A novel fluoro colorimetric Loop mediated isothermal amplification (LAMP) assay for detection of Trichomonasvaginalis.

BACKGROUND: Trichomoniasis is a curable, non-viral, sexually transmitted infection. Early diagnosis and treatment of cases can prevent complications and further spread of infection. Rapid diagnostics tests, which can be performed on-site, will help in early diagnosis. The study aims to develop a rapid diagnostic test based on the principle of fluoro-colorimetric LAMP for detecting Trichomonas vaginalis (TV). MATERIALS AND METHODS: T. vaginalis was grown in a modified CPLM medium, and DNA was extracted. Three pairs of LAMP primers targeting the actin gene were designed using the primerexplorer V.5 online tool. The LAMP assay was standardized for temperature and time. To determine the LAMP assay's detection limit, diluted TV DNA and spiked urine samples were used. Conventional PCR was done using previously published primers and compared with LAMP results. The sensitivity and specificity to detect TV from clinical specimens were assessed. RESULTS: The optimum performance of the LAMP assay was determined to be 63 °C for 60 min and terminated at 80 °C for 5 min. The LAMP assay could detect 60 fg/µl of diluted TV DNA and up to 1 parasite/ml of spiked samples. The assay was 1000 times more sensitive than PCR. The LAMP assay was 100% sensitive and specific with crude extract, and reactions were visually discernible. INTERPRETATION & CONCLUSIONS: The LAMP assay developed in the study is easy to perform and interpret, affordable, rapid, and highly sensitive to detect T. vaginalis. It is ideally suited for the point-of-care test, as it fulfills WHO's recommended ASSURED characteristics.

Molecular identification of Malassezia from the skin scrapings of clinically suspected cases of pityriasis versicolor.

BACKGROUND: Pityriasis versicolor (PV) is caused by a lipophilic fungus belonging to the genus Malassezia. Potassium hydroxide (KOH) mount is most frequently used for screening of cases and culture is the gold standard. KOH lacks sensitivity, and culture is time-consuming and technically demanding. AIM, SETTINGS, AND DESIGN: A cross-sectional study was conducted at a tertiary care teaching institution. We aimed to use multiplex-PCR for faster and accurate identification of Malassezia spp directly from skin scrapings of suspected cases of PV. MATERIALS AND METHODS: The study was conducted on suspected cases of PV over a period of 12 months. The clinical and demographic details were recorded. The skin scrapings were subjected to KOH mount and cultured on Sabouraud's dextrose agar with an olive oil overlay. Multiplex-PCR targeting 11 Malassezia spp was performed on DNA extracted from skin scrapings. RESULTS: A total of 69 suspected cases of PV were studied. Most patients belonged to metro cities and worked in hot and humid climates. The mean duration of lesions was 18 months, and most had macular and patchy lesions. The sensitivity of KOH and culture was found to be 82.6% and 91.3%, respectively. M. globosa (n = 60, 87%) and M. restricta (n = 3, 4.3%) were isolated in culture. Multiplex PCR detected 85.5% of M. globosa, 5.8% of M. restricta, and 8.7% of mixed infection with M. globosa and M. restricta. M-PCR detected Malassezia in all the samples. CONCLUSIONS: M-PCR could identify Malassezia species directly from skin specimens, eliminating the need for culture. M-PCR was accurate, dependable, and exhibited a rapid turnaround time.

In Vitro Efficacy of Biocompatible Zinc Ion Chelating Molecules as Metallo-ß-Lactamase Inhibitor among NDM Producing Escherichia coli.

Objective This article assesses the effectiveness of captopril, tetracycline, and ciprofloxacin as metallo-ß-lactamase (MBL) inhibitors against New Delhi metallo-ß-lactamase (NDM)-producing Escherichia coli. Materials and Methods Twenty-four well-characterized carbapenem-resistant E. coli isolates which produced NDM ( n = 21) and Oxa-48-like enzymes ( n = 3) were used to assess the inhibitors. The positive control organism was designed by cloning the NDM gene into pET-24a plasmid and transforming it into expression vector E. coli BL21. All the proposed inhibitors were assessed for their interaction with MBLs using checkerboard minimum inhibitory concentration (MIC) assay with imipenem and meropenem. The fractional inhibitory concentration (FIC) index was calculated to assess the activity of molecules. Results The E. coli BL21 (DE3) pET-24a- bla NDM showed carbapenem resistance upon isopropyl ß-D-1-thiogalactopyranoside induction and had MIC of 32 µg/mL for both imipenem and meropenem. For the test isolates, ∑FIC values of imipenem and meropenem with ethylenediaminetetraacetic acid (EDTA) ranged from 0.039 to 0.266 and 0.023 to 0.156, respectively. At a 256 µg/mL concentration, captopril had ∑FIC index value for imipenem and meropenem as 0.133 to 0.375 and 0.133 to 0.188, respectively. The tetracycline and ciprofloxacin in combination with meropenem/imipenem showed indifferent results. Conclusion Among the three molecules tested, captopril had MBL inhibitory activity, but the concentration required for inhibition was beyond the therapeutic safety levels. Ciprofloxacin and tetracycline had weak or no MBL inhibitory activity. Checkerboard MIC of EDTA with carbapenem antibiotic and control organism with NDM enzyme production helped us create a reference system for comparing and assessing the results of potential MBL inhibitors in future.

Non Diphtheritic Corynebacteria: An Emerging Nosocomial Pathogen in Skin and Soft Tissue Infection.

INTRODUCTION: Non-diphtheritic corynebacteria are normal inhabitants of skin and mucous membrane. When isolated from clinical specimens they are often considered as contaminants. Recent reports suggest their role as emerging nosocomial pathogens. AIM: To speciate non-diphtheritic corynebacteria isolated from wound specimens, to correlate their clinical significance and to determine their invitro antimicrobial susceptibilities to 9 antimicrobial agents. MATERIALS AND METHODS: Twenty five non-diphtheritic corynebacteria from skin and soft tissue infections were selected for study. Isolates were identified by battery of tests and minimum inhibitory concentration (MIC) was detected by Clinical & Laboratory Standards Institute (CLSI) described broth microdilution method. MIC was interpreted according CLSI and British Society for Antimicrobial Chemotherapy (BSAC) guidelines. RESULTS: C. amycolatum was the predominant species (20%) followed by C. striatum (16%). Penicillin was least effective invitro followed by clindamycin and ciprofloxacin. Excellent activities were shown by vancomycin, linezolid and imipenem. Multidrug resistance was found in all the species. CONCLUSION: Non-diphtheritic corynebacteria are potential nosocomial pathogens among acute/chronic complicated skin and soft tissue infection. Vancomycin or linezolid can be used empirically to treat such infections until the invitro susceptibility results are available.