Transcriptomic and proteomic analyses of Mycobacterium tuberculosis strains isolated from tuberculous meningitis patients.

Background: Tuberculous meningitis (TBM) is caused by the dissemination of Mycobacterium tuberculosis (MTB) from the primary site of infection to the central nervous system. However, the bacterial factors associated with the pathogenesis of TBM remain unclear. This study employed transcriptomic and proteomic methods to comprehensively analyze the changes in genes and proteins and their associated pathways in MTB strains isolated from cerebrospinal fluid (CSF) of TBM and sputum of pulmonary TB (PTB) cases. Methodology: Five MTB strains were subjected to OMICs (transcriptomic and proteomic) analysis. Among five MTB strains, two were isolated from CSF and sputum samples of the same patient with PTB and TBM infections, one from the sputum of a different PTB patient, and a strain obtained from the CSF of another TBM patient. H37Rv was used as a reference strain. The reliability of transcriptomic results was validated by real time polymerase chain reaction with selected genes from 100 MTB isolates (CSF, 50 and sputum, 50). Results: The transcriptomic study revealed that overlapping differentially expressed genes of MTB strains isolated from TBM patients showed featured enrichment in benzoate degradation, lysine degradation, tryptophan metabolism, fatty acid degradation, ATP binding cassette transporters, microbial metabolism in diverse environments, biosynthesis of antibiotics, and metabolic pathways. Eleven genes were upregulated, and four were downregulated in MTB strains isolated from TBM compared to PTB. From proteomic analysis, we identified three candidate proteins belonging to plasminogen binding proteins (PBP) (enolase, dnaK, and isocitrate lyase 1) that were significantly upregulated in MTB strains isolated from TBM. Conclusion: Overall, the transcriptomic and proteomic analyses provided an important base for understanding the unique feature of TBM pathogenesis. To the best of our knowledge, this is the first report highlighting the importance of PBPs on TBM pathogenesis.

Diagnostic performance of real time PCR for the detection of Mycobacterium tuberculosis in cerebrospinal fluid samples.

PURPOSE: We aimed this study to standardize real time - polymerase chain reaction (RT-PCR) for the detection of Mycobacterium tuberculosis (Mtb) in cerebrospinal fluid (CSF) samples and compare its diagnostic performance with GeneXpert (Xpert), Mycobacteria Growth Indicator Tube (MGIT) and Multiplex PCR (MPCR) for tuberculous meningitis (TBM). METHODOLOGY: A total of 217 CSF samples were obtained from patients with suspected TBM during the study period between January 2019 and December 2021. The optimal cycle threshold (CT) of RT-PCR was determined by comparing different gene targets of Mtb (IS6110, 16SrRNA, HSP65 and Ag85B). Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) was determined for RT-PCR, Xpert, MGIT960 and MPCR. Diagnostic accuracy of these assays was compared by using clinical diagnosis as reference standard. RESULTS: IS6110RT-PCR was found to be highly sensitive as compared to other gene targets. Sensitivities of IS6110RT-PCR, MPCR, Xpert and MGIT against a reference standard of definite, probable and possible TBM were 36.7%, 21.1%, 16.7% and 6.7%, respectively; specificities were 97.6%, 100%, 100% and 100%, respectively. Xpert, RT-PCR, MPCR and MGIT960 detected 6.91% (n = 15), 5.99% (n = 13), 5.99% (n = 13) and 2.76% (n = 6) of definite TBM, respectively. RT-PCR detected 6.45% (n = 14) and 2.76% (n = 6) of possible TBM and probable TBM, respectively and MPCR detected 1.38% (n = 3) of possible and probable TBM each. CONCLUSION: IS6110RT-PCR is highly sensitive for primary screening of suspected TB cases, which may help clinicians to start appropriate patient's treatment with clinical suspicion of TBM.

Prevalence and speciation of non-tuberculous mycobacteria among pulmonary and extrapulmonary tuberculosis suspects in South India.

BACKGROUND AND OBJECTIVES: Non tuberculous mycobacteria (NTM) is an emerging opportunistic pathogen increasing globally and indistinguishable from tuberculosis (TB), which remains a challenge particularly in developing countries. This study aimed to identify the prevalence and diversity of NTM among both pulmonary TB (PTB) and extrpulmonary TB (EPTB) clinical isolates from south India. METHODOLOGY: A total of 7633 specimens from TB suspects (PTB, n = 4327 and EPTB, n = 3306) were collected during the study period (July 2018-March 2020) in a tertiary care hospital. The study specimens were subjected to Ziehl Neelsen (ZN) staining and Auramine phenol (AP) staining followed by Lowenstein-Jensen (LJ) and mycobacteria growth indicator tube (MGIT) culture. The MPT64 immunochromatographic test (ICT) was performed among mycobacterial cultures and ICT negative isolates were subjected to Line Probe Assay (LPA). In addition, 53 (PTB, 48 and EPTB, 5) NTM MGIT positive cultures were collected from Intermediate Reference Laboratory (IRL), Puducherry and subjected to LPA for speciation. RESULTS: Of the 7633 TB suspects, 0.6% were diagnosed as NTM diseases and 5.5% with Mycobacterium tuberculosis (MTBC). NTM infection was observed among 0.7% (31/4327) of PTB and 0.4% (14/3306) of EPTB. MTBC was detected among 6.1% (264/4327) of PTB and 4.6% (153/3306) of EPTB. Among 98 NTM cultures, 80.6% of isolates were recovered from PTB and 19.4% from EPTB specimens. Among pulmonary specimens, Mycobacterium intracellulare (26.6%), Mycobacterium abscessus (17.7%) and Mycobacterium kansasii (12.7%) were the most frequently detected species, while Mycobacterium intracellulare (21.1%), Mycobacterium scrofulaceum (15.8%) and Mycobacterium fortuitum (10.5%) were common in extrapulmonary specimens. CONCLUSION: The frequency of NTM infection among TB suspects was low at a South Indian tertiary care hospital. The most predominant NTM species isolated from both pulmonary and extrapulmonary specimens was M. intracellulare.

Molecular diagnosis, genetic diversity and drug sensitivity patterns of Mycobacterium tuberculosis strains isolated from tuberculous meningitis patients at a tertiary care hospital in South India.

Tuberculous meningitis (TBM) is the most severe form of Mycobacterium tuberculosis (Mtb) infection in humans and is a public health concern worldwide. We evaluated the performance of GeneXpert MTB/RIF (GeneXpert) for the diagnosis of TBM. In addition, genetic diversity and drug susceptibility profiling of Mtb strains isolated from TBM patients were also investigated. A total of 293 TBM suspected cerebrospinal fluid (CSF) samples were collected and subjected to GeneXpert and Mycobacterial Growth Indicator Tube (MGIT 960) culture, respectively. Sensitivity and specificity of GeneXpert was 72.7% and 98.5%, respectively by using MGIT 960 as a gold standard (GeneXpert (n = 20, 6.8%) vs MGIT 960 (n = 22, 7.5%)). All Mtb positive cultures were subjected to 24-locus Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) typing, Line probe assay (LPA) and MGIT 960- Drug Susceptibility Testing (DST). The rpoB gene was amplified and sequenced for selected isolates. Among our TBM patients, East African Indian (EAI) lineage (n = 16, 72.7%) was most predominant followed by Beijing (n = 3, 13.6%), S-family (n = 2, 9.1%) and Delhi/CAS (n = 1, 4.5%). Three Mtb strains were found to be Isoniazid (INH) resistant by MGIT 960; however LPA revealed that two strains were INH resistant and one strain was multi drug resistant (MDR) (Resistant to Isoniazid and Rifampicin (RIF)). We identified rifampicin resistant isolate with the mutation D516F in rifampicin resistance-determining region (RRDR) and observed discordant results between LPA, GeneXpert and MGIT 960. In addition, GeneXpert showing false RIF resistance was identified (no mutation in RRDR). We conclude that GeneXpert is useful for the diagnostic confirmation of TBM; however a GeneXpert negative sample should be subjected to MGIT 960 culture or LPA to rule out TBM. EAI lineage was the most predominant among TBM patients in South India and associated with drug resistance. The discordance between GeneXpert, MGIT 960 and LPA with respect to rifampicin resistance has to be ruled out to avoid TB treatment failure or relapse.