This paper provides the first ever theoretical and modeling framework to support the view that assays of enzymes involved in the synthesis of DNA precursors may be applied to shorten the time-lines to positive identification of microbial cultures (and possibly of other cell lines including cancers). Specifically: if variations in TMKmyc during the S phase of the tubercle bacillus cell cycle are independent of the time taken by the tubercle in other stages of the cycle, then TMKmyc assays are a potential predictive biomarker of in vitro M.tb growth that may be applied to reducing the time-lines to positive identification of Mycobacterium tuberculosis (M.tb) cultures. There are several existing methods for detecting TB within cultures, but they mostly lack inexpensive platforms for routine use within resource-limited settings where the TB burden is highest . Moreover, many of these emerging TB assays  are not specific for detecting duplicative changes in M.tb DNA. We therefore felt it necessary to establish a highly specific M.tb DNA duplicative assay that may be mounted on inexpensive technology platforms that are currently widely used within resource-limited settings, such as lateral flow immunochromatography.
Overall, tmk-based assays are therefore likely to be cheap but still serve the purpose of accelerating diagnosis and drug resistance profiling of M.tb and possibly other infectious diseases in culture. First, we show here why variations in the durations of the G1, S and G2 phases of the M.tb cell cycle may account for a longer 'culture time' for M.tb (see Fig. 1 for illustration) than for other microbes. In principle, the generation time of any cell is determined by the time it spends in G1 and G2 . Some cells, however, are known to go into deeper states of latency or dormancy (designated G0 or even G00), only reverting to the G1 stage when a need for reproduction, proliferation or regeneration arises . Various environmental factors are known to initiate such deeper states of dormancy in microbes and eukaryotes alike including low temperature, absence of nutrients and low oxygenation states ; though the full range of factors that determine why tubercle bacilli go into related states of latency in vivo are yet to be fully established [1, 21]. It is therefore unclear whether these deeper states of dormancy explain the slowly-growing nature of M.tb (especially since host immune factors also seem to play a significant role in arresting M.tb proliferation in vivo ). What is evident from our theoretical models, though, is that experiments aimed at establishing the actual values of the durations tG1, ts and tG2 for various microbes including M.tb may yield significant knowledge about the rate-limiting steps in their "growth" activity when cultured. For instance, using our proposed TMKmyc antigen capture assays, together with radio-labeled phosphorylated (dTMP
) nucleotide bases as metabolites, the time ts required for DNA duplication by the tubercle bacillus may be estimated.
, from the theoretical model that the timeline for the G1
phase of the M.tb
cell cycle (tG1
) is constant at 2, 4, 6 or 8 days, we inferred a parallel correlation of tubercle bacillus "growth changes" with TMK levels in each scenario (although changes in TMKmyc levels were predicted to occur much earlier, as noted in Figure 3B
). These predictions are based on the hypothesis that since DNA synthesis precedes all cellular division, and TMKmyc is required to create the dTTP necessary for DNA synthesis, monitoring the levels of TMKmyc may predict actual growth changes well before such changes occur. Note, however, that the actual durations of the constituent phases of the M.tb
cell cycle remain uncertain as stated above. One may therefore be justified in arguing that this presumption regarding the timing of the G1
phase of the M.tb
cell cycle at days 2, 4,.... etc. is currently speculative, given that it is not yet clear which of the two stages, G1
, constitutes the larger proportion of the generation time of the tubercle bacillus (see Fig. 2
). Nevertheless, using this arbitrary presumption that DNA synthesis may occur only days after inoculation and that it is the G2
phase rather than G1
that accounts for much of the delay in physical manifestation of M.tb
growth changes in culture, we show here that TMKmyc assays could predict TB growth in vitro
as early as day 2 of culture. Moreover, we have successfully used it here to theorize that TMKmyc assays may also be exploited to distinguish drug-sensitive from – resistant forms of tuberculosis in culture (see Fig. 4
). In this context, those test samples with evident rises in TMKmyc levels would in a practical sense be resistant to the drug being tested, whereas isolates where TMKmyc levels fail to rise are sensitive. From the graphs 4 A and B
, one equally notes that: (1) tubercle bacillus drug sensitivity is an inverse factor of TMKmyc while existing resistance profiles are directly correlated to TMKmyc levels; (2) by measuring and quantifying the comparative difference between the areas under the TMKmyc curves (AUC) of resistant and sensitive isolates, it may be possible to quantify the existing levels of resistance (or number of resistant mutants). Assuming that the difference between TMKmyc levels in stationary phase between the tested M.tb
isolates and the standard tmk curve is arbitrarily denoted a window "wj" of which the numerical value is "WJ"; then:
Note that in equations I and II representing tmk variation in sensitive and resistant M.tb isolate scenarios respectively; the |tmk| levels within the test isolate culture are inversely correlated to the numerical value of the wayengera-joloba lag or "wj" window, also denoted "WJ".
The above Area Under Curve (AUC) method for drug resistance profiling offers a means of quantifying drug resistance. Such ability to quantify resistance is especially clinically significant within resource-limited settings where second-line TB chemotherapy options are limited and drugs with minimal evident resistance are useful in combination with other drugs to which the isolate being treated is not resistant. In other words, TMK assays would enable the derivation of "salvage" regimens from the limited available drug options. Supposing that tmk levels follow a quadratic curve as above, parallel to the "S" growth curve of microbes, then this AUC can be calculated as the integral (∫) of the equation: |tmk| = at2+ bt + c or simply 1/3 at3+1/2 bt2+ct. From this equation, it can also be derived that the drug sensitivity of tubercle bacilli is an inverse factor of the AUC while existing resistance profiles are directly correlated to the same Area. However, in order to compare the prevailing resistance profiles between two isolates, one would need first to define the time over which to make the above calculations (say t1-t2); although we recommend that future universal algorithms be based on the AUC between t = 0 and the t-value when the tmk levels first reach the stationery stage. Note also that TMKmyc assays have the advantage of being applicable to all drug scenarios without the need for modifications such as those required for colorimetric redox-indicator methods or nitrate reductase assays, which have been found to be highly sensitive only for rifampicin and isoniazid resistance testing . Moreover, targeted detection of other related microbial enzymes such as HIV thymidine kinase may enable drug resistance profiling for HAART to be conducted within resource-limited settings, where inexpensive platforms for phenotyping or genotyping for drug sensitivity have remained mostly lacking . In the case of the M.tb scenario, whether or not the proposed TMKmyc-based drug profiling assay would yield consistent results relative to several existing rapid drug resistance tests such as phage amplification, or line probe assays such as INNO-LipA Rif.TB(LiPA) or Genotype MTBDR, is a subject that requires future comparative evaluations once TMKmyc assays are in practical use .
Several limitations are evident in our theoretical model, which must be dealt with prior to the actualization of TMKmyc assays as a predictive biomarker for in vitro tubercle bacillus growth. First, it is currently unclear whether secreted TMKmyc is present in the culture medium at levels detectable by MAb(s). Specifically, existing data provide contradictory views about the possible secretory nature of TMKmyc. Munier-Lehmman et al.  have previously shown that TMKmt forms over 30% of all colony filtrate proteins in a strain of E. coli genetically engineered to express TMKmt. However, other studies on host sera using in vivo-induced antigen technology (IVIAT) [23–25] and crossed immunoelectrophoresis (CIE) , though identifying over 11 genes involved in M.tb metabolism, do not categorically list TMKmt among them. Note, however, that the absence to date of methods for specific monitoring of TMKmyc (such as the one we propose) among the latter studies [[23–25] and ] relative to work by Munier-Lehmann and colleagues  may possibly explain these discrepancies. Second, it is assumed that TB cultures constitute a homogenous mixture of actively proliferating cells. Within wild type M.tb cultures, however, this may not be the case as active and dormant tubercle may co-exist. Such heterogeneity of growth among the M.tb in culture may also affect TMKmyc secretory levels. We hold that since qualitative rather than quantitative measures of TMKmyc are required to determine M.tb culture positivity, TMKmyc-based assays are still valid for the purpose of reducing time-lines to the reading of TB cultures. What may be affected is drug resistance profiling, since this requires measurement of the levels of TMK in the culture. Therefore, for purposes of drug resistance profiling, more algorithms may need to be integrated into the TMKmyc assays to render them more standardized and reliable in the face of heterogeneity of M.tb proliferation in vitro. Third, in the absence of in vitro evidence to support the view that TMKmyc may be adequately and specifically detected by monoclonal antibodies, the proposed assays for TMKmyc levels in culture are still speculative. Whether TMKmyc-based assays will perform better than, worse than or the same as existing TB tests  therefore remains elusive and requires future comparative clinical trials. More work needs to be done to affirm the binding affinity and specificity of the antigen capture assays for TMKmt. This work is, however, complicated by the findings by Steingart and colleagues, who recently reported inconsistencies in most commercially available TB serodiagnostics . Fourth, although it is clear that DNA duplication precedes actual microbial division , the duration of this process among various microbes including M.tb is largely unclear. Studies such as that by Sherley and Kelly  are therefore needed for M.tb and other infectious pathogens alike. Overall, if the above-listed potential shortcomings are overcome, the proposed antibody-based TMKmyc assays provide the flexibility to be mounted on a cheap technology platform for use within resource-limited settings. For instance, incorporation of "chemiluminiscent or fluorescent" strategies may enable "photo-detection of TMKmyc changes in culture" and thereby automation of the entire assay. Specifically, if the initiation of light emission is tailored to antigen (TMKmyc) capture, then chemiluminiscent or fluorescent TMKmyc-specific antibodies (CAbs and FAbs respectively) may be integrated within the LJ medium so that visual observation of color change or photometric measurements may be used to detect the levels of TMKmyc secreted. This simple automation could remove the need to conduct laborious quantitative ELISA or antigen capture assays to monitor TMKmyc levels, and thereby allow use by persons without extensive specialized training. Lastly, as TMKmyc is a specific antigen of M.tb , targeted detection of TMKmyc in sputum may provide an alternative strategy for diagnosing symptomatic TB to that based on staining for AFBs.