The rise of fungal infections correlates with the widespread use of broad-spectrum antibacterial agents, prolonged hospitalization of critically ill patients

This study was conducted to compare of the yeasts identification results obtained with two new systems using the MALDI-TOF MS technique with the ones obtained using the routine identification methods of Candida spp. in clinical microbiology laboratories. All 124 Candida spp. isolates were recovered from the routine examination of clinical specimens in microbiological laboratories and collected in the Centre of Quality Control in Microbiology in Warsaw (Poland). Our findings confirm the high agreement (98%) of fungal identification using the standard, biochemistry laboratory methods and mass spectrometry technique. K e y w o r d s: Candida spp. – identification, chromogenic medium and biochemical methods, MALDI-TOF MS Stefaniuk E. et al. 1 112 Centre of Quality Control in Microbiology (CQCM) located in Warsaw (Poland) for further evaluation and deep frozen. They were deposited in the CQCM collection. The strains used during the study were transferred from storage at –70°C onto the non-selective Sabouraud agar medium (bioMérieux, France) and incubated for 48 h at 30°C. The biochemical identification of the strains was carried out at the same time using the API ID32C as based method of yeasts identification in routine laboratory and YST card system, according to the manufacturer’s instructions. In parallel with the biochemical identification the study strains were inoculated on CHROMagar Candida Medium which shows different colour colonies for C. albicans (green), C. tropicalis (dark blue, with a pink halo), C. krusei (pink and downy appearance). All tested Candida strains were simultaneously identified to species level by MALDI-TOF MS Biotyper and VITEK MS in line with the manufacturer’s instructions. The results of the pattern-matching process were expressed as log score values ranging from 0 to 3 using the MALDI-TOF MS Biotyper application and software. The score values ranging from 2.300 to 3.000 meant “highly probable species identification”; 2.000–2.299 – “secure genus identification, probable species identification”; 1.800–1.999 – “identification to the genus level”, and a score of ≤ 1.800 was interpreted as ‘‘no reliable identification’’. The resulting slides were then processed in the VITEK MS device with MYLA software offering the automated analysis of the obtained mass spectra against the built-in database. A single identification is displayed (green), with a confidence value (% probability) from 60.0 to 99.9 (good confidence level), when one significant yeast or yeast group is retained. “Low-discrimination” identifications are displayed (red) when two or four significant yeasts or yeast groups are retained. When no match is found, the yeast is considered unidentified (orange). The results of analysis are shown in Table I. The growth characteristics of 124 Candida spp. strains on CHROMagar Candida Medium enabled the unambiguous identification of isolates belonging to three species: green colonies as C. albicans (n = 68; 55%), steel-blue colonies – C. tropicalis (n = 21; 17%) and pink colonies – C. krusei (n = 35; 28%). Green colony colour suggested C. albicans strains, but different intensity of colour was difficult to evaluate objectively. Thirty five of strains (28.2%), which grew on chromogenic medium, formed pink/violet to pink colour colonies: pink colonies (n = 1), pink/violet-pink colonies (n = 24), white large glossy pink colonies (n = 2), matt pink colonies (n = 3), pink colonies with white “halo” (n = 3) or ivory to lavender colonies (n = 2). According to manufacturer instructions, pink colony colour suggested C. krusei strains. Biochemical analysis with the API ID 32C system identified 123 (99,2%) Candida isolates. The isolates, which grew on chromogenic medium forming pink/ violet-pink colonies (n = 24) and white large glossy pink colonies (n = 2), were identified by API ID 32C as C. glabrata (n = 26). All isolates growing as matt pale pink colonies (n = 3) were identified as C. krusei. Only four from five isolates which grew forming pink with a white “halo” or ivory colonies were identified as C. parapsilosis. The green colour colonies were identified by API ID 32C as C. albicans (n = 67) and C. dubliniensis (n = 1). The identification levels of VITEK 2 YST were: excellent – 58.1%, very good (34.7%), good (4.8%) and acceptable (only 2.4%). The results obtained with VITEK 2 YST card were correct with API identification for the majority of common clinical yeasts, C. albicans Study strains (n = 124) Dark green, green or light green colonies C. albicans (n = 67) C. albicans (n = 66) C. albicans (n = 66) C. albicans (n = 66) (n = 68) C. dubliniensis (n = 1) C. dubliniensis (n = 2) C. dubliniensis (n = 2) C. dubliniensis (n = 2) Dark blue colonies (n = 21) C. tropicalis (n = 21) C. tropicalis (n = 21) C. tropicalis (n = 21) C. tropicalis (n = 21) Pink/violet-pink colonies (n = 24) White large glossy pink colonies C. glabrata (n = 26) C. glabrata (n = 26) C. glabrata (n = 26) C. glabrata (n = 26)