new single nucleotide polymorphisms (SNPs), including SNPs of genes that may encode cell wall components and possibly influence virulence.

Variable number of tandem repeat sequences act as regulatory elements in Mycobacterium tuberculosis.

VNTR (variable number of tandem repeats) has been used extensively for Mycobacterium tuberculosis strain discrimination. In contrast, their biological roles have been rarely investigated. We, herewith, studied whether two VNTR could promote expression of a downstream reporter gene, gfp. The VNTR loci, VNTR0960c and VNTR4052, reside upstream of the translational start sites of Rv0861c (helicase gene, ercc3) and Rv3610c (ftsH), respectively. Both are highly polymorphic among clinical strains of M. tuberculosis. DNA fragments containing various numbers of the repeat units were amplified and inserted upstream of the gfp gene and transformed into M. smegmatis mc(2)155 and M. tuberculosis H37Ra. The levels of fluorescence were determined by microplate fluorometry and flow cytometry. It was found that VNTR0960c could promote the expression of gfp while VNTR4052 alone could not. However, VNTR4052 was needed for complementing the promoter activity of the downstream sequence. The effects were discernible in M. tuberculosis, even though only the incomplete copy of the repeat was present. The addition of a complete copy of the repeat augmented the promoter activity. The presence of more copies of the repeat had minimal effects or even decreased the expressions. Significance of the effects on the cellular metabolisms of M. tuberculosis warrants further studies.

Restriction fragment length polymorphism study of nationwide samples of Mycobacterium tuberculosis in Thailand, 1997-1998.

SETTING: During 1997-1998, a national anti-tuberculosis drug resistance survey was conducted in Thailand as a part of a global project. OBJECTIVE: To evaluate the IS6110 hybridisation patterns and the level of clustering, which was expected to be low due to the short duration of the sample collection. DESIGN: Eight hundred and twenty-eight bacterial isolates were available for fingerprinting by standard IS6110 hybridisation. RESULTS: The restriction fragment length polymorphism patterns varied with geographic locations, ages of the patients, and resistance to rifampicin and streptomycin. The Beijing strain was more common among younger patients, and their prevalence appeared to decrease with the distance from Bangkok, while the opposite was true for the single-banded isolates. Excluding isolates containing five or less copies of IS6110, 26.4% were clustered. Clustering was more common among females. The clustered isolates were sometimes from different provinces and, if resistant to drugs, usually possessed different resistance profiles. CONCLUSIONS: The results question the validity of inferring recent transmission from the clustering of IS6110 hybridisation patterns in some settings in Thailand. The level of recent transmission in a nationwide study in a country with a high incidence of tuberculosis should be evaluated with caution.

Antimycobacterial screening of some Turkish plants.

Extracts of 44 plant species distributed among 17 families from Turkey were screened against Mycobacterium tuberculosis H(37)Ra using microplate Alamar blue assay test. Six plants inhibited growth of M. tuberculosis H(37)Ra at 50 microg/ml concentrations.

Development of acquired drug resistance in recurrent tuberculosis patients with various previous treatment outcomes.

SETTING: Chiang Rai province, Northern Thailand. OBJECTIVE: To study the probability of acquiring drug resistance to isoniazid (H) and rifampicin (R) on recurrence after treatment success, default and failure, among sputum smear-positive pulmonary tuberculosis (TB) patients treated with standardised short-course chemotherapy. DESIGN: Retrospective analysis of registration records of TB patients from May 1996 to December 2000 in Chiang Rai, where routine drug susceptibility testing (DST) is conducted for surveillance purposes. Patients registered twice or more were examined. RESULTS: Of 59 cases treated with HRZE/HR who underwent DST at the time of registration, 31 were fully susceptible to H and R at first registration, of whom four acquired drug resistance to H or R. Of 13 cases resistant to H or R at first registration, 11 became multidrug-resistant (MDR). The remaining 15 patients were original MDR cases. Among 28 MDR or H- or R-resistant cases, six reverted from resistant to susceptible. DISCUSSION: A high proportion of patients with H- or R-resistant TB became MDR after treatment with 2HRZE/HR. Using this regimen, MDR may increase in a population with a high prevalence of H or R resistance. We are unable to explain why some drug-resistant cases became drug-susceptible. Further investigation is necessary.

Tuberculosis in Thai prisons: magnitude, transmission and drug susceptibility.

BACKGROUND: Because of the human immunodeficiency virus (HIV) epidemic, tuberculosis has reemerged as a major public health problem in Thailand. Prison inmates are at high risk for developing tuberculosis because of the high prevalence of HIV infection. OBJECTIVES: To determine the magnitude, transmission, and drug susceptibility of tuberculosis in Thai prisons. SETTINGS: Four provincial prisons in Southern Thailand. DESIGN: Cross-sectional, descriptive, clinical and molecular study. RESULTS: Miniature chest roentgenograms were performed on 304 (6.4%) of 4751 inmates screened for a > or = 2 week history of chronic cough and fever. At least 17 (35%) of 49 inmates who had a miniature chest roentgenogram compatible with tuberculosis were HIV-positive. The prevalence of smear-positive pulmonary tuberculosis was 568 per 100,000 inmates, which was eight times higher than that in the general population. Eight (38%) of 21 culture-positive Mycobacterium tuberculosis isolates had DNA fingerprints matching those of another inmate who was housed in the same room or in the same dormitory unit; 39% of the M. tuberculosis isolates were resistant to isoniazid; three of these isolates were also borderline resistant to rifampicin. CONCLUSION: The prevalence of pulmonary tuberculosis in these prisons was high. A substantial proportion were acquired in the prisons. Isoniazid (INH) resistance was common, and theoretically precludes the use of INH-preventive therapy for contacts of these cases. Active case finding should be done and directly observed therapy implemented to prevent the spread of tuberculosis into the community.

A variable number of tandem repeats result in polymorphic alpha -isopropylmalate synthase in Mycobacterium tuberculosis.

A locus of variable number of the tandem repeat, VNTR4155, resides in the putative leuA gene, encoding for alpha -isopropylmalate synthase (alpha -IPMS) of Mycobacterium tuberculosis, a repeat that is unique to the bacterium. The objective was to determine whether the polymorphic VNTR4155 was translated and resulted in a polymorphic protein. The putative leuA gene of the M. tuberculosis H37Rv strain was cloned by PCR and expressed in a His-tagged form in Escherichia coli. The enzymatic properties of the purified protein were studied. The protein was used as an antigen to immunize rabbits. Soluble proteins of several strains of M. tuberculosis were examined by Western blot analysis. The polymorphism of VNTR4155 was due to the presence of different copy number of the 57-bp tandem repeat. The putative alpha -IPMS of various strains of M. tuberculosis had different sizes, varying directly with the length of their VNTR4155.

Molecular evidence for independent occurrence of IS6110 insertions at the same sites of the genome of Mycobacterium tuberculosis in different clinical isolates.

Several characteristics of Mycobacterium tuberculosis (e.g., conserved genome and low growth rate) have severely restricted the study of the microorganism. The discovery of IS6110 raised hopes of overcoming these obstacles. However, our knowledge of this IS element is relatively limited; even its two basic characteristics (transposition mechanism and target site selection) are far from well understood. In this study, IS6110 insertions in ipl loci (iplA and iplB) in two collections of clinical isolates of M. tuberculosis from different geographic locations, one from Scotland and the other from Thailand, were investigated. Five different IS6110 insertions in the loci were identified: ipl-4::IS6110, ipl-5::IS6110, ipl-11::IS6110, ipl-12::IS6110, and ipl-13::IS6110. An attempt to establish the phylogenetic relationship of the isolates containing these insertions was unsuccessful, suggesting that some of these insertions may have arisen from more than one event. This possibility is further supported by the observation that IS6110 copies existed in the same site but with different orientations in different isolates, and the insertion site of ipl-1::IS6110 harbored IS6110 copies in both iplA and iplB in different strains. All these suggest the independent occurrence of IS6110 insertions at the same sites of the genome of M. tuberculosis in different clinical isolates. The implications of this finding are discussed.

A new antimycobacterial, 3 beta-acetoxy-15 alpha,22-dihydroxyhopane, from the insect pathogenic fungus Aschersonia tubulata.

Bioassay-guided fractionation of the cell extract of the insect pathogenic fungus Aschersonia tubulata BCC 1785 led to the isolation of dustanin (1), 3 beta,15 alpha,22-trihydroxyhopane (3), 5 alpha,8 alpha-epidioxy-24(R)-methylcholesta-6,22-diene-3 beta-ol (6), together with the new 3 beta-acetoxy-15 alpha,22-dihydroxyhopane (4). Chemical structures of these compounds were elucidated by spectral analyses as well as chemical transformation. Compounds 1 and 4 exhibited antimycobacterial activity with the minimum inhibitory concentration (MIC) of 12.5 micrograms/ml.

Identification of possible loci of variable number of tandem repeats in Mycobacterium tuberculosis.

Three VNTR loci were previously cloned from Mycobacterium tuberculosis in our laboratory. The VNTR sequences were used as queries to search for similar sequences in the GenBank database by the BLAST program. Direct and tandem repeats were identified visually. The search revealed 45 more loci of direct and tandem repeats. Comparison of the sequences to the ones in the genome sequence database of the M. tuberculosis CDC1551 strain revealed 22 different loci. Combining these results with previously reported experimental work, at least 24 loci should be polymorphic enough to be detected by simple PCR. The repeats are present both inside coding sequences and in intergenic regions on the 5' or 3' ends of genes. M. tuberculosis contains several VNTR. Studies of their functions may be useful for understanding the differences of phenotypes between strains.

Antimycobacterial and antiplasmodial cyclodepsipeptides from the insect pathogenic fungus Paecilomyces tenuipes BCC 1614.

Bioassay-guided fractionation of the crude extract from the insect pathogenic fungus Paecilomyces tenuipes BCC 1614 led to the isolation and identification of two antimycobacterial and antiplasmodial cyclodepsipeptides, beauvericin and beauvericin A.

Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility.

In this study, the currently known typing methods for Mycobacterium tuberculosis isolates were evaluated with regard to reproducibility, discrimination, and specificity. Therefore, 90 M. tuberculosis complex strains, originating from 38 countries, were tested in five restriction fragment length polymorphism (RFLP) typing methods and in seven PCR-based assays. In all methods, one or more repetitive DNA elements were targeted. The strain typing and the DNA fingerprint analysis were performed in the laboratory most experienced in the respective method. To examine intralaboratory reproducibility, blinded duplicate samples were included. The specificities of the various methods were tested by inclusion of 10 non-M. tuberculosis complex strains. All five RFLP typing methods were highly reproducible. The reliability of the PCR-based methods was highest for the mixed-linker PCR, followed by variable numbers of tandem repeat (VNTR) typing and spoligotyping. In contrast, the double repetitive element PCR (DRE-PCR), IS6110 inverse PCR, IS6110 ampliprinting, and arbitrarily primed PCR (APPCR) typing were found to be poorly reproducible. The 90 strains were best discriminated by IS6110 RFLP typing, yielding 84 different banding patterns, followed by mixed-linker PCR (81 patterns), APPCR (71 patterns), RFLP using the polymorphic GC-rich sequence as a probe (70 patterns), DRE-PCR (63 patterns), spoligotyping (61 patterns), and VNTR typing (56 patterns). We conclude that for epidemiological investigations, strain differentiation by IS6110 RFLP or mixed-linker PCR are the methods of choice. A strong association was found between the results of different genetic markers, indicating a clonal population structure of M. tuberculosis strains. Several separate genotype families within the M. tuberculosis complex could be recognized on the basis of the genetic markers used.

IS6110-mediated deletions of wild-type chromosomes of Mycobacterium tuberculosis.

The ipl locus is a site for the preferential insertion of IS6110 and has been identified as an insertion sequence, IS1547, in its own right. Various deletions around the ipl locus of clinical isolates of Mycobacterium tuberculosis were identified, and these deletions ranged in length from several hundred base pairs up to several kilobase pairs. The most obvious feature shared by these deletions was the presence of an IS6110 copy at the deletion sites, which suggested two possible mechanisms for their occurrence, IS6110 transposition and homologous recombination. To clarify the mechanism, an investigation was conducted; the results suggest that although deletion transpositionally mediated by IS6110 was a possibility, homologous recombination was a more likely one. The implications of such chromosomal rearrangements for the evolution of M. tuberculosis, for IS6110-mediated mutagenesis, and for the development of genetic tools are discussed. The deletion of genomic DNA in isolates of M. tuberculosis has previously been noted at only a few sites. This study examined the deletional loss of genetic material at a new site and suggests that such losses may occur elsewhere too and may be more prevalent than was previously thought. Distinct from the study of laboratory-induced mutations, the detailed analysis of clinical isolates, in combination with knowledge of their evolutionary relationships to each other, gives us the opportunity to study mutational diversity in isolates that have survived in the human host and therefore offers a different perspective on the importance of particular genetic markers in pathogenesis.

Differentiation between Mycobacterium tuberculosis and Mycobacterium avium by amplification of the 16S-23S ribosomal DNA spacer.

Differentiation between Mycobacterium tuberculosis and M. avium is helpful for the treatment of disseminated mycobacterial infection in AIDS patients. This can traditionally be done by time-consuming biochemical tests or with Accuprobe. Previously, PCR restriction enzyme analysis (PCR-REA) of the 16S-23S rRNA gene spacer was shown to be able to identify a limited number of strains of Mycobacterium. In this study the method was improved by using more specific primers and was tested with 50 clinical isolates of M. tuberculosis and 65 clinical isolates of M. avium complex. Probes specific to the spacers of M. tuberculosis and M. avium were also tested. Both M. tuberculosis and M. avium could be reliably identified either by PCR-REA or by PCR-hybridization, with the results completely agreeing with those obtained by biochemical tests and with the Accuprobe, respectively. The method may therefore be useful as an alternative in-house method for identification of the bacteria.

The genetic diversity of Mycobacterium tuberculosis strains in Thailand studied by amplification of DNA segments containing direct repetitive sequences.

OBJECTIVE: To develop and use a polymerase chain reaction (PCR) method for studying the genetic diversity of Mycobacterium tuberculosis. DESIGN: Two polymorphic DNA segments of M. tuberculosis H37Rv were identified and sequenced. Primers were then designed for simultaneous amplification of both polymorphic segments. The method was used for studying 179 clinical isolates of M. tuberculosis that had been previously characterized by Southern hybridization with IS6110. RESULTS: Both polymorphic segments contained direct repetitive sequences. In one segment the direct repetitive sequences were within the putative coding sequence of alpha-isopropylmalate synthase gene. After amplifying both segments of the 179 isolates, 40 patterns of PCR products could be identified. The method was able to differentiate 38 IS6110-single-banded isolates into 23 types. Most of the isolates belonging to the Beijing family had PCR products identical to the H37Rv strain. The PCR products of the members of the Nonthaburi group were similar to each other. CONCLUSION: These results agree with the hypothesis that the members of the Beijing family and the Nonthaburi group descended from two common ancestors. The PCR method might be useful for differentiating strains of M. tuberculosis that contain a single copy of IS6110.

Restriction fragment length polymorphism study of Mycobacterium tuberculosis in Thailand using IS6110 as probe.

SETTING: Three referral hospitals in central Thailand. OBJECTIVE: To determine the population structure of Mycobacterium tuberculosis isolated from the referral hospitals. DESIGN: Study of 211 isolates of the bacteria received from the hospitals in central Thailand by Southern hybridization, with IS6110 probe and other probes when indicated. RESULTS: In 43 isolates only one copy of IS6110 was observed. These could be further differentiated by DR- and PGRS-specific probes. Two large groups of isolates with similar hybridization patterns were identified. The Beijing family, comprising 80 isolates, was previously reported to be commonly found in China, Mongolia, Thailand and Korea. The Nonthaburi group, comprising 29 isolates, were local strains. The age, sex and HIV status of the patients did not significantly correlate with the chance of being infected by isolates of any particular hybridization pattern. However, clustered isolates were found more commonly among the members of both the Beijing family and the Nonthaburi group. CONCLUSION: Southern hybridization with IS6110 was found to be useful in studying the epidemiology of tuberculosis in Thailand. The existence of the Beijing family was confirmed. The unusually wide spread of the Beijing family in several countries in Asia merits further investigation.

Differentiation of Mycobacterium species by restriction enzyme analysis of amplified 16S-23S ribosomal DNA spacer sequences.

SETTING: Mycobacteriology research and service laboratories in Thailand. OBJECTIVE: To evaluate the possibility of differentiating species of mycobacteria by amplifying 16S-23S ribosomal deoxyribonucleic acid (DNA) spacer and restriction enzyme analysis of the products. DESIGN: DNA of 113 strains of mycobacteria belonging to 18 species of the genus Mycobacterium were amplified by primers PL1 (5'-GAAGTCGTAACAAGG) and PL2 (5'-CAAGGCATCCACCAT). The amplified products as well as their HaeIII-, MspI- and BstXI-digested products were visualized after agarose gel electrophoresis. RESULTS: The amplified products of rapid-growing mycobacteria were different from the slow-growing mycobacteria. The restriction profiles of members of M. tuberculosis complex were the same as each other but different from other investigated species. The restriction profiles of some species, such as M. avium, M. intracellulare and M. gordonae, were unique, while those of the other species had more than one pattern. However, the restriction profiles of most investigated species were different from each other. CONCLUSION: This preliminary study suggested that the method might be useful for species differentiation of some commonly isolated pathogenic mycobacteria.