Optimized Tube Dilution Technique and Sole Carbon Utilization Assay for Anti-leptospiral In Vitro Screening of Plant Extracts

Introduction@#Leptospirosis is one of the neglected reemerging zoonoses that is of public health concern globally. The need to discover novel therapeutic alternatives for leptospirosis through screening for and elucidating the mechanism/s of the anti-leptospiral activity of plant extracts is therefore necessary. This study analyzes the optimized tube dilution technique and the BiologTM sole carbon utilization phenotype microarray as screening tool for anti-leptospiral activity of plant extracts. @*Methods@#The suitability of the optimized tube dilution technique was evaluated by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and motility inhibition property of a plant extract and an antimicrobial control (pen G) against 4 dominantly circulating Leptospira serovars/serogroup in the Philippines. Likewise, the suitability of the BiologTM sole carbon utilization assay was evaluated using a plant extract and selected antimicrobials against L. interrogans serovar Manilae strain K64 and L. interrogans serovar Losbanos strain K37. @*Results@#The MIC, MBC, and motility inhibition property of a plant extract and the antibiotic controls as well as its effect on the carbon utilization phenome of the Leptospira serovars gave consistent results, within and between several runs. With standard deviation = 0 for all serovars. The MIC and MBC of the antimicrobial control (pen G), the positive control, was 10 ug/ml. The growth control (leptospires without treatment), the negative control, showed presence of motile leptospires. The MIC and the MBC of the test plant extract was 250 ug/ml – 500 ug/ml. Results of the carbon utilization phenome or pattern of carbon utilization were consistent within the 3 replicates and between two runs. @*Conclusion@#The optimized tube dilution technique and the BiologTM sole carbon utilization assay is a potential in vitro screening tool for determining anti-leptospiral activity of plant extracts.

HPLC method for ethylenethiourea in biological and environmental samples

@#<p style="text-align: justify;"><strong>OBJECTIVE:</strong> In view of both the economic importance of ethylenebisdithiocarbamate (EBDC) fungicides in the current agricultural practice and the potential health hazards associated with ethylenethiourea (ETU) exposure, this study aimed to develop and validate a high-pressure liquid chromatography (HPLC) method to determine ETU in biological and environmental samples.</p><p style="text-align: justify;"><strong>METHODS:</strong> The samples were pre-treated according to sample types and were analyzed for ETU using a reversed-phase HPLC system (JASCO?) equipped with UV detector set at 230 nm using C18 bonded silica column and a mobile phase of 0.05M ammonium acetate in methanol (95:5).</p><p style="text-align: justify;"><strong>RESULTS:</strong> The method showed a limit of detection of 0.2 ug/L, with a precision of 3.33 to 12.86 %CV and an accuracy of >90% at 1, 10 and 100 ug/L of ETU in all sample types. The calibration curve was linear from 1 to 200 ug/L for blood, air and water samples and 1 to 2000 ug/L for urine.</p><p style="text-align: justify;"><strong>CONCLUSION:</strong> This method showed an acceptable accuracy, precision, sensitivity and specificity and was used subsequently to determine ETU levels in blood, urine, air, soil and water samples among banana plantation workers.</p>