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Level of drug resistance among new TB patients indicates the efficacy of any control programme. A drug resistance survey, as a part of global study in new pulmonary tuberculosis (NPTB) patients, was conducted in Wardha district, India under WHO /IUTLD in 2001, before the implementation of the Revised National Tuberculosis Control Programme (RNTCP) which was implemented in the district in 2002. Objective: The present study was conducted in Wardha district in 2014-2015, thirteen years after the implementation of RNTCP, and the drug resistance in Mycobacterium tuberculosis isolates from NPTB patients was compared to the results of 2001 survey. The methodology used was same in both the surveys. Material and Methods: In addition to 132 isolates fromWardha, the study also included 112 isolates from adjoining city, Nagpur and total of 244 sputum isolates were subjected to drug sensitivity by standard 1% proportion method on Lowenstein Jensen’s medium. In addition molecular typing of resistant strains was done. Results: In Wardha, compared to 2001 survey, overall susceptibility to first line drugs was higher (94.7% vs 80.2%); and resistance to streptomycin (3% vs 7.6%) and isoniazid (2.2% vs 15.2%) were significantly lower (p 0.05). MDR was 0.75% against 0.50% in the earlier study but the difference was statistically not significant. Only two MDR isolate were recovered, of which only one was from Wardha. Conclusion: After the implementation of RNTCP in Wardha District, drug resistance in new PTB patients has shown a decline and MDR continues to be low reflecting upon the efficiency of the program. Keywords: Resurvey, New Pulmonary TB patients, Drug Resistance

Background & objectives: Early case detection is essential to interrupt transmission and to prevent further spread of tuberculosis (TB) in high endemic settings. Nucleic acid amplification tests (NAATs) with visual read-outs are ideal as point-of-care tests. Truenat™ MTB is an indigenous chip-based NAAT for detection of Mycobacterium tuberculosis, which involves extraction of DNA and real-time polymerase chain reaction (PCR) using portable, automated, battery-operated instruments. The current multicentric study was aimed to evaluate Truenat for detection of MTB in sputum samples obtained from patients with presumptive pulmonary TB with reference to culture as gold standard and Xpert as a comparator. Methods: The study was conducted at four sites, namely ICMR-National Institute for Research in Tuberculosis, Chennai; All India Institute of Medical Sciences, New Delhi; ICMR-National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra; and National Institute of TB and Respiratory Diseases, New Delhi. Patients suspected to have TB were screened for eligibility. Two sputum samples were collected from each patient. Tests included smear, Xpert and Truenat directly from the sputum sample and culture by Lowenstein-Jensen (L-J) medium and MGIT960 from decontaminated pellets. Sample used for Truenat assay was coded. Resolution of Truenat false positives was done using an in-house PCR with TRC4 primers. Results: The study enrolled 2419 presumptive TB patients after screening 2465 patients, and 3541 sputum samples were collected from the enrolled patients. Results of 2623 samples were available for analysis. Truenat showed a positivity rate of 48.5 per cent as compared to 37.0 per cent by Xpert. The sensitivities of Truenat and Xpert were was 88.3 and 79.7 per cent, respectively in comparison with culture. Interpretation & conclusions: Truenat MTB identified more positives among culture-confirmed samples than Xpert and had higher sensitivity. In addition, other advantageous operational features of Truenat MTB were identified which would be useful in field settings.

Background & objectives: There is a need for an affordable, easy, high-sensitivity test usable at the peripheral health facility for diagnosis of drug-resistant (DR) tuberculosis (TB) to interrupt disease transmission. Nucleic acid amplification tests (NAATs) for early detection of DR-TB are ideal to bring testing near to the patient. TruenatTM MTB (Mycobacterium tuberculosis) and TruenatTM MTB-RIF (rifampicin) is an indigenous chip-based real-time polymerase chain reaction (PCR) based test for detection of multidrug-resistant (MDR) TB. The test involves extraction of DNA using automated, battery operated Trueprep instrument and real-time PCR performed on the Truelab analyzer. We report here multicentric validation of Truenat MTB-RIF for detection of DR-TB in suspected DR-TB patients. Methods: Consecutive patients aged 18-65 yr, with symptoms suggestive of TB and with a history of previous treatment, reporting to the National TB Elimination Programme (NTEP) clinics under four national institutes, namely AIIMS (All India Institute of Medical Sciences, New Delhi), NITRD (National Institute of Tuberculosis and Respiratory Diseases, New Delhi), NIRT (National Institute for Research in Tuberculosis, Chennai) and ICMR-National JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra, were included in the study. Two sputum samples (one spot and one morning) were collected from each patient, after obtaining informed written consent. The samples were subjected to smear, GeneXpert and MGIT 960 culture (and drug susceptibility testing to RIF) (surrogate for MDR-TB) to serve as reference tests. The samples were coded to ensure blinding and subjected to Truenat MTB-RIF. Truenat MTB-RIF Version 1.5 was used for testing 1084 samples for RIF resistance, while Version 2.0 was used to test another 1201 samples. Results: Truenat MTB-RIF Version 1.5 in comparison with comprehensive laboratory reference standards yielded sensitivity and specificity of 76.2 and 94.7 per cent, respectively for the detection of RIF resistance in 1084 samples, collected across four sites. Based on the analysis of discordant samples, Version 2.0 of Truenat was developed by the manufacturer and this was further tested on additional 1201 samples, yielding a sensitivity of 87.5 per cent and specificity of 99.5 per cent. Interpretation & conclusions: Multicentric trial of TruenatTM MTB-RIF demonstrated a great potential of this point of care NAAT for detection of MDR-TB. The test would be useful in limited resource settings and inaccessible areas without need for any additional infrastructure.

Background Diagnosing tuberculosis (TB) in children is challenging due to paucibacillary disease, and lack of ability for microbiologic confirmation. Hence, we measured the plasma chemokines as biomarkers for diagnosis of pediatric tuberculosis. Methods We conducted a prospective case control study using children with confirmed, unconfirmed and unlikely TB. Multiplex assay was performed to examine the plasma CC and CXC levels of chemokines. Results Baseline levels of CCL1, CCL3, CXCL1, CXCL2 and CXCL10 were significantly higher in active TB (confirmed TB and unconfirmed TB) in comparison to unlikely TB children. Receiver operating characteristics curve analysis revealed that CCL1, CXCL1 and CXCL10 could act as biomarkers distinguishing confirmed or unconfirmed TB from unlikely TB with the sensitivity and specificity of more than 80%. In addition, combiROC exhibited more than 90% sensitivity and specificity in distinguishing confirmed and unconfirmed TB from unlikely TB. Finally, classification and regression tree models also offered more than 90% sensitivity and specificity for CCL1 with a cutoff value of 28 pg/ml, which clearly classify active TB from unlikely TB. The levels of CCL1, CXCL1, CXCL2 and CXCL10 exhibited a significant reduction following anti-TB treatment. Conclusion Thus, a baseline chemokine signature of CCL1/CXCL1/CXCL10 could serve as an accurate biomarker for the diagnosis of pediatric tuberculosis.

Mycobacterium kansasii, most virulent of all atypical mycobacteria, causes pulmonary disease identical to the disease caused by Mycobacterium tuberculosis. Early identification of the species and prompt initiation of treatment for M. kansasii is necessary to prevent morbidity and mortality due to this disease. This case series highlights the similarity in the clinical presentation of both M. tuberculosis and M. kansasii and response to direct observation of short-course chemotherapy with rifampicin, in the management of pulmonary M. kansasii disease. Larger studies are required to evaluate the long-term effect of short-course chemotherapy, especially use of moxifloxacin, in the management of pulmonary M. kansasii disease.

BACKGROUND: The performance of the Capilia test for rapid identification of Mycobacterium tuberculosis complex (MTC) in Mycobacterium Growth Indicator Tube (MGIT) positive samples with contaminating organisms is not well documented. OBJECTIVE: To assess the diagnostic yield of the Capilia test in the rapid identification of MTC in MGIT-positive cultures. DESIGN: A total of 459 selected sputum samples were cultured using BACTEC™ MGIT™ 960. Tubes flagged positive by the MGIT instrument (MGIT-positive) were examined for acid-fast bacilli and cording in smears, spotted on blood agar (BA), subcultured for biochemical tests and tested using the Capilia test. Based on smear and growth on BA, MGIT-positive tubes were grouped into MGIT true-positive, MGIT-positive with contamination and MGIT contamination. Performance parameters of Capiliatest such as sensitivity, specificity, efficiency, and positive and negative predictive values (PPV, NPV) for each of these groups were determined against biochemical tests as gold standard. RESULTS: Of the 346 MGIT-positives, respectively 233, 73 and 40 were MGIT true-positive, MGIT-positive with contamination and MGIT contamination. For the three groups, the PPV and NPV of the Capilia test were respectively 97%, 96% and 100%, and 32%, 27% and 60%. CONCLUSION: In settings with high contamination of MGIT cultures, the performance of the Capilia test is diminished.

Diagnosis of latent tuberculosis infection is a myth for want of a simple, direct tool. Simulation of hypoxic environment was done to create a novel hypothetical model for persistence using processed sputum samples. The adaptation of tubercle bacilli to hypoxic environment seems to be influenced by pre-existing clinical status of the patients at the time of sputum collection, resulting in varied growth pattern. Bacilli from 36 samples did not get adapted to latency of which 15 samples were from patients in whom the disease was well established and the tubercle bacilli in them probably did not experience any stress whatsoever. Similarly, 10 of the 37 samples showing the presence of cultivable cells in both aerobic and anaerobic conditions were from patients who had relapsed. The bacilli in these samples had been probably experiencing stress and thus were ready to adapt to the hypoxic environment. Diagnostic luciferase reporter phage assay for non-replicating persistors (DLRPA-NRP) identified 30 additional positives which failed to grow on Lowenstein–Jensen medium. Presence of viable bacilli in these samples was confirmed by reverse transcriptase-PCR (RT-PCR) for 16S rRNA indicating either the improved sensitivity of the assay to detect actively growing bacilli or its ability to detect non-replicating persistors. The utility of LRP assay to detect both dormant and active tubercle bacilli was explored in this work and was optimized using lysis inhibition to diagnose tuberculosis with rapidity, improved sensitivity and specificity. DLRPA-NRP, a rapid growth based assay is thus developed to detect both dormant and actively growing tubercle bacilli.

National Institute for Research in Tuberculosis clinics in Chennai and Madurai, India.To examine the pattern of serial smears (negative-negative [NN], negative-positive [NP], positive-negative [PN], positive-positive [PP]) during treatment follow-up of culture-confirmed new smear-positive tuberculosis (TB) patients, and the proportion of culture-negatives in each category.We reviewed the records and extracted follow-up smear (fluorescent microscopy) and culture (Löwenstein-Jensen) results of patients enrolled in clinical trials from January 2000 to August 2012 and treated with the Category I regimen (2EHRZ3/4HR3). Data entry and analysis were performed using EpiData.Among 520 patients (176 infected with the human immunodeficiency virus), the proportions of culture-negative patients with NN, discordant (PN or NP) and PP patterns were approximately 98%, 80% and 40%, respectively. The smear-positive culture-negative phenomenon was more frequent in follow-up smear results graded 1+, followed by 2+ and 3+.There is justification for discontinuing the examination of second specimens during treatment follow-up among TB patients. However, a positive result on the first smear needs to be confirmed by a second positive result before making clinical management decisions. The World Health Organization may need to reconsider its recommendation on this issue.Dispensaires de l’Institut National de Recherche sur la Tuberculose (NIRT) à Chennai et Madurai, Inde.Examiner le type des frottis successifs (négatif-négatif [NN], négatif-positif [NP], positif-négatif [PN], positif-positif [PP]) au cours du suivi du traitement des nouveaux cas de tuberculose (TB) à frottis positif confirmés par la culture ainsi que la proportion de cultures négatives dans chacune de ces catégories.Nous avons revu les dossiers et extrait les résultats des frottis de suivi (examen microscopique par fluorescence) et des cultures (Löwenstein-Jensen) chez les patients enrôlés dans des essais cliniques entre janvier 2000 et août 2012 et traités par un régime de Catégorie 1 (2EHRZSur 520 patients, dont 176 infectés par le virus de l’immunodéficience humaine, les proportions de cultures négatives parmi les patients avec le type NN, le type discordant (PN ou NP) et le type PP ont été respectivement d’environ 98%, 80% et 40%. Le phénomène de culture négative avec frottis positif est plus prononcé dans les résultats des frottis de suivi classés comme 1+, suivis par 2+ et 3.Il est justifié d’interrompre l’examen du deuxième échantillon au cours du suivi du traitement chez les patients TB. Toutefois un résultat positif du premier frottis impose une confirmation par un deuxième résultat positif avant de prendre des décisions de prise en charge clinique. A cet égard, l’Organisation Mondiale de la Santé pourrait devoir revoir ses recommandations.Los consultorios del Instituto Nacional de Investigación en Tuberculosis de Chennai y Madurai, en la India.Examinar el patrón de los resultados de los exámenes microscópicos seriados de esputo (NN, NP, PN, PP; [N = negativo, P = positivo]) durante el seguimiento del tratamiento de los casos nuevos de tuberculosis (TB) con baciloscopia positiva y determinar la proporción de casos con cultivo negativo en cada categoría.Se analizaron las historias clínicas y se extrajeron los resultados de las baciloscopias (por microscopia fluorescente) y los cultivos (en medio Löwenstein-Jensen) de los pacientes que participaron en estudios clínicos entre enero del 2000 y agosto del 2012 y que recibieron tratamiento con un régimen de Categoría I (2EHRZEn los 520 pacientes incluidos (176 infectados por el virus de la inmunodeficiencia humana), la proporción aproximada de cultivos negativos en los pacientes con baciloscopia NN fue 98%; en los casos discordantes, PN o NP fue 80%; y en los PP la proporción fue 40%. El fenómeno de obtener un resultado positivo de la baciloscopia con un cultivo negativo fue más frecuente con las baciloscopias clasificadas de grado 1+, seguidas por los grados 2+ y 3+.Está justificado suspender el examen de la segunda muestra de esputo durante el seguimiento del tratamiento de los pacientes con TB. Sin embargo, un resultado positivo en la primera baciloscopia se debe confirmar con un segundo resultado antes de tomar decisiones clínicas en materia de manejo. Tal vez sería necesario que la Organización Mundial de la Salud examinase de nuevo sus recomendaciones al aspecto.

Che12 is a temperate Chennai phage infecting Mycobacterium tuberculosis. The nucleotide sequence of the 52,047 bp linear double stranded DNA genome has a GC content of 62.9% with 70 putative ORFs identified. Functions are assigned to 24 genes based on the similarity of the predicted products to known proteins. Che12 genome is highly similar to mycobacteriophage L5 and D29 genomes. The overall genome similarity of Che12 to L5 is 82.5% and D29 is 81.5%. The genes attributing to lysogeny such as integrase, excisionase and repressor protein are identified. The attachment site of Che12 genome attP is homologous to attB sites of Mycobacterium smegmatis and M. tuberculosis. Similarities between certain phage gene products are noted, in particular, the terminases, DNA primase and endonucleases. The complete sequence clarifies the overall transcription map of Che12 and the positions of elements involved in the maintenance of lysogeny.

The mechanical pressure exerted during centrifugation and the chemical pressure experienced when sputum specimens are processed, leave the tubercle bacilli in the sputum unsuitable for rapid detection especially in phage based assays. Thus, growing Mycobacterium tuberculosis in broth, at least overnight, is mandatory for allowing the tubercle bacilli to recoup. During this time the surviving colonizing flora grow faster and overgrow tubercle bacilli interfering with TB diagnosis. In the present study normal flora surviving the action of 4% NaOH was isolated and characterized. Phages capable of killing 14 different species representing this normal flora were isolated from soil and sewage samples and characterized. A novel and bio-friendly approach to treat sputum samples with a cocktail of three phages capable of killing most of the 14 representative organisms and not infecting mycobacteria is explored to control the overgrowth of colonizing bacteria in broth culture. While 26 of the 100 sputum samples processed by modified Petroff's procedure showed growth of colonizing flora on blood agar, all of them when grown in broth overnight showed mixed, confluent growth. The addition of phagebiotics controlled them all, showing a significant reduction in colony forming units but resulting in few discrete colonies in 54 samples. Isolation of phages capable of controlling these surviving organisms and including them in the phagebiotics mixture should lead to the control of colonizing bacteria effectively.