Abstract
Tuberculosis (TB) is a global health problem with 480000 new cases being reported annually, 9 percent of which exhibit extensive drug resistant TB. With 9.6 cases per lac population early detection of drug resistance at peripheral levels is must. The study was planned to use Fluorescent microscopy (FM) with Fluorescein Diacetate Ethidium Bromide (FDA/EB) stain to evaluate the viability of the mycobacteria to predict the drug resistance. This cross-sectional analytical study was carried out on all sputum smear positive patients after obtaining the Institutional Ethics Committee clearance over a period of 3 months. Samples were obtained on day 0, 3, 7 and 14. After staining with the working solution, observation was made by two independent observers for viable verses non-viable mycobacteria. FDA will stain the live bacilli and fluoresce green whereas dead bacilli lack the esterase activity and are counterstained by ethidium bromide and appear red. Data was maintained in MS Excel and analysed using tests of proportion and significance. A total of 30 participants were included based on the inclusion criteria. There was a loss of follow up from 14 patients. 4/16 (25%) patients were found be harbor drug resistant mycobacteria strains bases on decreasing ratio of viable to non-viable bacteria on followup. Results when compared with gold standard of Line Probe Assay (LiPA) was found to be highly significant with Chi square value of 11.73 and p value <0.001This study establishes the utility of fluorescent microscopy using FDA and EB to detect drug resistance in patients of pulmonary tuberculosis by evaluation of viable and non-viable at peripheral microscopic centers Fluorescent Microscopy with FDA/EB staining holds a potential to predict drug resistance in sputum smear positive patients at peripheral designated microscopic centers which will reduce financial as well as human resource burden.
Highlights
Tuberculosis (TB) has existed for centuries and still remains the global health problem of the 21st century.Globally about ten million people are infected with TB
Under Revised National Tuberculosis Control Program (RNTCP), National Tuberculosis Elimination Program (NLEP) all the sputum smear positive samples are sent for detection of MDR status right on the first day by Line Probe Assay (LiPA) at Intermediate Reference Laboratories (IRL) as around 25-30% newly diagnosed cases are MDR TB cases
All the strains were stained with conventional AFB staining which could detect the AFB in all the samples but viability studies using Fluorescein Diacetate Ethidium Bromide (FDA/EB) could predict the resistant nature
Summary
Tuberculosis (TB) has existed for centuries and still remains the global health problem of the 21st century.Globally about ten million people are infected with TB. 1. The Multi Drug Resistant TB (MDR-TB) cases, which means the patient is resistant to two most important 1st line drugs, that is Rifampin and Isoniazid has been rapidly increasing along these years. Around 4,80,000 new cases of MDR-TB have been recorded, out of which about 9% exhibit extensive drug resistance TB. Again the patient is examined for sputum positivity. If still the sputum microscopy is positive, the patient is put under CAT II and are assumed to be drug resistant that is having MDR TB. It is important in view that globally only 5% cases of PTB are tested for drug susceptibility tests (DST). It is important in view that globally only 5% cases of PTB are tested for drug susceptibility tests (DST). 4
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