Impact of anthropogenic activities on the abundance of microplastics in copepods sampled from the southeast coast of India.

In recent year, the use of plastics has become inevitable due to its unique properties that allow for the production of durable and non-durable goods. Post use, plastics enter the waste stream and now can be found in all compartments of the biosphere as microplastics (MPs). This study presents the abundance of MPs in surface water and within copepods in the southwestern Bay of Bengal during dry (June 2022) and wet season (November 2022). The MPs in the surface water were found in all three regions [Chennai, Tuticorin and Nagapattinam (four locations in each region)] and maximum in wet season (53 particles/m3). Moreover, during dry season the mean ingestion of MPs by copepods in Chennai (0.103 ± 0.04 particles/individual), Tuticorin (0.11 ± 0.07 particles/individual) and Nagapattinam (0.036 ± 0.01 particles/individual) is high compared to the wet season. The maximum level of MPs found in both surface water and ingestion by copepods in Tuticorin and Chennai is subjective to the high maritime activities than Nagapattinam region. Whatever, the anthropogenic activities in the study region increase the bioavailability of MPs pollutant in the copepods and transported to higher trophic levels, endangering marine life and human health. Hence, further studies are needed to determine their potential impact on marine food chain in this alarming situation.

Production, purification and application of Cutinase in enzymatic scouring of cotton fabric isolated from Acinetobacter baumannii AU10.

Conventional cotton scouring in the textile industry using alkali results in huge environmental impact which can be overcome by using enzymes. Pectinase along with cutinase gives enhanced bioscouring results. Cutin was extracted from tomato peels and was used as substrate in the microbial media. The strain isolated from tomato peel was identified as Acinetobacter baumannii AU10 by 16S rDNA sequencing. The cutinase production was optimized by Placket-Burman and Response Surface Methodology (RSM) and the maximum production of 82.75 U/mL obtained at sucrose 6.68% (w/v), gelatin 2.74 g/L at a temperature of 35.93 °C. Cutinase was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography and ion exchange chromatography with a recovery of 25.6% and specific activity of 38030 U/mg. The confirmation test for the purity of cutinase was analyzed by RP-HPLC. The molecular mass of cutinase was determined as 28.9 kDa by SDS-PAGE technique. Scanning electron microscopic analysis showed a rough and open primary wall surface on the cutinase bioscoured fabric which confirmed its activity on cutin present in the cotton fabric. Additionally, the cutinase-bioscoured samples showed better absorbency than the untreated samples. Therefore, enzymatic scouring increases wetting capacity of scoured cotton and also helps to reduce environmental pollution.

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.

Could repeated prevalence surveys lead to decreasing tuberculosis prevalence in a community?

SETTING: Tiruvallur District, South India, where one baseline tuberculosis (TB) disease prevalence survey followed by three repeat prevalence surveys were conducted every 2.5 years between 1999 and 2008, and where the DOTS strategy was implemented in 1999. OBJECTIVE: To rule out the possibility that the observed decline in TB prevalence was influenced by conducting repeat prevalence surveys, we compared the findings from two surveys: the third repeat survey conducted in 2006-2008 and an independent single survey in a neighbouring area conducted in 2008-2009. DESIGN: An independent survey was conducted to estimate the prevalence of TB in the same district in 2008-2009 using a different set of villages and employing repeat survey methodology. The independent survey findings were compared with those of the third repeat survey. RESULTS: The estimated prevalence rate of culture- and smear-positive TB was respectively 401 per 100,000 and 186 per 100,000 population in the third repeat survey area. The corresponding rates were 340 and 184/100,000 in the independent survey area. The difference in prevalence was not significant (culture P = 0.09; smear P = 0.93). CONCLUSION: The estimated prevalence rates in the two different sample survey areas were comparable, indicating that the repeated prevalence surveys in the study area did not influence the observed decline in TB disease prevalence.

High rates of ofloxacin resistance in Mycobacterium tuberculosis among both new and previously treated patients in Tamil Nadu, South India.

Periodic drug resistance surveillance provides useful information on trends of drug resistance and effectiveness of tuberculosis (TB) control measures. The present study determines the prevalence of drug resistance among new sputum smear positive (NSP) and previously treated (PT) pulmonary TB patients, diagnosed at public sector designated microscopy centers (DMCs) in the state of Tamil Nadu, India. In this single-stage cluster-sampling prevalence survey, 70 of 700 DMCs were randomly selected using a probability-proportional to size method. A cluster size of 24 for NSP and a varying size of 0 to 99 for PT cases were fixed for each selected DMC. Culture and drug susceptibility testing was done on Lowenstein-Jensen medium using the economic variant of proportion sensitivity test for isoniazid (INH), rifampicin (RMP), ofloxacin (OFX) and kanamycin (KAN). Human Immunodeficiency Virus (HIV) status was collected from patient records. From June 2011 to August 2012, 1524 NSP and 901 PT patients were enrolled. Any RMP resistance and any INH resistance were observed in 2.6% and 15.1%, and in 10.4% and 30% respectively in NSP and PT cases. Among PT patients, multi drug resistant TB (MDR-TB) was highest in the treatment failure (35%) group, followed by relapse (13%) and treatment after default (10%) groups. Extensively drug resistant TB (XDRTB) was seen in 4.3% of MDR-TB cases. Any OFX resistance was seen in 10.4% of NSP, 13.9% of PT and 29% of PT MDR-TB patients. The HIV status of the patient had no impact on drug resistance levels. RMP resistance was present in 2.6% of new and 15.1% of previously treated patients in Tamil Nadu. Rates of OFX resistance were high among NSP and PT patients, especially among those with MDR-TB, a matter of concern for development of new treatment regimens for TB.

Investigation of Ser315 substitutions within katG gene in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis from south India.

Mutation at codon 315 of katG gene is the major cause for isoniazid (INH) resistance in Mycobacterium tuberculosis (M. tuberculosis). Substitution at codon 315 of katG gene was analyzed in 85 phenotypically resistant isolates collected from various parts of southern India by direct sequencing method. The obtained results were interpreted in the context of minimum inhibitory concentration (MIC) of INH. Of the 85 phenotypically resistant isolates, 56 (66%) were also correlated by the presence of resistance mutations in the katG gene; 47 of these isolates had ACC, 6 had AAC, 2 had ATC, and one had CGC codon. The frequency of Ser315 substitution in katG gene was found to be higher (70%) amongst multidrug-resistant (MDR) strains than among non-MDR (61%) INH-resistant isolates. Further, the frequency of mutations was found to be greater (74%) in isolates with higher MIC values in contrast to those isolates with low MIC values (58%). Therefore, the study identified high prevalence of Ser315Thr substitution in katG gene of INH-resistant isolates from south India. Also, isolates harboring this substitution were found to be associated with multidrug and high level INH resistance.

Carbon nanotube-based tandem absorber with tunable spectral selectivity: transition from near-perfect blackbody absorber to solar selective absorber.

CVD grown CNT thin film with a thickness greater than 10 µm behaves like a near-perfect blackbody absorber (i.e., α/ε = 0.99/0.99). Whereas, for a thickness ≤ 0.4 µm, the CNT based tandem absorber acts as a spectrally selective coating (i.e., α/ε = 0.95/0.20). These selective coatings exhibit thermal stability up to 650 °C in vacuum, which can be used for solar thermal power generation.

The Assessment of Viability of M. Tuberculosis after Exposure to CPC Using Different Methods.

Settings. National Institute for Research in Tuberculosis, Chennai. Objective. To assess the proportion of metabolically active cells of Mycobacterium tuberculosis after exposed to CPC using FDA-EB vital staining and viable counts on LJ medium. Mycolic acid content in M. tuberculosis after exposure to CPC was estimated using HPLC. Methods. Clinical isolates of M. tuberculosis and standard reference strain M. tuberculosis H37Rv were used for FDA-EB, viable count, and HPLC. Results. FDA/EB consistently stained 70-90% of log phase cells as green and the remaining cells as red-orange. After CPC treatment, 65-70% of the cells stained red-orange. The viability counts were comparable to 0-day controls. Synthesis of mycolic acids in mycobacteria was reduced when exposed to CPC using HPLC due to the decreased metabolic activity of the organisms. Conclusion. The cells are metabolically inactive during storage with CPC but these cells grew well on LJ medium after removal of CPC. The viability of M. tuberculosis was maintained in CPC with minimal reduction. Mycolic acid content was reduced if the cells of M. tuberculosis were treated with CPC for 7 days. All the above findings provide yet another evidence for the damage of cell wall of M. tuberculosis.

Trends in the prevalence of pulmonary tuberculosis over a period of seven and half years in a rural community in south India with DOTS.

SETTING: Tiruvallur district In Tamil Nadu where DOTS was implemented by the State Government as the tuberculosis control measure in 1999, and monitored by the National Institute for Research in Tuberculosis for over five years. OBJECTIVE: To estimate trends in TB prevalence in a rural community with DOTS. DESIGN: Surveys of pulmonary tuberculosis were undertaken in representative samples of subjects aged > or =15 years (N = 83,000 - 92,000), initially and after two and half, five and seven and half years of implementation of DOTS. Sputa were collected from those with abnormal radiograph and/or presence of chest symptoms, and examined by direct smear and culture. RESULTS: The prevalence of culture-positive tuberculosis was 607, 454, 309 and 388 per 100,000 in the four surveys, and that of smear-positive tuberculosis was 326, 259, 168 and 180. In the first five years; annual decrease was 12.4% (95% CI 10.4 - 14.4%) for culture-positive tuberculosis, and 12.2% (95% CI 8.0-16.2) for smear-positive tuberculosis. This was, however, followed by a significant increase in the next two and half years. The average new smear-positive case-notification rate was 75 per 100,000 during first four years but declined to 49 in subsequent years. There were no methodological differences during this period and information on changes in socio-economic indicators and nutritional standards was unavailable. CONCLUSION: Despite the average annual success rate (78%) in this tuberculosis unit being lower than the expected rate of 85%, the implementation of DOTS was followed by a substantial decrease in the prevalence of pulmonary tuberculosis over the seven and half year period. Our findings suggest that sustaining the high effectiveness of DOTS programme needs vigilant supervision.

Blinded rechecking of acid-fast bacilli smears by light-emitting diode microscopy.

BACKGROUND: Blinded rechecking of auramine-stained acid-fast bacilli (AFB) sputum smears using fluorescence microscopy (FM), especially FM using light-emitting diode (LED), is not well understood. OBJECTIVE: To examine the rechecking of auramine-stained sputum smears without restaining within a month using LED FM. METHODS: A total of 4799 centrifuged smears of sputum samples were stained by the auramine phenol method and examined using LED FM; 564 systematically selected smears were subjected to blinded rechecking without restaining by controllers. The initial results of the readers were compared to those of the controllers. Discrepancies were resolved by a referee. The quality of LED FM was assessed by the referee using the culture result as gold standard. RESULTS: Among the rechecked smears, one high false-negative error was made by a reader, while one high false-positive error and 19 high false-negative errors were made by the controllers. The errors were resolved by culture. Smear results for 18 slides were not available due to AFB fading. CONCLUSION: AFB colour fading using LED FM, which affected the accurate evaluation of blinded rechecking of AFB smears without restaining within a month, is confirmed in this large study.

Modification of proportion sensitivity testing method for ethionamide.

Standardized methodology for drug susceptibility testing of second line drugs is vital for treatment of multi/extensively drug resistant tuberculosis. Discrepancy between laboratory methods and clinical interpretation is well established for bacteriostatic drugs such as ethionamide. Optimization of the standard proportion sensitivity testing (PST) method for ethionamide was under taken in 235 Mycobacterium tuberculosis isolates from new and previously treated pulmonary tuberculosis patients. An additional higher concentration of 57 µg/ml was evaluated against at the standard 40 µg/ml concentration in PST method. Performance parameters and agreement between the two drug concentrations was higher indicating the efficiency of PST method at its present format at 40 µg/ml and additional higher concentration of 57 µg/ml as an alternative when required.

Is Duct to Duct biliary Anastomosis the Rule in Orthotopic Liver Transplantation?

Biliary complications after Liver Transplantation continue to be the major cause of morbidity in 11-25 % of patients. Biliary complications in patients who underwent orthotopic liver transplantation (OLT) at our institute between March 2007 and June 2010 were analyzed retrospectively. 32 patients underwent Deceased Donor Liver Transplantation (DDLT) and in 12 patients Living Donor Liver Transplantation (LDLT) was done. No patients were lost to follow up. Follow up ranged between 4 and 44 months. During the study period, 44 patients underwent orthotopic liver transplantation. Patients were divided into two groups: Biliary Complications group (BC) n = 5 and Non Biliary Complications group (NBC) n = 39. Biliary complications occurred in 15.9 % of patients. Bile leaks accounted for majority of biliary complications. Fifteen variables were analyzed as possible risk factors for biliary complications. Of these, split grafts, duct to duct biliary anastomosis and total blood loss were statistically significant (P < 0.05) for biliary complications. Endoscopic treatment was successful in managing biliary complications in 75 % of patients. Biliary complications are the most common major complications in orthotopic liver transplantation. Significant risk factors are split liver grafts and duct to duct biliary anastomosis. Increased blood loss is a predictor for post operative biliary complications. These complications should be managed by endoscopic interventions. Surgery is indicated following failure of endoscopic interventions.

Assessment of panel slides prepared by phenol ammonium sulphate and NALC methods for proficiency testing.

BACKGROUND: Existing methods for the preparation of panel slides necessitate handling high-grade acid-fast bacilli positive sputum samples. OBJECTIVE: To compare panel slides prepared using the phenol ammonium sulphate sediment (PhAS) method with those prepared using the N-acetyl-L-cysteine (NALC) method in proficiency testing. METHODS: Pooled sputum specimens of known smear-positives and -negatives were divided into two parts: one part was used for preparing panel slides using the NALC method and the other using PhAS, a non-hazardous method. Respectively 413 and 384 smears of different grades were prepared in three batches using the PhAS and NALC methods. Smear grade and quality were recorded by 121 microscopists during proficiency testing in different states. Agreement between reference and reported results was analysed using the kappa test. RESULTS: The overall agreement was 96% for the PhAS method and 91% for the NALC method. There were 37 errors using the NALC method compared to 21 for the PhAS method (P < 0.223). Smear quality was equally good in both methods; however, the cell count was significantly higher in the PhAS than in the NALC method. CONCLUSION: The PhAS method, a non-hazardous procedure with good-quality smears, may be further explored for the preparation of panel slides.

Capilia test for identification of Mycobacterium tuberculosis in MGIT™-positive cultures.

BACKGROUND: The performance of the Capilia test for rapid identification of Mycobacterium tuberculosis complex (MTC) in Mycobacterium Growth Indicator Tube (MGIT) positive samples with contaminating organisms is not well documented. OBJECTIVE: To assess the diagnostic yield of the Capilia test in the rapid identification of MTC in MGIT-positive cultures. DESIGN: A total of 459 selected sputum samples were cultured using BACTEC™ MGIT™ 960. Tubes flagged positive by the MGIT instrument (MGIT-positive) were examined for acid-fast bacilli and cording in smears, spotted on blood agar (BA), subcultured for biochemical tests and tested using the Capilia test. Based on smear and growth on BA, MGIT-positive tubes were grouped into MGIT true-positive, MGIT-positive with contamination and MGIT contamination. Performance parameters of Capilia test such as sensitivity, specificity, efficiency, and positive and negative predictive values (PPV, NPV) for each of these groups were determined against biochemical tests as gold standard. RESULTS: Of the 346 MGIT-positives, respectively 233, 73 and 40 were MGIT true-positive, MGIT-positive with contamination and MGIT contamination. For the three groups, the PPV and NPV of the Capilia test were respectively 97%, 96% and 100%, and 32%, 27% and 60%. CONCLUSION: In settings with high contamination of MGIT cultures, the performance of the Capilia test is diminished.

Recovery of Mycobacterium tuberculosis from Löwenstein-Jensen media contaminated with other organisms.

Growth of contaminating organisms along with Mycobacterium tuberculosis on Löwenstein-Jensen (LJ) medium is common. However, there is no documented evidence on the decontamination procedure adopted in mycobacteriology laboratories to recover M. tuberculosis from the contaminants grown on LJ medium. At the National Institute for Research in Tuberculosis, of 1048 LJ slopes with M. tuberculosis received from intermediate reference laboratories, 98 (9%) were contaminated. Of these, 87 (89%) M. tuberculosis cultures were retrieved after decontamination with 1% cetrimide. The use of cetrimide as a decontaminating agent to retrieve M. tuberculosis cultures grown with contaminants is documented.

Diagnostic luciferase reporter phage assay for active and non-replicating persistors to detect tubercle bacilli from sputum samples.

Diagnosis of latent tuberculosis infection is a myth for want of a simple, direct tool. Simulation of hypoxic environment was done to create a novel hypothetical model for persistence using processed sputum samples. The adaptation of tubercle bacilli to hypoxic environment seems to be influenced by pre-existing clinical status of the patients at the time of sputum collection, resulting in varied growth pattern. Bacilli from 36 samples did not get adapted to latency of which 15 samples were from patients in whom the disease was well established and the tubercle bacilli in them probably did not experience any stress whatsoever. Similarly, 10 of the 37 samples showing the presence of cultivable cells in both aerobic and anaerobic conditions were from patients who had relapsed. The bacilli in these samples had been probably experiencing stress and thus were ready to adapt to the hypoxic environment. Diagnostic luciferase reporter phage assay for non-replicating persistors (DLRPA-NRP) identified 30 additional positives which failed to grow on Lowenstein-Jensen medium. Presence of viable bacilli in these samples was confirmed by reverse transcriptase-PCR (RT-PCR) for 16S rRNA indicating either the improved sensitivity of the assay to detect actively growing bacilli or its ability to detect non-replicating persistors. The utility of LRP assay to detect both dormant and active tubercle bacilli was explored in this work and was optimized using lysis inhibition to diagnose tuberculosis with rapidity, improved sensitivity and specificity. DLRPA-NRP, a rapid growth based assay is thus developed to detect both dormant and actively growing tubercle bacilli.

Selected risk factors associated with pulmonary tuberculosis among Saharia tribe of Madhya Pradesh, central India.

Tuberculosis (TB) is a major public health problem among the Saharia, a marginalized tribal group in Madhya Pradesh state, central India. However, there is no information on the risk factors associated with the development of TB disease in this community. A cross-sectional TB prevalence survey was conducted among the Saharia residing in Sheopur district of Madhya Pradesh. Information on tobacco smoking and alcohol consumption was collected from all the individuals. Persons aged ≥45 years, males, smokers and alcohol consumers had higher risks of developing TB disease. There is an urgent need to develop and implement culturally appropriate awareness raising activities to target smoking and alcohol consumption to support the efforts to control TB in this community.

Quality indicators in a mycobacteriology laboratory supporting clinical trials for pulmonary tuberculosis.

BACKGROUND: Documentation of structured quality indicators for mycobacteriology laboratories supporting exclusively controlled clinical trials in pulmonary tuberculosis (PTB) is lacking. OBJECTIVE: To document laboratory indicators for a solid (Lowenstein-Jensen medium) culture system in a mycobacteriology laboratory for a period of 4years (2007-2010). METHODS: The sputum samples, collected from PTB suspects/patients enrolled in clinical trials, were subjected to fluorescence microscopy, culture and drug sensitivity testing (DST). Data was retrospectively collected from TB laboratory registers and computed using pre-formulated Microsoft Office Excel. Laboratory indicators were calculated and analyzed. RESULTS: The number of samples processed in a calendar year varied from 6261 to 10,710. Of the samples processed in a calendar year, specimen contamination (4.8-6.9%), culture positives (78.4-85.1%) among smear positives, smear positives (71.8-79.0%) among culture positive samples, smear negatives among culture negative samples (95.2-96.7%), and average time to report DST results (76-97days) varied as shown in parentheses. CONCLUSION: Values of quality indicators in mycobacteriology laboratories supporting exclusively clinical trials of PTB have to be defined and used for meaningful monitoring of laboratories.

Performance indicators of fluorescence microscopy for sputum samples in pulmonary tuberculosis.

To get insight into the sensitivity of fluorescence microscopy for diagnostic and follow-up sputum samples from pulmonary tuberculosis patients in clinical trials, the yield of smear positivity - among culture positive sputum samples - encountered in diagnostic and follow-up samples was retrospectively analyzed from the data available in a mycobacteriology laboratory in India. The sensitivity of fluorescence microscopy for diagnostic and follow-up samples respectively was found to be 94.3% and 60.7%. With these values as guidelines, the performance of fluorescence microscopy in the treatment of multi-drug resistant tuberculosis under DOTS plus program remains to be monitored and studied.