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.

Alcohol use and tuberculosis clinical presentation at the time of diagnosis in Puducherry and Tamil Nadu, India.

SETTING: Alcohol use increases the risk of tuberculosis (TB) disease and is associated with worse outcomes. OBJECTIVE: To determine whether alcohol use affects TB severity at diagnosis in a high-burden setting. DESIGN: Participants were smear-positive people living with TB (PLWTB) in India. Disease severity was assessed as 1) high versus low smear grade, 2) time to positivity (TTP) on liquid culture, 3) chest radiograph cavitation, and 4) percent lung affected. Alcohol use and being at-risk for alcohol use disorders (AUD) were assessed using the AUDIT-C. Univariable and multivariable analyses were conducted. RESULTS: Of 1166 PLWTB, 691 (59.3%) were drinkers; of those, 518/691 (75.0%) were at-risk for AUD. Drinkers had more lung affected than non-drinkers (adjusted mean difference 10.8%, p<0.0001); this was not significant for those at-risk for AUD (adjusted mean difference 3.7%, p = 0.11). High smear grade (aOR 1.0, 95%CI: 0.7-1.4), cavitation (aOR 0.8, 95%CI 0.4-1.8), and TTP (mean difference 5.2 hours, p = 0.51) did not differ between drinkers and non-drinkers, nor between those at-risk and not at-risk for AUD. CONCLUSIONS: A large proportion of PLWTB were drinkers and were at-risk for AUD. Alcohol drinkers had more lung affected than non-drinkers. Studies are needed to explore mechanisms of this association.

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.

Evaluation of factors influencing Mycobacterium tuberculosis complex recovery and contamination rates in MGIT960.

BACKGROUND: Tuberculosis (TB) is a major public health problem worldwide. Contamination rate and poor recovery of Mycobacterium tuberculosis complex (MTBC) in MGIT960 culture may affect the early diagnosis of TB. Evidence is needed to determine the factors associated with contamination rates and MTBC recovery in MGIT960. Hence, we undertook this study to compare the factors influencing MTBC culture positivity and contamination rates in MGIT960 in patients with Pulmonary tuberculosis (PTB). METHODS: A total of 849 sputum samples from newly diagnosed smear-positive TB cases enrolled into the Regional Prospective Observational Research for Tuberculosis India cohort between May 2014 to March 2017 were analyzed. Samples were inoculated into MGIT960 and positive cultures were examined for the presence of MTBC by immunochromatographic test for detection of MPT64 antigen. RESULTS: Of the 849 cases, 811 (95.5%) were culture positive for MTBC, 23 (2.7%) were culture negative and 15 (1.8%) were contaminated. Salivary sputum showed significantly less culture yield compared to mucopurulent/blood stained samples (p = 0.021). Sputum from individuals <20 or ≥60 years showed lower culture yield of 93.9%, compared to those aged 20-59years (98.2%) (p = 0.002). Based on smear grading, culture isolation of MTBC by MGIT960 was 86.1%, 93.6% and 99.5% for negative, scanty and positive (1+/2+/3+) samples, respectively (p ≤ 0.0001). Sputum from HIV negative patients showed higher culture yield, compared to HIV positive patients (p ≤ 0.0001). Chest X-Ray revealed that patient with cavity showed higher culture isolation of MTBC compared to patients without cavity (p = 0.035). Contamination rates were higher in smear negatives (6.0%), compared to scanty (2.1%) and smear positives (1.1%) (p = 0.007). However, delay in transport of the specimen to the laboratory was the only independent factor significantly associated with increase in culture contamination. CONCLUSION: Our results highlight that extremes of age, smear negativity, HIV infection, sputum quality and cavitation significantly influence the culture yield of MTBC, whereas transport duration and smear grading affected the contamination rates in MGIT960. Hence, addressing these factors may improve the diagnostic performance of MGIT960.

Diagnostic performance of real time PCR and MALDI-TOF in the detection of nontuberculous mycobacteria from clinical isolates.

This study aimed to evaluate the performance of real-time PCR (qPCR) and MALDI-TOF for accurate and timely detection of nontuberculous mycobacterium (NTM) from clinical isolates. We collected fifty NTM suspected Mycobacteria Growth Indicator Tube (MGIT) cultures and analysed the diagnostic performance of qPCR and VITEK MS using Line Probe Assay (LPA) GenoType CM (Common Mycobacteria) as gold standard. The qPCR assays targeting 16S rRNA, ITS and IS6110 genes were developed for the identification of NTM and Mycobacterium tuberculosis complex (MTBC). LPA GenoType CM, a PCR technique targeting 23S rRNA gene, followed by reverse hybridization and line probe technology identified 90% of Mycobacterium species including M. fortuitum (16%,n = 8), M. intracellulare (10%,n = 5), M. gordonae (10%,n = 5), M. xenopi (4%,n = 2), M. scrofulaceum (4%,n = 2), Mycobacterium additional species (AS) (32%,n = 16) and MTBC (14%,n = 7), qPCR detected 80% of Mycobacterium species (NTM, 66% (n = 33) and MTBC, 14% (n = 7)) and MALDI-TOF, 52% (M. fortuitum (12%,n = 6), M. intracellulare (10%, n = 5), M. simiae (8%,n = 4), M. gordonae (8%,n = 4), and MTBC (14%,n = 7)). Sensitivity of qPCR and MALDI-TOF was 88.9% and 57.8%, respectively with 100% specificity. The combination of qPCR and MALDI-TOF remains an appropriate test for timely diagnosis of Mycobacterium species. This may eventually assist to detect the cases that may have been missed by phenotypic tests and enhance the NTM diagnosis capability to improve effective patient management.