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No. 1, 2014

LABORATORY BRANCH UPDATES

Southeastern Mycobacteria Meeting

Members of the Applied Research Team from DTBE's Laboratory Branch participated in the 5th Southeastern Mycobacteria Meeting in Birmingham, Alabama, January 24—26, 2014. This conference is designed to showcase current research, build relationships, and foster collaborations among attendees.

The Applied Research Team presented seven posters, three of which focused on the team's current work aimed at improving the detection of pyrazinamide (PZA) resistance. PZA resistance is problematic for growth-based assays. Alexandra Mercante described a modified protocol for determining PZA resistance via the widely used MGIT system. This protocol uses a reduced inoculum size, leading to a decrease in false resistance. Kristin Birkness presented data describing development of a colorimetric assay in E. coli for detection of the effects of mutations in pncA, the gene which encodes pyrazinamidase (PZase) activity and can predict resistance to PZA. The assay was applied to 88 isolates with pncA mutations. Prediction of resistance based on PZase activity as determined in the E. coli assay correlated well with MGIT results. The Applied Research Team's ongoing effort to construct over 1,000 mutations in pncA and assess these mutations in the previously described E. coli assay was detailed on Kelsey Hughes' poster. The team has thus far completed construction and evaluation of 121 pncA mutations, observing highly reduced levels of PZase activity in a number of the mutations assessed.

The Ion Torrent Personal Genome Machine (PGM) can generate up to 6 million bases of sequence data from as many as 96 individual samples in about 2 hours. Melisa Willby's poster presented the team's efforts to simplify large-scale drug-resistance surveillance efforts through development of a multiplexed assay for detecting mutations associated with drug resistance. In initial experiments comparing Ion Torrent results with traditional sequencing, the Ion Torrent Suite software did not find all expected mutations. However, when the data were examined directly by the investigators, all expected mutations were found. This highlighted the need for improvements in the current sequence analysis software programs. These experiments also emphasized the need for a more streamlined approach to sample preparation. The poster described preliminary efforts to improve efficiency and decrease cost.

Spoligotyping is an assay used to genotype M. tuberculosis strains. Paige Gupton presented results of a pilot study in which the M. tuberculosis spoligotyping assay was transferred from the currently used Luminex platform to the Ion Torrent PGM. Using the Ion Torrent assay, 270 previously spoligotyped samples were reanalyzed, with a resulting 100% concordance between the two platforms. The Applied Research Team began spoligotyping on the Ion Torrent in October 2013 for routine surveillance.

Improved vaccination strategies against TB are urgently needed, for example, approaches to boost immune responses induced by the current vaccine, BCG. Design of these strategies has been hampered by a lack of knowledge of the kinetics of the host immune responses induced by BCG vaccination. Subhadra Nandakumar described longitudinal changes in the effector functional abilities of BCG vaccination induced T-cells and their association with the protection against M. tuberculosis over a period of 2 years in the mouse model. Her presentation highlighted the lifelong BCG persistence and its association with the attrition and exhaustion of T-cell response and waning of protective efficacy against M. tuberculosis in mice. Her results questioned the empirical development of BCG-booster vaccines and emphasized the pursuit of strategies that maintain superior T-cell functional capacity.

Expanding on this topic, Suraj Sable presented prime-boost vaccination strategies using BCG and an effective subunit vaccine based on the adhesin, Apa. His presentation described the ability of Apa subunit vaccine to boost waning BCG immunity in older mice regardless of its native or recombinant form. These results have implications for the development of effective prime-boost vaccination strategies against tuberculosis.

The posters and talks presented by other attendees provided an important glimpse into the state of current research in the mycobacteria field. Additionally, this conference provided a valuable forum for not only sharing our current research with the mycobacterial community, but also for discussing future directions and possible collaborations. As we pursue a greater understanding of M. tuberculosis from these and other activities, we hope to be part of achieving the U.S. goal of TB elimination.

Submitted by Melisa Willby, PhD,
and Suraj Sable, DVM, PhD,
Div of TB Elimination

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