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.

In-vitro anti-Mycobacterium tuberculosis effect of Eugenol.

BACKGROUND/OBJECTIVES: Mycobacterium tuberculosis, the causative agent of tuberculosis has developed resistance to most of the available antimicrobials. Therefore research on the detection of new antimicrobials against Mycobacterium tuberculosis is needed urgently. Essential oils extracted from plants have been shown to have anti-Mycobacterium tuberculosis effect in in-vitro experiments. Essential oil contains many chemicals and any one or more than one chemical may have the anti-Mycobacterium tuberculosis effect. Eugenol is one such chemical in the essential oil and the anti-Mycobacterium tuberculosis effect of eugenol is investigated. METHODS: The anti-Mycobacterium tuberculosis effect of eugenol was evaluated against H37Rv and twelve clinical isolates of Mycobacterium tuberculosis in the BD BACTEC MGIT instrument using different volumes of eugenol. RESULTS: H37Rv and all the twelve clinical isolates of Mycobacterium tuberculosis were inhibited by eugenol. The minimal inhibitory concentration of H37Rv was 2.5 µl (2.67 mg) and those of the clinical isolates of Mycobacterium tuberculosis ranged from to 2.5 µl (2.67 mg) to 10 µl (10.68 mg). CONCLUSION: Eugenol has anti-Mycobacterium tuberculosis effect in the in-vitro BD BACTEC MGIT method.

Development and Validation of a Parsimonious Tuberculosis Gene Signature Using the digital NanoString nCounter Platform.

BACKGROUND: Blood-based biomarkers for diagnosing active tuberculosis (TB), monitoring treatment response, and predicting risk of progression to TB disease have been reported. However, validation of the biomarkers across multiple independent cohorts is scarce. A robust platform to validate TB biomarkers in different populations with clinical end points is essential to the development of a point-of-care clinical test. NanoString nCounter technology is an amplification-free digital detection platform that directly measures mRNA transcripts with high specificity. Here, we determined whether NanoString could serve as a platform for extensive validation of candidate TB biomarkers. METHODS: The NanoString platform was used for performance evaluation of existing TB gene signatures in a cohort in which signatures were previously evaluated on an RNA-seq dataset. A NanoString codeset that probes 107 genes comprising 12 TB signatures and 6 housekeeping genes (NS-TB107) was developed and applied to total RNA derived from whole blood samples of TB patients and individuals with latent TB infection (LTBI) from South India. The TBSignatureProfiler tool was used to score samples for each signature. An ensemble of machine learning algorithms was used to derive a parsimonious biomarker. RESULTS: Gene signatures present in NS-TB107 had statistically significant discriminative power for segregating TB from LTBI. Further analysis of the data yielded a NanoString 6-gene set (NANO6) that when tested on 10 published datasets was highly diagnostic for active TB. CONCLUSIONS: The NanoString nCounter system provides a robust platform for validating existing TB biomarkers and deriving a parsimonious gene signature with enhanced diagnostic performance.

In-vitro anti-Mycobacterium tuberculosis effect of essential oil of Ocimum sanctum L. (Tulsi/Basil) leaves.

BACKGROUND/OBJECTIVES: Mycobacterium tuberculosis, the causative agent of tuberculosis has developed resistance to most of the available antimicrobials. Consequently, it is difficult to cure all the patients with tuberculosis and in future, the incidence of tuberculosis by drug resistant M. tuberculosis is likely to increase, worldwide. Therefore detection and development of new antimicrobials against M. tuberculosis is needed urgently. METHODS: Essential oil from the leaves of Ocimum sanctum L (Tulsi/Basil) was obtained by hydro distillation. The anti-mycobacterial effect of essential oil was evaluated against H37Rv and nine clinical isolates of M. tuberculosis in the BD BACTEC MGIT instrument using different volumes of essential oil. RESULTS: The essential oil inhibited the growth of H37Rv and all the nine clinical isolates of M. tuberculosis. The minimal inhibitory concentration of H37Rv was 3 µl (2.931 µg) and those of the clinical isolates of M. tuberculosis ranged from 1.5 µl (1.4655 µg) to 6 µl (5.862 µg). CONCLUSION: The Essential oil from the leaves of O. sanctum L.(Tulsi/Basil) has anti-M. tuberculosis effect in the in-vitro BD BACTEC MGIT method.

Prevalence and factors associated with multidrug-resistant tuberculosis in South India.

India accounts for about one-fourth of the global burden of MDR-TB. This study aims to assess  the prevalence and factors associated with tuberculosis drug resistance among patients from South India. MTBDRplus assay and MGIT liquid culture performed on 20,245 sputum specimens obtained from presumptive MDR-TB cases during a six-year period from 2013 to 2018 were analyzed retrospectively. Univariate and multivariate logistic regression analysis was carried out to evaluate factors associated with MDR, Rifampicin mono-resistance, and Isoniazid mono-resistance. MDR, Rifampicin mono- resistant and Isoniazid mono-resistant TB were  found in 5.4%, 2.5%, and 11.4% cases of presumptive MDR-TB, respectively. Based on the rpoB gene, true resistance, hetero-resistance, and inferred resistance to Rifampicin was found in 38%, 29.3%, and 32.7% of the 1582 MDR cases, respectively. S450L (MUT3) was the most common rpoB mutation present in 59.4% of the Rifampicin resistant cases. Of the 3390 Isoniazid resistant cases, 72.5% had mutations in the katG gene, and 27.5% had mutations in the inhA gene. True resistance, heteroresistance, and inferred resistance accounted for 42.9%, 22.2%, and 17.3% of the 2459 katG resistant cases, respectively. True resistance, heteroresistance, and inferred resistance for the inhA gene were found in 54.5%, 40.7%, and 4.7% cases, respectively. MDR-contact (AOR 3.171 95% CI: 1.747-5.754, p-0.000) treatment failure (AOR 2.17595% CI: 1.703-2.777, p-0.000) and female gender (AOR 1.315 95% CI: 1.117-1.548, p-0.001), were positively associated with MDR-TB. Previous TB treatment did not show a significant positive association with MDR (AOR 1.113 95% CI: 0.801-1.546, p-0.523). Old age (AOR 0.994 95% CI: 0.990-0.999, p-0.023) and HIV seropositivity (AOR 0.580 95% CI: 0.369-0.911, p-0.018) were negatively associated with MDR-TB. Although Rifampicin mono-resistance had a positive association with treatment failure (AOR 2.509 95% CI: 1.804-3.490, p < .001), it did not show any association with previous TB treatment (AOR 1.286 95% CI: 0.765-2.164, p-0.342) or with history of contact with MDR-TB (AOR 1.813 95% CI: 0.591-5.560, p-0.298). However, INH mono-resistance showed a small positive association with the previous history of treatment for TB (AOR 1.303 95% CI: 1.021-1.662, p-0.033). It was also positively associated (AOR 2.094 95% CI: 1.236-3.548, p-0.006) with MDR-TB contacts. Thus INH resistance may develop during treatment if compliance has not adhered too and may be easily passed on to the contacts while Rifampicin resistance is probably due to factors other than treatment compliance. MDR-TB, i.e. resistance to both Rifampicin and Isoniazid, is strongly correlated with treatment failure, spread through contact, and not to treatment compliance. The temporal trend in this region shows a decrease in MDR prevalence from 8.4% in 2015 to 1.3% in 2018. A similar trend is observed for Rifampicin mono-resistance and Isoniazid mono-resistance, pointing to the effectiveness of the TB control program. The higher proportion of inferred resistance observed for Rifampicin compared with INH may indicate a surfeit of mechanisms that enable rifampicin resistance. Association of MDR-TB with age, gender, and HIV status suggest the role of the immune system in the emergence of the MDR phenotype.

Predictors of Loss to Follow-Up among Men with Tuberculosis in Puducherry and Tamil Nadu, India.

Identifying predictors of loss to follow-up (LTFU; treatment lapse ≥ 2 months) among people with tuberculosis (TB) may assist programmatic efforts in controlling the spread of TB. Newly diagnosed smear-positive TB patients were enrolled in the Regional Prospective Observational Research for TB study in Puducherry and Tamil Nadu, India. Treatment records were used to identify LTFU of those who were enrolled from May 2014 through December 2017. This nested case-control study evaluated male TB patients. Predictors were assessed using multivariable logistic regression. Of 425 men with TB, 82 (19%) were LTFU. In the adjusted analyses of males, divorced/separated marital status (adjusted odds ratio [aOR] 3.80; 95% CI: 1.39-10.38) and at-risk alcohol use (aOR 1.92; 95% CI: 1.12-3.27) were significant predictors for increased risk of LTFU, and diabetes was a significant predictor for decreased risk of LTFU (aOR 0.52; 95% CI: 0.29-0.92). Of 53 men with recorded date of last treatment visit, 23 (43%) and 43 (81%) had LTFU within the first 2 and first 4 months of treatment, respectively. Addressing at-risk alcohol use and providing more intensive follow-up could lead to improved treatment completion.

Effect of malnutrition on radiographic findings and mycobacterial burden in pulmonary tuberculosis.

BACKGROUND: The relationship between malnutrition and tuberculosis (TB) severity is understudied. We investigated the effect of malnutrition on radiographic findings and mycobacterial burden. METHODS: Subjects included newly diagnosed, smear-positive, culture-confirmed, pulmonary TB cases enrolled in the Regional Prospective Observational Research for TB (RePORT) cohort. Multivariate regression models were used to evaluate the relationship at start of treatment between body mass index (BMI) and chest radiograph (CXR) findings of cavitation and percentage of lung affected and mycobacterial growth indicator tube (MGIT) time to positive (TTP). Severe malnutrition was defined as BMI<16 kg/m2, moderate malnutrition as 16-18.4kg/m2, and "normal"/overweight as ≥18.5 kg/m2. RESULTS: Of 173 TB cases with chest x-ray data, 131 (76%) were male. The median age was 45 years (range 16-82); 42 (24%) had severe malnutrition and 58 (34%) moderate malnutrition. Median percentage of lung affected was 32% (range 0-95), and 132 (76%) had cavitation. Individuals with severe malnutrition had, on average, 11.1% [95% CI: 4.0-13.3] more lung affected, compared to those with normal BMI, controlling for diabetes and cavitation. In multivariable analyses, cases with severe malnutrition had a 4.6-fold [95% CI, 1.5-14.1] increased odds of cavitation compared to those with normal BMI, controlling for smoking. Median MGIT TTP was 194.5 hours. Neither severe (aRR 0.99; 95% CI, 0.9-1.2) nor moderate (aRR 0.97; 95% CI, 0.8-1.1) malnutrition was associated with MGIT TTP. CONCLUSION: We found that malnutrition was associated with increased extent of disease and cavitation on CXR. These findings may reflect the immunomodulatory effect of malnutrition on pulmonary pathology.

Prevalence of mutations in genes associated with isoniazid resistance in Mycobacterium tuberculosis isolates from re-treated smear-positive pulmonary tuberculosis patients: A meta-analysis.

OBJECTIVES: The prevalence of isoniazid (INH) monoresistance is high in India. In this study, molecular epidemiological characteristics associated with INH resistance mutations in Mycobacterium tuberculosis in codon katG315 and in the promoter region of the inhA gene were investigated. METHODS: Sputum specimens of smear-positive tuberculosis (TB) patients were subjected to GenoType MTBDRplus assay to identify katG and inhA mutations in M. tuberculosis. In addition to the current study, 17 publications assessed 14100 genotypically resistant M. tuberculosis isolates for mutations in katG inclusive of codon 315. RESULTS: In total, 1821 (11.8%) of 15438 INH-resistant strains had detectable mutations: 71.0% in katG315 and 29.0% in the inhA promoter region. The prevalence of IHN monoresistance was 89.1% in the economically-active age group, 0.4% in the paediatric age group and 10.5% in those aged >60years; the rate in males and females was 12.0% and 10.8%, respectively. Meta-analysis derived a pooled katGS315T resistant TB prevalence of 67.3% (95% CI 59.3-75.4%) with Q=732.19, I2=98.35% and P=0.000 for treated TB cases. CONCLUSION: INH resistance was spread widely and transmission of INH-resistant isolates, especially with katG315T mutation, was confirmed. Therefore, it is important to diagnose katG315T mutants among INH-resistant strains as it may be a risk factor for subsequent development of multidrug-resistant TB (MDR-TB). Prompt detection of patients with INH-resistant strains would expedite modification of treatment regimens, and appropriate infection control measures could be taken in time to diminish the risk of further development and transmission of MDR-TB.

Prevalence of mutations in genes associated with rifampicin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates.

Purpose: To analyze prevalence of mutations in genes associated with rifampicin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates from patients with possible MDR TB of Puducherry, South India and to explore the association of specific mutations conferring rifampicin (RIF) resistance. Methods: We performed a commercial Genotype MDBDRplus V.2.0 assay for the rapid detection of rifampicin and isoniazid resistance directly on sputum specimens of patients with possible MDR TB. Results: Totally 558 multidrug resistant, 293 RIF mono resistant and 923 INH mono resistant tuberculosis were detected from the 12,786 patients with possible MDR TB samples. The 50.5% mutations were observed in the region of S531L in MDR TB patients and 55.6% in rifampicin monoresistant cases. In total isoniazid monoresistant, 68.0% mutations were detected in katG gene, which is more prevalent in comparison to inhA gene 32.0%. There were about 57.9% and 32.2% MDR TB cases diagnosed in the age group of > 15 to ≤ 45 years and > 45 to ≤ 60 years respectively. Conclusions: The rate of occurrences of mutations were found widely in the Rifampicin Resistant Determination Region (81 bp) of rpoB gene and the hypervariable region 530-533 codons of rpoB gene is alarming in the specification. The higher frequency of mutation in codons of rpoB (S531L) and katG (S315T) gene help to design simple, new and less expensive molecular techniques to use in peripheral laboratories.

Molecular typing and differentiation of Mycobacterium tuberculosis clinical isolates using Double Repetitive Element PCR and Duplex PCR.

BACKGROUND: To date, the advancements in polymerase chain reaction (PCR) assures accurate, fast identification and mycobacterial speciation in clinical settings, which promotes a better tuberculosis (TB) treatment regimen. METHODS: In this study, a total of 78 clinically suspected cases of TB were processed for the detection of Mycobacterial infections by standard Ziehl Neelsen (ZN) staining, conventional Lowenstein-Jensen (LJ) and BACTEC MGIT-960™ liquid culture. Strain typing was performed by using Double Repetitive Element PCR (DRE-PCR) and Duplex PCR (DPCR) to differentiate Mycobacterium tuberculosis complex (MTB) from non-tuberculous mycobacteria (NTM), respectively. RESULTS: Of 78 clinical isolates, 25 (32%) were drug-susceptible, and 53 (68%) were resistant to at least one drug. The BACTEC MGIT-960™ showed the highest (88.5%) positivity rate, compared with conventional LJ (82%) and ZN smear (61.5%). The mean time detection and drug susceptibility for MTB was 28 and 40days in LJ culture, and 10 and 13 days in BACTEC MGIT-960™ culture. Using DPCR, Mycobacterium avium infection was identified in HIV-positive (2.56%) and MTB in HIV-negative patients (97.4%), and the DRE-PCR system divulged 15 unique genotype patterns, and an institutional-based epidemiology database was created. CONCLUSIONS: The combination of an in-house DRE-DPCR system could possibly identify and differentiate MTB from other mycobacterial species in a single reaction. In addition, restriction polymorphism analysis and DNA sequencing of NTM could assist in species identification directly from clinical isolates.

Molecular analysis of rpoB gene mutations in rifampicin resistant Mycobacterium tuberculosis isolates by multiple allele specific polymerase chain reaction in Puducherry, South India.

BACKGROUND: rpoB gene mutations in Mycobacterium tuberculosis (MTB) make the bacteria resistant to rifampicin. Thus, these mutations are surrogate markers for multi-drug resistance (MDR). The objective of this study was to evaluate an allele-specific multiplex-polymerase chain reaction (MAS-PCR) assay to detect mutations at codons 516, 526 and 531 of the rpoB gene. METHODS: In total, 127 M. tuberculosis clinical isolates were subjected to standard drug susceptibility tests. A MAS-PCR assay was then performed to detect mutations in the rpoB gene. Three different allele-specific PCR assays were performed (single-step MAS-PCR) and the amplified products were sequenced. RESULTS: Of the 127 isolates, 69 (54.3%) were multidrug resistant M. tuberculosis (MDR-TB), 21 (16.5%) were rifampicin mono-resistant and 37 (29.1%) were drug susceptible. The frequency of mutations at codons 531, 526 and 516 was 54.4%, 18.9% and 5.6%, respectively. A triple mutation was found in 4 (4.4%) isolates. Mutations in regions other than the 81-bp region were observed at codons 413 (11.1%), 511 (12.2%) and 521 (15.6%) of the rpoB gene. CONCLUSIONS: The simplicity and specificity of the MAS-PCR assay allows for easy implementation in clinical laboratories to detect rifampicin drug resistance in MDR-TB strains.

Clinical evaluation of mtp40 polymerase chain reaction for the diagnosis of extra pulmonary tuberculosis.

Rapid and sensitive detection of Mycobacterium tuberculosis from patient samples is vital for clinical diagnosis and treatment. The emergence of M. tuberculosis strains with either no copies or only a single copy of IS6110 in Asian countries makes the standard PCR based diagnosis of M. tuberculosis using IS6110 not reliable. We studied the diagnostic efficacy of the in-house PCR amplification of the candidate gene mtp40 as an alternative to IS6110 element based diagnosis. Clinical samples included pulmonary and extra-pulmonary specimens from TB suspected patients residing in Puducherry, South India and were analyzed using in-house PCR procedures targeting IS6110 element and mtp40 genes. Out of 317 clinical specimens analyzed, 132 (41.6 %) and 114 (36 %) were found positive for mtp40 PCR and IS6110 PCR, respectively. However, 18 specimens that were found to negative for IS6110 PCR were found positive for mtp40 PCR, which was further confirmed by DNA sequencing method. PCR amplification of mtp40 gene for the diagnosis of M. tuberculosis in clinical samples is fast, sensitive, and further identified clinical strains that lack IS6110 element in this region. It is clearly demonstrated that there is a significant difference between the two PCR procedures and the sensitivity and specificity levels of mtp40 PCR were found to be higher when compared with DNA sequencing method. Thus, mtp40 based PCR technique will be beneficial in diagnosis of TB where M. tuberculosis strains lack of IS6110 element is predominant.

Molecular epidemiological study of pyrazinamide-resistance in clinical isolates of mycobacterium tuberculosis from South India.

Pyrazinamide (PZA) has been in use for almost 50 years as a first-line drug for short-course chemotherapy against Mycobacterium tuberculosis. In this study, PCR mediated automated DNA sequencing is used to check the prevalence of PZA resistance among treatment failure cases of pulmonary tuberculosis. Out of 50 clinical isolates examined, 39 had mutations in the pncA gene that encodes Pyrazinamidase, an enzyme required to activate PZA. Of these, 31 (79.5%) were localized to three regions of pncA. We found two isolates with hitherto unreported mutation at amino acid 26 (Ala-->Gly) of pncA.