Abstract
BackgroundLaboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read—thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB.Methods and resultsModelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × 10−4–1 × 10−5 (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × 105) patient sample.ConclusionsDetection of TMKmt expressed mRNA and Ag offers us opportune for instant diagnosis of M. tb in sputum, and reduction of timelines for positive pathogen detection in cultures to within 3 h.
Highlights
Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture
Modelling of Mycobacterium tuberculosis thymidylate kinase (TMKmt) secretion in vitro using purified recombinant TMKmt cloned and expressed in E. coli In order to model the in vitro expression of the target M. tb growth and proliferation dependent antigen or biomarker (TMKmt), sodium dodecyl sulphate (SDS) Poly-Acrylamide Gel Electrophoresis (PAGE) and MALDI-TOF analysis were done on pooled fractions of urea dissolved precipatates from centrifugal cell-lysates of clones of E. coli filtered through a 0.22 uM sterilizing filter revealed that expressed rTMKmt has a molecular weight of 22, 635 and Isoelectric point of 7.66
A rise in rTMKmt antigen levels was observed by MALDI-TOF at 3 h post-innoculation among in vitro cultures of the E. coli BL21 (DE) strain transformed with pET30a plasmid vector transducing the rTMKmt gene
Summary
Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. Application of these nucleic acids amplification tests (NAATS) either directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as and reduce time-lines for positive culture detection, the need for microscopy or culture remains [3]. Previous efforts to reduce timelines for M. tb culture have been based on radio-labelled carbon, acridinium-ester labelled DNA probes and other nucleic acids amplification tests (NAATS) [6,7,8] These assays are nonethless, laborous to precisely undertake or replicate, and most take over 5 days to yield positive result. The low sensitivity of several immuno-assays for detecting M. tb in sputum at low bacillary loads (as is the case seen in HIV coinfection), has relegated several potentially emerging assays such as the Inducible Protein of 10Kda(IP-10), lipoarabinomannan (LAM) antigen and Adenosine-deaminase (ADA), and protein MPT64 (Capilia®) test to use strictly for purposes of reducing timelines of positive cultures [9,10,11,12,13]
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