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.

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.

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.

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.