Rama Ramani

Flow Cytometry Antifungal Susceptibility Testing of Pathogenic Yeasts other than Candida albicans and Comparison with the NCCLS Broth Microdilution Test

ABSTRACT Candida species other than Candida albicansfrequently cause nosocomial infections in immunocompromised patients. Some of these pathogens have either variable susceptibility patterns or intrinsic resistance against common azoles. The availability of a rapid and reproducible susceptibility-testing method is likely to help in the selection of an appropriate regimen for therapy. A flow cytometry (FC) method was used in the present study for susceptibility testing ofCandida glabrata, Candida guilliermondii,Candida krusei, Candida lusitaniae,Candida parapsilosis, Candida tropicalis, andCryptococcus neoformans based on accumulation of the DNA binding dye propidium iodide (PI). The results were compared with MIC results obtained for amphotericin B and fluconazole using the NCCLS broth microdilution method (M27-A). For FC, the yeast inoculum was prepared spectrophotometrically, the drugs were diluted in either RPMI 1640 or yeast nitrogen base containing 1% dextrose, and yeast samples and drug dilutions were incubated with amphotericin B and fluconazole, respectively, for 4 to 6 h. Sodium deoxycholate and PI were added at the end of incubation, and fluorescence was measured with a FACScan flow cytometer (Becton Dickinson). The lowest drug concentration that showed a 50% increase in mean channel fluorescence compared to that of the growth control was designated the MIC. All tests were repeated once. The MICs obtained by FC for all yeast isolates except C. lusitaniae were in very good agreement (within 1 dilution) of the results of the NCCLS broth microdilution method. Paired ttest values were not statistically significant (P = 0.377 for amphotericin B; P = 0.383 for fluconazole). Exceptionally, C. lusitaniae isolates showed higher MICs (2 dilutions or more) than in the corresponding NCCLS broth microdilution method for amphotericin B. Overall, FC antifungal susceptibility testing provided rapid, reproducible results that were statistically comparable to those obtained with the NCCLS method.

MOLDS IN ONYCHOMYCOSIS

Background. Onychomycosis is a major cause of nail dystrophy. The causative organisms in onychomycosis are dermatophytes, Candida and molds. A variety of molds have been isolated from nails.

Collaborative Study of the NCCLS and Flow Cytometry Methods for Antifungal Susceptibility Testing of Candida albicans

ABSTRACT One hundred clinical isolates of Candida albicans were tested for amphotericin B and fluconazole susceptibilities by the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution test at center 1 (C1). The same isolates were tested blinded at center 2 (C2) by NCCLS and flow cytometry (FC) methods. The agreement between NCCLS and FC methods ranged from 96 to 99%.

Flow Cytometry Antifungal Susceptibility Testing of Aspergillus fumigatus and Comparison of Mode of Action of Voriconazole vis-à-vis Amphotericin B and Itraconazole

ABSTRACT Aspergillus fumigatus isolates were tested with three antifungals by flow cytometry (FC) and fluorescence-activated cell sorting. FC results after 4 h correlated well with MICs obtained by the NCCLS M38-A method; voriconazole exhibited fungicidal activity, albeit to a lesser extent than amphotericin B, but to a greater extent than itraconazole.

Chemical and Pharmacological Investigations of Limnophila conferta and Limnophila heterophylla

AbstractFifteen components of the essential Oil of L. conferta were identified by GC-MS analysis. The main components were identified to be a-phcllandrene (52.2%) and thymol (38.2%). The antifungal activity at 1:50 dilution in ethylene glycol was found to be of the same order as that of griseofulvin (1001Jg/ 0.1 ml). The oil also possessed good anthelmintic activity. The crude alcoholic extract after the removal of the oil showed significant reduction in the epithelisation period compared to that of controls in excision wound models. Nevadensin isolated from the plant showed good anti-inflammatory activity in an acute inflammation model, and exhibited moderate cytotoxic activity and anti-tubercular activity. L. heterophylla on chemical analysis afforded hymenoxin.

Proficiency Testing Program for Clinical Laboratories Performing Antifungal Susceptibility Testing of Pathogenic Yeast Species

ABSTRACT Antifungal susceptibility testing is expected to facilitate the selection of adequate therapy for fungal infections. The general availability of antifungal susceptibility testing in clinical laboratories is low, even though a number of standard methods are now available. The objective of the present study was to develop and evaluate a proficiency testing program (PTP) for the antifungal susceptibility testing of pathogenic yeasts in laboratories licensed by the New York State Department of Health. A number of quality control standards, and methods for documenting laboratory performance, were developed in consultation with the laboratory directors. The participating laboratories were provided with five American Type Culture Collection strains of pathogenic yeasts for which the minimum inhibitory concentrations (MICs) of amphotericin B and fluconazole were well defined. A majority of laboratories (14 of 17) used broth microdilution, and these were evenly split between the NCCLS M-27A protocol and the Sensititre YeastOne method. The other three laboratories performed susceptibility testing with Etest. Overall, the levels of agreement between MIC reference ranges and the reported MICs were 85 and 74% for amphotericin B and for fluconazole, respectively. All laboratories except one successfully detected fluconazole resistance in a Candida krusei strain. However, amphotericin B resistance in a Candida lusitaniae strain was not detected by any of the participating labs. It is concluded that a suitably designed PTP could adequately monitor the competence of clinical laboratories performing antifungal susceptibility testing.

Collaborative Study of Antibiotic Medium 3 and Flow Cytometry for Identification of Amphotericin B-Resistant Candida Isolates

ABSTRACT Center 1 used the National Committee for Clinical Laboratory Standards M27-A2 method and antibiotic medium 3 (AM3) test to determine amphotericin B resistance in 5 of 30 Candida isolates. These isolates were tested at center 2 by AM3 test and flow cytometry (FC). The agreements (C1-C2) were 90% for AM3 test and FC and 73% for the AM3 tests.

Cokeromyces recurvatus as a human pathogenic fungus: case report and critical review of the published literature.

During the past few years several rarely encountered taxa of zygomycete fungi (Mucorales and Entomophthorales) have been implicated in the etiology of human disease. Prominent among these is Cokeromyces recurvatus Poitras, which is usually isolated from soil and from feces of the lizard and certain rodents. 2 This fungus has been implicated as a potential etiologic agent in seven human cases in which the fungus was isolated from clinical specimens including vaginal secretions, alimentary tract, bladder, urine, pleura and peritoneal fluids. Recently C. recurvatus was isolated from stool samples in two cases of severe diarrhea in which the fungal elements were also noted in the tissue sections, thus providing the first evidence of its pathogenic potential in humans. 9 Because tissue invasion was noted in only two instances, it is still not unequivocally established whether the fungus is a colonizer of the affected tissue or a chance contaminant. We describe a case in which C. recurvatus was isolated on two occasions from an abscess and drainage fluid from a 9-year-old patient with a perforated Meckel’s diverticulum. The patient responded to surgical intervention and antifungal therapy leading to full recovery.

Evaluation of New Medium for Identification of Dermatophytes and Primary Dimorphic Pathogens

ABSTRACT Only 25 of 77 dermatophytic isolates caused dermatophyte identification medium (DIM) to turn purple after incubation at the recommended temperature (37°C); the accuracy of the results was improved at 30°C (71 of 77 isolates yielded positive results). Many dimorphic pathogenic fungi also tested positive at both incubation temperatures. Thus, DIM has limited usefulness for presumptive identification of dermatophytes.