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Molecular epidemiology and antifungal susceptibility profiles of clinical Cryptococcus neoformans/Cryptococcus gattii species complex.

ABSTRACT: Introduction. Limited data regarding the epidemiology and susceptibility profiles of cryptococcosis are available in the Middle East.Aim. Our study aimed to evaluate the molecular diversity, mating types and antifungal susceptibility pattern of Cryptococcus species (n=14) isolated from 320 suspected patients with cryptococcosis.Methodology. The URA5 gene was subjected to restriction fragment length polymorphism and sequence analysis. In addition, in vitro antifungal susceptibility testing was performed by Clinical and Laboratory Standards Institute (CLSI) M27-A4 and M59 guidelines.Results. Overall, 14 (4.4?%) patients were confirmed as cryptococcosis. Based on molecular type, 85.7 and 14.3?% of the isolates were C. neoformans VN I and VN II, respectively. Phylogenetic analysis of URA5 gene sequences revealed clustering of VN I and VN II isolates into two distinct clades with a substantial difference within each molecular type. Voriconazole and 5-fluorocytosine, respectively, had the lowest (0.031 ?g ml-1) and highest (8 µg ml-1) MICs. The epidemiological cutoff values (ECVs) for amphotericin B, fluconazole, voriconazole and 5-fluorocytosine encompassed ?97?% of all 14 C. neoformans VN I species. However, according to the CLSI document M59, ECVs for itraconazole (7; 50?% of the isolates) and for posaconazole (1; 7.1?% of the isolate), were one log2 dilution higher than the wild type range. Combinations of amphotericin B with 5-fluorocytosine, amphotericin B with fluconazole and fluconazole with 5-fluorocytosine exhibited synergistic effects against 37, 31 and 12.5?% of the isolates, respectively.Conclusion. Our findings may significantly contribute to the development of management strategies for patients at a higher risk of cryptococcosis, particularly HIV-positive individuals.

SUBMITTER: Bandalizadeh Z

PROVIDER: S-EPMC7137767 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Molecular epidemiology and antifungal susceptibility profiles of clinical Cryptococcus neoformans/Cryptococcus gattii species complex.

Bandalizadeh Zainab Z Shokohi Tahereh T Badali Hamid H Abastabar Mahdi M Babamahmoudi Farhang F Davoodi Lotfolah L Mardani Masoud M Javanian Mostafa M Cheraghmakani Hamed H Sepidgar Ali Asghar AA Badiee Parisa P Khodavaisy Sadegh S Afshari Setareh Agha Kuchak SAK Ahmadikia Kazem K Seyedmousavi Seyedmojtaba S

Journal of medical microbiology 20200101 1

Introduction. Limited data regarding the epidemiology and susceptibility profiles of cryptococcosis are available in the Middle East.Aim. Our study aimed to evaluate the molecular diversity, mating types and antifungal susceptibility pattern of Cryptococcus species (n=14) isolated from 320 suspected patients with cryptococcosis.Methodology. The URA5 gene was subjected to restriction fragment length polymorphism and sequence analysis. In addition, in v ...[more]

PMID: 31750814

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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.

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:Cryptococcosis is an infectious disease of worldwide distribution, caused by encapsulated yeasts belonging to the phylum Basidiomycota. The genus Cryptococcus includes several species distributed around the world. The C. gattii/neoformans species complex is largely responsible for most cases of cryptococcosis. However, clinical series have been published of infections caused by Papiliotrema (Cryptococcus) laurentii and Naganishia albida (Cryptococcus albidus), among other related genera. Here, we examined the pathogenic potential and antifungal susceptibility of C. gattii/neoformans species complex (clades I and II) and related genera (Papiliotrema and Naganishia) isolated from environmental and clinical samples. P. laurentii (clade III), N. liquefasciens/N. albidosimilis (clade IV); and N. adeliensis/N. albida (clade V) strains produced higher levels of phospholipase and hemolysins, whereas the C. gattii/neoformans species complex strains (clades I and II) had markedly thicker capsules, produced more biofilm biomass and melanin, which are known virulence attributes. Interestingly, 40% of C. neoformans strains (clade II) had MICs above the ECV established for this species to amphotericin B. Several non-C. gattii/neoformans species complex (clades III to V) had MICs equal to or above the ECVs established for C. deuterogattii and C. neoformans for all the three antifungal drugs tested. Finally, all the non-C. gattii/neoformans clinical isolates (clades III to V) produced more melanin than the environmental isolates might reflect their particularly enhanced need for melanin during in vivo protection. It is very clear that C. gattii/neoformans species complex (clades I and II) strains, in general, show more similar virulence phenotypes between each other when compared to non-C. gattii/neoformans species complex (clades III to V) isolates. These observations together with the fact that P. laurentii and Naganishia spp. (clades III to V) strains were collected from the outside of a University Hospital, identify features of these yeasts important for environmental and patient colonization and furthermore, define mechanisms for infections with these uncommon pathogens.

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Molecular epidemiology and antifungal susceptibility profiles of clinical Cryptococcus neoformans/Cryptococcus gattii species complex.

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Molecular epidemiology and antifungal susceptibility profiles of clinical <i>Cryptococcus neoformans/Cryptococcus gattii</i> species complex.

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