Polymerase chain reaction targeting insertion sequence for the diagnosis of extrapulmonary tuberculosis.

Background & objectives: Diagnosis of extrapulmonary tuberculosis (EPTB) is difficult using conventional diagnostic methods. This study was conducted to evaluate the use of polymerase chain reaction (PCR) in diagnosis of definitive and probable extrapulmonary tuberculosis patients, and to assess the performance of insertion sequence (IS) 6110 based PCR assay as compared to conventional culture by Lowenstein-Jensen (LJ) method for the diagnosis of EPTB. Methods: A total of 178 non repeated clinical specimens were collected from clinically suspected extrapulmonary tuberculosis patients. The specimens included 59 ascitic fluid, 54 pleural fluid, 25 cerebrospinal fluid (CSF), 12 fine needle aspiration (FNA), 8 urine, 7 pus, 6 synovial fluid, 2 skin tissue, one pericardial fluid, one liver abscess, one pancreatic cyst fluid, one omental biopsy and one semen sample. All these clinical samples were subjected to Ziehl-Neelsen staining (ZN) for acid fast bacilli (AFB) and culture on LJ medium. PCR was performed by targeting 123bp fragment of insertion sequence IS6110 of Mycobacterium tuberculosis (MTB). Results: Of the 178 specimens, 10 (5.61%) were ZN smear positive for AFB, six (3.37%) were L-J culture positive from 10 AFB smear positive cases and 48 (26.96%) were PCR IS 6110 positive for M. tuberculosis. Interpretation & conclusions: PCR using IS6110 primer was able to pick up more EPTB patients compared to conventional L-J culture method for detection of M. tuberculosis. False positive PCR IS6110 in three CSF samples may be due to latent TB infection which was limitation in this study.

Occurrence of Non-Tuberculous Mycobacterium in clinical samples - a potential pathogen.

Background: Silent presence of non-tuberculous mycobacterium (NTM) has been observed since the last 100 years, but now the increasing incidence of NTM is of great concern for clinical microbiologists as well as clinicians. Although many advanced efforts are being made for identification and control of Mycobacterium tuberculosis, still the silently growing menace of non-tuberculous mycobacteria is receiving negligible attention. Objectives: This study was aimed to find NTMs in positive cultures and identify them up to species level. Material & Methods: During the study period, i.e. from January 2009 to June 2011, a total of 4104 positive cultures were subjected to species identification by different morphological and biochemical tests. All the tests for identification were performed as per standard procedure along with the standard strains of NTM provided by JALMA, Agra. Results: The identification of positive cultures showed 4044/15581 (25.95%) Mycobacterium tuberculosis complex and 60/15581(0.38%) NTM. The mycobacterium species identification results showed that out of total 60 NTM, 21 different species of NTM were found and they belonged to all the four groups of runyon. The most common species identified in this study was M.simiae (07) followed by M.avium(06), M.gordonae(05), M.kansasii(05), M.fortuitum(05), M.chelonae(05), M.pheli(05), M.terrae(04), M.szulgai(02), M.vaccae(02), M.flavescens(02), M. trivale(02), M.malmoense(01), M.scrofulaceum(01), M.intracellulare(01), M.xenopi(01), M.ulcerans(01), M.tusciae(01), M.triplex(01), M.septicum(01), M.mucogenicum(01). Conclusion: The isolation of NTMs from different clinical samples indicated that they may be the causative agents for pulmonary and extra-pulmonary non-tuberculous diseases. Elaborate and focused studies are needed to differentiate NTMs amongst commensal/colonizer, pathogen and laboratory contaminants.

Ethambutol resistance of indigenous Mycobacterium tuberculosis isolated from human patients

The present study was conducted to find out the ethambutol resistance pattern of indigenous isolates of Mycobacterium tuberculosis from Tuberculosis diagnosed human patients. A total of 172 specimens were collected from six different sources and comprised of 84.9 percent sputum, 10.5 percent pus and 4.7 percent bronchial washings. There were 70.9 percent males and 29.1 percent females with 84.30 percent pulmonary and 15.69 percent extra-pulmonary tuberculosis. The Mycobacterium tuberculosis isolates collected from primary culture were further studied to determine their pattern and level of resistance. The inoculums were prepared using 0.5 Mac Farland turbidity standards. Five different concentration of ethambutol were used in Lowenstein Jensen (LJ) medium i.e. 2μg/ml, 4μg/ml, 6μg/ml, 8μg/ml and 10μg/ml for sensitivity testing. Data showed 10 (5.8 percent) resistant and 162 (94.2 percent) sensitive Mycobacterium tuberculosis out of total 172 clinical isolates. The growth was not inhibited at 1st (2μg/ml) and 2nd (4μg/ml) drug levels, while growth of 50 percent isolates inhibited at 3rd level (6μg/ml), 30 percent inhibited at 4th level (8μg/ml) and 20 percent at 5th level (10μg/ml). The last three levels are above the therapeutic index and not recommended in actual clinical practice. It is thus conceivable to explore some other more effective chemotherapeutic agents, modify combinations or find more effective procedures to stop morbidity and mortality due to ethambutol resistant Mycobacterium tuberculosis.