ASSOCIATION OF GENERATION TIME WITH ANTI-TUBERCULAR DRUG(S) RESISTANCE PATTERN OF MYCOBACTERIUM TUBERCULOSIS ISOLATES AMONG TREATMENT FAILURE PULMONARY TUBERCULOSIS PATIENTS

ABSTRACTObjective: The emergence of drug resistance has complicated tuberculosis (TB) scenario and is associated to treatment failure. The causative agent,Mycobacterium tuberculosis is usually slow growing and has been implicated as a contributing factor for drug tolerance and development of resistantstrains. On the other hand, if rapidly growing bacilli, with shorter generation time emerge, mutations may lead to the development of drug resistance.From the hypothesis, this study was aimed to explore the whether there is any association between the generation time of Mycobacteria with theirdistinct drug resistant pattern.Methods: In-vitro generation time was determined from 77 mycobacterial isolates with varied drug resistance pattern, i.e. rifampicin resistant (RIFR),isoniazid resistant, multi-drug resistant (MDR), the sensitive clinical strains along with reference strains. The minimal inhibitory concentration wasalso determined for the respective resistant groups.Results: Among the individual group of clinical isolates, there was a significant negative association between generation time and drug resistancepattern of RIFR isolates.Conclusion: Keeping the current upsurge of the MDR-TB epidemic in India and the influence of generation time on dosing schedule and treatmentstrategy, necessary customization of dosing and therapeutic planning seemed urgent to minimize the operational and clinical potential for developmentof drug resistance among treatment failure pulmonary TB patients in this country.Keywords: Mycobacterium tuberculosis, Generation time, Multi-drug resistant, Treatment failure

Th1 and Th2 cytokine response of peripheral blood macrophages-lymphocyte co-culture on challenge with Bacillus Calmette-Guérin and Mycobacterium tuberculosis H37RV in different categories of tuberculosis patients

Background: The quantitative analysis of Th1 and Th2 cytokines by in vitro challenged macrophages from tuberculosis (TB) patients of different clinical setting and healthy controls would enlighten our knowledge of macrophage efficiency at different ages and status of disease, facilitating new treatment feasibility by immunotherapy. Aims and Objectives: The aim of the study was to study the pattern of release of interferon-gamma (IFN-γ), interleukin-4 (IL-4), and IL-10 by macrophages, from pulmonary TB cases compared to normal individuals, in in vitro culture, on the challenge with Bacillus Calmette-Guérin (BCG) and Mycobacterium tuberculosis H37Rv. Materials and Methods: Fifteen sputum smear-positive newly diagnosed TB and 15 relapsed TB cases fulfilling the inclusion and exclusion criteria were recruited. Fifteen tuberculin skin test (TST) positive and 15 TST negative age- and sex-matched healthy volunteers were included as a control. Isolated macrophages were cultured and pretreated with BCG and H37Rv. After 24 h, the cell-free supernatant was collected and subjected to a sandwich enzyme-linked immunosorbent assay for IL-4, IL-10, and IFN-γ. Results: It was found that monocytes behave differently toward the virulent and avirulent strains of Mycobacterium in IFN-γ production. Th1 cytokine response was found to be higher by BCG challenge followed by H37Rv among all the study groups. However, Th2 cytokine responses with IL-10 and IL-4 were found to be higher in the patients as compared to healthy controls. Conclusion: Capacity to mount an inflammatory response determines the outcome of TB infection. Further study will enrich our knowledge in understanding the individual cell function and immunopathological mechanisms associated with the different clinical forms of TB

Interferon-gamma and nitric oxide burst response in different categories of pulmonary tuberculosis patients

Background: Anti-tuberculous cellular immunity involves the critical interplay of T lymphocytes, macrophages, and cytokines. The quantitative analysis of pro-inflammatory cytokine (interferon-gamma [IFN-γ]) release and nitric oxide (NO) production by in vitro challenged macrophages from tuberculosis (TB) patients of different clinical settings and healthy controls would enlighten our knowledge of macrophage efficiency at different age and status of disease, thereby paving the way for immunotherapy. Aims and Objectives: To study the pattern of release of IFN-γ and NO burst response by macrophages, from pulmonary TB cases compared to normal individuals, in vitro culture, on challenge with Bacillus Calmette-Guérin (BCG) and Mycobacterium tuberculosis H37Rv. Materials and Methods: Fifteen consecutive sputum smear-positive newly diagnosed TB and 15 relapsed TB cases were recruited. All cases were pulmonary with no BCG vaccination history. 15 tuberculin skin test (TST) positive, 15 TST negative healthy volunteer, and 5 newborn antigen naïve cases were included as control. Isolated macrophages were cultured and pretreated with BCG and H37Rv. IFN-γ and NO were measured from the cell free culture supernatant after 24 h. Results: The monocytes of the patients are functionally different than the healthy subjects toward the same antigenic stimulation. The level of NO release correlates with the extent of IFN-γ production. The presence of Th1 response and associated factors like NO burst are some of the main regulators of the outcome of the disease. Conclusion: Capacity to mount inflammatory response, effective NO generation determines the outcome of TB infection and may contribute to better understanding of the underlying immunopathology

Performance of SNAPPE-II score in neonatal sepsis: an experience from a tertiary care center

Background and Objectives. The Score for Neonatal Acute Physiology II with Perinatal Extension (SNAPPE-II) is a vital tool for prognostication in newborns. The study was conducted with the hypothesis that the performance of the SNAPPE-II score might be affected by the presence of sepsis in newborns admitted with possible early onset septicemia and whether score performance varies between culture positive and culture negative sepsis. Methods. The prospective observational study was conducted over a period of 1 year (January 2014 to January 2015) in neonates presenting with clinical suspicion of sepsis to the Sick Newborn Care Unit (SNCU) of a tertiary care hospital in Eastern India. Results. SNAPPE-II score cut-off of ≥20 offered the highest sensitivity of 74.5% with specificity 48.3%, PPV 27.6% and NPV 87.7%. Comparison of mortality proportions between the two subgroups defined by this cut-off returned p= 0.005 with OR 3.47 (95% 1.40 to 8.64). No significant association was found between SNAPPE-II score and blood culture results; mean scores for culture positive (25.16 ± 15.6) and negative groups (24.49 ± 15.6) were comparable (p= 0.920). Conclusions. At a cut-off value of ≥20 in presence of sepsis, SNAPPE-II score offers acceptable indices to predict mortality outcome. Prediction of outcome by SNAPPE-II score is not affected by positive or negative blood culture sepsis

Immunomodulation in host-protective immune response against murine tuberculosis through regulation of the T regulatory cell function

Abstract Tuberculosis, caused by the bacteria Mycobacterium tuberculosis, is characterized by an infection in lung and spleen. In the present study, we have elucidated the mechanism by which Mycobacterium indicus pranii renders protection in in vivo Mycobacterium tuberculosis infection. We observed that Mycobacterium indicus pranii treated infected C57BL/6 mice showed a strong host-protective Th1 immune response along with a marked decrease in immunosuppressive cytokines, TGF-β, and IL-10-secreting CD4+ T cells. This Mycobacterium indicus pranii mediated decrease in immunosuppressive cytokines was correlated with the reduction in the elevated frequency of CD4+CD25+ T regulatory cells, along with the reduced TGF-β production from these T regulatory cells in tuberculosis-infected mice. This reduction in the T regulatory cell population was a result of effective modulation of STAT4–STAT5 transcription factor counter-regulation by Mycobacterium indicus pranii, which in turn, reduced the immunosuppressive activity of T regulatory cells. Thus, these findings put forward a detailed mechanistic insight into Mycobacterium indicus pranii mediated regulation of the T regulatory cell functioning during experimental murine tuberculosis, which might be helpful in combating Mycobacterium-induced pathogenesis.</jats:p

Correlates of Treatment Outcomes and Drug Resistance among Pulmonary Tuberculosis Patients Attending Tertiary Care Hospitals of Kolkata, India

Worldwide highest number of new pulmonary tuberculosis (PTB) cases, was reported from India in 2012. Adverse treatment outcomes and emergence of drug resistance further complicated the prevailing scenario owing to increased duration, cost and toxicity associated with the treatment of drug-resistant cases. Hence to reinforce India's fight against TB, identification of the correlates of adverse treatment outcomes and drug resistance, seemed critical.To estimate the associations between diagnostic findings, patient types (based on treatment outcomes), drug resistance and socio-demographic characteristics of PTB patients, a cross-sectional study was conducted in two tertiary-care hospitals in Kolkata between April 2010 and March 2013. Altogether, 350 consenting Mycobacterium tuberculosis sputum-culture positive PTB patients were interviewed about their socio-demographic background, evaluated regarding their X-ray findings (minimal/moderately advanced/far advanced/cavities), sputum-smear positivity, and treatment history/outcomes (new/defaulter/relapse/treatment-failure cases). Multiple-allele-specific polymerase chain reaction (MAS-PCR) was conducted to diagnose drug resistance.Among all participants, 31.43% were newly diagnosed, while 44%, 15.43% and 9.14% patients fell into the categories of relapsed, defaulters and treatment-failures, respectively. 12.29% were multi-drug-resistant (MDR: resistant to at least isoniazid and rifampicin), 57.71% had non-MDR two-drug resistance and 12% had single-drug resistance. Subjects with higher BMI had lower odds of being a relapse/defaulter/treatment failure case while females were more likely to be defaulters and older age-groups had more relapse. Elderly, females, unmarried, those with low BMI and higher grade of sputum-smear positivity were more likely to have advanced X-ray features. Higher grade of sputum-smear positivity and advanced chest X-ray findings were associated with relapse/treatment-failures. Elderly, unmarried, relapse/defaulter/treatment-failure cases had higher odds and those with higher BMI and moderately/far advanced X-ray findings had lower odds of having MDR/non-MDR two-drug resistant PTB.Targeted intervention and appropriate counseling are needed urgently to prevent adverse treatment outcomes and development of drug resistance among PTB patients in Kolkata