Prahlad Kumar

Proteogenomic Analysis of Mycobacterium tuberculosis By High Resolution Mass Spectrometry

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ∼80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ∼250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.

Risk factors associated with default among new smear positive TB patients treated under DOTS in India.

Background Poor treatment adherence leading to risk of drug resistance, treatment failure, relapse, death and persistent infectiousness remains an impediment to the tuberculosis control programmes. The objective of the study was to identify predictors of default among new smear positive TB patients registered for treatment to suggest possible interventions to set right the problems to sustain and enhance the programme performance. Methodology & Principal Findings Twenty districts selected from six states were assigned to six strata formed, considering the geographic, socio-cultural and demographic setup of the area. New smear positive patients registered for treatment in two consecutive quarters during III quarter 2004 to III quarter 2005 formed the retrospective study cohort. Case control analysis was done including defaulted patients as “cases” and equal number of age and sex matched patients completing treatment as “controls”. The presence and degree of association between default and determinant factors was computed through univariate and multivariate logistic regression analysis. Data collection was through patient interviews using pre-tested semi structured questionnaire and review of treatment related records. Information on a wide range of socio demographic and patient related factors was obtained. Among the 687 defaulted and equal numbers of patients in completed group, 389 and 540 patients respectively were satisfactorily interviewed. In the logistic regression analysis, factors independently associated with default were alcoholism [AOR-1.72 (1.23–2.44)], illiteracy [AOR-1.40 (1.03–1.92)], having other commitments during treatment [AOR-3.22 (1.1–9.09)], inadequate knowledge of TB [AOR-1.88(1.35–2.63)], poor patient provider interaction [AOR-1.72(1.23–2.44)], lack of support from health staff [AOR-1.93(1.41–2.64)], having instances of missed doses [AOR-2.56(1.82–3.57)], side effects to anti TB drugs [AOR-2.55 (1.87–3.47)] and dissatisfaction with services provided [AOR-1.73 (1.14–2.6)]. Conclusion Majority of risk factors for default were treatment and provider oriented and rectifiable with appropriate interventions, which would help in sustaining the good programme performance.

Appropriate DevR (DosR)-Mediated Signaling Determines Transcriptional Response, Hypoxic Viability and Virulence of Mycobacterium tuberculosis

Background The DevR(DosR) regulon is implicated in hypoxic adaptation and virulence of Mycobacterium tuberculosis. The present study was designed to decipher the impact of perturbation in DevR-mediated signaling on these properties. Methodology/Principal Findings M. tb complemented (Comp) strains expressing different levels of DevR were constructed in Mut1* background (expressing DevR N-terminal domain in fusion with AphI (DevRN-Kan) and in Mut2ΔdevR background (deletion mutant). They were compared for their hypoxia adaptation and virulence properties. Diverse phenotypes were noted; basal level expression (∼5.3±2.3 µM) when induced to levels equivalent to WT levels (∼25.8±9.3 µM) was associated with robust DevR regulon induction and hypoxic adaptation (Comp 9* and 10*), whereas low-level expression (detectable at transcript level) as in Comp 11* and Comp15 was associated with an adaptation defect. Intermediate-level expression (∼3.3±1.2 µM) partially restored hypoxic adaptation functions in Comp2, but not in Comp1* bacteria that co-expressed DevRN-Kan. Comp* strains in Mut1* background also exhibited diverse virulence phenotypes; high/very low-level DevR expression was associated with virulence whereas intermediate-level expression was associated with low virulence. Transcription profiling and gene expression analysis revealed up-regulation of the phosphate starvation response (PSR) in Mut1* and Comp11* bacteria, but not in WT/Mut2ΔdevR/other Comp strains, indicating a plasticity in expression pathways that is determined by the magnitude of signaling perturbation through DevRN-Kan. Conclusions/Significance A minimum DevR concentration of ∼3.3±1.2 µM (as in Comp2 bacteria) is required to support HspX expression in the standing culture hypoxia model. The relative intracellular concentrations of DevR and DevRN-Kan appear to be critical for determining dormancy regulon induction, hypoxic adaptation and virulence. Dysregulated DevRN-Kan-mediated signaling selectively triggers the PSR in bacteria expressing no/very low level of DevR. Our findings illustrate the important role of appropriate two-component- mediated signaling in pathogen physiology and the resilience of bacteria when such signaling is perturbed.

Treatment Outcome and Mortality at One and Half Year Follow-Up of HIV Infected TB Patients Under TB Control Programme in a District of South India

Background There is paucity of data from India on the impact of HIV related immunosuppression in response to TB treatment and mortality among HIV infected TB patients. We assessed the TB treatment outcome and mortality in a cohort of HIV infected TB patients treated with intermittent short course chemotherapy under TB control programme in a high HIV prevalent district of south India. Methodology/ Findings Among 3798 TB patients registered for treatment in Mysore district from July 2007 to June 2008, 281 HIV infected patients formed the study group. The socio-demographic and treatment related data of these patients was obtained from TB and HIV programme records and patient interviews 19 months after TB treatment initiation by field investigators. Treatment success rate of 281 patients was 75% while in smear positive pulmonary tuberculosis cases it was 62%, attributable to defaults (16%) and deaths (19%). Only 2 patients had treatment failure. Overall, 83 (30%) patients were reported dead; 26 while on treatment and 57 after TB treatment. Association of treatment related factors with treatment outcome and survival status was studied through logistic regression analysis. Factors significantly associated with ‘unfavourable outcome’ were disease classification as Pulmonary [aOR-1.96, CI (1.02–3.77)], type of patient as retreatment [aOR-4.78, CI (2.12–10.76)], and non initiation of ART [aOR-4.90, CI (1.85–12.96)]. Factors associated with ‘Death’ were non initiation of ART [aOR-2.80, CI (1.15–6.81)] and CPT [aOR-3.46, CI (1.47–8.14)]. Conclusion Despite the treatment success of 75% the high mortality (30%) in the study group is a matter of concern and needs immediate intervention. Non initiation of ART has emerged as a high risk factor for unfavourable treatment outcome and mortality. These findings underscore the importance of expanding and improving delivery of ART services as a priority and reconsideration of the programme guidelines for ART initiation in HIV infected TB patients.

Prevalence of Pulmonary Tuberculosis among Adults in a Rural Sub-District of South India

Background We conducted a survey to estimate point prevalence of bacteriologically positive pulmonary TB (PTB) in a rural area in South India, implementing TB program DOTS strategy since 2002. Methods Survey was conducted among persons ≥15 years of age in fifteen clusters selected by simple random sampling; each consisting of 5–12 villages. Persons having symptoms suggestive of PTB or history of anti-TB treatment (ATT) were eligible for sputum examination by smear microscopy for Acid Fast Bacilli and culture for Mycobacterium tuberculosis; two sputum samples were collected from each eligible person. Persons with one or both sputum specimen positive on microscopy and/or culture were labeled suffering from PTB. Prevalence was estimated after imputing missing values to correct for bias introduced by incompleteness of data. In six clusters, registered persons were also screened by X-ray chest. Persons with any abnormal shadow on X-ray were eligible for sputum examination in addition to those with symptoms and ATT. Multiplication factor calculated as ratio of prevalence while using both screening tools to prevalence using symptoms screening alone was applied to entire study population to estimate prevalence corrected for non-screening by X-ray. Results Of 71,874 residents ≥15 years of age, 63,362 (88.2%) were screened for symptoms and ATT. Of them, 5120 (8.1%) - 4681 (7.4%) with symptoms and an additional 439 (0.7%) with ATT were eligible for sputum examination. Spot specimen were collected from 4850 (94.7%) and early morning sputum specimens from 4719 (92.2%). Using symptom screening alone, prevalence of smear, culture and bacteriologically positive PTB in persons ≥15 years of age was 83 (CI: 57–109), 152 (CI: 108–197) and 196 (CI :145–246) per 100,000 population respectively. Prevalence corrected for non-screening by X-ray was 108 (CI: 82–134), 198 (CI: 153–243) and 254 (CI: 204–301) respectively. Conclusion Observed prevalence suggests further strengthening of TB control program.

PknE, a serine/threonine protein kinase from Mycobacterium tuberculosis has a role in adaptive responses

Serine/threonine protein kinases (STPK) play a major role in the physiology and pathogenesis of Mycobacterium tuberculosis. Here, we have examined the role of pknE, a STPK in the adaptive responses of M. tuberculosis using a deletion mutant ΔpknE. The survival of ΔpknE was assessed in the presence of stress (pH, surfactant and cell wall–damaging agents) and anti-tuberculosis drugs. ΔpknE had a defective growth in pH 7.0 and lysozyme (a cell wall–damaging agent) with better survival in pH 5.5, SDS and kanamycin (a second-line anti-tuberculosis drug). Furthermore, ΔpknE was reduced in cell size during growth in liquid media and exhibited hypervirulence in a guinea pig model of infection. In conclusion, our data suggest that pknE plays a role in adaptive response of M. tuberculosis regulating cellular integrity and survival.

Feasibility of Provider-Initiated HIV Testing and Counselling of Tuberculosis Patients Under the TB Control Programme in Two Districts of South India

Background Provider-initiated HIV testing and counselling (PITC) is internationally recommended for tuberculosis (TB) patients, but the feasibility, effectiveness, and impact of this policy on the TB programme in India are unknown. We evaluated PITC of TB patients across two districts in India considered to have generalized HIV epidemics, Tiruchirappalli (population 2.5 million) and Mysore (population 2.8 million). Methodology/Principal Findings Starting June 2007, healthcare providers in both districts were instructed to ascertain HIV status for all TB patients, and refer those with unknown HIV status to the nearest Integrated Counselling and Testing Centre (ICTC)—often in the same facility—for counselling and voluntary HIV testing. All TB patients registered from June 2007 to March 2008 were followed prospectively. Field investigators assessed PITC practices and abstracted data from routine TB programme records and HIV counselling registers to determine the proportion of TB patients appropriately evaluated for HIV infection. Patient records were traced to determine the efficiency of referral links to HIV care and antiretroviral treatment (ART). Between July 2007 and March 2008, 5299 TB patients were registered in both study districts. Of the 4701 with unknown HIV status at the time of TB treatment initiation, 3368 (72%) were referred to an ICTC, and 3111 (66%) were newly tested for HIV. PITC implementation resulted in the ascertainment of HIV status for 3709/5299 (70%) of TB patients, and detected 200 cases with previously undiagnosed HIV infection. Overall, 468 (8.8%) of all registered TB patients were HIV-infected; 177 (37%) were documented to have also received any ART. Conclusions With implementation of PITC in India, HIV status was successfully ascertained for 70% of TB patients. Previously undiagnosed HIV-infection was detected in 6.4% of those TB patients newly tested, enabling referral for life-saving anti-retroviral treatment. ART uptake, however, was poor, suggesting that PITC implementation should include measures to strengthen and support ART referral, evaluation, and initiation.

Co-Expression of DevR and DevRN-Aph Proteins Is Associated with Hypoxic Adaptation Defect and Virulence Attenuation of Mycobacterium tuberculosis

Background The DevR response regulator is implicated in both hypoxic adaptation and virulence of Mycobacterium tuberculosis (M. tb). DevR regulon genes are powerfully induced in vivo implicating them in bacterial adaptation to host control strategies. A better understanding of DevR function will illumine the way for new strategies to control and treat tuberculosis. Methodology/Principal Findings Towards this objective, we used a combination of genetic, microbiological, biochemical, cell biological tools and a guinea pig virulence assay to compare the hypoxic adaptation and virulence properties of two novel M. tb strains, namely, a devR disruption mutant, Mut1, that expresses C-terminal truncated N-terminal domain of DevR (DevRNTD) as a fusion protein with AphI (DevRN-Kan), and its complemented strain, Comp1, that expresses intact DevR along with DevRN-Kan. Comp1 bacteria exhibit a defect in DevR-mediated phosphosignalling, hypoxic induction of HspX and also hypoxic survival. In addition, we find that Comp1 is attenuated in virulence in guinea pigs and shows decreased infectivity of THP-1 cells. While Mut1 bacilli are also defective in hypoxic adaptation and early growth in spleen, they exhibit an overall virulence comparable to that of wild-type bacteria. Conclusions/Significance The hypoxic defect of Comp1 is associated to a defect in DevR expression level. The demonstrated repression of DevR function by DevRN-Kan suggests that such a knockdown approach could be useful for evaluating the activity of DevRS and other two-component signaling pathways. Further investigation is necessary to elucidate the mechanism underlying Comp1 attenuation.

Relationship of nutritional status with tuberculin sensitivity

ObjectiveTo estimate the prevalence of under- nutrition among school children and to find out the relationship between nutritional status and tuberculin sensitivity.MethodsA cross sectional study was carried out among 3335 children between 5–8 years of age attending 60 schools in Bangalore city selected by stratified random sampling. The nutritional anthropometric indices were calculated using reference median as recommended by World Health Organization, classified according to standard deviation units termed as Z-scores. The nutritional status of the children was assessed by Weight for age, Height for age and Bio-mass-index (BMI).ResultsDepending upon the method for classifying nutritional status, the prevalence of under-nutrition (including mild and severe under-nutrition) varied between 14.9–29.8%. The prevalence of severe under-nutrition varied from 2.9–6.7%. The frequency distributions of reaction sizes were found to be similar among children classified by nutritional status. The differences in proportions of significant reactions (=10mm) and mean tuberculin reaction sizes between children classified by nutritional status were not found to be statistically significant.ConclusionTuberculin sensitivity was not influenced by nutritional status among apparently healthy school children.

Index-TB guidelines: Guidelines on extrapulmonary tuberculosis for India.

Extrapulmonary tuberculosis (EPTB) is frequently a diagnostic and therapeutic challenge. It is a common opportunistic infection in people living with HIV/AIDS and other immunocompromised states such as diabetes mellitus and malnutrition. There is a paucity of data from clinical trials in EPTB and most of the information regarding diagnosis and management is extrapolated from pulmonary TB. Further, there are no formal national or international guidelines on EPTB. To address these concerns, Indian EPTB guidelines were developed under the auspices of Central TB Division and Directorate of Health Services, Ministry of Health and Family Welfare, Government of India. The objective was to provide guidance on uniform, evidence-informed practices for suspecting, diagnosing and managing EPTB at all levels of healthcare delivery. The guidelines describe agreed principles relevant to 10 key areas of EPTB which are complementary to the existing country standards of TB care and technical operational guidelines for pulmonary TB. These guidelines provide recommendations on three priority areas for EPTB: (i) use of Xpert MTB/RIF in diagnosis, (ii) use of adjunct corticosteroids in treatment, and (iii) duration of treatment. The guidelines were developed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) criteria, which were evidence based, and due consideration was given to various healthcare settings across India. Further, for those forms of EPTB in which evidence regarding best practice was lacking, clinical practice points were developed by consensus on accumulated knowledge and experience of specialists who participated in the working groups. This would also reflect the needs of healthcare providers and develop a platform for future research.