Biomedical subjects
Publications and source records attributed to R Guinet.
A rapid method has been standardized for extraction and SDS-PAGE analyses of mycobacterial cellular protein. Complex patterns were reproducibly obtained of the 31 reference strains or clinical isolates studied, belonging to 11 species. These patterns were species specific; thus this method could be an easy tool for mycobacterial strain characterization.
The standardized micromethod Mycototal is based on ready-to-use microtitration plates and the same culture medium for sensitivity testing of all antifungal agents. Since the method was very reproducible for yeasts it was applied for the determination of minimal inhibitory concentrations of Aspergillus to itraconazole and ketoconazole. Again the reproducibility for itraconazole was excellent since for 19 strains tested two times 18 showed no more than two dilutions different results and this was also observed for 6 strains over 7 tested 4 or 5 times. Over 31 strains tested belonging to different Aspergillus species, itraconazole was much more active than ketoconazole since for 2.5 mg/l all the strains were sensitive to itraconazole and only one to ketoconazole. Moreover no strains were sensitive to ketoconazole for 0.62 mg/l versus 9 strains sensitive to itraconazole. These results showed on one hand the reproducibility of the method and on the other hand the potency of itraconazole against Aspergillus according to the literature and let us hope to a better chemotherapy of aspergillosis.
The minimal inhibitory concentrations of 200 yeast strains (48 reference strains and 152 recently isolated from pathological products) were evaluated with a new standardized micromethod using a liquid medium comparatively for ketoconazole, itraconazole and fluconazole. The ready-to-use microtitration plates contained the antifungal agents in concentrations ranging from 100 to 0.10 mg/l. The cumulative MIC curves clearly showed the superiority of ketoconazole, fluconazole being the less active product. These results were confirmed after categorization in sensitive (S: MIC less than 0.78 mg/l), intermediate (I: 0.78 less than MIC less than 6.25) or resistant (R: MIC greater than 6.25). These results are discussed particularly for fluconazole showing the largest half-life and for which higher plasmatic levels could be achieved. Important variations were observed depending on the species and as example for Candida tropicalis the sensitivities were ketoconazole 100%, itraconazole 55% and fluconazole 0%. The less sensitive species to the 3 azoles were Saccharomyces cerevisiae and Torulopsis glabrata. This new micromethod being very easy to use and allowing the determination of fungicidal activities should be introduced in routine.
A simple electrophoretic method for yeast identification was evaluated. Whole cells were extracted by SDS and the protein profiles obtained in SDS-PAGE after Coomassie blue staining were compared for 52 strains from 9 species of yeast or yeast-like fungi commonly isolated from man (Candida albicans, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, C. pseudotropicalis, C. tropicalis, Geotrichum candidum, Saccharomyces cerevisiae). The corresponding patterns showed 30 to 45 polypeptides in the range 95-20 kDa and were clearly different for the 9 species. No differences could be detected between strains from the same species. The characteristic patterns were obtained within 24 h allowing rapid identification of the most commonly encountered clinical yeast isolates.
Cytotoxin assay and culture for Clostridium difficile were performed on 303 diarrhoeic stools from 261 hospitalized patients. Specimens from 42 patients were positive by at least one of the methods, and 40 of them had an antibiotic-associated diarrhoea. The cytotoxin assay was positive in 5 of 7 patients with pseudomembranous colitis. Thirteen had an appropriate response to specific therapy and the remainder have resolved of diarrhoea without C. difficile directed chemotherapy. These findings show the lack of reliability of the cytotoxin assay for the diagnosis of C. difficile antibiotic-associated diarrhoea. The 6 strains isolated from patients with pseudomembranous colitis were examined for enterotoxin by the rabbit ileal loop test: 4 produced both toxins, 2 only enterotoxin. Both toxins could therefore not be essential for the clinical expression of the disease.
Explore the source record for details and available documents.
The new micromethod for yeast susceptibility testing, MYCOTOTAL, was evaluated with 10 reference strains in seven laboratories. Ready-to-use microtitration plates and the same synthetic medium were used with two dilutions of imidazoles, flucytosine, and amphotericin B, permitting the categorization of each strain as susceptible, intermediate, or resistant. The results were compared with the MIC for each reference strain, and the repeatability and reproducibility were evaluated. The yeasts tested presenting different patterns of susceptibilities in reference MICs included six strains of Candida albicans, two strains of Candida tropicalis, one strain of Candida parapsilosis, and one strain of Torulopsis glabrata. For 4,200 antifungal agent-yeast results, the repeatability was 99.3% and the reproducibility was 96.3%. The correlation between the reference MICs and the category results was 91.5% for seven laboratories (and 92.7% for six laboratories excluding the laboratory which did not follow exactly the same protocol). We observed only 7.9% minor discrepancies, 0.5% (0.29% for six laboratories) major discrepancies, and 0.1% uninterpretable results. The percentages of concording results were similar for each strain and each antifungal agent tested. The overall results indicated that MYCOTOTAL was a reliable and reproducible method, well correlated with reference MICs. This ready-to-use micromethod with the same medium for all antifungal agents would be an important step in the necessary standardization of yeast susceptibility testing.
A simple, rapid SDS-Page method for protein profiles analysis of Actinomyces species was developed. Eighteen strains (12 reference strains and 6 fresh isolates) representing four species were used in this study. Eight detergents were tested for protein extraction. Cell extracts were obtained by shaking the bacteria suspended in a solution containing a detergent and glass beads. After electrophoresis, according to the Laemmli's technique, two protein stain procedure were tested (Coomassie blue and silver stain). Best results were obtained with 0.5% Triton X-100 for 4 h and the silver stain procedure of Oakley. Protein profiles analysis showed that the method is reproducible and distinguishes not only species but sometimes also subspecies.
Twenty three Clostridium difficile strains isolated from diarrheic faecal samples of 22 patients in a neurosurgical department of the Pellegrin Hospital in Bordeaux between January 1984 and May 1987 (15 isolated in 1984, 2 in 1985, 3 in 1986 and 3 in 1987) and 15 strains isolated from 15 patients in other departments of the same hospital in 1984 have been compared using their antimicrobial susceptibility, cytotoxin production and electrophoretic pattern. Twelve of the 15 strains isolated in the neurosurgical department and 4 of the 15 strains isolated in other departments in 1984 were phenotypically identical. These findings suggest that the origin of C. difficile associated-diarrheas can be nosocomial.
A non-competitive avidin-biotin immuno-enzymatic assay (NABA) for lysozyme is described. The assay was found to be more sensitive than a competitive assay with biotinylated lysozyme. The lower detection limit of NABA was 0.1 ng lysozyme/ml compared to 1 ng/ml for the competitive assay. The intra-assay and inter-assay coefficients of variation for NABA were 5.9 and 9.1%, respectively. The total time for NABA can be decreased (to less than 1 h) without influencing the detection limit or the analytical range. Serum lysozyme levels measured by NABA and the enzymatic assay in 32 samples showed a correlation coefficient of r = 0.97.
Explore the source record for details and available documents.
The comparative susceptibility of 1 850 yeast strains belonging to 8 species was determined. The standardized micromethods used allows determinations of minimal inhibitory concentrations (MICs) or categorized sensitivities for two different concentrations (AB). Overall results showed that amphotericin B (AMB) is the most active agent, followed by the various imidazoles. 5-fluorocytosine (5FC) was the least effective drug, with 68% susceptible strains. However, results varied widely across species and drugs. For instance, among Candida albicans and Torulopsis glabrata strains, none were resistant to AMB and only 6% were resistant to 5-FC; in contrast, Candida albicans was highly susceptible to imidazoles (0.8 to 2.5% resistant strains) whereas Torulopsis glabrata showed much higher resistance rates (18% of strains for tioconazole and 70% for ketoconazole). Variations in susceptibility were also recorded across imidazoles: clotrimazole, tioconazole and ketoconazole were much more effective against Candida tropicalis and Candida parapsilosis than miconazole and econazole, whereas almost no strains were resistant to AMB and more than 50% of strains were resistant to 5-FC. Results obtained by AB (967 strains) and MIC (455 strains) were consistent for the 1 422 Candida albicans strains. Our results show that standardized micromethods should be used to determine the susceptibility of yeasts to antifungal agents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Serological methods can be applied to the diagnosis of systemic candidiasis, but cell wall mannans can detect antibodies occurring in several cases unrelated to candidiasis. The present study proposes a procedure for the preparation of specific antigens obtained from an experimental infectious model. The specific immunoglobulins were obtained from rabbits with chronic systemic candidiasis. After precipitation by ammonium sulfate and purification by ion-exchange chromatography on DEAE-Sephadex A-50, these immunoglobulins were fixed on cyanogen bromide-activated Sepharose 4B and used for the preparation of an affinity chromatography column. This column allowed isolation of specifically bound fractions of Candida albicans soluble antigens. When analyzed by quantitative immunoelectrophoresis with a polyvalent hyperimmune antiserum, these fractions showed eight precipitation lines, whereas the complete soluble antigen exhibited 48 lines. Possible applications of these antigens to specific serodiagnosis of systemic candidiasis are being evaluated.
Minimum inhibitory concentrations (MIC) of amphotericin B, 5-fluorocytosine, miconazole, tioconazole, econazole and ketoconazole were determined for 310 Aspergillus strains belonging to four different species isolated from clinical specimens. Econazole exhibited the best in vitro activity with MIC less than or equal to 3.12 micrograms/ml for 96% of strains and less than or equal to 1.56 micrograms/ml for 68%. For amphotericin B, 69% of strains were less than or equal to 1.56 micrograms/ml and, for 5-fluorocytosine, 50% were less than or equal to 25 micrograms/ml. Miconazole and ticonazole exhibited comparable in vitro activities with MIC less than or equal to 6.25 micrograms/ml for 66% and 58% of strains respectively. Ketoconazole was the less active agent in vitro, with MIC less than or equal to 6.25 micrograms/ml for only 27% of strains. Moreover, this study demonstrated significant differences in susceptibility from species to species. A. fumigatus was susceptible to amphotericin B (75%) and 47% of strains were susceptible to 5-fluorocytosine. A. niger showed high susceptibility to amphotericin B (82%) and 5-fluorocytosine (82%). A. flavus exhibited the highest resistance to 5-fluorocytosine (14%), the highest susceptibility to ketoconazole (72%), and average susceptibility to amphotericin B (41%). A. nidulans was rarely susceptible to amphotericin B (21%) or 5-fluorocytosine (29%) and 46% of strains were susceptible to miconazole and ketoconazole. These results provide useful guidelines for the choice of antifungal agents in the difficult treatment of aspergillosis.
We report a new case of primary pulmonary mucormycosis. The radiographic evidence and anatomopathological facts demonstrate an intra-cavitary mycetoma of Fowler associated with a chronic pneumonia in the left basal segment. The patient was not immunosuppressed but had undergone a left upper lobectomy 25 years before. This appears to be the first case in which a serological study has confirmed the diagnosis.
Five yeast strains, causally associated with septicemia and death in a patient after peritonitis, were identified as Candida lusitaniae van Uden et do Carmo-Sousa by standard methods. The organism was initially susceptible to 5-fluorocytosine but strongly resistant to amphotericin B, requiring 50 micrograms/ml for complete inhibition at 48 h.