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

Wayengera M - Theor Biol Med Model (2009)

Bottom Line: Mycobacterial thymidylate kinase (TMKmyc) is a phosphotransferase critical for the synthesis of the thymidine triphosphate precursor necessary for M.tb DNA synthesis.Two drug resistance profiling scenarios are offered: isoniazid (INH) resistance and sensitivity.According to our current model, the areas under TMKmyc curves (AUC, calculated as the integral integral(at2+ bt + c)dt or approximately 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Pathology, Department of Pathology, School of Biomedical Sciences, College of Health Sciences, Makerere University, PO Box 7072, Kampala, Uganda. wmisaki@yahoo.com

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 integral(at2+ bt + c)dt or approximately 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.

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 "photo-detection of TMKmyc changes" and hence automation of the entire assay.

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Projected patterns of variation of TMKmyc curves in the drug sensitive and resistance scenario against a normal TMKmyc curve. This figure shows theorized patterns of variation for TMKmyc curves in the drug sensitive (A) and resistance (B) scenarios against a background normal TMKmyc curve. Note that, from this illustration, one could say that the extent of resistance (or number of mutant phenotypes) present in scenario B is the difference between the areas under both curves shaded light yellow. Note also that for any drug resistance profiling based on tmk assays, resistance may be viewed as inversely correlated to TMKmyc levels, while sensitivity is directly proportional to levels of TMK. The difference between TMKmyc levels of test versus standard TMKmyc curve is denoted by a window "wj" or wayengera-joloba lag, the numerical value of which is "WJ". Overall, drug sensitivity ~WJ and resistance ~1/WJ.
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Figure 4: Projected patterns of variation of TMKmyc curves in the drug sensitive and resistance scenario against a normal TMKmyc curve. This figure shows theorized patterns of variation for TMKmyc curves in the drug sensitive (A) and resistance (B) scenarios against a background normal TMKmyc curve. Note that, from this illustration, one could say that the extent of resistance (or number of mutant phenotypes) present in scenario B is the difference between the areas under both curves shaded light yellow. Note also that for any drug resistance profiling based on tmk assays, resistance may be viewed as inversely correlated to TMKmyc levels, while sensitivity is directly proportional to levels of TMK. The difference between TMKmyc levels of test versus standard TMKmyc curve is denoted by a window "wj" or wayengera-joloba lag, the numerical value of which is "WJ". Overall, drug sensitivity ~WJ and resistance ~1/WJ.

Mentions: Two drug resistance profiling scenarios were modelled, isoniazid (INH) resistance and sensitivity. In the INH resistance scenario, we projected that although the dose of INH in LJ is optimal for stopping M.tb proliferation, tubercle bacilli will continue to grow. Because the time-line for observing these growth changes (colonies/unit) is long, we believe that predictions of such growth changes based on TMKmyc assays (which parallel growth changes but occur earlier) may be a relatively quick way of determining drug resistance. In the INH sensitive scenario, since the drug inhibits or slows microbial growth, these changes may equally be predicted by TMKmyc levels recorded in the presence of the drug (growth is bound to be absent or at least much lower than in the absence of drug or in the drug resistance scenario) (see Fig. 4A and 4B for illustrations of INH sensitive and resistant scenarios respectively).


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

Wayengera M - Theor Biol Med Model (2009)

Projected patterns of variation of TMKmyc curves in the drug sensitive and resistance scenario against a normal TMKmyc curve. This figure shows theorized patterns of variation for TMKmyc curves in the drug sensitive (A) and resistance (B) scenarios against a background normal TMKmyc curve. Note that, from this illustration, one could say that the extent of resistance (or number of mutant phenotypes) present in scenario B is the difference between the areas under both curves shaded light yellow. Note also that for any drug resistance profiling based on tmk assays, resistance may be viewed as inversely correlated to TMKmyc levels, while sensitivity is directly proportional to levels of TMK. The difference between TMKmyc levels of test versus standard TMKmyc curve is denoted by a window "wj" or wayengera-joloba lag, the numerical value of which is "WJ". Overall, drug sensitivity ~WJ and resistance ~1/WJ.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2662806&req=5

Figure 4: Projected patterns of variation of TMKmyc curves in the drug sensitive and resistance scenario against a normal TMKmyc curve. This figure shows theorized patterns of variation for TMKmyc curves in the drug sensitive (A) and resistance (B) scenarios against a background normal TMKmyc curve. Note that, from this illustration, one could say that the extent of resistance (or number of mutant phenotypes) present in scenario B is the difference between the areas under both curves shaded light yellow. Note also that for any drug resistance profiling based on tmk assays, resistance may be viewed as inversely correlated to TMKmyc levels, while sensitivity is directly proportional to levels of TMK. The difference between TMKmyc levels of test versus standard TMKmyc curve is denoted by a window "wj" or wayengera-joloba lag, the numerical value of which is "WJ". Overall, drug sensitivity ~WJ and resistance ~1/WJ.
Mentions: Two drug resistance profiling scenarios were modelled, isoniazid (INH) resistance and sensitivity. In the INH resistance scenario, we projected that although the dose of INH in LJ is optimal for stopping M.tb proliferation, tubercle bacilli will continue to grow. Because the time-line for observing these growth changes (colonies/unit) is long, we believe that predictions of such growth changes based on TMKmyc assays (which parallel growth changes but occur earlier) may be a relatively quick way of determining drug resistance. In the INH sensitive scenario, since the drug inhibits or slows microbial growth, these changes may equally be predicted by TMKmyc levels recorded in the presence of the drug (growth is bound to be absent or at least much lower than in the absence of drug or in the drug resistance scenario) (see Fig. 4A and 4B for illustrations of INH sensitive and resistant scenarios respectively).

Bottom Line: Mycobacterial thymidylate kinase (TMKmyc) is a phosphotransferase critical for the synthesis of the thymidine triphosphate precursor necessary for M.tb DNA synthesis.Two drug resistance profiling scenarios are offered: isoniazid (INH) resistance and sensitivity.According to our current model, the areas under TMKmyc curves (AUC, calculated as the integral integral(at2+ bt + c)dt or approximately 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Pathology, Department of Pathology, School of Biomedical Sciences, College of Health Sciences, Makerere University, PO Box 7072, Kampala, Uganda. wmisaki@yahoo.com

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 integral(at2+ bt + c)dt or approximately 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.

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 "photo-detection of TMKmyc changes" and hence automation of the entire assay.

Show MeSH
Related in: MedlinePlus