Molecular Techniques for Detection of Candida Species

Candida species are a major cause of invasive infections, in both critically ill and immunocompromised patients. Thus, rapid identification of these pathogens may facilitate specific therapy and patient management. The development of rapid and specific diagnostic methods remains a challenge. Different methods for the detection of Candida species including simplex and multiplex PCRs, PCR/RLB (Reverse Line Blot) hybridization technique, and finally a novel pan-fungal (yeast) specific primers for the detection of medically important Candida species and other fungal yeasts have been developed in this study. ATCC strains of Candida species (n=13), Aspergillus species (n=4), Pichia species (n=1), and two clinical isolates of Cryptococcus species were used in this study to confirm the specificity of the designed primers. For evaluation of the developed simplex and multiplex PCRs and PCR/RLB, 23 clinical strains of the following Candida species; C. albicans (n=5), C. glabrata (n=5), C. parapsilosis (n=5),and C. tropicalis (n=8) obtained from University Malaya Medical Centre (UMMC)between 2003 and 2004 were used (Table 3.2). In addition, 44 blood samples taken from BacT/Alert blood culture bottles were obtained from UMMC during the period from December 2009 to August 2010 were used for further validation. Simplex and multiplex PCR methods were developed for the detection of the four most common Candida species isolated from clinical samples: C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis. The developed methods target the phospholipase B gene (PLB) as a novel target. The alignment software surprisingly showed that this gene contain large sequence variation among Candida species. The nucleotide sequence variability between C. tropicalis and C. glabrata was as high as 75%. Four pairs of species-specific primers were designed for the corresponding Candida species and used in the simplex PCR. New primer sets were designed for multiplexing purpose for C.albicans, C. parapsilosis, and forward primer for C. glabrata. While the reverse primer of C. glabrata and primer site of C. tropicalis used in the simplex PCR were also suitable for the multiplex PCR. On the other hand, novel species-specific primers and probes were designed for the development of specific PCR and PCR/RLB hybridization techniques for the detection of the less commonly isolated Candida species, namely; (C. famata, C. guilliermondii, C. lusitaniae, C. metapsilosis, and C. orthopsilosis). The methods developed targeted the internal transcribed spacer (ITS) region. The validity tests showed that the designed primers and probes achieved highly specific identification of the selected species, with no cross- reaction with the other tested fungal species. The methods were able to detect