Prabha Desikan

Molecular surveillance of hepatitis and tuberculosis infections in a cohort exposed to methyl isocyanate

ObjectiveThe potential toxic effects on the immune system exerted by occupational and accidental environmental exposures and underlying molecular regulatory mechanisms involved in the etiology and progression of infectious diseases are now being characterized. The Bhopal gas tragedy is undoubtedly one of the worst industrial disasters in the history of mankind. After 25 years of accidental exposure to methyl isocyanate (MIC), severe systemic ailments still continue to preoccupy the lives of the affected population that survived this tragedy. We have performed a molecular surveillance study to characterize hepatitis and tuberculosis infections amongst the first and the second generation of survivors exposed to MIC.Materials and MethodsBoth outdoor and indoor patients referred for molecular diagnosis of hepatitis B virus (HBV), hepatitis C virus (HCV) and Mycobacterium tuberculosis (MTB) were examined. Qualitative analysis for HBsAg, anti-HBc, anti-HCV through ELISA was performed, while BacT/ALERT and Ziehl-Neelson technique were utilized for the assessment of tuberculosis. Detection and quantification of viral and bacterial nucleic acid and characterization of hepatitis genotypes were analyzed using real-time and end-point PCR techniques.ResultsThe results suggest that HBV infections are most common among the MIC-exposed cohort, followed by extra-pulmonary and pulmonary MTB and HCV infections. Genotype 3 is the most prevalent HCV genotype among the survivors. Failure to detect HBsAg, anti-HBc and anti-HCV through ELISA, and tuberculosis by culture and Ziehl-Neelson stain, indicates higher prevalence of occult hepatitis and latent tuberculosis in the affected population.ConclusionsOur study underscores the importance of hospital-based records used as a data source for monitoring possible environmental health hazards. As the risk of progress of infection is often influenced by conditions and periods of environmental chemical exposure, therefore, insights of interconnected molecular pathways will further illuminate the gene-environment association and might offer valuable information for rational drug design.

Clinical presentation, etiology, and survival in adult acute encephalitis syndrome in rural Central India

BACKGROUND Acute encephalitis syndrome (AES) is a constellation of symptoms that includes fever and altered mental status. Most cases are attributed to viral encephalitis (VE), occurring either in outbreaks or sporadically. We conducted hospital-based surveillance for sporadic adult-AES in rural Central India in order to describe its incidence, spatial and temporal distribution, clinical profile, etiology and predictors of mortality. METHODS All consecutive hospital admissions during the study period were screened to identify adult-AES cases and were followed until 30-days of hospitalization. We estimated incidence by administrative sub-division of residence and described the temporal distribution of cases. We performed viral diagnostic studies on cerebrospinal fluid (CSF) samples to determine the etiology of AES. The diagnostic tests included RT-PCR (for enteroviruses, HSV 1 and 2), conventional PCR (for flaviviruses), CSF IgM capture ELISA (for Japanese encephalitis virus, dengue, West Nile virus, Varicella zoster virus, measles, and mumps). We compared demographic and clinical variables across etiologic subtypes and estimated predictors of 30-day mortality. RESULTS A total of 183 AES cases were identified between January and October 2007, representing 2.38% of all admissions. The incidence of adult AES in the administrative subdivisions closest to the hospital was 16 per 100,000. Of the 183 cases, a non-viral etiology was confirmed in 31 (16.9%) and the remaining 152 were considered as VE suspects. Of the VE suspects, we could confirm a viral etiology in 31 cases: 17 (11.2%) enterovirus; 8 (5.2%) flavivirus; 3 (1.9%) Varicella zoster; 1 (0.6%) herpesvirus; and 2 (1.3%) mixed etiology); the etiology remained unknown in remaining 121 (79.6%) cases. 53 (36%) of the AES patients died; the case fatality proportion was similar in patients with a confirmed and unknown viral etiology (45.1 and 33.6% respectively). A requirement for assisted ventilation significantly increased mortality (HR 2.14 (95% CI 1.0-4.77)), while a high Glasgow coma score (HR 0.76 (95% CI 0.69-0.83)), and longer duration of hospitalization (HR 0.88 (95% CI 0.83-0.94)) were protective. CONCLUSION This study is the first description of the etiology of adult-AES in India, and provides a framework for future surveillance programs in India.

Diagnosis of gastrointestinal tuberculosis: Using cytomorphological, microbiological, immunological and molecular techniques — A study from Central India

The present study included three groups: (A) age and gender matched control (n=24) with no previous signs of M. tuberculosis complex (MTBC) infection, (B) patients (n=28) diagnosed with gastro-intestinal TB (GITB), (C) patients (n=50) with clinical and histo-pathological signs of GITB, but were culture and AFB negative. Real time assay performed using fluorescence resonance energy transfer hybridization probes showed a positivity index of 36 % in group C, i.e. 18 were found reactive from the total 50 cases studied. In addition, immune characterization of these 18 cases showed depleted CD4+ count and increased levels of IFN-γ and TNF-α cytokines. No positive case was found in group A, while in group B, out of total 28 cases studied 27 were found positive. A combinatorial diagnostic approach for rapid detection and characterization of GITB might provide specific therapeutic strategies for prevention and treatment of the infection in future.

Pulmonary nocardiosis mimicking relapse of tuberculosis.

The present report concerns a case of pulmonary nocardiosis in an immunocompetent host. This patient was diagnosed as having smear positive pulmonary tuberculosis and received supervised antitubercular treatment for 6 months from a government run tuberculosis centre (Directly Observed Therapy, Short-Course (DOTS) centre). At 3 months after completion of treatment, she presented with fever and cough with posterior–anterior (PA) view chest x ray showing a cavitary lesion on left upper zone. She was subsequently diagnosed as having a case of pulmonary nocardiosis and responded to oral cotrimoxazole.

Frequency of mutations in rifampicin and isoniazid resistant isolates of M. tuberculosis: an analysis from Central India.

BACKGROUND The spread of drug-resistant tuberculosis has challenged tuberculosis control strategies globally. The present study aims to analyze the frequency of mutations in rpoB, katG and inhA genes in strains of M. tuberculosis complex (MTBC) circulating in Central India. It is anticipated that the findings may provide a starting point to understand the evolutionary success of drug-resistant strains of MTBC in this region. METHODS Line probe assay was carried out on 720 consecutive sputum samples of MDR suspects from June 2012 to May 2013. Mutation frequencies in the rpoB, katG and inhA genes were analyzed. RESULTS Mutations were identified in 269 (37.6%) samples, as follows: 55 (7.6%) samples had mutations conferring resistance to only isoniazid, 84 (11.6%) had mutations conferring resistance to only rifampicin and 130 (18%) isolates had mutations conferring resistance to both isoniazid and rifampicin. The most frequent mutation in the rpoB gene was at codon S531L, seen in 141 (19.5%) isolates. The most frequent mutation in the katG gene was at codon S315T1, seen in 151 (20.9%) isolates; and in the inhA gene at codon C15T, seen in 21 (2.9%) isolates. Some unidentified mutations were also observed. CONCLUSION The patterns and the frequency of the mutations identified in this study indicate the most frequent mutations at S531L codon in the rpoB gene, S315T1 codon in the katG gene and C15T codon in the promoter region of the inhA gene. Controlling the emergence and spread of MDR TB requires an understanding of the evolution of these mutations.

The incredible journey of mankind: Helicobacter pylori as the narrator

Over the past decade, sequence differences between microbes from various geographical areas have been studied with the intent to interpret population movements of their hosts. An organism that is a reliable storehouse of such data, by virtue of its long association with its human host, is Helicobacter pylori. Functional and comparative analyses of its genome provide fascinating insights into human behaviour in the ancient past.

Genetic diversity of Mycobacterium tuberculosis isolates from central India

Background & objectives: There is a paucity of data available on genetic biodiversity of Mycobacterium tuberculosis isolates from central India. The present study was carried out on isolates of M. tuberculosis cultured from diagnostic clinical samples of patients from Bhopal, central India, using spoligotyping as a method of molecular typing. Methods: DNA was extracted from 340 isolates of M. tuberculosis from culture, confirmed as M. tuberculosis by molecular and biochemical methods and subjected to spoligotyping. The results were compared with the international SITVIT2 database. Results: Sixty five different spoligo international type (SIT) patterns were observed. A total of 239 (70.3%) isolates could be clustered into 25 SITs. The Central Asian (CAS) and East African Indian (EAI) families were found to be the two major circulating families in this region. SIT26/CAS1_DEL was identified as the most predominant type, followed by SIT11/EAI3_IND and SIT288/CAS2. Forty (11.8%) unique (non-clustered) and 61 (17.9%) orphan isolates were identified in the study. There was no significant association of clustering with clinical and demographic characteristics of patients. Interpretation & conclusions: Well established SITs were found to be predominant in our study. SIT26/CAS1_DEL was the most predominant type. However, the occurrence of a substantial number of orphan isolates may indicate the presence of active spatial and temporal evolutionary dynamics within the isolates of M. tuberculosis.

Unusual cause of chronic cough in an immunocompetent host.

Aspergillus infections cause a spectrum of pulmonary diseases depending on the immune status of the host. We report a rare case of a young, immunocompetent, patient who developed Aspergillus tracheobronchitis. Owing to primary resistance to itraconazole, the disease progressed to aspergilloma despite treatment. The condition was successfully treated with oral voriconazole.

Line probe assay for detection of Mycobacterium tuberculosis complex: An experience from Central India

Background & objectives: Mycobacterium tuberculosis complex may sometimes not be detected in sputum samples of suspected multidrug-resistant tuberculosis (MDR-TB) patients by line probe assay (LPA) even though they are smear positive for acid-fast bacilli (AFB). This retrospective analysis was attempted to understand and document our experience with LPA for detection of M. tuberculosis complex and diagnosis of MDR-TB under programmatic conditions. Methods: One thousand two hundred and ninety four sputum samples of MDR-TB suspects that were smear positive for AFB, and received from February to November 2013, were tested by LPA for the presence of M. tuberculosis complex and resistance to isoniazid (INH) and rifampicin as per the diagnostic mandate of an accredited reference laboratory. As per the mandate, those samples that were negative for M. tuberculosis complex were cultured, and the growth again tested by LPA. A retrospective analysis of the results was carried out. Results: M. tuberculosis complex could be detected in 1217 (94.04%) but not in 77 (5.9%) of smear-positive sputum samples. Of the 1217 positive samples, 232 (19.1%) were MDR, 130 (10.6%) were rifampicin monoresistant and 101 (8.3%) were INH monoresistant. Seven hundred and fifty four (61.9%) strains were found to be pansensitive. Overall, 5.1 per cent of the sputum samples were negative for M. tuberculosis complex by LPA and culture. In at least 10 (0.77%) sputum samples smear positive for AFB, M. tuberculosis complex could not be identified by LPA though M. tuberculosis was present, as evidenced by culture positivity. Interpretation & conclusions: LPA is a robust technique for diagnosis of drug-resistant TB that has provided the basis for rapid and effective control of drug-resistant TB in India. While the reasons for concomitantly negative LPA and culture results of smear-positive sputum samples from MDR-TB suspects may be many, the possible presence of non-tubercular mycobacteria in these samples and the likelihood of inappropriate therapy in these patients cannot be ruled out. Addition of culture to the diagnostic algorithm may enhance the diagnostic yield.

Public health relevance of non-tuberculous mycobacteria among AFB positive sputa

BACKGROUND Sputum smear microscopy for acid fast bacilli (AFB) is used by most public health programmes to detect tuberculosis. While most AFB in countries endemic for tuberculosis are Mycobacterium tuberculosis (MTB), some may also be non-tuberculous mycobacteria (NTM). The inability to differentiate NTM from MTB by sputum smear microscopy may lead to erroneous diagnoses of tuberculosis, leading in turn to inappropriate therapy. METHODS This was a retrospective study of consecutive sputum samples received from November 2013 to March 2015 in the Department of Microbiology, Bhopal Memorial Hospital & Research Centre, Bhopal, India. Samples underwent smear microscopy, line probe assay (LPA) for MTB complex, culture, biochemical tests and LPA for NTM. RESULTS Of 4095 sputum samples, 2886 were AFB smear positive (70.5%). Of these, MTB complex was detected in 2611 (90.5%) samples by LPA. Of the remaining 275 samples, 47 grew AFB on culture. Nine strains belonged to the MTB complex. The remaining 38 (1.3%) were NTM, and could be speciated in 26 strains; 14 (53.8 %) were M. abscessus; 10 (38.4%) M. intracellulare, one (3.8%) M. kansasii and one (3.8%) M. fortuitum. The remaining 12 NTM could not be speciated. CONCLUSION NTM were present in at least 1.3% of all smear positive samples. It is important for public health programs to recognize the avoidable burden on logistics, infrastructure and finances caused by this. Detection and quantification of this burden would help design an appropriate strategy for optimal tuberculosis control.