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.

FIG. 1

Multiplex PCR using primers ITS1, ITS2, CA3, and CA4. Lane 1, 50-bp DNA ladder. Lanes 2 to 4, C. krusei CCRC 20514, C. neoformans CCRC 20528, and C. albicans CCRC 20512, respectively. Lane 5, species markers formulated from amplicons of the seven major yeast species; the bands from top to bottom were PCR products of C. glabrata, C. guilliermondii, C. parapsilosis, C. tropicalis and C. albicans, C. neoformans, C. krusei, and C. albicans, respectively. Lanes 6 to 9, C. tropicalis CCRC 20520, C. parapsilosis CCRC 20515, C. guilliermondii CCRC 21500, and C. glabrata CCRC 20586, respectively.

FIG. 2

Identification of yeasts present in mixed cultures by the multiplex PCR. Lane 1, 50-bp DNA ladder. Lane 2, C. albicans and C. glabrata. Lane 3, C. albicans and C. parapsilosis. Lane 4, C. tropicalis and C. glabrata. Lane 5, species markers. Lane 6, C. albicans and E. coli. Lane 7, C. tropicalis and E. cloacae. Lane 8, sample of human blood. Lane 9, negative control.

FIG. 3

Identification of minor yeast species present in positive blood cultures by the multiplex PCR. Lane 1, 50-bp DNA ladder. Lane 2, C. pelliculosa. Lane 3, C. famata. Lane 4, species markers. Lane 5, R. rubra. Lane 6, T. beigelii. Lane 7, species markers. Lane 8, C. lusitaniae. Lane 9, Candida sp. R. rubra (lane 5) was misidentified as C. parapsilosis.