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Application of a whole blood mycobacterial growth inhibition assay to study immunity against Mycobacterium tuberculosis in a high tuberculosis burden population.

ABSTRACT: The determinants of immunological protection against Mycobacterium tuberculosis (M.tb) infection in humans are not known. Mycobacterial growth inhibition assays have potential utility as in vitro surrogates of in vivo immunological control of M.tb. We evaluated a whole blood growth inhibition assay in a setting with high burden of TB and aimed to identify immune responses that correlate with control of mycobacterial growth. We hypothesized that individuals with underlying M.tb infection will exhibit greater M.tb growth inhibition than uninfected individuals and that children aged 4 to 12 years, an age during which TB incidence is curiously low, will also exhibit greater M.tb growth inhibition than adolescents or adults. Neither M.tb infection status, age of the study participants, nor M.tb strain was associated with differential control of mycobacterial growth. Abundance and function of innate or T cell responses were also not associated with mycobacterial growth. Our data suggest that this assay does not provide a useful measure of age-associated differential host control of M.tb infection in a high TB burden setting. We propose that universally high levels of mycobacterial sensitization (through environmental non-tuberculous mycobacteria and/or universal BCG vaccination) in persons from high TB burden settings may impart broad inhibition of mycobacterial growth, irrespective of M.tb infection status. This sensitization may mask the augmentative effects of mycobacterial sensitization on M.tb growth inhibition that is typical in low burden settings.

SUBMITTER: Baguma R

PROVIDER: S-EPMC5590973 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Tuberculosis (TB), caused by Mycobacterium tuberculosis bacteria, is a leading infectious cause of mortality worldwide, including in Pakistan. Drug resistant M. tuberculosis is an emerging threat for TB control, making it important to detect the underlying genetic mutations, and thereby inform treatment decision making and prevent transmission. Whole genome sequencing has emerged as the new diagnostic to reliably predict drug resistance within a clinically relevant time frame, and its deployment will have the greatest impact on TB control in highly endemic regions. To evaluate the mutations leading to drug resistance and to assess for evidence of the transmission of resistant strains, 81 M. tuberculosis samples from Khyber Pakhtunkhwa province (North West Pakistan) were subjected to whole genome sequencing and standard drug susceptibility testing for eleven anti-TB drugs. We found the majority of M. tuberculosis isolates were the CAS/Delhi strain-type (lineage 3; n = 57; 70.4%) and multi-drug resistant (MDR; n = 62; 76.5%). The most frequent resistance mutations were observed in the katG and rpoB genes, conferring resistance to isoniazid and rifampicin respectively. Mutations were also observed in genes conferring resistance to other first and second-line drugs, including in pncA (pyrazinamide), embB (ethambutol), gyrA (fluoroquinolones), rrs (aminoglycosides), rpsL, rrs and giB (streptomycin) loci. Whilst the majority of mutations have been reported in global datasets, we describe unreported putative resistance markers in katG, ethA (ethionamide), gyrA and gyrB (fluoroquinolones), and pncA. Analysis of the mutations revealed that acquisition of rifampicin resistance often preceded isoniazid in our isolates. We also observed a high proportion (17.6%) of pre-MDR isolates with fluoroquinolone resistance markers, potentially due to unregulated anti-TB drug use. Our isolates were compared to previously sequenced strains from Pakistan in a combined phylogenetic tree analysis. The presence of lineage 2 was only observed in our isolates. Using a cut-off of less than ten genome-wide mutation differences between isolates, a transmission analysis revealed 18 M. tuberculosis isolates clustering within eight networks, thereby providing evidence of drug-resistant TB transmission in the Khyber Pakhtunkhwa province. Overall, we have demonstrated that drug-resistant TB isolates are circulating and transmitted in North West Pakistan. Further, we have shown the usefulness of whole genome sequencing as a diagnostic tool for characterizing M. tuberculosis isolates, which will assist future epidemiological studies and disease control activities in Pakistan.

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Application of a whole blood mycobacterial growth inhibition assay to study immunity against Mycobacterium tuberculosis in a high tuberculosis burden population.

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