Gopa Banerjee

Comparative Study of Biofilm Formation in Pseudomonas aeruginosa Isolates from Patients of Lower Respiratory Tract Infection.

BACKGROUND This study assessed biofilm formations of P.aeruginosa which was isolated from patients with Lower Respiratory Tract Infections (LRTIs). OBJECTIVE This study was conducted to compare different methods of biofilm formations seen in P. aeruginosa which was obtained from LRTI patients. MATERIALS AND METHODS In this cross-sectional study, we investigated a total of 80 P. aeruginosa isolates obtained from LRTI patients by different methods. Tube method (TM), tissue culture plate (TCP) method and modified tissue culture plate (MTCP) method. They were subjected to biofilm detection methods. RESULTS The MTCP method produced a higher accuracy ratio than TCP method. In terms of sensitivity and specificity, the MTCP method was considered to be superior to TM. We observed a higher antibiotic resistance in biofilm producing bacteria than in non-biofilm producers. CONCLUSION In our study, MTCP was found to be more sensitive and specific method for biofilm detection than TCP and TM.

Comparative evaluation of pan-fungal real-time PCR, galactomannan and (1-3)-β-D-glucan assay for invasive fungal infection in paediatric cancer patients

Limited specific data and investigations are available for the diagnosis of Invasive Fungal Infection (IFI) in paediatrics cancer patients. Three non‐invasive tests; Platelia Aspergillus EIA for galactomannan (GM), β‐D‐glucan (BDG) assay and pan‐fungal real‐time PCR for fungal DNA in blood were evaluated. One hundred twenty‐five paediatrics cancer patients at the high risk of IFI were enrolled. Single blood and serum samples were evaluated by all the three methods. Patients were classified into 10 proven, 52 probable and 63 no IFI cases in accordance with EORTC MSG 2008 revised guidelines. The sensitivity, specificity, PPV and NPV of all the three tests in proven, probable and no IFIs cases were analysed singly and in combination. The sensitivity, specificity, PPV and NPV of GM, BDG and pan‐fungal real‐time PCR were: 87%, 61%, 81%, 69.5% for GM, 88%, 59.5%, 81%, 71.4% for BDG and 89%, 69.2%, 85%, 67.5% for PCR (95% CI). Among different combinations, best combination was found to be GM and PCR with sensitivity, specificity, PPV and NPV of 98.2%, 89.3%, 97.1% and 90% respectively. Single samples must be evaluated by combination of tests.

Comparative Evaluation of Disc Diffusion and E-test with Broth Micro-dilution in Susceptibility testing of Amphotericin B, Voriconazole and Caspofungin against Clinical Aspergillus isolates

BACKGROUND Clinical importance of Aspergillus has increased over the past few decades because of rise in immunosuppressive drugs and immune-modulating diseases. Antifungal susceptibility of Aspergillus is rarely performed by clinical laboratories because of lack of easier method. This study has investigated and compared susceptibility pattern of Aspergillus isolates by disc diffusion, E-test and broth micro-dilution for amphotericin B, voriconazole and caspofungin. MATERIALS AND METHODS Disk diffusion (DD) method of antifungal susceptibility (AFS) was evaluated for three different classes of antifungals: amphotericin B (AMB), voriconazole (VCZ) and caspofungin (CAS). Forty four clinical isolates of Aspergillus were selected; these included 34 A.fumigatus, 8 A.flavus and 2 A. terreus. AFS by DD and E-test was done on non-supplemented Mueller Hinton Agar (MHA) and was compared to Clinical Laboratory Standard Institute(CLSI) broth micro-dilution (BMD) method of AFS. RESULTS Disk diffusion method for amphotericin B showed 87.5% agreement while E-test showed 93.8% agreement with broth micro-dilution. The agreement with broth micro-dilution was similar for both disk diffusion and E-test in case of voriconazole (93.8%) and caspofungin (100%). 31.8% and 9.1% Aspergillus isolates were found to have amphotericin B and voriconazole MIC values above epidemiological cut off value (ECV) respectively. All isolates were within ECV for caspofungin. CONCLUSION CLSI method of DD promises to be easier, reproducible and cost effective method of susceptibility testing, but this method must be interpreted with caution in case of amphotericin B susceptibility testing. E-test correlates better than DD with BMD.

In-vitro Inhibition of Biofilm Formation in Candida albicans and Candida tropicalis by Heat Stable Compounds in Culture Filtrate of Aspergillus flavus

BACKGROUND Invasive candidiasis, caused mostly by Candida albicans and C. tropicalis is one of the most common causes of bloodstream infection with a substantial attributable mortality. This disease is associated with formation of structured, multilayered microbial communities known as biofilms over indwelling devices. Treatment is rendered difficult owing to factors like poor drug penetration through biofilms and high cost of the available antifungal drugs. Hence there is imminent need of developing low-cost natural compounds inhibiting Candidal biofilm formation in vitro. Organohalgen compounds derived from crude culture filtrate of Aspergillus flavus have been documented to impair in vitro Candidal survival. AIM We aimed to detect the effect of preheated and unheated crude culture filtrate of Aspergillus flavus on biofilm formation of Candida albicans and C. tropicalis in vitro. Setting and Designs: Ours was a laboratory-based observational study with clinical isolates of the microorganisms selected randomly. MATERIAL AND METHODS In this study, we showed for the first time by microtitre plate method that heat stable compounds which were present in preheated and unheated culture filtrates of Aspergillus flavus inhibited biofilm formation of Candida albicans and C. tropicalis and also lipase activities of these pathogens, and filtrate was non-toxic on human cell line as checked microscopically. STATISTICAL ANALYSIS USED Z-test of significance was used to calculate significant difference between Candidal biofilm formation in normal liquid medium and culture filtrate, respectively. RESULTS AND CONCLUSION Heat stable compounds present in culture filtrate of Aspergillus flavus inhibit biofilm formation of Candida albicans and C. tropicalis and also in-vitro lipase activity of these pathogens and could pave the way for development of low-cost alternatives to treat invasive candidiasis.

Comparative Analysis of Disc Diffusion and E-test with Broth Micro-dilution for Susceptibility Testing of Clinical Candida Isolates Against Amphotericin B, Fluconazole, Voriconazole and Caspofungin.

BACKGROUND Antifungal susceptibility testing remains an area of intense interest because of the increasing number of clinical isolates resistant to antifungal therapy. Clinical and Laboratory Standards Institute has proposed reference broth micro dilution (BMD) method for susceptibility testing. The reference method is time-consuming and poorly suited for the routine clinical laboratory setting. Agar-based susceptibility testing methods, disk diffusion (DD) method and the E-test method can be an easier, reliable and less time consuming alternative for the BMD method. AIM To compare the results of Amphotericin B, fluconazole, voriconazole, and Caspofungin susceptibility testing by DD, and the E-test method with the CLSI reference method for clinical Candida isolates. MATERIALS AND METHODS Broth Microdilution (BMD), E-test and Disk diffusion testing of the various clinical Candida isolates was performed in accordance with CLSI documents. The results obtained were analysed and compared. RESULTS The categorical agreement for Amphotericin B, fluconazole, voriconazole, and Caspofungin susceptibility results by E-test and DD method was 65.2%, 67.4%; 100%, 82.6%; 100%, 100%; 100%, 97.8% respectively. CONCLUSION The agar-based E-test and disk diffusion methods are reliable alternatives to the BMD method for Candida isolates when test susceptible to fluconazole, voriconazole, and Caspofungin, however the susceptibility testing results must be interpreted with caution in case of Amphotericin B.

Inhibition of biofilm formation and lipase in Candida albicans by culture filtrate of Staphylococcus epidermidis in vitro

Background: Candida spp. are fourth most common cause of bloodstream infection in developed countries and emerging agents of fungemia in developing countries, with considerable attributable mortality. Candidemia is associated with the formation of complex, structured microbial communities called biofilms. Biofilm formation makes treatment difficult due to improper drug penetration and factors like high cost and adverse effects of antifungal drugs available. Hence, low-cost alternatives are urgently required to treat device-associated invasive candidiasis. Objectives: To study the effect of culture filtrate of Staphylococcus epidermidis on biofilm formation and lipase expression of Candida albicans in vitro. Materials and Methods: Yeast cells isolated from clinical samples were suspended to a turbidity of 106 in (a) Yeast extract-peptone-dextrose (YPD) broth and (b) culture filtrate, and 100 μl of each were dispensed in separate wells of microtiter plate. After repeated washing and reloading with respective liquid media, readings were taken spectrophotometrically. To check for lipase inhibition, yeasts were incubated overnight in YPD and filtrate and subcultured on media containing Tween-80 and CaCl2. Positive lipase activity was denoted by haziness around colonies. Results: Mean reading of C. albicans in YPD broth was 0.579 while the same when yeasts were suspended in S. epidermidis culture filtrate was 0.281 (P < 0.05 by Z-test of significance). Lipase of C. albicans was inhibited by culture filtrate. Filtrate was found to be nontoxic to human cell line. Conclusions: Culture filtrate of S. epidermidis can hence pave the way for development of new strategies to inhibit biofilm formation in device-associated candidemia.

CTX-M and PER-1 group extended spectrum β-lactamases-producing Pseudomonas aeruginosa from the patients of lower respiratory tract infection.

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