Separation And Identification Of Candida Dubliniensis With PCR-RAPD Method In Genital Candidiasis

Objectives: With germ tubes test, CHROMagar tes, 42℃ temperature test, chlamdospore production tes, the suger assimilation pattern and RAPD(Randomly Amplified Polymorphic DNA )to seperate and identificate Candida dubliniensis. Investigating the incidence of Candida dubliniensis in genital candidiasis in health population.Methods: A collection of 700 clinical isolates were sampled from the patients of genital candidiasis in the dermatology department of Tianjin Medical University General Hospital since 2002 to 2006. One standard isolate of C. albicans (ATCC-11006) and one standard isolate C.dublinniensis (CBS-8501) were presented by Beijing Medical University.All isolates could produce germ tube after 2h of incubation in serum at 37℃.The yeast isolates were cultured on CHROM agar with 48h of incubation. The colonies appeared blue-green color.A small portion of a single colony of each isolate was removed from the CHROMagar plate and incubated at 42℃, The growth condition was observed after 48h.The 108 clinical isolates and the C.dubliniensis standard isolate grew poorly at 42℃ on SDA.According to the instructions of the manufacturer, 100ul of inoculum suspension was transferred to the API 20C AUX system basal medium ampoules. The API trays were inoculated and incubated for 48-72h at 25℃.Cupules showingturbitidy significantly greater than that of negative control cupule were considered positive. The C.dubliniensis (CBS-8501) failed to assimilate XYL and MDG.But almost all C. albicans could utilize XYL and MDG at one time in the API 20C AUX system.In addition, all clinic C. albicans and the standard isolate of C. albicans (ATCC-11006) isolates formed longer filaments and prouduced fewer chlamydospores, with usually only one chlamydospore at the end of the pseudohyphae. But the sandard isolate of C.dublinniensis (CBS-8501) demonetrated abundant chlamydospores frequently and arranged in triplets or in contiguous pairs on short branching pseudohyphae.At last, all C. albicans and the standard isolate of C. albicans (ATCC-11006) isolates and the sandard isolate of C.dubliniensis (CBS-8501) were digested with proteinaseK solution and extracted with 1:1 phenol/chloroform and deposited with isopropyl alcohol. PCR-RAPD were performed with 5ul template from Candida strains extraction. The primer was only M2. PCR prouducts were separated in agarose 2% gel electrophoresis lh at 70V with 0.5ug/ul ethidium bromide. The results were analyzed using Eagle Eye illuminator and compared with the standard strains.Resullts: The unequivocal PCR-RAPD method for discrimination of the two Candida species had not found C.dubliniensis infection among the clinical genital candidiasis in dermatology department of Tianjin Medical University General Hospital. Conclusions:1. 15.4% isolates showed no growth or poor growth in the patients of genital candidiasis.2. The chlamydospores test and the API 20C AUX test were helpful to identificate the C.dubliniensis and the C. albicans.3. Combining the phenotypic methods and RAPD method to identificate the C. dubliniensis was accuracy.4. At last, we had not find out the C. dubliniensis in clinical genital candidiasis.The incidence of C.dubliniensis infection among the clinical genital candidiasis in dermatology department of Tianjin Medical University General Hospital is zero.