Phage lysin to control the overgrowth of normal flora in processed sputum samples for the rapid and sensitive detection of Mycobacterium tuberculosis by luciferase reporter phage assay

BACKGROUND: Phage lysin, extracted from three bacteriophages was used in place of antibiotics to control the overgrowth of normal flora in processed sputum samples leading to the sensitive detection of Mycobacterium tuberculosis using diagnostic luciferase reporter phage assay (DLRPA). METHODS: A total of 129 sputum samples were processed by modified Petroff’s method. Two Lowenstein Jensen slopes were inoculated from the processed sputum deposit thus obtained. The remaining deposits were transferred to 7 ml of Middlebrook 7H9 complete medium supplemented with phage lysin and incubated at 37°C. DLRPA was done using phAE129 at days 7, 9, 14 and 21. At the end of day 21, the samples were centrifuged and the pellets were inoculated on to 2 more LJ slopes to validate DLRPA results. RESULTS: The sensitivity and specificity of DLRPA in detecting M. tuberculosis from sputum specimens was 90% and 81% respectively compared to conventional LJ culture. The agreement between the methods was 87%. The rate of contamination for DLRPA using phage lysin was 9.3%. CONCLUSION: Phage lysin can be used to decontaminate sputum samples for the detection of M. tuberculosis by DLRPA directly from processed sputum specimens

Luciferase reporter phage phAE85 for rapid detection of rifampicin resistance in clinical isolates of Mycobacterium tuberculosis

AbstractObjectiveTo evaluate luciferase reporter phage (LRP) phAE85 in rapid detection of rifampicin resistance in a region where TB is endemic.MethodsOne hundred and ninety primary isolates on Lowenstein-Jensen medium were tested. Middlebrook 7H9 complete medium with and without rifampicin at 2 μg/mL was inoculated with standard inoculum from suspensions of the clinical isolate. After incubation for 72 h, LRP was added. Following 4 h of further incubation, light output from both control and test was measured as relative light units. Strains exhibiting a reduction of less than 50% relative light units in the drug containing vial compared to control were classified as resistant. Results were compared with the conventional minimum inhibitory concentration method (MIC) of drug susceptibility testing.ResultsThe two methods showed high level of agreement of 97% (CI 0.94, 0.99) and P value was 0.000 1. The sensitivity and specificity of LRP assay for detection of rifampicin resistance were 91% (CI 0.75, 0.98) and 99% (CI 0.95, 1.00) respectively. Time to detection of resistance by LRP assay was 3 d in comparison with 28 d by the minimum inhibitory concentration method.ConclusionsLRP assay with phAE85 is 99% specific, 91% sensitive and is highly reproducible. Thus the assay offers a simple procedure for drug sensitivity testing, within the scope of semi-automation

Acquired rifampicin resistance in thrice-weekly antituberculosis therapy: impact of HIV and antiretroviral therapy

Risk factors for acquired rifampicin resistance (ARR) among tuberculosis patients on thrice-weekly antituberculosis therapy were baseline isoniazid resistance and HIV. Among HIV-infected patients, higher mycobacterial burden and lower CD4 count, but not highly active antiretroviral therapy, were significantly associated with ARR. Background: Risk factors for acquired rifampicin resistance (ARR) in human immunodeficiency virus (HIV)/tuberculosis coinfection, in the highly active antiretroviral therapy (HAART) era, needs evaluation. We studied the impact of HIV and HAART on ARR among patients taking thrice-weekly antituberculosis therapy. Methods: This cross-protocol analysis included patients with newly diagnosed, rifampicin-susceptible pulmonary tuberculosis, with and without HIV, enrolled in clinical trials (who took >80% of medication) at the National Institute for Research in Tuberculosis between 1999 and 2013. All patients received rifampicin and isoniazid for 6 months reinforced with pyrazinamide and ethambutol in the first 2 months, given thrice-weekly throughout the study along with HAART in one of the groups. Outcomes were categorized and multivariate logistic regression analysis performed to identify risk factors for ARR. Results: The per-protocol results included patients with tuberculosis: 246 HIV-uninfected patients (HIV–TB+), 212 HIV patients not on HAART (non-HAART), and 116 HIV-infected patients on HAART. Median CD4 counts of the latter 2 groups were 150 and 93 cells/μL, respectively, and the median viral loads were 147 000 and 266 000 copies/mL, respectively. Compared with HIV–TB+, the relative risks (RRs) for an unfavorable response in the coinfected, non-HAART and HAART groups were 2.1 (95% confidence interval [CI], 1.7–14.8; P<.0001) and 2.1 (95% CI, .9–5.2; P=.3), whereas for ARR, the RRs were 21.1 (95% CI, 2.6–184; P<.001) and 8.2 (95% CI, .6–104; P=.07), respectively. Conclusions: HIV-infected patients with tuberculosis treated with a thrice-weekly antituberculosis regimen are at a higher risk of ARR, compared with HIV-uninfected patients, in the presence of baseline isoniazid resistance. HAART reduces but does not eliminate the risk of ARR

Smoking, alcohol use disorder and tuberculosis treatment outcomes: A dual co-morbidity burden that cannot be ignored

BackgroundMore than 20% of tuberculosis (TB) disease worldwide may be attributable to smoking and alcohol abuse. India is the second largest consumer of tobacco products, a major consumer of alcohol particularly among males, and has the highest burden of TB globally. The impact of increasing tobacco dose, relevance of alcohol misuse and past versus current or never smoking status on TB treatment outcomes remain inadequately defined.MethodsWe conducted a multi-centric prospective cohort study of newly diagnosed adult pulmonary TB patients initiated on TB treatment and followed for a minimum of 6 months to assess the impact of smoking status with or without alcohol abuse on treatment outcomes. Smokers were defined as never smokers, past smokers or current smokers. Alcohol Use Disorder Identification Test (AUDIT) scores were used to assess alcohol misuse. The association between smoking status and treatment outcomes was assessed in univariate and multivariate random effects poisson regression models.ResultsOf 455 enrolled, 129 (28%) had a history of smoking with 94 (20%) current smokers and 35 (8%) past smokers. Unfavourable treatment outcomes were significantly higher among past and current smokers as compared to never smokers. Specifically, the risk of treatment failure was significantly higher among past smokers (aIRR = 2.66, 95% CI: 1.41-4.90, p = 0.002), recurrent TB among current smokers (aIRR = 2.94, 95% CI: 1.30-6.67, p = 0.010) and death among both past (2.63, 95% CI: 1.11-6.24, p = 0.028) and current (aIRR = 2.59, 95% CI: 1.29-5.18, p = 0.007) smokers. Furthermore, the combined effect of alcohol misuse and smoking on unfavorable treatment outcomes was significantly higher among past smokers (aIRR: 4.67, 95% CI: 2.17-10.02, pConclusionPast and current smoking along with alcohol misuse have combined effects on increasing the risk of unfavourable TB treatment outcomes. Innovative interventions that can readily address both co-morbidities are urgently needed