Rapid Identification of Yeasts in Positive Blood Cultures by a Multiplex PCR Method

Abstract

Yeasts are emerging as important etiological agents of nosocomial bloodstream infections. A multiplex PCR method was developed to rapidly identify clinically important yeasts that cause fungemia. The method amplified the internal transcribed spacer 1 (ITS1) region between the 18S and 5.8S rRNA genes and a specific DNA fragment within the ITS2 region of Candida albicans. With this method, C. albicans produced two amplicons, whereas other species produced only one. Through sequence analysis, the precise lengths of the PCR products were found to be as follows: C. glabrata (482 or 483 bp), C. guilliermondii (248 bp), C. parapsilosis (229 bp), C. albicans (218 or 219 and 110 bp), C. tropicalis (218 bp), Cryptococcus neoformans (201 bp), and C. krusei (182 bp). The PCR products could be effectively separated by disk polyacrylamide gel electrophoresis. The method was used to test 249 positive blood cultures (255 isolates), from which the following species (strain number) were isolated: C. albicans (128), C. tropicalis (51), C. glabrata (28), C. parapsilosis (23), C. neoformans (9), C. krusei (5), C. guilliermondii (3), and other, minor species (8). The test sensitivity of the method was 96.9% (247 of 255 isolates). The eight minor species were either misidentified (one strain) or not identified (seven strains). From the time at which a positive bottle was found, the multiplex PCR could be completed within 8 h; the present method is simpler than any previously reported molecular method for the identification of blood yeasts.

Identification of Candida spp. isolated from vaginal swab by phenotypic methods and multiplex PCR in Duhok, Iraq

“…However, methods such as single and direct PCR or Multiplex PCR have not been used extensively despite being highly sensitive and specific with a shorter turn-around time. [11][12][13][14] Multiplex PCR is a rapid diagnostic assay which combines many specific species primers in one PCR tube. Hence, it could be used to identify more than one species in a specimen simultaneously.…”

Systemic infection by Spencermartinsiella sp. in a Nile crocodile (Crocodylus niloticus)

“…Culture and isolation tests are time-consuming, but most importantly, fungal isolation is not doable when only formalin-fixed samples are available, as in this case. Indeed, ribosomal DNA amplification and sequencing has been successfully applied in this case to confirm the etiologic diagnosis, as previously reported (MILDE et al, 2000;CHANG et al, 2001;KHLIF et al, 2007).…”

In Vitro Antidrug Susceptibility Testing of Candida Species Isolated from Aseptic Body Fluids

“…and Ct2 (5'-TGGCCACTAGCAAAATAAGCGT-3') (11)(12)(13). PCR reactions were carried out in a GeneAmp PCR system 2400 (PerkinElmer, US) and performed in 0.2 mL microcentrifuge tubes with a final reaction mixture containing 2 µL of the prepared template, 12.5 µL of 2 × Taq PCR master mix (Tiangen Biotech (Beijing) Co., Ltd., China), 0.5 µL of each primer (10 mM)(BGI, China), and 7.5 µL ddH 2 O. Amplification conditions were as follows: 5 minutes initial denaturation at 96°C, repeated for 40 cycles of 30 seconds denaturation at 94°C, 30 seconds of primer annealing at 53°C, and elongation at 72°C for 1 minute.…”