Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay.

Purpose: There is scarcity of prevalence data of multi-drug–resistant tuberculosis (MDR-TB) data and common mutations responsible in North India. This study aimed to detect MDR-TB among MDR-TB suspects from Delhi and mutation patterns using GenoType MTBDRplus assay. Materials and Methods: All MDR suspects in fi ve districts of New Delhi were referred to the laboratory from 1st October 2011 to 31st December 2012 as per criterion defi ned by Programmatic Management of Drug Resistant Tuberculosis (PMDT). GenoType MTBDRplus assay was performed on 2182 samples or cultures and mutations in the rpoB gene for rifampicin (RIF) and katG and inhA genes for isoniazid (INH) were analyzed. Results: A total of 366 (16.8%) MDR-TB cases were diagnosed. MDR rate was found to be 32%, 16.6% and 10.2% during criterion A, B and C respectively. The most common mutation detected for RIF was S531L (59.0%) and for INH was S315T1 (88.3%). Mutations S531L and S315T1 occurred signifi cantly higher in MDR strains as compared to RIF mono-resistant and INH mono-resistant strains, respectively. Average laboratory turn-around time (TAT) for dispatch of result to districts for test conducted on samples was 4.4 days. Conclusion: GenoType MTBDRplus is a useful assay for rapid detection of MDR-TB. The common mutations for RIF and INH were similar to those seen in other regions. However, mutations determining MDR strains and mono-resistant strains differed signifi cantly for both RIF and INH.

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.

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.

Presumptive identification of Mycobacterium tuberculosis complex based on cord formation in BACTEC MGIT 960 medium.

We considered samples received for culture of mycobacteria using BACTEC MGIT 960 system over a period of 1 year. Tubes flagged positive by MGIT were evaluated for presence of serpentine cording. The cord formation was compared with isolates identified as Mycobacterium tuberculosis complex (MTC) based on p-nitrobenzoic acid (PNB) test. Cords were found in 591 isolates of which 584 (98.8%) were confirmed as MTC. The sensitivity and specificity of cord formation were found to be 99.7% and 89.9%, respectively.

A pilot study of same day sputum smear examination, its feasibility and usefulness in diagnosis of Pulmonary TB.

Introduction'. A large number of tuberculosis cases are continuously being reported from India and other developing countries leading to high morbidity and mortality. In spite of many newer tests available for diagnosing a case of tuberculosis, smear microscopy of sputum is still the preferred test under programmatic conditions. The current national and international guidelines recommend two sputum smear examinations in two days for diagnosing cases of tuberculosis, which is time consuming, tedious, needs multiple visits, leading to high dropout of infectious cases. In the background of existing limitations of smear microscopy, we attempted to complete the diagnosis of tuberculosis on same day by serial collection of the spot sputum specimen and analyze its advantages, feasibility and viability. Material & Methods: The study was undertaken by the Department of Microbiology, Lala Ram Sarup Institute of Tuberculosis and Respiratory Diseases during May 2010 to April 2011. Sputum specimens were collected from 330 randomly selected tuberculosis suspects who attended OPD of hospital, patients submitted spot and home collected morning sputum sample in a standard method and spot and additional spot sputum(X- spot) collected one hour after the flrst spot sample as per the proposed front loading method. All the samples received were stained by acid fast Ziehl-Neelsen (ZN) stain and examined on the same day. The sputum sample was pooled and cultured in Lowenstein Jensen (U) media in duplicate set of bottles. The results of two different microscopic methods were compared with the gold standard culture test. Results: Out of the total 330 TB suspects, 70.60% were males and 29.39% females. The most common complaint was of cough with sputum (88.18%), chest pain (70.21%), fever (55.15%) and loss of appetite (43.03%). Upon examining the total sputum slides, 18.48 % were positive for acid fast bacilli. The smear positivity was 61/330(l8.48%) by standard methods and in proposed new method 43/330(13.03%). Sensitivity of the standard and proposed new method smear microscopy was 58.25% and 40.07% respectively and specificity was 99.55% in both the methods. Conclusion: Same day smear microscopy for diagnosing tuberculosis by a proposed new method of smear examination in the case of suspected tuberculosis seems not a promising step towards improving the quality of sputum smear examination. The results of sensitivity and specificity of the two approaches were not similar. More than eighty per cent responded in favour of same day sputum delivery system and getting result on same day. This study can be confirmed on larger scale and preference of patients can be examined in peripheral laboratory also before taking it up for consideration in the national tuberculosis programme.

Prevalence of XDR TB cases - A retrospective study from a tertiary care TB Hospital.

Background: The emergence of XDR –TB strains is a major roadblock in the successful implementation of TB control programmes. This further leads to high morbidity and mortality, especially in immuno-compromised patients. Identification and observation of resistance patterns of XDR-TB strains may help clinicians manage MDR-TB cases, the treatment line of which is expensive, time-taking and involves intake of toxic drugs with many side-effects. Our study is aimed to find out the prevalence of XDR-TB among the MDR-TB strains isolated in a tertiary care hospital. Material & Methods: The study population consisted of 223 patients of tuberculosis who were culture positive and Mycobacterium tuberculosis was resistant to Rifampicin and Isoniazid during January 2007 to December 2009. Each patient had submitted two sputum samples i.e. spot and morning. The identified Mycobacterium tuberculosis complex was subjected to drug sensitivity testing by first and second line drugs by proportion and absolute concentration methods as per standard procedure . Results : The results showed that 20.17% strains (45/223) were XDR-TB strains. Most of these strains showed resistance to four drug combination viz. KM, ETH, OFX & PAS (5.82%), KM & OFX (3.13%), OFX, KM and ETH (1.79%), 1.34% strains showed resistance to all the drugs i.e. pan resistance and other combinations in the remaining strains. Nearly 80% of the XDR-TB strains showed resistance to three or more drugs combination pattern. Conclusion: The multidrug resistant TB cases need urgent and timely sensitivity report for second line ATT drugs to help clinicians start proper drug combinations to treat MDR-TB patients.

A thyroid tubercular abscess and bilateral symmetrical hilar lymphadenopathy: a rare association.

An 18-year-old boy presented with a rare association of a thyroid tubercular abscess and bilateral symmetrical hilar lymphadenopathy. He was put on a Category I regimen with standard short course daily chemotherapy of four anti-tubercular drugs under the National Tuberculosis Programme. After a six-month of anti-tubercular treatment (ATT), the boy showed clinical and bacteriological improvement. The thyroid scan with Technetium 99 (Tc 99) and the chest skiagram also became normal.