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CryptoType - Public Datasets for MALDI-TOF-MS Based Differentiation of Cryptococcus neoformans/gattii Complexes.

ABSTRACT: Yeasts of the Cryptococcus neoformans/gattii species complexes are human pathogens mostly in immune compromised individuals, and can cause infections from dermal lesions to fungal meningitis. Differences in virulence and antifungal drug susceptibility of species in these complexes indicate the value of full differentiation to species level in diagnostic procedures. MALDI-TOF MS has been reported to sufficiently discriminate these species. Here, we sought to re-evaluate sample pre-processing procedures and create a set of publicly available references for use with the MALDI Biotyper system. Peak content using four different pre-processing protocols was assessed, and database entries for 13 reference strains created. These were evaluated against a collection of 153 clinical isolates, typed by conventional means. The use of decapsulating protocols or mechanical disruption did not sufficiently increase the information content to justify the extra hands-on-time. Using the set of 13 reference entries created with the standard formic acid extraction, we were able to correctly classify 143/153 (93.5%) of our test isolates. The majority of the remaining ten isolates still gave correct top matches; only two isolates did not give reproducible identifications. This indicates that the log score cut-off can be lowered also in this context. Ease to identify cryptococcal isolates to the species level is improved by the workflow evaluated here. The database references are freely available from https://github.com/oliverbader/BioTyper-libraries for incorporation into local diagnostic systems.

SUBMITTER: Bernhard M

PROVIDER: S-EPMC8089388 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe Cryptococcus neoformans/C. gattii species complex comprises two sibling species that are divided into eight major molecular types, C. neoformans VNI to VNIV and C. gattii VGI to VGIV. These genotypes differ in host range, epidemiology, virulence, antifungal susceptibility and geographic distribution. The currently used phenotypic and molecular identification methods for the species/molecular types are time consuming and expensive. As Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) offers an effective alternative for the rapid identification of microorganisms, the objective of this study was to examine its potential for the identification of C. neoformans and C. gattii strains at the intra- and inter-species level.MethodologyProtein extracts obtained via the formic acid extraction method of 164 C. neoformans/C. gattii isolates, including four inter-species hybrids, were studied.ResultsThe obtained mass spectra correctly identified 100% of all studied isolates, grouped each isolate according to the currently recognized species, C. neoformans and C. gattii, and detected potential hybrids. In addition, all isolates were clearly separated according to their major molecular type, generating greater spectral differences among the C. neoformans molecular types than the C. gattii molecular types, most likely reflecting a closer phylogenetic relationship between the latter. The number of colonies used and the incubation length did not affect the results. No spectra were obtained from intact yeast cells. An extended validated spectral library containing spectra of all eight major molecular types was established.ConclusionsMALDI-TOF MS is a rapid identification tool for the correct recognition of the two currently recognized human pathogenic Cryptococcus species and offers a simple method for the separation of the eight major molecular types and the detection of hybrid strains within this species complex in the clinical laboratory. The obtained mass spectra provide further evidence that the major molecular types warrant variety or even species status.

Project description:BackgroundCryptoccocal meningitis continues to present high incidence among AIDS patients. The treatment of choice is the synergistic combination of flucytosine (5-FC) with amphotericin B deoxycholate (AmBd) or its lipid formulations. However, 5-FC is unavailable in many countries and AmB demands hospitalization. The combination of AmB with the fungistatic fluconazole (FLC) or the use of high FLC daily doses alone became the choice. Nonetheless, sterilization of cerebrospinal fluid is delayed with FLC monotherapy, mainly with high fungal burden. These findings suggest the search for new antifungal compounds, such as liriodenine.MethodsLiriodenine antifungal activity was evaluated by three procedures: determining the minimum inhibitory concentration (MIC) on 30 strains of the Cryptococcus neoformans (C. neoformans) complex and 30 of the Cryptococcus gattii (C. gattii) complex, using EUCAST methodology and amphotericin B deoxycholate as control; performing the time-kill methodology in two strains of the C. neoformans complex and one of the C. gattii complex; and injury to cryptococcal cells, evaluated by transmission electron microscopy (TEM). Liriodenine absorption and safety at 0.75 and 1.50 mg.kg-1 doses were evaluated in BALB/c mice.ResultsLiriodenine MICs ranged from 3.9 to 62.5 μg.mL-1 for both species complexes, with no differences between them. Time-kill methodology confirmed its concentration-dependent fungicidal effect, killing all the strains below the limit of detection (33 CFU.mL-1) at the highest liriodenine concentration (32-fold MIC), with predominant activity during the first 48 hours. Liriodenine induced severe Cryptococcus alterations - cytoplasm with intense rarefaction and/or degradation, injury of organelles, and presence of vacuoles. Liriodenine was better absorbed at lower doses, with no histopathological alterations on the digestive tract.ConclusionThe fungicidal activity confirmed by time-kill methodology, the intense Cryptococcus injury observed by TEM, the absorption after gavage administration, and the safety at the tested doses indicate that the liriodenine molecule is a promising drug lead for development of anticryptococcal agents.

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CryptoType - Public Datasets for MALDI-TOF-MS Based Differentiation of Cryptococcus neoformans/gattii Complexes.

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