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OBJECTIVE To investigate the resistant mechanisms of Candida albicans to azoles at molecular level.METHODS NCCLS M-27 protocols were used to test the in vitro susceptibilities of 102 C.albicans strains isolated from the patients with recurrent vulvovaginal candidiasis(RVVC) against fluconazole(FLC) and itraconazole(ITC) to screen the FLC-and ITC-resistant C.albicans isolates;six pairs of primers,A1-A2,B1-B2,C1-C2,D1-D2,E1-E2 and F1-F2 were respectively to amplify gene CYP51 of 4 strains with FLC-and ITC-resistance.The PCR products were sequenced and analyzed to identify the mutation sites by compared with the sequence of gene CYP51 of referenced C.albicans strain in NCBI site of Internet.RESULTS The analysis of full length sequence of CYP51 from 4 FLC-and ITC-resistant strains showed that from total 32 mutation sites there were 4 significant site mutations,where the mutation of GAT to GAC at 116 caused the substitution of D by E(E266D in two strains);GCC to GGT at 117 caused the substitution of A by G(A117G in 1 strain);GAA to GAC at 266 caused the substitution of E by D(E266D in 2 strains);and GTT to ATT at 488 caused the substitution of I by V(V488I in 1 strain).The site mutations of 266 and 488 were tested in 1 strain of 4 strains.CONCLUSIONS The CYP51 total gene of 4 strains has been checked out.Of FLC and ITC-resistant C.albicans alignment in this time,find out 4 significant bp mutations.Causing its amino acide change,among them,A117G has not be interrelated report still now.The details of mechanism need to be further studied.
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Objective To study the morphology, physiology, biochemistry and molecular biologic characteristics of Ochroconis gallopava which was firstly isolated from a pulmonary phaeohyphomycosis in China and compare it with a standard strain to assist in the clinical diagnosis and treatment. Methods The clinical isolate and standard strain were inoculated on SDA, PDA, CMA, CDA, BHIA and OA, respectively to observe the colony morphology and growth rate, and slide cultures were prepared and were examined under light microscope and electron microscope. Thermo-tolerance study, cycloheximide resistance test, API 20C, antifungal susceptibility test and PCR for the internal transcribed spacer of rDNA were also performed. Results Ochroconis gallopava grew rapidly on most media. The colonies appeared to be brown in color with smooth surface. There were aerial mycelia on SDA and PDA. Microscopic observations revealed pale brown and septate hyphae with club-shaped conidia. The growth of O. gallopava was optimal at 35 ℃ and was inhibited by cycloheximide. The two strains assimilated the same 14 glycogens, but 1 different, in API 20C AUX. MICs of fluconazole, itraconazole, amphotericin B and terbinafine were 32 ~ 64 ?g/mL, 1 ?g/mL, 2 ?g/mL and 0.25 ~ 0.5 ?g/mL, respectively. PCR results showed that the ITS segment was 743 bp with 3 bp difference in both strains. The length of 18S region was 506 bp, their sequences were the same entirely. Conclusions The clinical isolate is proved as a domestic strain of Ochroconis gallopava at the level of morphology, physiology, biochemistry and molecular biologic characteristics.
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Objective To investigate the cause of an outbreak of tinea capitis in a primary school.Methods All of the exposed children were surveyed by physical examinations and mycological examina-tions,including KOH preparation and fungal culture.The environment was also investigated in the school.With molecular typing of M.canis isolated from the patients and the environment,random primer amplifica-tion polymorphic DNA(RAPD)method,specific amplification of subrepeat elements in ribosomal DNA non-transcribed spacer(NTS),and DNA sequencing in intertranscribed spacer(ITS)of rDNA were performed.Results The total number of exposed children was seventy-one,among which fourty-two were suffered from tinea capitis.The ratio between boys and girls was13∶1.The age of the patients ranged from3.5-10years old.Four patients bred cats or dogs as pets.Fourty-one patients had noninflammatory type of tinea capitis and one had inflammatory type.Under microscopy all affected hairs showed ectothrix infection.M.canis was isolated from the patients,as well as from carpets in gymnasium and pillowcases in the school.The patterns of all strains of M.canis were identical in both RAPD and PCR amplification of rDNA NTS region.The same DNA sequence was found in the ITS region of isolates from the patients and the environment.Conclusion The outbreak of tinea capitis was caused by M.canis.M.canis strains isolated from the patients and from the environment were probably the same origin.
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Objectives To evaluate the application of M38-P method recommend by NCCLS to dematiaceous fungi, and detect the susceptibility of itraconazole, fluconazole, miconazole, econazole, flucytosine, clotrimazole, nystatin, amphotericin B and terbinafine against dematiaceous fungi. Methods Using M38-P method forty-five strains of dematiaceous fungi were studied, which included 2 strains of Bipolaris spp., 1 strain of Drechslera spp., 1 strain of Curvularia spp.,14 strains of C. carrionii, 8 strains of F. pedrosoi, 3 strains of F. compacta and 16 strains of P. verrucosa. The media containing the fungi in the concentrations of (0.4 ~ 5) ? 106 CFU/mL were incubated at 35℃ for 5 ~ 7 days. Results The tested dematiaceous fungi were highly susceptible to itraconazole and terbinafine. Some of them were resistant to itraconzole, fluconazole, flucytosine and amphotericin B. Conclusions The data suggest that M38-P standards could be applied as the susceptibility testing of dematiaceous fungi, and it is necessary to monitor the resistant strains in antifungal treatment.