Detection of tuberculosis in cynomolgus macaques (Macaca fascicularis) using a supplementary Monkey Interferon Gamma Releasing Assay (mIGRA).

Cynomolgus monkeys (Macaca fascicularis; MF) are commonly used as nonhuman primate models for pharmaceutical product testing. In their habitat range, monkeys have close contact with humans, allowing the possibility of bidirectional transmission of tuberculosis (TB) between the two species. Although the intradermal tuberculin skin test (TST) is used for TB detection in MF, it has limitations. Herein, we established the mIGRA, combining human QuantiFERON-TB Gold-Plus and monkey IFN-γ ELISApro systems, and used it to investigate 39 captive MF who were cage-mates or lived in cages located near a monkey who died from the naturally TB infection. During a 12-month period of study, 14 (36%), 10 (26%), and 8 (21%) monkeys showed TB-positive results using the mIGRA, the TST, and TB culture, respectively. Among the 14 mIGRA-positive monkeys, 8 (57.1%) were TST-positive and 7 (50%) were culture-positive, indicating early TB detection in the latent and active TB stages with the mIGRA. Interestingly, 3 (37.5%) of the TST-negative monkeys were culture-positive. Our study showed that the mIGRA offers many advantages, including high sensitivity and high throughput, and it requires only one on-site visit to the animals. The assay may be used as a supplementary tool for TB screening in MF.

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.

Comparing genomes within the species Mycobacterium tuberculosis.

The study of genetic variability within natural populations of pathogens may provide insight into their evolution and pathogenesis. We used a Mycobacterium tuberculosis high-density oligonucleotide microarray to detect small-scale genomic deletions among 19 clinically and epidemiologically well-characterized isolates of M. tuberculosis. The pattern of deletions detected was identical within mycobacterial clones but differed between different clones, suggesting that this is a suitable genotyping system for epidemiologic studies. An analysis of genomic deletions among an extant population of pathogenic bacteria provided a novel perspective on genomic organization and evolution. Deletions are likely to contain ancestral genes whose functions are no longer essential for the organism's survival, whereas genes that are never deleted constitute the minimal mycobacterial genome. As the amount of genomic deletion increased, the likelihood that the bacteria will cause pulmonary cavitation decreased, suggesting that the accumulation of mutations tends to diminish their pathogenicity. Array-based comparative genomics is a promising approach to exploring molecular epidemiology, microbial evolution, and pathogenesis.

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.

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.