Peptide microarray-based identification of dormancy-associated Mycobacterium tuberculosis antigens inducing immune responses among latent tuberculosis infection individuals in Thailand.

Multi-stage tuberculosis (TB) vaccines composed of active- and dormancy-associated antigens are promising to trigger the immune protection against all TB stages. However, scientists are still in quest of the suitable vaccine candidates. In this study, we identified the potential targets for this vaccine in a high TB burden country, Thailand. Peptide microarray was applied to gauge IgA and IgG antibodies specific to 16,730 linear epitopes of 52 dormancy-associated Mycobacterium tuberculosis (M. tb) proteins in three study groups: active tuberculosis (ATB), latent tuberculosis infection (LTBI) and endemic healthy control (EHC). Preferential IgA recognition against epitopes of dormancy-associated proteins was identified in LTBI group. Validation of these findings revealed that LTBI subjects exhibited the greater levels of Rv2659c- and Rv1738-specific IgA than those of household contacts, but less than did ATB subjects. Frequencies of IFNγ-producing CD4+ and CD8+ T cells induced by proteins Rv2659c and Rv1738 were higher in LTBI than ATB individuals. The results indicated that LTBI group in a high TB burden country demonstrated cell-mediated immune response to proteins Rv2659c and Rv1738 stronger than those of ATB. These immune responses likely contribute to natural protection against dormant M. tb and might be potential targets for a multi-stage TB vaccine.

Circulating IgA/IgG memory B cells against Mycobacterium tuberculosis dormancy-associated antigens Rv2659c and Rv3128c in active and latent tuberculosis.

OBJECTIVE: To elucidate the antigenic potential of dormancy-associated antigens Rv2659c and Rv3128c of Mycobacterium tuberculosis by examining the persistence of specific IgG and IgA memory B cells (MBCs) among patients with active tuberculosis (TB), household contacts with latent tuberculosis (LTBI), and an endemic healthy control group. METHODS: Fresh peripheral blood mononuclear cells from the three study groups were used to enumerate the numbers of IgG and IgA MBCs specific to recombinant protein Rv2659c and Rv3128c by ELISpot assay. The composition of MBC subsets IgA+ and IgG + was analyzed by flow cytometry. RESULTS: The number of IgA MBCs specific to antigen Rv2659c was significantly higher in the LTBI group than the TB group. In contrast, no significant difference was found in IgA or IgG MBCs against antigen Rv3128c. The number of IgA+ MBCs was significantly higher than that of IgG+ MBCs in the classical MBC subset of the LTBI group. CONCLUSION: The results indicated that the dormancy-associated antigen Rv2659c induced an IgA MBCs response in individuals with latent TB, and IgA+ classical MBCs formed a major portion of the MBCs subset. This new knowledge will be beneficial for the development of novel TB vaccines and their control of latent TB.

Mitogenomic phylogeny of the Asian colobine genus Trachypithecus with special focus on Trachypithecus phayrei (Blyth, 1847) and description of a new species.

Trachypithecus, which currently contains 20 species divided into four groups, is the most speciose and geographically dispersed genus among Asian colobines. Despite several morphological and molecular studies, however, its evolutionary history and phylogeography remain poorly understood. Phayre's langur ( Trachypithecus phayrei) is one of the most widespread members of the genus, but details on its actual distribution and intraspecific taxonomy are limited and controversial. Thus, to elucidate the evolutionary history of Trachypithecus and to clarify the intraspecific taxonomy and distribution of T. phayrei, we sequenced 41 mitochondrial genomes from georeferenced fecal samples and museum specimens, including two holotypes. Phylogenetic analyses revealed a robustly supported phylogeny of Trachypithecus, suggesting that the T. pileatus group branched first, followed by the T. francoisi group, and the T. cristatus and T. obscurus groups most recently. The four species groups diverged from each other 4.5-3.1 million years ago (Ma), while speciation events within these groups occurred much more recently (1.6-0.3 Ma). Within T. phayrei, we found three clades that diverged 1.0-0.9 Ma, indicating the existence of three rather than two taxa. Following the phylogenetic species concept and based on genetic, morphological, and ecological differences, we elevate the T. phayrei subspecies to species level, describe a new species from central Myanmar, and refine the distribution of the three taxa. Overall, our study highlights the importance of museum specimens and provides new insights not only into the evolutionary history of T. phayrei but the entire Trachypithecus genus as well.

New elephant crisis in Asia-Early warning signs from Myanmar.

In the southern Bago Yoma mountain range in Myanmar, Asian elephants are being killed at a disturbing rate. This emerging crisis was identified initially through a telemetry study when 7 of 19 of collared elephants were poached within a year of being fitted with a satellite-GPS collar. Subsequent follow up of ground teams confirmed the human caused death or disappearance of at least 19 elephants, including the seven collared individuals, within a 35 km2 area in less than two years. The carcasses of 40 additional elephants were found in areas located across south-central Myanmar once systematic surveys began by our team and collaborators. In addition to the extreme rate of loss, this study documents the targeting of elephants for their skin instead of the more common ivory, an increasing trend in Myanmar. Intensive research programs focused on other conservation problems identified this issue and are now encouraging local authorities to prioritize anti-poaching efforts and improve conservation policies within the country. Myanmar represents one of the last remaining countries in Asia with substantial wildlands suitable for elephants. Increasing rates of human-elephant conflict and poaching events in this country pose a dire threat to the global population.

Sensitive detection of the IS6110 sequence of Mycobacterium tuberculosis complex based on PCR-magnetic bead ELISA.

Tuberculosis (TB) is ranked as the top killer among infectious diseases worldwide. Early and accurate diagnosis of the disease is crucial to end the global TB epidemic. The current commercially available molecular tests are still unaffordable by most TB affected communities. Herein, we developed a novel rapid and sensitive diagnostic method to detect the IS6110 sequence of Mycobacterium tuberculosis (M. tuberculosis) complex using PCR-magnetic bead ELISA. PCR amplification ofa 123 bp repetitive sequence of the IS6110 gene was performed by using digoxigenin (DIG) and biotin-labelled primers. Streptavidin-conjugated magnetic beads were used to collect the dual-labelled amplicons and subsequently, colourimetric detection was done by using horseradish peroxidase (HRP)-conjugated anti-DIG antibody. This method is able to detect M. tuberculosis DNA down to 0.5 fg per reaction within 3 hours. The sensitivity of IS6110 PCR detection by magnetic bead ELISA is 100 times higher than that of conventional agarose gel electrophoresis. The assay specificity was determined using a panel of DNA extracted from 10 common bacteria causing lower respiratory tract infections. No cross-reactivity was detected from those bacteria by IS6110 PCR-magnetic bead ELISA. Thus, the novel highly sensitive and specific, reduced assay time and simplicity of this PCR-magnetic bead ELISA for the detection of the specific gene of M. tuberculosis complex makes it an attractive diagnostic tool for large-scale screening of tuberculosis in standard clinical laboratories.

Study of malaria in a village of lower Myanmar.

Malaria endemicity in lower Myanmar has been studied to identify the causes for the prevalence of malaria in Yeasitkan village of lower Myanmar. Vector mosquitoes were collected by mosquito net in cattlesheds and in human dwellings (indoor and outdoor) by biting and catching procedure for the identification of species, insecticide susceptibility test and sporozoites detection. Larvae of mosquitoes were also collected in and around the village for vector identification and for breeding sources. Malaria infection in humans was examined by blood examination and blood antibody detection by ELISA method. Results showed that malaria infection was 43.2% in children under 10 years of age and An. dirus and An. minimus were found as main vectors. Total parasite positive rate was found to be 41.28% and in this 78.87% were P. falciparum infections and remaining 18.31% were of P. vivax. Spleen positive rate has been found very high in children between 2 and 9 years (52.94%). Study indicates that villages near to dam areas are more prone to malaria infection.