Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-017-2649-y) contains supplementary material, which is available to authorized users.

Methods and approaches to disease mechanisms using systems kinomics

“…In addition, the field of infectious diseases has been impacted by the discovery of kinases that are unique to infectious organisms; this can allow for rapid, sensitive and specific diagnosis testing as was recently described by Wayengera et al. for Mycobacterium [ 92 ]. Some kinases are well-known biomarkers of disease and many studies have actively screened serum, tissue and urine samples to identify new markers.…”

Classification and characterisation of extracellular vesicles‐related tuberculosis subgroups and immune cell profiles

“…Tregs, a specialized subset of T cells, play an important role in preventing the development of autoimmune diseases by suppressing harmful immune cell activity 38 . Monocytes are the major innate immune cells at the onset of Mtb infection and protect the host from intracellular pathogens 39,40 . Monocyte heterogeneity and differentiation into monocyte‐derived dendritic cells or monocyte‐derived macrophages have established a link between innate and adaptive immune responses 41 .…”

“… 38 Monocytes are the major innate immune cells at the onset of Mtb infection and protect the host from intracellular pathogens. 39 , 40 Monocyte heterogeneity and differentiation into monocyte‐derived dendritic cells or monocyte‐derived macrophages have established a link between innate and adaptive immune responses. 41 Mast cells play a critical role in the establishment and perpetuation of the inflammatory response leading to granuloma generation during tuberculosis.…”

Instrumental analytical tools for mycobacteria characterisation

“…As for M. tuberculosis identification, usually, these M. tuberculosis-secreted antigens such as mycobacterial protein from species tuberculosis-64 (MPT64), early secreted antigenic target 6 kDa protein (ESAT-6), 10 kDa culture filtrate protein (CFP-10), and antigen 85B (Ag85B) serve as markers for the diagnosis (Mustafa et al 2006;Zhang et al 2015). Enzyme-linked immunosorbent assay (ELISA) is the most common immunoassay for mycobacteria identification from clinical samples (Verstijnen et al 1991;Ahmad et al 1995;Attallah et al 2003;Wayengera et al 2020). During ELISA, an inoculum of a sample is introduced to a well coated with primary antibodies, which correspond to the antigen of interest.…”