Prevalence of tuberculosis in Faridabad district, Haryana State, India.

Background & objectives: Epidemiological information on tuberculosis (TB) has always been vital for planning control strategies. It has now gained further importance for monitoring the impact of interventions to control the disease. The present study was done to estimate the prevalence of bacillary tuberculosis in the district of Faridabad in Haryana State of India among persons aged older than 15 years. Methods: In this cross-sectional study, residents of Faridabad district were assessed for the prevalence of tuberculosis. Twelve rural and 24 urban clusters with estimated populations of 41,106 and 64,827 individuals were selected for the study. Two sputum samples were collected from individuals found eligible for inclusion. The samples were also cultured by modified Petroff’s method and were examined for growth of Mycobacterium tuberculosis once a week for eight weeks. A person found positive by smear and/or culture was identified as sputum-positive pulmonary TB positive. Results: A total of 105,202 subjects were enumerated in various clusters of the Faridabad district. There were 50,057 (47.58%) females and 55,145 (52.42%) males. Of these 98,599 (93.7%) were examined by the study group (47,976 females; 50,623 males). The overall prevalence of sputum smear or culture positive pulmonary tuberculosis in our study was found to be 101.4 per 100,000 population. Interpretation & conclusions: The present results showed that the prevalence of sputum positive pulmonary tuberculosis was higher in Faridabad district than the notification rates recorded by the World Health Organization for the contemporary period, a disparity that could be explained by a difference in case detection strategy employed for the study.

Association of mycobacterium tuberculosis in the causation of Eales' disease: An institutional experience.

Background: Eales’ disease is an idiopathic retinal vasculitis characterized by retinal infl ammation, ischemia, and neo-vascularisation. It frequently causes massive vitreous haemorrhage and retinal detachment leading to blindness. Although the exact etiology is unknown, this condition is considered to be a consequence of hypersensitivity reaction to tubercular protein due to previous Mycobacterium tuberculosis (M. tuberculosis) infection. This study is aimed at the detection of association of M. tuberculosis in patients with Eales’ disease. Materials and Methods: A prospective case-control study was undertaken in 65 clinically diagnosed cases of Eales’ disease. Patients with proliferative diabetic retinopathy, neo-vascular proliferation, macular oedema, premacular fi brosis and tractional retinal detachment were taken as controls. M. tuberculosis DNA was detected (MPT64 gene by polymerase chain reaction, PCR) in patients with Eales’ disease. Clinical symptoms along with tuberculin skin test (TST) and erythrocyte sedimentation rate (ESR) were used as gold standard for comparing results of PCR. Result: PCR positivity was found in 12 (38.7%) patients with Eales’ disease. The PCR positivity was signifi cantly associated with the patients with high TST reading and high ESR values. Conclusion: Patients with a high TST reading and ESR value and a positive PCR in vitreous samples have a high likelihood of having M. tuberculosis as an etiology.

Rapid identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry.

Purpose: The purpose of this study was to evaluate the identification of Mycobacterium tuberculosis which is often plagued with ambiguity. It is a time consuming process requiring 4-8 weeks after culture positivity, thereby delaying therapeutic intervention. For a successful treatment and disease management, timely diagnosis is imperative. We evaluated a rapid, proteomic based technique for identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Materials and Methods: Freshly grown mycobacterial isolates were used. Acetonitrile/trifluoroacetic acid extraction procedure was carried out, following which cinnamic acid charged plates were subjected to identification by MALDI-TOF MS. Results: A comparative analysis of 42 clinical mycobacterial isolates using the MALDI-TOF MS and conventional techniques was carried out. Among these, 97.61% were found to corroborate with the standard methods at genus level and 85.36% were accurate till the species level. One out of 42 was not in accord with the conventional assays because MALDI-TOF MS established it as Mycobacterium tuberculosis (log (score) >2.0) and conventional methods established it to be non-tuberculous Mycobacterium. Conclusions: MALDI-TOF MS was found to be an accurate, rapid, cost effective and robust system for identification of mycobacterial species. This innovative approach holds promise for early therapeutic intervention leading to better patient care.

Study of dihydropteroate synthase (DHPS) gene mutations among isolates of Pneumocystis jiroveci.

BACKGROUND & OBJECTIVES: Pneumocystis jiroveci (also known as P. carinii) causes fatal pneumonia in patients with AIDS and other immunocompromised patients. Co-trimoxazole (trimethoprim + sulphamethoxazole, TMP-SMZ) is the drug of choice for treatment and prophylaxis. Widespread use of sulpha medication has raised the possible selection of resistant P. jiroveci strains worldwide. Non-synonymous polymorphisms associated with sulpha resistance have been observed in P. jiroveci dihydropteroate synthase (DHPS) gene at codons 55 and 57. In view of this, we investigated mutation at DHPS locus amongst P. jiroveci isolates obtained at a tertiary care hospital in north India. METHODS: Microscopic examination of P. jiroveci in 69 clinical samples obtained from patients suspected to have P. carinii pneumonia (PCP), was performed by Grocott's Gomori methenamine silver and direct fluorescent antibody staining. Molecular studies were carried out by polymerase chain reaction (PCR) using major surface glycoprotein (MSG) as the target gene. Investigations for DHPS mutations were carried at specific 55th and 57th codon using PCR-RFLP (restriction fragment length polymorphism) assay. RESULTS: Microscopic examination detected P. jiroveci in four cases and MSG gene was amplified in five cases. Further, amplification of DHPS gene was successful in four of the five cases positive by MSG gene PCR. No point mutation was observed and all four isolates presented wild-type sequences at DHPS gene by RFLP analysis. INTERPRETATION & CONCLUSION: Although our findings suggest that in Indian subpopulation, point mutations in DHPS gene of P. jiroveci are not as common as in other parts of the developed world, further studies are needed.

DOTS at a tertiary care center in northern India: successes, challenges and the next steps in tuberculosis control.

BACKGROUND AND OBJECTIVES: The past decade has seen a rapid expansion of directly observed treatment, short-course (DOTS) centers throughout India, under the guidance of the Revised National Tuberculosis Control Programme (RNTCP). While expansion has been rapid and extensive, few reports exist detailing individual DOTS centers' experiences, their challenges, and their successes. We present a brief report on the status of a DOTS center being run at a large tertiary care center in northern India for almost four years (2001-2005). METHODS: The DOTS center followed RNTCP guidelines for the evaluation and treatment of suspected TB cases. A register carrying detailed information of all patients seen at the DOTS center was kept by the senior clinician. Data from this register were extracted and analyzed for descriptive measures. RESULTS: A total of 1490 patients were evaluated. Of the 768 patients with cough, 27 per cent (211) were found to be sputum positive for acid-fast bacilli (AFB). Among patients who were initiated on anti-tuberculosis medications, cure was achieved in 92 per cent (71 of 77) of new sputum smear positive patients; treatment completion was achieved in 91 per cent (91 of 100) of extrapulmonary TB (EPTB) and 75 per cent (46 of 61) of sputum-negative pulmonary TB patients. Overall treatment success was achieved in 86 per cent (229 of 266). INTERPRETATION AND CONCLUSION: Treatment results were in keeping with the RNTCP guidelines. Tertiary care centers appear to be excellent place for education of medical students and operational research. The latter is much needed, as HIV-TB co-infection, multi-drug resistant TB, and EPTB continue to be major public health threats even in the era of DOTS.

DOTS centre at a tertiary care teaching hospital: lessons learned and future directions.

BACKGROUND: In 1993, Government of India started the Revised National Tuberculosis Control Programme (RNTCP). A model Directly Observed Therapy, Short-Course (DOTS) centre was established at the All India Institute of Medical Sciences (AIIMS) to (i) identify the challenges and opportunities in establishing DOTS centres at tertiary care facilities, (ii) to teach the strategies of RNTCP to medical and paramedical staff, and (iii) to undertake relevant operational research connected with tuberculosis (TB) treatment and control. In this communication, we describe the experience of establishing a DOTS centre at India's premier medical institute and discuss the lessons learned. METHODS: Since September 2001 through November 2002 AIIMS employees and their dependants diagnosed with tuberculosis were enrolled for treatment at AIIMS DOTS centre. One hundred sixty-eight patients were diagnosed as suffering from tuberculosis. Of these 49 patients were referred out and remaining 119 patients were treated at AIIMS DOTS centre. RESULTS: Treatment success was achieved in 80% (20/25) of new smear positive cases and the DOTS centre achieved other targets set up by the RNTCP. As the results of the pilot study at AIIMS DOTS centre were favourable, the facilities of AIIMS DOTS centre were extended to the general public from September 2002 onwards. CONCLUSION: Despite tremendous patient load at tertiary care facilities, it is possible to achieve targets established by the RNTCP. However, additional research needs to be conducted especially relating to drug resistance and surrogate markers of failure under RNTCP.

Comparative evaluation of various commercial assays for diagnosis of dengue fever.

Dengue fever (DF) is endemic in India and dengue hemorrhagic fever (DHF) has been reported with increasing frequency in the last decade. We evaluated three commercial assays for detection of antibodies to dengue virus, to assess their performance in a diagnostic laboratory. Sera from 58 patients collected during a febrile outbreak in New Delhi in 1997 were studied. The methods evaluated were MRL Diagnostic Dengue Fever Virus IgM Capture ELISA, Pan Bio Dengue Duo IgM and IgG Capture ELISA and Pan Bio Rapid Immunochromatographic test. The MRL ELISA correctly identified 97.8% (43 of 44) of samples as dengue positive while the Pan Bio Duo ELISA and Pan Bio RIT identified 95.45% (42 of 44). The sensitivities of both Pan Bio Duo ELISA and Pan Bio RIT for primary dengue and secondary dengue were 100% and 93.54% respectively. The specificity of three assays were MRL IgM ELISA 100%, Pan Bio Duo ELISA 92.8% and Pan Bio RIT 85.7%.

Use of nucleotide sequencing of the genomic cDNA fragments of the capsid/premembrane junction region for molecular epidemiology of dengue type 2 viruses.

The recent emergence of dengue hemorrhagic fever/dengue shock syndrome (DHF/ DSS) in India has been a source of concern. In the present study a quantitative comparison of 406 nucleotide long sequence from the capsid-premembrane junction region (C-PrM) of 9 dengue virus type 2 (DEN-2) isolates from Delhi with 10 DEN-2 isolates from diverse geographic areas provided sufficient information for estimating genetic relationships. The data indicated that the 1996 epidemic of DHF in Delhi was caused by genotype IV strains of DEN-2. This genotype, perhaps, displaced genotype V strains of DEN-2, which was circulating genotype in 1967. The period during which this displacement had occurred is not clear from the present study. Nonetheless, similar experience in four countries in Latin America and in Sri Lanka suggest that the introduction of new genotypes of DEN-2 displacing the circulating genotype may be associated with the appearance of DHF/DSS. More work is required to elucidate this hypothesis. Transitions at nucleotide positions 406 and 431 resulted in amino acid substitutions near (aa position 104, methionine --> valine) and at the hinge region (aa position 112, valine --> alanine) of C-PrM, respectively in all/most genotypes of group III and IV DEN-2 viruses analysed. Most of these virus strains have been isolated from DHF/DSS outbreaks. Significance of this observation is discussed. The data presented in this study suggest the utility of C-PrM sequence analysis for molecular epidemiology of dengue viruses.