Theoretical basis for reducing time-lines to the determination of positive Mycobacterium tuberculosis cultures using thymidylate kinase (TMK) assays

<p>Abstract</p> <p>Background</p> <p><it>In vitro </it>culture of pathogens on growth media forms a "pillar" for both infectious disease diagnosis and drug sensitivity profiling. Conventional cultures of <it>Mycobacterium tuberculosis </it>(M.<it>tb</it>) on Lowenstein Jensen (LJ) medium, however, take over two months to yield observable growth, thereby delaying diagnosis and appropriate intervention. Since DNA duplication during interphase precedes microbial division, "para-DNA synthesis assays" could be used to predict impending microbial growth. Mycobacterial thymidylate kinase (TMKmyc) is a phosphotransferase critical for the synthesis of the thymidine triphosphate precursor necessary for M.<it>tb </it>DNA synthesis. Assays based on high-affinity detection of secretory TMKmyc levels in culture using specific antibodies are considered. The aim of this study was to define algorithms for predicting positive TB cultures using antibody-based assays of TMKmyc levels <it>in vitro</it>.</p> <p>Methods and results</p> <p>Systems and chemical biology were used to derive parallel correlation of "M.<it>tb </it>growth curves" with "TMKmyc curves" theoretically in four different scenarios, showing that changes in TMKmyc levels in culture would in each case be predictive of M.<it>tb </it>growth through a simple quadratic curvature, |tmk| = at²+ bt + c, consistent with the "S" pattern of microbial growth curves. Two drug resistance profiling scenarios are offered: isoniazid (INH) resistance and sensitivity. In the INH resistance scenario, it is shown that despite the presence of optimal doses of INH in LJ to stop M.<it>tb </it>proliferation, bacilli grow and the resulting phenotypic growth changes in colonies/units are predictable through the TMKmyc assay. According to our current model, the areas under TMKmyc curves (AUC, calculated as the integral ∫(at²+ bt + c)dt or ~1/3 at³+ 1/2 bt²+ct) could directly reveal the extent of prevailing drug resistance and thereby aid decisions about the usefulness of a resisted drug in devising "salvage combinations" within resource-limited settings, where second line TB chemotherapy options are limited.</p> <p>Conclusion</p> <p>TMKmyc assays may be useful for reducing the time-lines to positive identification of <it>Mycobacterium tuberculosis </it>(M.<it>tb</it>) cultures, thereby accelerating disease diagnosis and drug resistance profiling. Incorporating "chemiluminiscent or fluorescent" strategies may enable "photo-detection of TMKmyc changes" and hence automation of the entire assay.</p