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

Abstract

Background: In vitro culture of pathogens on growth media forms a "pillar" for both infectious disease diagnosis and drug sensitivity profiling. Conventional cultures of Mycobacterium tuberculosis (M.tb) 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.tb 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 in vitro. Methods and results: Systems and chemical biology were used to derive parallel correlation of "M.tb 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.tb 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.tb 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+ ½ 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. Conclusion: TMKmyc assays may be useful for reducing the time-lines to positive identification of Mycobacterium tuberculosis (M.tb) cultures, thereby accelerating disease diagnosis and drug resistance profiling. Incorporating "chemiluminiscent or fluorescent" strategies may enable "photodetection of TMKmyc changes" and hence automation of the entire assay.