The recent trend in mycobacterial strain diversity among extra pulmonary lymph node tuberculosis and their association with drug resistance and the host immunological response in South India.

BACKGROUND: Tuberculosis (TB) though primarily affects the lungs it may also affect the other parts of the body and referred as extra pulmonary (EPTB). This study is focused on understanding the genetic diversity and molecular epidemiology of Mycobacterium tuberculosis (M.tb) among tuberculous lymphadenitis (TBL), a form of EPTB patients identified in Chennai, Tamil Nadu. METHODS: The genetic diversity was identified by performing spoligotyping on the M.tb clinical isolates that were recovered from lymph node samples. A total of 71 M.tb isolates were recovered from extra pulmonary lymph node samples and subjected to Drug susceptibility testing and spoligotyping was carried out. In addition, immunological characterization from blood of same individuals from whom M.tb was isolated was carried out between the two major lineages groups East African Indian 3 (EAI3) and non-EAI3 strains by ELISA. The results of spoligotyping patterns were compared with the world Spoligotyping Database of Institute Pasteur de Guadeloupe (SpolDB4). RESULTS: We found 41 spoligotype patterns and their associated lineages. Out of 41 spoligotype pattern, only 22 patterns are available in the spoldB4 database with Spoligotype international Type (SIT) number and remaining patterns were orphan strains without SIT number. The most predominant spoligotype lineage that was found in lymph node sample in this region of India was EAI (36), followed by central Asian strain (CAS) (6), T1 (5), Beijing (3), Latin American & Mediterranean (LAM) (2), U (1), X2 (1) and orphan (22). In addition to EAI, CAS and Beijing, our study identified the presence of orphan and unique spoligotyping patterns in Chennai region. We observed six drug resistant isolates. Out of six drug resistant isolates, four were resistant to isoniazid drug and associated with EAI family. Moreover, we observed increased levels of type 2 and type 17 cytokine profiles between EAI3 and non-EAI family, infected individuals. CONCLUSIONS: The study confirms that EAI lineage to be the most predominant lineages in EPTB patients with lymphadenitis and were found to have increased type 1 and type 17 proinflammatory cytokine profiles.

Spoligotype Diversity of Mycobacterium tuberculosis over Two Decades from Tiruvallur, South India.

Geographically, most tuberculosis (TB) cases in 2018 were reported from India. This TB burden is compounded by MDR-TB and XDR-TB. The strategies for the management and control of TB in the community depend on an understanding of the mode of spread of the different strains of TB isolates in the community. To determine the distribution and trends of M. tb strains over the time period in the community due to treatment, we carried out the present study on changes over two decades. Design/Methods. A total of 1218 M. tb isolates (year: 2001-2018) from Tiruvallur, India, were genotyped by spoligotyping after DNA extraction and subjected to anti-TB drug susceptibility testing for the first-line anti-TB drugs. Results. On analysis with the SpolDB4 database, majority (2001-2003: 53.32% and 2015-2018: 46.3%) of the isolates belonged to East African Indian (EAI) lineage, and the orphans designated in comparison to SpolDB4 stood 33% among 2001-2003 strain collection and 46.3% among 2015-2018 strain collection. 10.2% (2001-2003) and 9.26% (2015 to 2018) of isolates were monoresistant to isoniazid (H). MDR strains were less common among EAI strains (3.2%) compared to non-EAI strains (10.32%). Conclusions. EAI is the most predominant lineage in Tiruvallur, despite the presence of highly transmissible lineages like Beijing for the last two decades. The prevalence of MDR-TB is below the national average of 2-3% among the new TB cases in the last two decades. The reason can be attributed to the well-established nature of the locally circulating strains in this region which are not associated with drug resistance.

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.

PknB remains an essential and a conserved target for drug development in susceptible and MDR strains of M. Tuberculosis.

BACKGROUND: The Mycobacterium tuberculosis (M.tb) protein kinase B (PknB) which is now proved to be essential for the growth and survival of M.tb, is a transmembrane protein with a potential to be a good drug target. However it is not known if this target remains conserved in otherwise resistant isolates from clinical origin. The present study describes the conservation analysis of sequences covering the inhibitor binding domain of PknB to assess if it remains conserved in susceptible and resistant clinical strains of mycobacteria picked from three different geographical areas of India. METHODS: A total of 116 isolates from North, South and West India were used in the study with a variable profile of their susceptibilities towards streptomycin, isoniazid, rifampicin, ethambutol and ofloxacin. Isolates were also spoligotyped in order to find if the conservation pattern of pknB gene remain consistent or differ with different spoligotypes. The impact of variation as found in the study was analyzed using Molecular dynamics simulations. RESULTS: The sequencing results with 115/116 isolates revealed the conserved nature of pknB sequences irrespective of their susceptibility status and spoligotypes. The only variation found was in one strains wherein pnkB sequence had G to A mutation at 664 position translating into a change of amino acid, Valine to Isoleucine. After analyzing the impact of this sequence variation using Molecular dynamics simulations, it was observed that the variation is causing no significant change in protein structure or the inhibitor binding. CONCLUSIONS: Hence, the study endorses that PknB is an ideal target for drug development and there is no pre-existing or induced resistance with respect to the sequences involved in inhibitor binding. Also if the mutation that we are reporting for the first time is found again in subsequent work, it should be checked with phenotypic profile before drawing the conclusion that it would affect the activity in any way. Bioinformatics analysis in our study says that it has no significant effect on the binding and hence the activity of the protein.

Tuberculous Lymphadenitis Is Associated with Enhanced Baseline and Antigen-Specific Induction of Type 1 and Type 17 Cytokines and Reduced Interleukin-1ß (IL-1ß) and IL-18 at the Site of Infection.

Tuberculous lymphadenitis (TBL) is characterized by an expansion of Th1 and Th17 cells with altered serum levels of proinflammatory cytokines. However, the cytokine profile at the site of infection, i.e., the affected lymph nodes, has not been examined in detail. To estimate the baseline and mycobacterial antigen-stimulated concentrations of type 1, type 17, and other proinflammatory cytokines in patients with TBL (n = 14), we examined both the baseline and the antigen-specific concentrations of these cytokines before and after chemotherapy and compared them with those in individuals with pulmonary tuberculosis (PTB) (n = 14). In addition, we also compared the cytokine responses in whole blood and those in the lymph nodes of TBL individuals. We observed significantly enhanced baseline and antigen-specific levels of type 1 cytokines (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]) and a type 17 cytokine (interleukin-17 [IL-17]) and significantly diminished baseline and antigen-specific levels of proinflammatory cytokines (IL-1ß and IL-18) in the whole blood of TBL individuals compared to those in the whole blood of PTB individuals. Moreover, we also observed a pattern of baseline and antigen-specific cytokine production at the site of infection (lymph node) similar to that in the whole blood of TBL individuals. Following standard antituberculosis (anti-TB) treatment, we observed alterations in the baseline and/or antigen-specific levels of IFN-γ, TNF-α, IL-1ß, and IL-18. TBL is therefore characterized by enhanced baseline and antigen-specific production of type 1 and type 17 cytokines and reduced baseline and antigen-specific production of IL-1ß and IL-18 at the site of infection.

Tuberculosis among the homeless in Chennai city, South India.

Background: In India from a national perspective, the incidence/prevalence of active tuberculosis (TB) among the homeless are unknown. Methods: Homeless individuals, aged 15 years and above, were screened for TB by radiography and smear examination in Chennai city. Results: 301 individuals were enrolled and screened for TB; 8% (24/301) had chest symptoms; 5.6% (17/301) found X-ray abnormalities. The overall prevalence of TB was 1661/100 000; prevalence of culture-positive TB was 997/100 000 and smear-positive TB was 664/100 000 population. Conclusion: There is a need to address TB control among homeless populations. The current pilot study showed that the prevalence of TB was disproportionately high and there is a need for a larger study with an adequately powered sample size.

Reduced systemic and mycobacterial antigen-stimulated concentrations of IL-1ß and IL-18 in tuberculous lymphadenitis.

BACKGROUND: Type 1, Type 17 and other pro-inflammatory cytokines are known to play an important role in resistance to pulmonary tuberculosis. The role of these cytokines in tuberculous lymphadenitis (TBL) is not well characterized. METHODS: To estimate the systemic and mycobacterial antigen - stimulated cytokine concentrations of Type 1, Type 17, other pro-inflammatory and regulatory cytokines in TBL, we examined both the systemic and the antigen-specific concentrations of these cytokines in TBL (n=31) before and after chemotherapy, and compared them with those with latent tuberculosis infection (LTB, n=31). RESULTS: We observed significantly reduced systemic concentrations of the pro-inflammatory cytokines - IL-1ß and IL-18 but not other Type 1 or Type 17 cytokines in TBL compared to LTB. Following standard anti-tuberculosis (TB) treatment, we observed a significant increase in the concentrations of both IL-1ß and IL-18. In addition, we also observed significantly reduced baseline or mycobacterial - antigen or mitogen stimulated concentrations of IL-1ß and IL-18 in TBL individuals. Similar to systemic cytokine concentrations, anti-TB treatment resulted in significantly increased concentrations of these cytokines following antigen stimulation. CONCLUSIONS: TBL is therefore, characterized by reduced systemic and antigen-specific concentrations of IL-1ß and IL-18, which are reversible following anti-TB treatment, indicating that these cytokines are potential correlates of protective immunity in TBL.

Adequacy of examining one sputum specimen in tuberculosis drug resistance surveys.

OBJECTIVE/BACKGROUND: Collection of one spot and one morning sputum specimen is recommended for tuberculosis (TB) drug resistance surveys. This was a retrospective analysis of Mycobacterium tuberculosis cultures isolated from two spot sputum specimens collected from smear positive TB patients in a TB drug resistance survey. It was conducted to understand the value of a second specimen. METHODS: A TB drug resistance survey was conducted in the state of Tamil Nadu, India, to estimate the prevalence of drug resistance among new sputum smear-positive (NSP) and previously treated (PT) patients diagnosed in Revised National Tuberculosis Control Program microscopy centers. A total of 2425 patients (1524 NSP and 901 PT cases) were enrolled in the study. From these patients, two spot sputum specimens (C and D) were collected within a period of 2h. No preservative was added to sputum. The samples were transported at ambient conditions without cold storage to the central laboratory for culture of M. tuberculosis. Culture yield from each sample was computed and analyzed. RESULTS: The proportion of cultures retrieved from C and D specimens among NSP cases (89.3% and 89.7%) and PT cases (90.8% and 90.3%) were similar. The culture grades of C and D samples were comparable (chi-square test, 3560.135; p<.001) and the agreement was moderate (kappa test, 0.454). CONCLUSION: The findings of the study reveal the adequacy of single spot sputum specimen from smear positive pulmonary TB patients for bacteriological examination in a quality-assured TB laboratory to determine precisely the level of drug resistance in a province of India.

Drug resistance among extrapulmonary TB patients: Six years experience from a supranational reference laboratory.

BACKGROUND & OBJECTIVES: There is limited information available about the drug resistance patterns in extrapulmonary tuberculosis (EPTB), especially from high burden countries. This may be due to difficulty in obtaining extrapulmonary specimens and limited facilities for drug susceptibility testing. This study was undertaken to review and report the first and second-line anti-TB drug susceptibility patterns in extrapulmonary specimens received at the National Institute for Research in Tuberculosis (NIRT), Chennai, India, between 2005 and 2012. METHODS: Extrapulmonary specimens received from referring hospitals were decontaminated and cultured using standard procedures. Drug susceptibility testing (DST) for Mycobacterium tuberculosis was done by absolute concentration or resistance ratio methods for the first and the second line anti-TB drugs. RESULTS: Between 2005 and 2012, of the 1295 extrapulmonary specimens, 189 grew M. tuberculosis, 37 (19%) cases were multidrug resistant (MDR) while one was extensively drug resistant (XDR). Specimen-wise MDR prevalence was found to be: CSF-10 per cent, urine-6 per cent, fluids and aspirates-27 per cent, pus-23 per cent, lymph nodes-19 per cent. Resistance to isoniazid and ethionamide was found to be high (31 and 38%, respectively). INTERPRETATION & CONCLUSIONS: Drug resistance including MDR-TB was observed in a significant proportion of extrapulmonary specimens referred for DST. Access to culture and DST for extrapulmonary specimens should be expanded. Guidelines for MDR-TB management should have explicit sections on extra-pulmonary tuberculosis and training on laboratory techniques is urgently required.

Prevalence and risk factors for adult pulmonary tuberculosis in a metropolitan city of South India.

BACKGROUND: The present study measured the community prevalence and risk factors of adult pulmonary tuberculosis (PTB) in Chennai city, and also studied geographical distribution and the presence of different M. tuberculosis strains in the survey area. METHODS: A community-based cross sectional survey was carried out from July 2010 to October 2012 in Chennai city. Prevalence of bacteriologically positive PTB was estimated by direct standardization method. Univariate and multivariate analyses were carried out to identify significant risk factors. Drug susceptibility testing and spoligotyping was performed on isolated M. tuberculosis strains. Mapping of PTB cases was done using geographic positioning systems. RESULTS: Of 59,957 eligible people, 55,617 were screened by X-ray and /or TB symptoms and the prevalence of smear, culture, and bacteriologically positive PTB was estimated to be 228 (95% CI 189-265), 259 (95% CI 217-299) and 349 (95% CI 330-428) per 100,000 population, respectively. Prevalence of smear, culture, and bacteriologically positive PTB was highest amongst men aged 55-64 years. Multivariate analysis showed that occurrence of both culture and bacteriologically positive PTB disease was significantly associated with: age >35 years, past history of TB treatment, BMI <18.5 Kgs/m2, solid cooking fuel, and being a male currently consuming alcohol. The most frequent spoligotype family was East African Indian. Spatial distribution showed that a high proportion of patients were clustered in the densely populated north eastern part of the city. CONCLUSION: Our findings demonstrate that TB is a major public health problem in this urban area of south India, and support the use of intensified case finding in high risk groups. Undernutrition, slum dwelling, indoor air pollution and alcohol intake are modifiable risk factors for TB disease.

High rates of ofloxacin resistance in Mycobacterium tuberculosis among both new and previously treated patients in Tamil Nadu, South India.

Periodic drug resistance surveillance provides useful information on trends of drug resistance and effectiveness of tuberculosis (TB) control measures. The present study determines the prevalence of drug resistance among new sputum smear positive (NSP) and previously treated (PT) pulmonary TB patients, diagnosed at public sector designated microscopy centers (DMCs) in the state of Tamil Nadu, India. In this single-stage cluster-sampling prevalence survey, 70 of 700 DMCs were randomly selected using a probability-proportional to size method. A cluster size of 24 for NSP and a varying size of 0 to 99 for PT cases were fixed for each selected DMC. Culture and drug susceptibility testing was done on Lowenstein-Jensen medium using the economic variant of proportion sensitivity test for isoniazid (INH), rifampicin (RMP), ofloxacin (OFX) and kanamycin (KAN). Human Immunodeficiency Virus (HIV) status was collected from patient records. From June 2011 to August 2012, 1524 NSP and 901 PT patients were enrolled. Any RMP resistance and any INH resistance were observed in 2.6% and 15.1%, and in 10.4% and 30% respectively in NSP and PT cases. Among PT patients, multi drug resistant TB (MDR-TB) was highest in the treatment failure (35%) group, followed by relapse (13%) and treatment after default (10%) groups. Extensively drug resistant TB (XDRTB) was seen in 4.3% of MDR-TB cases. Any OFX resistance was seen in 10.4% of NSP, 13.9% of PT and 29% of PT MDR-TB patients. The HIV status of the patient had no impact on drug resistance levels. RMP resistance was present in 2.6% of new and 15.1% of previously treated patients in Tamil Nadu. Rates of OFX resistance were high among NSP and PT patients, especially among those with MDR-TB, a matter of concern for development of new treatment regimens for TB.

The Assessment of Viability of M. Tuberculosis after Exposure to CPC Using Different Methods.

Settings. National Institute for Research in Tuberculosis, Chennai. Objective. To assess the proportion of metabolically active cells of Mycobacterium tuberculosis after exposed to CPC using FDA-EB vital staining and viable counts on LJ medium. Mycolic acid content in M. tuberculosis after exposure to CPC was estimated using HPLC. Methods. Clinical isolates of M. tuberculosis and standard reference strain M. tuberculosis H37Rv were used for FDA-EB, viable count, and HPLC. Results. FDA/EB consistently stained 70-90% of log phase cells as green and the remaining cells as red-orange. After CPC treatment, 65-70% of the cells stained red-orange. The viability counts were comparable to 0-day controls. Synthesis of mycolic acids in mycobacteria was reduced when exposed to CPC using HPLC due to the decreased metabolic activity of the organisms. Conclusion. The cells are metabolically inactive during storage with CPC but these cells grew well on LJ medium after removal of CPC. The viability of M. tuberculosis was maintained in CPC with minimal reduction. Mycolic acid content was reduced if the cells of M. tuberculosis were treated with CPC for 7 days. All the above findings provide yet another evidence for the damage of cell wall of M. tuberculosis.

Prevalence of tobacco use in urban, semi urban and rural areas in and around Chennai City, India.

BACKGROUND: Tobacco use leads to many health complications and is a risk factor for the occurrence of cardio vascular diseases, lung and oral cancers, chronic bronchitis etc. Almost 6 million people die from tobacco-related causes every year. This study was conducted to measure the prevalence of tobacco use in three different areas around Chennai city, south India. METHODS: A survey of 7510 individuals aged > = 15 years was undertaken covering Chennai city (urban), Ambattur (semi-urban) and Sriperumbudur (rural) taluk. Details on tobacco use were collected using a questionnaire adapted from both Global Youth Tobacco Survey and Global Adults Tobacco Survey. RESULTS: The overall prevalence of tobacco use was significantly higher in the rural (23.7%) compared to semi-urban (20.9%) and urban (19.4%) areas (P value <0.001) Tobacco smoking prevalence was 14.3%, 13.9% and 12.4% in rural, semi-urban and urban areas respectively. The corresponding values for smokeless tobacco use were 9.5%, 7.0% and 7.0% respectively. Logistic regression analysis showed that the odds of using tobacco (with smoke or smokeless forms) was significantly higher among males, older individuals, alcoholics, in rural areas and slum localities. Behavioural pattern analysis of current tobacco users led to three groups (1) those who were not reached by family or friends to advice on harmful effects (2) those who were well aware of harmful effects of tobacco and even want to quit and (3) those are exposed to second hand/passive smoking at home and outside. CONCLUSIONS: Tobacco use prevalence was significantly higher in rural areas, slum dwellers, males and older age groups in this region of south India. Women used mainly smokeless tobacco. Tobacco control programmes need to develop strategies to address the different subgroups among tobacco users. Public health facilities need to expand smoking cessation counseling services as well as provide pharmacotherapy where necessary.

Phage cocktail to control the exponential growth of normal flora in processed sputum specimens grown overnight in liquid medium for rapid TB diagnosis.

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.