Identification of candida species by restriction enzyme analysis

Yeasts are the most common and important pathogens in immunosupressive patients and it has been reported that Candida spp. are the most commonly isolated ones among them. Identification of Candida species isolated from clinical specimens gives information about the antifungal susceptibility as well as sheds light to the choice of ampirical treatment. Because of this reason, rapid, reliable and accurate identification of the isolates is very important. This study was planned to apply restriction enzyme analysis for more rapid and reliable identification of Candida albicans and non Candida albicans species isolated from serious clinical cases which had been previously identified by conventional methods and to compare the results. In this study one hundred and fortysix Candida strains (40 C. albicans, 27 C. parapsilosis, 26 C. tropicalis, 25 C. glabrata, 11 C. kefyr, 10 C. krusei, and 7 C. guilliermondii) isolated from systemical specimens at Dokuz Eylul University Hospital Mycology Laboratory and C. albicans ATCC 14053, C. parapsilosis ATCC 90018 and C. krusei ATCC 6258 were included. All Candida isolates were kept at -80°C in 50% glycerol and brain heart infusion broth as stock cultures. The strains which were subcultured onto Sabouraud dextrose agar were incubated at 37°C for 24- 48 hours. The strains were identified according to germ tube test, morphology at cornmeal tween 80 agar and CHROMagar Candida (CHROMagar, France) and API 20C AUX (Biomérieux, France) automatized system. DNA extraction of all Candida species was performed with ?Nucleospin Tissue Kit? (Macherey-Nagel, Germany) using EDTA, lyticase and sorbitol buffer according to the manufacturer?s recommendations. For the polymerase chain reaction, primer1 and primer3 which targeted ITS1, 5.8S rDNA and ITS2 regions were used. The amplification products were purified with ?GeneMark PCR Clean-Up Kit? and following this restriction enzyme analysis was performed with MwoI enzyme. Thirty six of the C. albicans strains produced a restriction enzyme analysis pattern with three 141, 184 and 261 bp bands while four of them produced four 184 bp, 141 bp and approximately 165 bp and 95 bp bands. The restriction of C. parapsilosis and C. tropicalis strains with the same enzyme resulted in three 336, 146, 88 bp and 325, 154, 97 bp fragments, respectively. In order to distinguish these two species, further restriction was performed with BslI enzyme and the lengths of restriction enzyme analysis products were 413, 94 and 63 bp for C. parapsilosis, and 326, 187 and 63 bp for C. tropicalis. Restriction of C. glabrata isolates with MwoI enzyme resulted in five bands of 414, 174, 171, 86, 80 bp. Calculated lengths of 5 restriction enzyme analysis products were 370 and 430 for C. kefyr; 289, 134, 83 and 54 bp for C. krusei isolates. Restriction of five C. guilliermondii isolates with MwoI enzyme showed two bands at 355 and 302 bp while the remaining two strains showed a restriction enzyme analysis pattern with two bands at 390 and 300 bp. Additional digestion with BslI enzyme established that these two strains showed totally different restriction patterns from the other five strains. The sensitivity, specifity, positive and negative predictive values of restriction enzyme analysis performed by MwoI enzyme for C. albicans strains were 90%, 100%, 100%, %96,4, respectively. These values were detected as 100%, 78,2%-81,5%, 49,1%-50,9%, 100% respectively for C. parapsilosis and C. tropicalis isolates. When BslI enzyme was used for restriction, all values for these two species increased to 100%. The sensitivity, specifity, positive and negative predictive values of MwoI enzyme restriction for C. glabrata, C. kefyr, C. krusei and C. guilliermondii strains were found to be 100%, 98,6%- 100%, 71,4%- 100%, 100%, respectively. Sequence analysis of both strands of four C. albicans isolates which resulted in different restriction patterns and two C. albicans isolates which produced three bands with the same enzyme and two C. guilliermondii isolates showing a different restriction enzyme analysis pattern were performed. The evaluation of the sequence analysis results was done by Bio-Edit version 7.0 programme. Three C. albicans isolates which produced a different restriction pattern had point mutations (guanine instead of adenine); one isolate had insertion/deletion type mutation and these mutations were the reason of a different pattern. In addition, sequence analysis of two C. guilliermondii isolates showing various fragments with different lengths by two enzymes were 66.6% similar with GenBank C. guilliermondii var guilliermondii and 91.2% similar with C. guilliermondii var membranaefaciens (nucleotide seqeunce). Because of this high nucleotide sequence similarity, these isolates were accepted as C. guilliermondii var membranaefaciens. As a result, we can conclude that restriction enzyme analysis with MwoI and BslI enzymes can be used for the identification of Candida species which need rapid identification or which are problematic with conventional methods that are still accepted as gold standard although some variations can be observed with this method. Key Words: Candida species, identification, restriction enzyme analysis