Efficacy and tolerability of a 4-month ofloxacin-containing regimen compared to a 6-month regimen in the treatment of patients with superficial lymph node tuberculosis: a randomized trial.

BACKGROUND: Tuberculosis (TB) lymphadenitis is the most common form of extra-pulmonary TB, and the treatment duration is six months. This non-inferiority based randomized clinical trial in South India evaluated the efficacy and safety of a four-month ofloxacin containing regimen in tuberculosis lymphadenitis (TBL) patients. METHODS: New, adult, HIV-negative, microbiologically and or histopathologically confirmed superficial lymph node TB patients were randomized to either four-month oflaxacin containing test regimen [ofloxacin (O), isoniazid (H), rifampicin (R), pyrazinamide (Z) -2RHZO daily/ 2RHO thrice-weekly] or a six-month thrice-weekly control regimen (2HRZ, ethambutol/4RH). The treatment was directly observed. Clinical progress was monitored monthly during and up to 12 months post-treatment, and thereafter every three months up to 24 months. The primary outcome was determined by response at the end of treatment and TB recurrence during the 24 months post-treatment. RESULTS: Of the 302 patients randomized, 298 (98.7%) were eligible for modified intention-to-treat (ITT) analysis and 294 (97%) for per-protocol (PP) analysis. The TB recurrence-free favourable response in the PP analysis was 94.0% (95% CI: 90.1-97.8) and 94.5% (95% CI: 90.8-98.2) in the test and control regimen respectively, while in the ITT analysis, it was 92.7% and 93.2%. The TB recurrence-free favourable response in the test regimen was non-inferior to the control regimen 0.5% (95% CI: -4.8-5.9) in the PP analysis based on the 6% non-inferiority margin. Treatment was modified for drug toxicity in two patients in the test regimen, while one patient had a paradoxical reaction. CONCLUSION: The 4-month ofloxacin containing regimen was found to be non-inferior and as safe as the 6-month thrice-weekly control regimen.

Evaluation of molecular diagnostic test for detection of adult pulmonary tuberculosis: A generic protocol.

BACKGROUND OBJECTIVES: Tuberculosis (TB) continues to be the second most-leading cause of death due to a single infectious agent as of 2022 after COVID-19. Many affordable new molecular diagnostic tools are being developed for early and more accurate diagnosis, especially for low-resource settings in low- and middle-income countries. In this context, there is a need to develop a standardized protocol for validation of new diagnostic tools. Here, we describe a generic protocol for multi-centric clinical evaluation of molecular diagnostic tests for adult pulmonary TB. METHODS: This protocol describes a cross-sectional study in TB reference laboratories in India. Adults (>18 yr) visitng the chest clinics or outpatient departments with symptoms of TB need to be enrolled consecutively till the required sample size of 150 culture positives and 470 culture negatives are met. Mycobacterium tuberculosis (Mtb) culture (mycobacteria growth indicator tube liquid culture) to be used under this protocol as the gold standard and Xpert MTB/RIF molecular test will be used as the comparator. The sputum samples will be tested by smear microscopy, Mtb culture, Xpert MTB/RIF and index molecular test as per the proposed algorithm. The specificity sensitivity, and positive/ negative predictive values are to be calculated for the index test with reference to the gold standard. DISCUSSION: TB diagnosis poses many challenges as it differs with type of disease, age group, clinical settings and type of diagnostic tests/kits used. Globally, different protocols are used by several investigators. This protocol provides standard methods for the validation of molecular tests for diagnosis of adult pulmonary TB, which can be adopted by investigators.

Xpert MTB/RIF assay in the diagnosis of pulmonary tuberculosis in children in tertiary care setting in South India.

Xpert MTB/RIF is recommended for the diagnosis of tuberculosis (TB) in children. We determined the performance of Xpert MTB/RIF in the diagnosis of pulmonary TB in children. The characteristics of children influencing Xpert MTB/RIF positivity were explored. Children aged <15 years with symptoms suggestive of pulmonary TB were prospectively enrolled from 2013 to 2019. Two sputum/early morning gastric aspirate specimens were collected for examination by smear (fluorescence microscopy), Xpert MTB/RIF, and culture [Mycobacteria growth indicator tube (MGIT)/Lowenstein-Jensen (LJ) medium]. Diagnostic performance of Xpert MTB/RIF was evaluated using LJ and or MGIT culture positivity as the reference standard. Sensitivity, specificity with 95% confidence interval (CI) were calculated. Stratified analysis was done; P < .05 was considered statistically significant. Of the total 1727 enrolled children, 1674 (97%) with complete results for at least one sputum/gastric aspirate sample were analyzed. The sensitivity of Xpert MTB/RIF was 68.5% in sputum and 53.6% in gastric aspirate while the specificity was 99% for both. The sensitivity compared to smear was 68.5% vs. 33.7% (P < .001) and 53.6% vs. 14.5%; (P < .001) in sputum and gastric aspirate, respectively. The sensitivity of Xpert MTB/RIF was 23.9% with decision to treat as reference standard. Xpert MTB/RIF positivity was significantly influenced by sex, age, nutritional status, chest X-ray abnormality, TB infection status, and symptoms suggestive of TB. Xpert MTB/RIF as an upfront test compared to smear improves diagnosis of pulmonary TB in children yet the sensitivity is suboptimal. Newer TB diagnostic tools with improved sensitivity is warranted in children.

We evaluated the performance of Xpert MTB/RIF in the diagnosis of pulmonary TB in children and explored the characteristics influencing Xpert MTB/RIF positivity. Sputum and or early morning gastric aspirate specimen was collected from children aged <15 years with symptoms suggestive of pulmonary TB. This was examined by smear (fluorescence microscopy), Xpert MTB/RIF, and culture (Mycobacteria growth indicator tube (MGIT)/Lowenstein­Jensen (LJ) medium). Diagnostic performance of Xpert MTB/RIF was evaluated using LJ and or MGIT culture positivity as the reference standard. Of the total 1727 enrolled children, 1674 (97%) with complete results for at least one sputum/gastric aspirate sample were analyzed. The sensitivity of Xpert MTB/RIF was 68.5% in sputum and 53.6% in gastric aspirate which was higher than smear and the specificity was 99%. The sensitivity of Xpert MTB/RIF was 23.9% with decision to treat for TB as reference standard. The Xpert MTB/RIF positivity was influenced by sex, age, nutritional status, chest X-ray abnormality, TB infection status, and symptoms suggestive of TB. Xpert MTB/RIF as an upfront test compared to smear improves the diagnosis of pulmonary TB in children yet the sensitivity is suboptimal. Newer TB diagnostic tools with improved sensitivity is warranted in children.

Resistance Profiles to Second-Line Anti-Tuberculosis Drugs and Their Treatment Outcomes: A Three-Year Retrospective Analysis from South India.

Background: Patients with first-line drug resistance (DR) to rifampicin (RIF) or isoniazid (INH) as a first-line (FL) line probe assay (LPA) were subjected to genotypic DST using second-line (SL) LPA to identify SL-DR (including pre-XDR) under the National TB Elimination Program (NTEP), India. SL-DR patients were initiated on different DR-TB treatment regimens and monitored for their outcomes. The objective of this retrospective analysis was to understand the mutation profile and treatment outcomes of SL-DR patients. Materials and Methods: A retrospective analysis of mutation profile, treatment regimen, and treatment outcome was performed for SL-DR patients who were tested at ICMR-NIRT, Supra-National Reference Laboratory, Chennai between the years 2018 and 2020. All information, including patient demographics and treatment outcomes, was extracted from the NTEP Ni-kshay database. Results: Between 2018 and 2020, 217 patients out of 2557 samples tested were identified with SL-DR by SL-LPA. Among them, 158/217 were FQ-resistant, 34/217 were SLID-resistant, and 25/217 were resistant to both. D94G (Mut3C) of gyrA and a1401g of rrs were the most predominant mutations in the FQ and SLID resistance types, respectively. Favorable (cured and treatment complete) and unfavorable outcomes (died, lost to follow up, treatment failed, and treatment regimen changed) were recorded in a total of 82/217 and 68/217 patients in the NTEP Ni-kshay database. Conclusions: As per the testing algorithm, SL- LPA is used for genotypic DST following identification of first-line resistance, for early detection of SL-DR in India. The fluoroquinolone resistance pattern seen in this study population corelates with the global trend. Early detection of fluoroquinolone resistance and monitoring of treatment outcome can help achieve better patient management.

Subtherapeutic Rifampicin Concentration Is Associated With Unfavorable Tuberculosis Treatment Outcomes.

BACKGROUND: The relationships between first-line drug concentrations and clinically important outcomes among patients with tuberculosis (TB) remain poorly understood. METHODS: We enrolled a prospective cohort of patients with new pulmonary TB receiving thrice-weekly treatment in India. The maximum plasma concentration of each drug was determined at months 1 and 5 using blood samples drawn 2 hours postdose. Subtherapeutic cutoffs were: rifampicin <8 µg/mL, isoniazid <3 µg/mL, and pyrazinamide <20 µg/mL. Factors associated with lower log-transformed drug concentrations, unfavorable outcomes (composite of treatment failure, all-cause mortality, and recurrence), and individual outcomes were examined using Poisson regression models. RESULTS: Among 404 participants, rifampicin, isoniazid, and pyrazinamide concentrations were subtherapeutic in 85%, 29%, and 13%, respectively, at month 1 (with similar results for rifampicin and isoniazid at month 5). Rifampicin concentrations were lower with human immunodeficiency virus coinfection (median, 1.6 vs 4.6 µg/mL; P = .015). Unfavorable outcome was observed in 19%; a 1-µg/mL decrease in rifampicin concentration was independently associated with unfavorable outcome (adjusted incidence rate ratio [aIRR], 1.21 [95% confidence interval {CI}, 1.01-1.47]) and treatment failure (aIRR, 1.16 [95% CI, 1.05-1.28]). A 1-µg/mL decrease in pyrazinamide concentration was associated with recurrence (aIRR, 1.05 [95% CI, 1.01-1.11]). CONCLUSIONS: Rifampicin concentrations were subtherapeutic in most Indian patients taking a thrice-weekly TB regimen, and low rifampicin and pyrazinamide concentrations were associated with poor outcomes. Higher or more frequent dosing is needed to improve TB treatment outcomes in India.

4-month moxifloxacin containing regimens in the treatment of patients with sputum-positive pulmonary tuberculosis in South India - a randomised clinical trial.

BACKGROUND: Shortening tuberculosis (TB) treatment duration is a research priority. We tested the efficacy and safety of 3- and 4-month regimens containing moxifloxacin in a randomised clinical trial in pulmonary TB (PTB) patients in South India. METHODS: New, sputum-positive, adult, HIV-negative, non-diabetic PTB patients were randomised to 3- or 4-month moxifloxacin regimens [moxifloxacin (M), isoniazid (H), rifampicin (R), pyrazinamide (Z) and ethambutol (E)] or to a control regimen (2H3 R3 Z3 E3 /4R3 H3 ) [C]. The 4 test regimens were 3R7 H7 Z7 E7 M7 [M3], 2R7 H7 Z7 E7 M7 /2R7 H7 M7 [M4], 2R7 H7 Z7 E7 M7 /2R3 H3 M3 [M4-I] or 2R7 H7 Z7 E7 M7 /2R3 H3 E3 M3 [M4-IE]. Treatment was directly observed. Clinical and bacteriological assessments were done monthly during treatment and for 24 months post-treatment. The primary end point was TB recurrence post-treatment. RESULTS: Of 1371 patients, randomised, modified intention-to-treat (ITT) analysis was done in 1329 and per-protocol (PP) analysis in 1223 patients. Regimen M3 was terminated due to high TB recurrence rates. 'Favourable' response at end of treatment was 96-100% in the moxifloxacin regimens and 93% in the control  regimen. Among these, the TB recurrence occurred in 4.1% in the M4 regimen and in 4.5% in the control regimen and demonstrated equivalence within a 5% margin (95% CI -3.68, 4.55). Similar findings were observed in modified ITT analysis. The TB recurrence rates in the M4-I and M4-IE regimens did not show equivalence with the control regimen. Sixteen (1.4%) of 1087 patients in the moxifloxacin regimens required treatment modification. CONCLUSION: The 4-month daily moxifloxacin regimen [M4] was found to be equivalent and as safe as the 6-month thrice-weekly control regimen.

CONTEXTE: La réduction de la durée du traitement de la tuberculose (TB) est une priorité de recherche. Nous avons testé l'efficacité et la sécurité de schémas thérapeutiques contenant de la moxifloxacine pendant 3 et 4 mois dans un essai clinique randomisé chez des patients atteints de TB pulmonaire (PTB) dans le sud de l'Inde. MÉTHODES: De nouveaux patients PTB, adultes, non diabétiques, positifs pour les expectorations, VIH négatifs ont été randomisés pour des schémas thérapeutiques contenant de la moxifloxacine pendant 3 mois ou 4 mois [moxifloxacine (M), isoniazide (H), rifampicine (R), pyrazinamide (Z), l'éthambutol (E)] ou pour un régime témoin (2H3 R3 Z3 E3 /4R3 H3 ) [C]. Les 4 régimes de l'essai étaient 3R7 H7 Z7 E7 M7 [M3], 2R7 H7 Z7 E7 M7 /2R7 H7 M7 [M4], 2R7 H7 Z7 E7 M7 /2R3 H3 M3 [M4-I] ou 2R7 H7 Z7 E7 M7 /2R3 H3 E3 M3 [M4-IE]. Le traitement a été directement observé. Les évaluations cliniques et bactériologiques ont été effectuées mensuellement au cours du traitement et durant 24 mois après le traitement. Le critère d'évaluation principal était la récidive de la TB après le traitement. RÉSULTATS: Des 1.371 patients randomisés, une analyse en intention de traiter (ITT) modifiée a été effectuée sur 1.329 et une analyse par protocole (PP) sur 1.223 patients. Le régime M3 a été interrompu en raison de taux élevés de récidive de la TB. La réponse «favorable¼ à la fin du traitement était de 96 à 100% dans les bras moxifloxacine et 93% dans le bras témoin. Parmi ceux-ci, la récidive de la TB est survenue chez 4,1% dans le schéma M4 et chez 4,5% dans le schéma témoin et a démontré une équivalence dans une marge de 5% (IC95%: −3,68, 4,55). Des résultats similaires ont été observés dans l'analyse ITT modifiée. Les taux de récidive de la TB dans les schémas M4-I et M4-IE n'ont pas montré d'équivalence avec le schéma témoin. 16 (1,4%) des 1.087 patients dans les régimes à moxifloxacine ont nécessité une modification du traitement. CONCLUSION: Le régime quotidien de moxifloxacine pendant 4 mois [M4] s'est avéré équivalent et aussi sûr que le régime témoin de trois fois par semaine pendant 6 mois.

Sputum culture conversion with moxifloxacin-containing regimens in the treatment of patients with newly diagnosed sputum-positive pulmonary tuberculosis in South India.

BACKGROUND: Rapid sputum culture conversion at 2 months indicates the sterilizing capacity and potential of regimens to shorten duration of tuberculosis treatment. We compared results of sputum culture conversion by moxifloxacin and control regimens and identified factors affecting sputum culture positivity after 2 months of treatment. METHODS: Human immunodeficiency virus-uninfected adults with newly diagnosed smear-positive pulmonary tuberculosis were randomized to receive a 3- or 4-month moxifloxacin regimen (moxifloxacin [M], isoniazid [H], rifampicin [R], pyrazinamide [Z], ethambutol [E]) or the control regimen (RHZE thrice weekly). Bacteriological assessments were done at 15, 30, 45, and 60 days of treatment. Because all patients in the moxifloxacin groups received 2 months of daily RHZEM, they were grouped together for analysis. Statistical methods included χ(2) test and logistic regression analysis. RESULTS: Sputum culture conversion was analyzed in 780 (616 in the moxifloxacin group and 164 in the control group) of 801 enrolled patients. Ninety-five percent of 590 patients in the moxifloxacin group and 81% of 151 patients in the control group had negative sputum cultures at month 2 (P < .001). The control regimen, age (≥35 years), initial sputum culture grade (2+ or 3+), and male sex were significantly associated with higher odds of positive sputum cultures at 2 months. CONCLUSIONS: A 5-drug daily regimen with moxifloxacin results in significantly higher sputum culture conversion in the first 2 months compared with a thrice-weekly, 4-drug regimen in patients with newly diagnosed sputum-positive pulmonary tuberculosis.

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.

Pulmonary Mycobacterium abscessus and response to treatment in an outpatient setting: Case series.

Pulmonary disease due to Mycobacterium abscessus (Mab) has become an increasing cause of health concern, particularly among individuals infected with nontuberculous mycobacteria. Since Mab is intrinsically resistant to many antibiotics, it is very challenging to treat patients with symptomatic disease. In this case series, we report four patients with symptomatic pulmonary Mab who had prior history of antituberculosis treatment intake and declared cured at the end of treatment. The current episode was confirmed to be due to Mab infection by molecular and clinical diagnosis and received species specific-antibiotics therapy. All were periodically monitored for the sputum smear and culture conversions throughout the treatment period. The clinical course was variable though all received similar antibiotic regimen and showed varied treatment outcomes. The time of diagnosis and the treatment outcome indicate that a better understanding of host-pathogen interactions is essential for the successful treatment of pulmonary Mab infection.

Reprocessing of Contaminated MGIT 960 Cultures to Improve Availability of Valid Results for Mycobacteria.

Optimal recovery of mycobacteria from the contaminated liquid culture is a challenge. While alternative methods have been suggested to reduce the rate of contamination in the BACTEC MGIT 960 system, reprocessing the contaminated liquid culture improves recovery of Mycobacterium tuberculosis. Among 793 MGIT cultures raised from as many sputum specimens after primary decontamination by the standard NaLC-NaOH method, valid results were available for 687 (86.6%) as 106 (13.4%) were contaminated. Reprocessing and reculturing of the contaminated cultures increased valid results to 739 (93.2%) and reduced the contamination rate to 6.8%. Both values were statistically significant. Recovery of the Mycobacterium tuberculosis complex increased from 45.6% to 48.4%. Valid negative results were available for an additional 3.4%. The method may be adopted to reduce the rate of contamination and to improve the valid culture results for mycobacteria.

Validation of bedaquiline drug-susceptibility testing by BACTEC MGIT 960 system for Mycobacterium tuberculosis.

Background: Bedaquiline (BDQ) is a new antituberculosis (TB) drug effectively used for the treatment of multidrug-resistant and extensively drug-resistant TB. However, the reports on drug-susceptibility testing (DST) for BDQ are scarce. The study aimed to validate and standardize BDQ DST by BACTEC MGIT 960 system for Mycobacterium tuberculosis. Methods: A panel of ten M. tuberculosis isolates comprising 8 BDQ sensitive and 2 BDQ resistant strains were used to test accuracy, repeatability, and reproducibility of BDQDST by MGIT 960. BDQ DST by Middlebrook 7H11 agar method using polystyrene tubes was used as a standard method to calculate the accuracy of the validation. Results: DST by MGIT for BDQ showed 100% accuracy, repeatability, and reproducibility, although variations were observed in the growth units of the "test" MGIT tubes between technologist and drug stocks while testing for reproducibility. Conclusion: BDQ DST by MGIT 960 system is accurate, repeatable, and reproducible and hence can be implemented in certified laboratories routinely performing DST by MGIT 960 system.

Pulmonary Mycobacterium kyorinense disease: A case report and review of literature.

We report here the first case of pulmonary infection due to Mycobacterium kyorinense in a 55-year-old hypertensive woman treated for pulmonary tuberculosis earlier on two occasions. She presented with productive cough, intermittent episode of left-sided chest pain, loss of appetite, low-grade fever, and breathlessness. Sputum cultures revealed non-tuberculous mycobacteria (NTM). She remained persistently symptomatic with sputum cultures positive for acid-fast bacilli even after 6 months of treatment. Hence, a 16SrRNA gene amplification and sequencing were done that revealed M. kyorinense. Based on the guidelines of the American Thoracic Society, she was started on weight-based dosing of clarithromycin, levofloxacin, ethambutol, isoniazid and injection amikacin daily. The patient improved symptomatically and became culture-negative after 3 months of therapy with the above regimen and continued to be culture negative for 12 months of treatment. She continues to remain symptom-free without evidence of any clinical or bacteriological relapse.

Rapid, point-of-care diagnosis of tuberculosis with novel Truenat assay: Cost-effectiveness analysis for India's public sector.

BACKGROUND: Truenat is a novel molecular assay that rapidly detects tuberculosis (TB) and rifampicin-resistance. Due to the portability of its battery-powered testing platform, it may be valuable in peripheral healthcare settings in India. METHODS: Using a microsimulation model, we compared four TB diagnostic strategies for HIV-negative adults with presumptive TB: (1) sputum smear microscopy in designated microscopy centers (DMCs) (SSM); (2) Xpert MTB/RIF in DMCs (Xpert); (3) Truenat in DMCs (Truenat DMC); and (4) Truenat for point-of-care testing in primary healthcare facilities (Truenat POC). We projected life expectancy, costs, incremental cost-effectiveness ratios (ICERs), and 5-year budget impact of deploying Truenat POC in India's public sector. We defined a strategy "cost-effective" if its ICER was

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

BACKGROUND: More 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. METHODS: We 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. RESULTS: Of 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, p<0.001) and current smokers (aIRR: 3.58, 95% CI: 1.89-6.76, p<0.001). CONCLUSION: Past 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.

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

[This corrects the article DOI: 10.1371/journal.pone.0220507.].

Infection free "resisters" among household contacts of adult pulmonary tuberculosis.

Despite substantial exposure to infectious pulmonary tuberculosis (TB) cases, some household contacts (HHC) never acquire latent TB infection (LTBI). Characterizing these "resisters" can inform who to study immunologically for the development of TB vaccines. We enrolled HHCs of culture-confirmed adult pulmonary TB in India who underwent LTBI testing using tuberculin skin test (TST) and QuantiFERON TB Gold Test-in-tube (QFT-GIT) at baseline and, if negative by both (<5mm TST and <0.35IU/mL QFT-GIT), underwent follow-up testing at 4-6 and/or 12 months. We defined persons with persistently negative LTBI tests at both baseline and followup as pLTBI- and resisters as those who had a high exposure to TB using a published score and remained pLTBI-. We calculated the proportion of resisters overall and resisters with complete absence of response to LTBI tests (0mm TST and/or QFT-GIT <0.01 IU/ml). Using random effects Poisson regression, we assessed factors associated with pLTBI-. Of 799 HHCs in 355 households, 67 (8%) were pLTBI- at 12 months; 52 (6.5%) pLTBI- in 39 households were resisters. Complete absence of response to LTBI tests was found in 27 (53%) resisters. No epidemiological characteristics were associated with the pLTBI- phenotype. LTBI free resisters among HHC exist but are uncommon and are without distinguishing epidemiologic characteristics. Assessing the genetic and immunologic features of such resister individuals is likely to elucidate mechanisms of protective immunity to TB.

Construction and evaluation of luciferase reporter phages for the detection of active and non-replicating tubercle bacilli.

The luciferase reporter phages (LRP) show great promise for diagnostic mycobacteriology. Though conventional constructs developed from lytic phages such as D29 and TM4 are highly specific, they lack sensitivity. We have isolated and characterized Che12, the first true temperate phage infecting M. tuberculosis. Since the tuberculosis (TB) cases among HIV infected population result from the reactivation of latent bacilli, it would be useful to develop LRP that can detect dormant bacteria. During dormancy, pathogenic mycobacteria switch their metabolism involving divergent genes than during normal, active growth phase. Since the promoters of these genes can potentially function during dormancy, they were exploited for the construction of novel mycobacterial luciferase reporter phages. The promoters of hsp60, isocitrate lyase (icl), and alpha crystallin (acr) genes from M. tuberculosis were used for expressing firefly luciferase gene (FFlux) in both Che12 and TM4 phages and their efficiency was evaluated in detecting dormant bacteria from clinical isolates of M. tuberculosis. These LRP constructs exhibited detectable luciferase activity in dormant as well as in actively growing M. tuberculosis. The TM4 ts mutant based constructs showed about one log increase in light output in three of the five tested clinical isolates and in M. tuberculosis H37Rv compared to conventional lytic reporter phage, phAE129. By refining the LRP assay format further, an ideal rapid assay can be designed not only to diagnose active and dormant TB but also to differentiate the species and to find their drug susceptibility pattern.

Tuberculin skin test and QuantiFERON-Gold In Tube assay for diagnosis of latent TB infection among household contacts of pulmonary TB patients in high TB burden setting.

BACKGROUND: World Health Organization (WHO) recommends systematic screening of high-risk populations, including household contacts (HHCs) of adult pulmonary tuberculosis (TB) patients, as a key strategy for elimination of TB. QuantiFERON-TB Gold In-Tube (QFT-GIT) assay and tuberculin skin test (TST) are two commonly used tools for the detection of latent tuberculosis infection (LTBI) but may yield differential results, affecting eligibility for TB preventive therapy. MATERIALS AND METHODS: A prospective cohort study of adult pulmonary TB patients and their HHCs were recruited in 2 cities of India, Pune and Chennai. HHCs underwent QFT-GIT (QIAGEN) and TST (PPD SPAN 2TU/5TU). A positive QFT-GIT was defined as value ≥0.35 IU/ml and a positive TST as an induration of ≥5 mm. A secondary outcome of TST induration ≥10mm was explored. Proportion positive by either or both assays, discordant positives and negatives were calculated; test concordance was assessed using percentage agreement and kappa statistics; and risk factors for concordance and discordance including age categories were assessed using logistic regression. Sensitivity and specificity was estimated by latent class model. RESULTS: Of 1048 HHCs enrolled, 869 [median (IQR) age: 27 years (15-40)] had both TST and QFT-GIT results available and prevalence of LTBI by QFT-GIT was 54% [95% CI (51, 57)], by TST was 55% [95% CI (52, 58)], by either test was 74% [95% CI (71, 77) and by both tests was 35% [95% CI (31, 38)]. Discordance of TST+/QFT-GIT- was 21% while TST-/QFT-GIT+ was 26%. Poor to fair agreement occurred with TST 5mm or 10mm cutoff (60 and 61% agreement with kappa value of 0.20 and 0.25 respectively). Test agreement varied by age, TST strength and induration cut-off. In multivariate analysis, span PPD was a risk factor for QFT-GIT+ and TST- while absence of BCG scar was for TST+ and QFT-GIT-. Being employed and exposure to TB case outside the household case were associated with positivity by both the tests. Sensitivity of TST and QFT-GIT to diagnose LTBI was 77% and 69%. Probability of having LTBI was >90% when both tests were positive irrespective of exposure gradient. CONCLUSION: Prevalence of LTBI among HHCs of adult pulmonary TB patients in India is very high and varies by test type, age, and exposure gradient. In our high TB burden setting, a strategy to treat all HHCs or a targeted strategy whereby an exposure index is used should be assessed in future preventive therapy and vaccine studies as HHCs have several factors that place them at high risk for progression to TB disease.

Daily vs Intermittent Antituberculosis Therapy for Pulmonary Tuberculosis in Patients With HIV: A Randomized Clinical Trial.

Importance: The benefit of daily over thrice-weekly antituberculosis therapy among HIV-positive patients with pulmonary tuberculosis (TB) who are receiving antiretroviral therapy remains unproven. Objective: To compare the efficacy and safety of daily, part-daily, and intermittent antituberculosis therapy regimens in the treatment of HIV-associated pulmonary TB. Design, Setting, and Participants: This open-label, randomized clinical trial was conducted by the National Institute for Research in Tuberculosis, south India. Adults infected with HIV with newly diagnosed, culture-positive, pulmonary TB were enrolled between September 14, 2009, and January 18, 2016. Interventions: Patients were randomized to daily, part-daily, and intermittent antituberculosis therapy regimens, stratified by baseline CD4 lymphocyte count and sputum smear grade. Antiretroviral therapy was initiated as per national guidelines. Clinical and sputum microbiological examinations of patients were performed monthly until 18 months after randomization. Adverse events were recorded using standard criteria. Main Outcomes and Measures: The primary outcome was favorable response, defined as treatment completion with all available sputum cultures negative for Mycobacterium tuberculosis during the last 2 months of treatment. Unfavorable responses included treatment failures, dropouts, deaths, and toxic effects among regimens. Results: Of 331 patients (251 [76%] male; mean [SD] age, 39 [9] years; mean [SD] HIV viral load, 4.9 [1.2] log10 copies/mL; and median [interquartile range] CD4 lymphocyte count, 138 [69-248] cells/µL), favorable responses were experienced by 91% (89 of 98), 80% (77 of 96), and 77% (75 of 98) in the daily, part-daily, and intermittent regimens, respectively. With the difference in outcome between daily and intermittent regimens crossing the O'Brien-Fleming group sequential boundaries and acquired rifampicin resistance emergence (n = 4) confined to the intermittent group, the data safety monitoring committee halted the study. A total of 18 patients died and 18 patients dropped out during the treatment period in the 3 regimens. Six, 4, and 6 patients in the daily, part-daily, and intermittent regimens, respectively, had TB recurrence. Conclusions and Relevance: Among HIV-positive patients with pulmonary TB receiving antiretroviral therapy, a daily anti-TB regimen proved superior to a thrice-weekly regimen in terms of efficacy and emergence of rifampicin resistance. Trial Registration: clinicaltrials.gov Identifier: NCT00933790.

Recurrence of tuberculosis among newly diagnosed sputum positive pulmonary tuberculosis patients treated under the Revised National Tuberculosis Control Programme, India: A multi-centric prospective study.

INTRODUCTION: There is lack of information on the proportion of new smear-positive pulmonary tuberculosis (PTB) patients treated with a 6-month thrice-weekly regimen under Revised National Tuberculosis Control Programme (RNTCP) who develop recurrent TB after successful treatment outcome. OBJECTIVE: To estimate TB recurrence among newly diagnosed PTB patients who have successfully completed treatment and to document endogenous reactivation or re-infection. Risk factors for unfavourable outcomes to treatment and TB recurrence were determined. METHODOLOGY: Adult (aged ≥ 18 yrs) new smear positive PTB patients initiated on treatment under RNTCP were enrolled from sites in Tamil Nadu, Karnataka, Delhi, Maharashtra, Madhya Pradesh and Kerala. Those declared "treatment success" at the end of treatment were followed up with 2 sputum examinations each at 3, 6 and 12 months after treatment completion. MIRU-VNTR genotyping was done to identify endogenous re-activation or exogenous re-infection at TB recurrence. TB recurrence was expressed as rate per 100 person-years (with 95% confidence interval [95%CI]). Regression models were used to identify the risk factors for unfavourable response to treatment and TB recurrence. RESULTS: Of the1577 new smear positive PTB patients enrolled, 1565 were analysed. The overall cure rate was 77% (1207/1565) and treatment success was 77% (1210 /1565). The cure rate varied from 65% to 86%. There were 158 of 1210 patients who had TB recurrence after treatment success. The pooled TB recurrence estimate was 10.9% [95%CI: 0.2-21.6] and TB recurrence rate per 100 person-years was 12.7 [95% CI: 0.4-25]. TB recurrence per 100 person-years varied from 5.4 to 30.5. Endogenous reactivation was observed in 56 (93%) of 60 patients for whom genotyping was done. Male gender was associated with TB recurrence. CONCLUSION: A substantial proportion of new smear positive PTB patients successfully treated with 6 -month thrice-weekly regimen have TB recurrence under program settings.