Risk factors for childhood illness and death in rural Uttar Pradesh, India: perspectives from the community, community health workers and facility staff.

BACKGROUND: Uttar Pradesh (UP), India continues to have a high burden of mortality among young children despite recent improvement. Therefore, it is vital to understand the risk factors associated with under-five (U5) deaths and episodes of severe illness in order to deliver programs targeted at decreasing mortality among U5 children in UP. However, in rural UP, almost every child has one or more commonly described risk factors, such as low socioeconomic status or undernutrition. Determining how risk factors for childhood illness and death are understood by community members, community health workers and facility staff in rural UP is important so that programs can identify the most vulnerable children. METHODS: This qualitative study was completed in three districts of UP that were part of a larger child health program. Twelve semi-structured interviews and 21 focus group discussions with 182 participants were conducted with community members (mothers and heads of households with U5 children), community health workers (CHWs; Accredited Social Health Activists and Auxiliary Nurse Midwives) and facility staff (medical officers and staff nurses). All interactions were recorded, transcribed and translated into English, coded and clustered by theme for analysis. The data presented are thematic areas that emerged around perceived risk factors for childhood illness and death. RESULTS: There were key differences among the three groups regarding the explanatory perspectives for identified risk factors. Some perspectives were completely divergent, such as why the location of the housing was a risk factor, whereas others were convergent, including the impact of seasonality and certain occupational factors. The classic explanatory risk factors for childhood illness and death identified in household surveys were often perceived as key risk factors by facility staff but not community members. However, overlapping views were frequently expressed by two of the groups with the CHWs bridging the perspectives of the community members and facility staff. CONCLUSION: The bridging views of the CHWs can be leveraged to identify and focus their activities on the most vulnerable children in the communities they serve, link them to facilities when they become ill and drive innovations in program delivery throughout the community-facility continuum.

Readiness to provide child health services in rural Uttar Pradesh, India: mapping, monitoring and ongoing supportive supervision.

BACKGROUND: In 2018, 875 000 under-five children died in India with children from poor families and rural communities disproportionately affected. Community health centres are positioned to improve access to quality child health services but capacity is often low and the systems for improvements are weak. METHODS: Secondary analysis of child health program data from the Uttar Pradesh Technical Support Unit was used to delineate how program activities were temporally related to public facility readiness to provide child health services including inpatient admissions. Fifteen community health centres were mapped regarding capacity to provide child health services in July 2015. Mapped domains included human resources and training, infrastructure, equipment, drugs/supplies and child health services. Results were disseminated to district health managers. Six months following dissemination, Clinical Support Officers began regular supportive supervision and gaps were discussed monthly with health managers. Senior pediatric residents mentored medical officers over a three-month period. Improvements were assessed using a composite score of facility readiness for child health services in July 2016. Usage of outpatient and inpatient services by under-five children was also assessed. RESULTS: The median essential composition score increased from 0.59 to 0.78 between July 2015 and July 2016 (maximum score of 1) and the median desirable composite increased from 0.44 to 0.58. The components contributing most to the change were equipment, drugs and supplies and service provision. Scores for trained human resources and infrastructure did not change between assessments. The number of facilities providing some admission services for sick children increased from 1 in July 2015 to 9 in October 2016. CONCLUSIONS: Facility readiness for the provision of child health services in Uttar Pradesh was improved with relatively low inputs and targeted assessment. However, these improvements were only translated into admissions for sick children when clinical mentoring was included in the support provided to facilities.

Mycobacterial origin protein Rv0674 localizes into mitochondria, interacts with D-loop and regulates OXPHOS for intracellular persistence of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) employs diverse strategies to survive inside the host macrophages. In this study, we have identified a conserved hypothetical protein of Mtb; Rv0674, which is present in the mitochondria of the host cell. The genetic knock-out of rv0674 (Mtb-KO) showed increased growth of Mtb. The intracellular infection with recombinant Mycobacterium smegmatis (MSMEG) expressing Rv0674 (MS_Rv0674), established that the protein is involved in promoting the apoptotic cell death of the macrophage. To investigate the mechanism incurred in mitochondria, we observed that the protein physically interacts with the control region (D-loop) of the mitochondrial DNA (LSP and HSP promoters of the loop) of the macrophages and facilitates the increased expression of mRNA in all the complexes of mitochondrial encoded OXPHOS subunits. The changes in OXPHOS levels corroborated with the ATP synthesis, mitochondrial membrane potential and superoxide production. The infection with MS_Rv0674 confirmed the role of this protein in effecting the intracellular infection. The fluorescent and confocal microscopy confirmed that the protein is localized in the mitochondria of infected macrophages and in the cells of BAL of TB patients. Together these findings indicate towards the novel function of the protein which is unlike to the earlier established mechanisms of mycobacterial physiology.

Clinical, demographic and radiological profile of smoker COPD versus nonsmoker COPD patients at a tertiary care center in North India.

INTRODUCTION: Cigarette smoking is the most predominant risk factor for development of chronic obstructive pulmonary disease (COPD). However, a considerable amount of patients do develop COPD without exposure to cigarette smoking. We aimed to analyze the incidence, demographic and clinical profile of nonsmoker COPD subjects at a tertiary care center. METHODS: In this prospective observational study, 410 patients were screened for dyspnea. On the basis of spirometry findings, 360 patients were diagnosed as COPD and enrolled into the study. Patients were categorized into 2 groups on the basis of smoking habits (smoker and nonsmoker COPD). Clinical and demographic attributes were compared in between these two groups. This study was conducted over a period of one year, from August 2014 to July 2015. All statistical analyses were performed using Statistical Package for the Social Sciences version 19.0 (SPSS Inc, Chicago, IL, USA). Values were considered to be statistically significant at P < 0.05. RESULTS: Out of 360 COPD cases, about 2/3rd (60%) were smokers and the rest nonsmokers. Majority of the patients were in the age group of 51-70 years. The mean age of smokers with COPD was significantly higher than nonsmokers with COPD (59.29 ± 10.28 years vs. 53.90 ± 8.77 years; P = 0.0001). Overall, males were predominant (57.2%) but there were higher number of female patients in nonsmoker group (25% vs. 70%; P = 0.001). At presentation, majority of nonsmoker with COPD were in GOLD severity grade II while in the smoking cohort majority were in GOLD severity Grade III. Among the 144 nonsmoker COPD patients, the most important and statistically significant risk factor was exposure to biomass smoke (68.06%). Other risk factors were long-standing asthma (37.50%), lower respiratory tract infection in childhood (32.60%), exposure to outdoor air pollution (17.92%). CONCLUSION: Nonsmoker COPD is emerging as a distinctive phenotype. They have less impairment in airflow limitation, and a lower prevalence of emphysema, chronic cough, and sputum compared with their smoking counterparts.

Association of Nat2 Gene Polymorphism with Antitubercular Drug-induced Hepatotoxicity in the Eastern Uttar Pradesh Population.

Introduction Tuberculosis (TB) remains an important cause of morbidity and mortality worldwide. There are more than 20 drugs available for TB treatment. Hepatotoxicity is the most serious adverse drug reaction of anti-TB drugs. Various pathogenesis and genetic factors are associated with antituberculosis drug-induced hepatotoxicity (ATDIH). Antituberculosis drugs (ATDs) are mostly metabolized by N-acetyltransferase 2 (NAT2). Therefore, in this study, we aim to evaluate the role of the NAT2 genotype in ATDIH in the eastern Uttar Pradesh population. Methods A total of 100 TB patients who had been treated with anti-TB drugs were enrolled in this studied. In this group, 70 TB patients did not develop drug-induced hepatotoxicity (tolerant control group) and 30 TB patients developed ATDIH (ATDIH group). The genetic polymorphisms of the NAT2 genes were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Genotype and allele frequencies were evaluated by the t-test and odds ratio (OR) with 95% confidence intervals (CIs) were used to evaluate the strength of the associations. Results There is a high percentage of slow acetylators in the Eastern Uttar Pradesh population. Four percent of people are fast acetylators, 34% are intermediate acetylators, and 62% are slow acetylators. The frequency of slow acetylators in the NAT2 genotype was commonly present and was not significantly different between the ATDIH (73.33%) and tolerant control groups (61.40%). However, the genotypic distribution of variants of slow-acetylator genotypes (NAT2*6/7, NAT2*5/7, and NAT2*5/6) was also not significantly different in ATDIH. Conclusion In the present study, the slow acetylators of the NAT2 genotype did not contribute to the elevated risk of ATDIH development in tuberculosis patients.

Exploration of some new secretory proteins to be employed for companion diagnosis of Mycobacterium tuberculosis.

Tuberculosis (TB) is a highly infectious disease and its early and precise diagnosis is essential to reduce morbidity and mortality of patients. Since the routine diagnostic tests (like Monteux, AFB smear microscopy, chest X-Ray) do not give infallible results, additional tests are always recommended. Therefore to address the concerns about non-specificity of the present battery of diagnostic tests, we have attempted to analyze some unique secretory antigens which could be able to identify the stage specific infection of MTB. In this study, we have used recombinant proteins CFP-10, ESAT-6, Ag85 A, Ag85B, Ag85C, PE3, PE4 and Mycp1 to eliminate heterogeneity and cross reactivity in clinical diagnosis. Amplified genes were cloned and over-expressed in Escherichia coli BL21 (DE3). The recombinantly purified proteins were used as antigens against 158 sera samples of TB patients. Secretory proteins showed better response than the PPD control. Among all the used antigens PE3 and PE4 proteins showed better reactivity levels among all the groups of TB patients. The secretions of CFP-10 and ESAT-6 were also higher as compared to other secretory proteins like Ag85 A, Ag85B, Ag85C and MycP1.The clinical use of these newly identified secretory antigens could be of significant value for the confirmatory, rapid, simple and low-cost diagnosis of TB patients.

Biophysical and immunological characterization of the ESX-4 system ESAT-6 family proteins Rv3444c and Rv3445c from Mycobacterium tuberculosis H37Rv.

The ESAT-6 family proteins of Mycobacterium tuberculosis are regarded as the key mediators in mycobacterial virulence and are largely considered as antigens that can improve TB vaccines and diagnostics. We have characterized Rv3444c and Rv3445c proteins of the ESX-4 system of ESAT-6 family of M. tuberculosis H37Rv, and have experimentally established that these two proteins interact to form a heterodimeric complex. Complex formation resulted in induction of α-helical conformation and stability against chemical denaturation. To evaluate the immunogenic potential, we have immunized mice with Rv3444c or Rv3445c along with Freund's incomplete adjuvant (FIA). Immunization with Rv3444c-FIA or Rv3445c-FIA resulted in long term humoral responses. Re-stimulation of splenocytes from immunized mice resulted in significant lymphocyte proliferation with induction of TNF-α and IL-6. Further, the humoral responses to Rv3444c and Rv3445c antigens in Indian patients with active pulmonary TB (n = 44), and healthy individuals (n = 20), were investigated. Compared to healthy individuals, high levels of IgG against Rv3444c and Rv3445c were observed in TB patient's sera, indicating that these proteins are actively produced during the active phase of TB. Cellular immune responses to these proteins in active pulmonary TB patients (n = 5) were also investigated using peripheral blood mononuclear cells (PBMCs). Both the proteins induce significant lymphocyte proliferation and up-regulate the induction of TNF-α and IL-6 in TB patients.

Peroxiredoxin-1 of macrophage is critical for mycobacterial infection and is controlled by early secretory antigenic target protein through the activation of p38 MAPK.

Early secretory antigenic target protein (ESAT-6) is an important virulent factor which plays a crucial role in Mycobacterium tuberculosis (MTB) pathogenesis. Here, we demonstrate the role of ESAT-6 in phagocytosis and intracellular survival of mycobacteria through a mechanism mediated by regulation of a host protein; Peroxiredoxin-1 (Prdx-1). Prdx-1 is an anti-apoptotic and stress response protein which protects cells from damage by ROS and H2O2. The J774 A.1 cells infected with MTB or over-expressing ESAT-6 through eukaryotic promoter vector showed elevated expression of Prdx-1. Further investigation revealed that the up-regulation of Prdx-1 is mediated through the activation of one of the MAP kinases, p38. The NRF-2, a transcriptional activator of Prdx-1 is translocated to the nucleus upon phosphorylation by p38 and subsequently, regulates expression of Prdx-1. Inhibition of the p38 MAPK by a specific inhibitor, SB203580, abrogates the ESAT-6 mediated induction of Prdx-1 expression as well as the phosphorylation of NRF-2 in a time-dependent manner. The inhibition of Prdx-1 expression by specific siRNA in J774 A.1 cells resulted in the reduced bacterial uptake and intracellular survival of the mycobacteria. This is the first report proclaiming that the ESAT-6 regulates Prdx-1 which is involved in the increase of mycobacterial uptake and survival. The intermediate mechanisms involve the increased Prdx-1 production in macrophages through the activation of p38 and NRF-2 dependent signaling.

A study on Beijing genotype in the clinical isolates of pulmonary drug-resistant tuberculosis.

BACKGROUND: Mycobacterium tuberculosis (MTB) Beijing strains are spread worldwide and are responsible for major outbreaks of tuberculosis (TB), sometimes spreading multidrug resistance (MDR). AIM: The aim of this study was to explore clinical features associated with the infection with Beijing strains among MDR patients of pulmonary TB in Lucknow and surrounding areas. MATERIALS AND METHODS: It was a hospital-based epidemiological study. Our study population was selected from all the newly diagnosed patients attending outpatient department and admitted patients of Department of Respiratory Medicine, King George's Medical University, Lucknow, Uttar Pradesh, India. Those isolates which were characterized to be MTB by morphological and molecular techniques were tested for their resistance against the first-line drugs; after which each patient's isolate was genotyped. RESULTS: The results suggested that the presence of Beijing genotype in 31.78% of strains. CONCLUSION: Our results predicted that genotypic patterns reveal a large diversity among the MTB Beijing strain population. Increasing frequency of Beijing strains demands further research to unravel the factors behind its propensity to prevail.

Assess drug resistance pattern and genetic profile of Mycobacterium tuberculosis clinical isolates by molecular typing methods using direct repeats and IS6110 in pulmonary tuberculosis cases.

BACKGROUND: Tuberculosis (TB), a highly contagious disease that sees no gender, age, or race is mainly a disease of lungs. According to World Health Organization, a TB patient can be completely cured with 6-9 months of anti-TB treatment under directly observed treatment short course. OBJECTIVES: The aim of this study was to check the mono, multi- and triple-drug resistance to first line drugs (FLDs) among TB patients and to access their genetic profile using DR 3074, DR 0270, DR 0642, DR 2068, and DR 4110 using molecular techniques. MATERIAL AND METHODS: To gain a better understanding of drug resistant TB, we characterized 121 clinical isolates recovered from 159 drug resistant pulmonary tuberculosis patients by IS6110 genotyping. MTB isolates recovered from HIV- negative, and smear positive cases of both genders, age varied from 18 to 70 years with drug resistant-TB that was refractory to chemotherapy given for > 12 months. Of a total of 159 sputum smear positive patients sum number of male and female patients was 121 (76.10%) and 38 (23.89%), respectively. Among these patients, number of literate and illiterate patients were 123 (77.3%) and 36 (22.6%). 25 (15.7%) patients had farming as their occupation, 80 (50.3%) had nonagricultural occupation and 54 (33.9%) women were housewives. RESULTS: Mono drug resistant, multi-drug resistant, and totally drug resistant (TDR) cases of TB were calculated as 113.83%, 125.1%, and 67.9%. Isoniazid showed the highest percentage of resistance among the patients. CONCLUSION: Any noncompliance to TB medications, lack of knowledge, and poor management in health centers, etc., results in the emergence of deadly direct repeat forms of TB, which are further complicated and complex to treat.

Characterization of culture filtrate proteins Rv1197 and Rv1198 of ESAT-6 family from Mycobacterium tuberculosis H37Rv.

BACKGROUND: We have characterized two immunogenic proteins, Rv1197 and Rv1198, of the Esx-5 system of the ESAT-6 family of Mycobacterium tuberculosis H37Rv. METHODS: The complex formation between Rv1197 and Rv1198 was characterized by biophysical techniques. The reactivity of serum from TB patients towards these proteins was characterized by ELISA. Lymphocyte proliferation and cytokine induction were followed in restimulated splenocytes from immunized mice by using MTT assay and CBA flowcytometry, respectively. RESULTS: Rv1197 and Rv1198 strongly interact to form a heterodimeric complex under reducing conditions, which is characterized by a dissociation constant of 97×10-9M and melting temperature, Tm, of 50.5°C. Strong humoral responses to Rv1197, Rv1198, CFP-10 and MoaC1 (Rv3111) antigens were found in Indian patients with active pulmonary tuberculosis (n=44), in comparison to non-infected healthy individuals (n=20). The seroreactivity to Rv1198 was characterized by a sensitivity of 75% and specificity of 90%. In BALB/c mice, immunization with Rv1198-FIA induced a pro-inflammatory response with elevated levels of TNF and IL-6, along with low induction of IFN-γ, IL-2 and IL-10, but no induction of IL-4. CONCLUSION: Rv1197 and Rv1198 form a stable complex, which is regulated by the redox state of Rv1198. Rv1198 is immunogenic with highly specific seroreactivity towards TB patients' serum. Rv1198 elicits a pro-inflammatory recall response in immunized mice. GENERAL SIGNIFICANCE: This study characterizes the interaction of Rv1197 and Rv1198, and establishes the immunogenic nature of Rv1198.

The M. tuberculosis HAD phosphatase (Rv3042c) interacts with host proteins and is inhibited by Clofazimine.

Mycobacterium tuberculosis codes for a HAD-phosphatase, Rv3042c (MtSerB2), that has earlier been characterized as a metabolic enzyme. Here we demonstrate that MtSerB2 is secreted into the cytosol of infected macrophages and is found in bronchoalveolar lavage samples of tuberculosis patients. MtSerB2 induces significant cytoskeleton rearrangements through cofilin activation and affects the expression of genes that regulate actin dynamics. It specifically interacts with HSP90, HSP70 and HSP27 that block apoptotic pathways and not with other HSPs. It actively dephosphorylates MAPK-p38 and NF-kappa B p65 that play crucial roles in inflammatory and immune responses. This in turn leads to down-regulation of Interleukin 8, a chemotactic and inflammatory cytokine. Finally, during evaluation of inhibitors against MtSerB2 we found that Clofazimine, a drug being evaluated for XDR and MDR tuberculosis, inhibits MtSerB2 phosphatase activity and reverses the above effects and interactions with host proteins. Overall, the study identifies that MtSerB2 has new functions that might help the pathogen to evade the host's immune response.

Pattern of drug resistance of Mycobacterium tuberculosis clinical isolates to first-line antituberculosis drugs in pulmonary cases.

CONTEXT: Mycobacterium tuberculosis (MTB), the human pathogen causes Tuberculosis (TB). It is a highly infectious and globally pandemic disease. The severity increases when the MTB becomes resistant to antituberculosis drugs. India is reported to be in the second place, with the highest number of drug-resistant TB cases. The treatment of drug-resistant TB is even more complicated. MATERIALS AND METHODS: The present study comprises of 159 TB patients, in which 88 are reported to have drug-resistant TB (55.3%). All the patients are in the age group of 18-70 years. Patients having extrapulmonary TB and diabetes were excluded from the study. The collected samples were processed and stained for acid fastness and smear positivity. They were subjected to inoculation on Lowenstein-Jensen (LJ) slants. RESULTS: The results showed that out of the four drugs - Streptomycin, Isoniazid, Rifampicin, and Ethambutol - the resistant cases reported in Streptomycin were 45 (24.9%), whereas, in Isoniazid, Rifampicin, and Ethambutol, the resistant cases were 62 (34.2%), 27 (14.9%), and 47 (26.0%), respectively. Isoniazid showed the highest percentage of resistance among the patients. CONCLUSION: Effective measures such as convincing the patients to take the prescribed drugs and follow the five major strategies under the Directly Observed Treatment, Short Course (DOTS), could help in managing such cases.

Familial Behcet's disease.

There are very few reports of Behetaet's disease from India. Familial aggregation of Behetaet's disease has been reported with restricted geographical distribution. We report here familial Behcet's disease from India in two brothers aged 30 and 32 years. Both patients had recurrent oral and genital ulcers for approximately five years. They also had arthralgias on and off along with fever. Pathergy test was positive in both cases. Their younger brother and a sister had recurrent oral aphthous ulcers.