Rapid Identification of Cryptococcus neoformans and Cryptococcus gattii by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Abstract

Compared to DNA sequence analysis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) correctly identified 100% of Cryptococcus species, distinguishing the notable pathogens Cryptococcus neoformans and C. gattii. Identification was greatly enhanced by supplementing a commercial spectral library with additional entries to account for subspecies variability.Cryptococcosis is typically a severe infection of the central nervous system characterized by meningoencephalitis and other neurological complications, primarily in patients with AIDS or other forms of immune compromise (26). Cryptococcus isolates must be distinguished and characterized at the species or subspecies level due to differences in epidemiology, virulence, and antifungal drug susceptibility. Cryptococcus gattii has a greater propensity to infect immunocompetent people (26), with some strains being more virulent (3,16,27) or less susceptible to fluconazole and other triazoles (6,20,42). Cryptococcus neoformans consists of two varieties, var. grubii and var. neoformans. While C. neoformans var. grubii causes the majority of clinical infections worldwide, C. neoformans var. neoformans infections are more prevalent in India (18) and certain areas of Europe (11,24) and are strongly correlated with infections in the elderly, the presence of skin lesions, and corticosteroid use (12). Infections due to other Cryptococcus species are rare, but the incidence is increasing, requiring greater vigilance by clinical laboratories (21).Currently, identification of Cryptococcus to the genus or subgenus level from clinical specimens relies upon the microscopic examination of yeast cells in conjunction with biochemical tests, differential media, and/or DNA sequence analysis (9,22,23,31,33,(44)(45)(46). These tests may require multiday incubation or labor-intensive and costly protocols that may delay diagnosis.Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been used to successfully identify various species of bacteria and fungi (17, 28, 34) By identifying species based on characteristic protein spectra extracted from whole cells, MALDI-TOF MS produces a highly discriminatory identification of a pure culture in 5 to 20 min at minimal cost, depending on the sample preparation procedure utilized. In this study, we tested the ability of wholecell MALDI-TOF MS to identify clinical isolates of Cryptococcus spp. and to further differentiate them at the species or subspecies level.We analyzed a total of 160 yeast isolates, including 137 Cryptococcus strains and 23 non-Cryptococcus yeast strains. Twenty-five were type and reference strains (Table 1) As a reference "gold standard" for this evaluation, all isolates were identified by DNA sequence analysis of the rRNA internal transcribed spacer (ITS) region (7), which differentiated all yeast species except for C. neoformans and C. gattii. Clinical isolates demonstrated Ͼ99% similarity to type strains, with a between-species diverge...

Comparison between the Biflex III-Biotyper and the Axima-SARAMIS Systems for Yeast Identification by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

“…ficulties in identification were reported by other studies using the manufacturers' standard databases (9,10,18,19). Good performances were obtained for Cryptococcus by enriching the databases (20,21). Of the 6 Trichosporon isolates tested, 3 isolates of T. mucoides were correctly identified by both systems, but T. asahii, T. inkin, and T. cutaneum posed problems of nonidentification or misidentification as other species of the genus Trichosporon.…”

Identification of Medically Relevant Species of Arthroconidial Yeasts by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

“…(20,29,(35)(36)(37). MALDI-TOF MS has been applied successfully to identify Cryptococcus species, including varieties and hybrids, Candida albicans and non-albicans Candida species, and rare pathogenic yeasts (29,30,(38)(39)(40)(41)(42)(43)(44). Non-Candida species isolates, such as Geotrichum, Galactomyces, Saprochaete, Magnusiomyces, and Trichosporon spp., are underrepresented in the reference library and thus MALDI-TOF MS identification runs were less successful, resulting in low identification rates or failure of reliable analysis (36,45).…”

Comparison of the Microflex LT and Vitek MS Systems for Routine Identification of Bacteria by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

“…The ability of MALDI-TOF MS to identify yeasts and fungi has been demonstrated many times (1,14,23,26,29). In a large comparison, Bader et al found no significant difference between the Saramis and Biotyper databases when identifying yeasts (2).…”