In vitro antioxidant activity and antimicrobial activity against biofilm forming bacteria by the pigment from Desert soil Streptomyces sp D25.

Streptomyces from rare ecosystems is the promising source for secondary metabolites exhibiting diverse biological activities. The present study reports the antioxidant activity and antimicrobial activity against biofilm forming bacteria by the pigment produced from Desert soil Streptomyces sp. D25. Crude pigment from the Streptomyces sp D25 was produced by agar surface fermentation using yeast extract malt extract agar and extracted using ethyl acetate. Antioxidant activity of pigment was tested at 100-500μg/ml concentration by DPPH and nitric acid scavenging assay. Antimicrobial activity against the biofilm forming bacteria was tested by disc diffusion method. The Streptomyces pigment showed 35.63% and 96.19% free radical scavenging activity in DPPH assay and nitric oxide assay, respectively. The results of radical scavenging activity of pigment in DPPH and nitric oxide assay showed its antioxidant potential. In antimicrobial assay, the pigment showed 10-20 mm inhibition against biofilm forming bacteria at 25μg/ml. Further in vivo studies on this Streptomyces pigment pave the way for its biomedical applications.

Drug resistance among extrapulmonary TB patients: Six years experience from a supranational reference laboratory.

Background & objectives: There is limited information available about the drug resistance patterns in extrapulmonary tuberculosis (EPTB), especially from high burden countries. This may be due to difficulty in obtaining extrapulmonary specimens and limited facilities for drug susceptibility testing. This study was undertaken to review and report the first and second-line anti-TB drug susceptibility patterns in extrapulmonary specimens received at the National Institute for Research in Tuberculosis (NIRT), Chennai, India, between 2005 and 2012. Methods: Extrapulmonary specimens received from referring hospitals were decontaminated and cultured using standard procedures. Drug susceptibility testing (DST) for Mycobacterium tuberculosis was done by absolute concentration or resistance ratio methods for the first and the second line anti-TB drugs. Results: Between 2005 and 2012, of the 1295 extrapulmonary specimens, 189 grew M. tuberculosis, 37 (19%) cases were multidrug resistant (MDR) while one was extensively drug resistant (XDR). Specimen-wise MDR prevalence was found to be: CSF-10 per cent, urine-6 per cent, fluids and aspirates-27 per cent, pus-23 per cent, lymph nodes-19 per cent. Resistance to isoniazid and ethionamide was found to be high (31 and 38%, respectively). Interpretation & conclusions: Drug resistance including MDR-TB was observed in a significant proportion of extrapulmonary specimens referred for DST. Access to culture and DST for extrapulmonary specimens should be expanded. Guidelines for MDR-TB management should have explicit sections on extra-pulmonary tuberculosis and training on laboratory techniques is urgently required.

Revisiting the susceptibility testing of Mycobacterium tuberculosis to ethionamide in solid culture medium.

Background & objectives: Increase in the isolation of drug resistant phenotypes of Mycobacterium tuberculosis necessitates accuracy in the testing methodology. Critical concentration defining resistance for ethionamide (ETO), needs re-evaluation in accordance with the current scenario. Thus, re-evaluation of conventional minimum inhibitory concentration (MIC) and proportion sensitivity testing (PST) methods for ETO was done to identify the ideal breakpoint concentration defining resistance. Methods: Isolates of M. tuberculosis (n=235) from new and treated patients were subjected to conventional MIC and PST methods for ETO following standard operating procedures. Results: With breakpoint concentration set at 114 and 156 μg/ml, an increase in specificity was observed whereas sensitivity was high with 80 μg/ml as breakpoint concentration. Errors due to false resistant and susceptible isolates were least at 80 μg/ml concentration. Interpretation & conclusions: Performance parameters at 80 μg/ml breakpoint concentration indicated significant association between PST and MIC methods.

Effect of critical medium components and culture conditions on antitubercular pigment production from novel Streptomyces sp D25 isolated from Thar desert, Rajasthan.

Effect of culture conditions and critical medium components on antitubercular pigment production from novel Streptomyces sp D25 isolated from Thar desert, Rajasthan was investigated. Antitubercular pigment from Streptomyces sp. D25 was produced by Agar Surface Fermentation (ASF) using yeast extract malt extract agar (YEME) as a basal medium. Effect of incubation period, temperature, carbon source, nitrogen source, minerals and sodium chloride concentration was studied by adopting one-variable-at-a-time method. Crude pigment produced under different conditions was extracted solid-liquid extraction method using ethyl acetate. Responses measured include the growth rate, quantity of crude extract and activity against S. aureus MTCC96 and M. tuberculosis H37Rv at 100 μg concentrations. Growth and pigment production was correlated with the bioactivity. Of the various conditions tested, maximum growth, pigmentation and bioactivity was observed on 6th day of incubation. Of the various medium components tested, 1% glucose, fructose and malt extract, pH 7 and 9, temperature 300C and 400C, 0.1% KNo3 and 0 – 5% NaCl was found to influence the growth, bioactive pigment production and antimicrobial bioactivity. Further statistical based optimization is in progress to prove the effect of interaction of the above variables on antitubercular pigment production from Streptomyces sp D25.

Equivalence of acid alone or acid-alcohol as decolourizing agent in Ziehl - Neelsen method.

Background: Microscopists opine that acid-alcohol decolourized slides may enhance acid-fast bacilli (AFB) smear positivity, and published documents on equivalence of acid and acid-alcohol in ZN staining method are not easily accessible. Setting: National Institute for Research in Tuberculosis, Chennai, India. Objective: To document the equivalence of 25% sulphuric acid (ZN-acid method) and 3% hydrochloric acid-alcohol (ZNalcohol method) as decolourizing agents in ZN method for detection of acid-fast bacilli. Methods: Two smears from each of 253 sputum samples from pulmonary tuberculosis patients, prepared and allocated, one to ZN-acid method and another to ZN-alcohol method were read blind. All the specimens were cultured for Mycobacterium tuberculosis by modified Petroff’s method. Culture of M. tuberculosis was gold standard. Results: The concordance between the methods was 85% (kappa 0.68), and the sensitivity (79%) and specificity (89%) were same for both the methods. Conclusion: In conclusion, the common belief that acid-alcohol decolourized slides give enhanced smear positivity stands void.

Consistency of standard laboratory strain Mycobacterium tuberculosis H37Rv with ethionamide susceptibility testing.

Drug susceptibility pattern of standard Mycobacterium tuberculosis strain H37Rv showed discrepancy in minimum inhibitory concentration method for ethionamide and consistent results were obtained for the other second line drugs namely, kanamycin and ofloxacin. It is, therefore, necessary to revisit the susceptibility testing method for ethionamide for effective clinical management of patients with drug resistant tuberculosis.

Pot method for storage & detection of acid-fast bacilli from sputum samples.

BACKGROUND & OBJECTIVES: Sputum acid-fast bacilli (AFB) microscopy services are not available in all health facilities. Alternative procedures are needed to transport sputum samples to the diagnostic centres for detection of AFB. The objective of the present study was to evaluate sputum smears made by pot-method with the direct smears made immediately after sputum collection by Ziehl-Neelsen (ZN) method. METHODS: Ninety three sputum samples from 49 pulmonary tuberculosis suspects were studied. Their direct smears (ZN smears) were stained by hot ZN method. The samples were then mixed with phenol ammonium sulphate basic fuchsin solution and stored at ambient conditions. The smears (pot smears), made on day 7, were then, decolourized and counter-stained for detection of AFB (pot method). The ZN and pot smears were read blind. After excluding 18 samples for various reasons, the results of pot and ZN smears of 63 samples from smear positive (2 of 3 direct smears were positive) and 12 from smear negative (3 of 3 direct smears were negative) patients were analysed. ZN method was the gold standard. RESULTS: Pot and ZN smears were positive in 61 of 63 samples from smear-positive patients and negative in 11 of 12 smear-negative patients (kappa = 0.87). The sensitivity and specificity of pot method were 96.8 and 91.7 per cent respectively. INTERPRETATION & CONCLUSION: Sputum samples can be stored for up to seven days in the sputum container with phenol ammonium sulphate basic fuchsin solution. However, a comprehensive study needs to be done confirm the accuracy of the pot method for storage and transportation of sputum to microscopy centres for detection of AFB.

Phenol ammonium sulphate basic fuchsin staining of sputum in pot for the detection of acid-fast bacilli.

BACKGROUND & OBJECTIVES: Improper practices of making direct smears of sputum for detection of acid-fast bacilli (AFB) and of disposing sputum cups are hazardous. The present study was undertaken with the objective to stain sputum samples in their containers by 'phenol (10%) ammonium sulphate (4%) basic fuchsin (2%) solution' and to decolourize and counterstain their smears for detection of AFB- (henceforth called pot method) and to compare the smear results of pot method with the standard Ziehl-Neelsen (ZN) method. METHODS: A total of 575 selected sputum samples from pulmonary tuberculosis patients were stained by the standard ZN and pot methods and the proportions of AFB positive smears were compared. RESULTS: Of the 575 samples, 126 were AFB positive for both the staining methods and the difference was not statistically significant. Pot method missed 9 ZN positive smears (8 scanty and one 1+) and ZN method missed 9 pot positive smears (9 scanty) and the difference was not significant. High grade smears (3+) were seen more in pot method (42) than in ZN method (25) and the difference was significant. INTERPRETATION & CONCLUSION: Our findings showed that pot method was comparable to standard ZN method and had many advantages. Pot method can be explored further for the detection of AFB in sputum samples obtained from pulmonary tuberculosis suspects.

Sensitivity of Ziehl-Neelsen method for centrifuged deposit smears of sputum samples transported in cetyl-pyridinium chloride.

BACKGROUND & OBJECTIVES: Sensitivity of Ziehl-Neelsen (ZN) method is known to be low for liquefied sputum smears. Information on the ZN sensitivity for centrifuged deposit smears is not known. This study was carried out to determine the sensitivity of ZN method for acid fast bacilli (AFB) in centrifuged deposit smears and liquefied sputum smears made from sputum samples transported in cetyl-pyridinium chloride (CPC) solution. METHODS: Liquefied sputum smears and the corresponding centrifuged deposit smears from each of the 607 consecutive sputum samples collected from tuberculosis patients admitted to receive treatment transported in CPC were read by the same readers and their results compared with culture results. RESULTS: A significantly (P<0.001) higher proportion of samples were positive in centrifuged deposit smears (40%) compared to liquefied sputum smears (30%). The results of 341 culture-positive specimens revealed that the sensitivity of ZN method was 47 per cent using liquefied sputum smears and 63 per cent using centrifuged deposit smears (P<0.001). INTERPRETATION & CONCLUSION: Our study demonstrated that the sensitivity of ZN method for AFB in centrifuged deposit smears and liquefied sputum smears was reduced if sputum samples are transported in CPC solution.

Proficiency to read sputum AFB smears by senior tuberculosis laboratory supervisors under training at a reference laboratory in India.

Background: A national reference laboratory imparting training on sputum AFB smear microscopy to fresh Senior Tuberculosis Laboratory Supervisors (STLS). Aim: To assess the proficiency of STLSs under training to read sputum AFB smears. Methods: Each of 342 trainees read the same set of 15 to 20 Ziehl Neelsen stained smears in a blinded fashion on day– 1 and on day-15 of the training programme. The smear results were matched with the original results. Observations: The sensitivity, specificity, positive predictive value and negative predictive value of smear reading were 75%, 88%, 93%, 63% and 94%, 99%, 99%, 89% respectively on day-1 and day –15. Conclusion: The sensitivity to read sputum AFB smears by fresh STLSs with little or no experience increased from 75% to 94% during the carefully planned training programme; the specificity increased from 88% to 99%. The study highlights the importance of training in improving the microscopy results.