Massimo Cogliati

Consensus multi-locus sequence typing scheme for Cryptococcus neoformans and Cryptococcus gattii

This communication describes the consensus multi-locus typing scheme established by the Cryptococcal Working Group I (Genotyping of Cryptococcus neoformans and C. gattii) of the International Society for Human and Animal Mycology (ISHAM) using seven unlinked genetic loci for global strain genotyping. These genetic loci include the housekeeping genes CAP59,GPD1, LAC1, PLB1, SOD1, URA5 and the IGS1 region. Allele and sequence type information are accessible at http://www.mlst.net/ .

Autochthonous and dormant Cryptococcus gattii infections in Europe

Dormant infections can become reactivated years after having been acquired on another continent.

Origin of Cryptococcus neoformans var.neoformans Diploid Strains

ABSTRACT The basidiomycetous yeast Cryptococcus neoformans is an important human fungal pathogen. Two varieties, C. neoformans var. neoformans and C. neoformans var. gattii, have been identified. Both are heterothallic with two mating types, MATa andMATα. Some rare isolates are self-fertile and are considered occasional diploid or aneuploid strains. In the present study, 133 isolates, mostly from Italian patients, were investigated to detect the presence of diploid strains in the Igiene UniversitàMilano culture collection. All of the diploid isolates were further investigated by different methods to elucidate their origins. Forty-nine diploid strains were identified by flow cytometry. PCR fingerprinting using the (GACA)4 primer showed that the diploid state was associated with two specific genotypes identified as VN3 and VN4. Determination of mating type on V8 juice medium confirmed that the majority of the strains were sterile. PCR and dot blotting using the two pheromone genes (MFa andMFα) as probes identified 36 of the 49 diploid isolates as MATa/α. The results of pheromone gene sequencing showed that two allelic MFα genes exist and are distinct for serotypes A and D. In contrast, the MFagene sequence was conserved in both serotype alleles. Amplification of serotype-specific STE20 alleles demonstrated that the diploid strains contained one mating locus inherited from a serotype A parent and one inherited from a serotype D parent. The present results suggest that diploid isolates may be common among the C. neoformans population and that in Italy and other European countries serotype A and D populations are not genetically isolated but are able to recombine by sexual reproduction.

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Method for Discrimination between Molecular Types of Cryptococcus neoformans and Cryptococcus gattii

ABSTRACT We evaluated the usefulness of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for Cryptococcus identification at the species and subspecies levels by using an in-house database of 25 reference cryptococcal spectra. Eighty-one out of the 82 Cryptococcus isolates (72 Cryptococcus neoformans and 10 Cryptococcus gattii) tested were correctly identified with respect to their molecular type designations. We showed that MALDI-TOF MS is a practicable alternative to conventional mycology or DNA-based methods.

Identification by polymerase chain reaction fingerprinting of Cryptococcus neoformans serotype AD

Seventy-three Cryptococcus neoformans isolates and eight other yeast strains were studied. Fingerprints produced by priming with (GACA)4 differentiated C. neoformans from all other yeasts tested and identified the five C. neoformans serotypes. Four major bands of molecular size 800, 540, 475 and 410 bp were recognized for serotypes A, AD and D. Two of them were specific for serotype A and the other two for serotype D isolates. Serotype AD strains were identified by five different genotypic patterns in which at least one of the two bands specific for serotype A and D were present in different combinations. On repeated and simultaneously performed genotype and serotype testing of nine strains, the genotypic pattern did not change, whereas serotyping was unstable in three cases. PCR-fingerprinting using (GACA)4 as a primer proved more stable than serology in discriminating among C. neoformans serotypes A, D and AD and was able to distinguish among serotype AD strains.

The case for adopting the "species complex" nomenclature for the etiologic agents of cryptococcosis

ABSTRACT Cryptococcosis is a potentially lethal disease of humans/animals caused by Cryptococcus neoformans and Cryptococcus gattii. Distinction between the two species is based on phenotypic and genotypic characteristics. Recently, it was proposed that C. neoformans be divided into two species and C. gattii into five species based on a phylogenetic analysis of 115 isolates. While this proposal adds to the knowledge about the genetic diversity and population structure of cryptococcosis agents, the published genotypes of 2,606 strains have already revealed more genetic diversity than is encompassed by seven species. Naming every clade as a separate species at this juncture will lead to continuing nomenclatural instability. In the absence of biological differences between clades and no consensus about how DNA sequence alone can delineate a species, we recommend using “Cryptococcus neoformans species complex” and “C. gattii species complex” as a practical intermediate step, rather than creating more species. This strategy recognizes genetic diversity without creating confusion.

Prevalence of serotype D in Cryptococcus neoformans isolates from HIV positive and HIV negative patients in Italy

Summary. Cryptococcus neoformans strains isolated from 207 HIV positive and HIV negative patients hospitalized in Northern Italy were serotyped by slide agglutination. One Brazilian HIV negative woman was infected by var. gattii serctype B and all the other patients by var. neoformans, serotype D in 71%, serotype A in 24.6% and serotype AD in 3.4%. No difference was observed between subjects with serotypes A and D in HIV coinfection, exposure categories for AIDS, age, sex, and CD4 count of HIV positive patients. Meningeal and respiratory tract involvements and prostatic reservoir occurred with comparable frequency in AIDS patients infected by serotypes A and D. Skin lesions were observed only in serotype D infections, occurring in 12.6% of HIV positive and 58.3% of HIV negative patients infected by this serotype. Serotype A was found less susceptible to fluconazole than serotype D: 53.7% of serotype A strains had a MIC 25 μg ml‐1 compared to 17.7% of the serotype D isolates. On the other hand, both serotypes were highly susceptible to itraconazole.

Environmental distribution of Cryptococcus neoformans and C. gattii around the Mediterranean basin

In order to elucidate the distribution of Cryptococcus neoformans and C. gattii in the Mediterranean basin, an extensive environmental survey was carried out during 2012-2015. A total of 302 sites located in 12 countries were sampled, 6436 samples from 3765 trees were collected and 5% of trees were found to be colonized by cryptococcal yeasts. Cryptococcus neoformans was isolated from 177 trees and C. gattii from 13. Cryptococcus neoformans colonized 27% of Ceratonia, 10% of Olea, Platanus and Prunus trees and a lower percentage of other tree genera. The 13 C. gattii isolates were collected from five Eucalyptus, four Ceratonia, two Pinus and two Olea trees. Cryptococcus neoformans was distributed all around the Mediterranean basin, whereas C. gattii was isolated in Greece, Southern Italy and Spain, in agreement with previous findings from both clinical and environmental sources. Among C. neoformans isolates, VNI was the prevalent molecular type but VNII, VNIV and VNIII hybrid strains were also isolated. With the exception of a single VGIV isolate, all C. gattii isolates were VGI. The results confirmed the presence of both Cryptococcus species in the Mediterranean environment, and showed that both carob and olive trees represent an important niche for these yeasts.

Azole-resistant Aspergillus fumigatus in the environment of northern Italy, May 2011 to June 2012

In recent years acquired azole resistance in Aspergillus fumigatus has been increasingly reported and a dominant mechanism of resistance (TR34/L98H) was found in clinical and environmental isolates. The aim of the present study was to investigate the prevalence of azole resistance in environmental A. fumigatus isolates collected in northern Italy. A. fumigatus grew from 29 of 47 soil samples analysed. Azole-resistant isolates were detected in 13% (6/47) of the soil samples and in 21% (6/29) of the soil samples containing A. fumigatus. High minimal inhibitory concentrations (MIC) of itraconazole (≥16 mg/L) and posaconazole (≥0.5 mg/L) were displayed by nine isolates from six different soil samples, namely apple orchard (1 sample), rose pot compost (2 samples), and cucurbit yields (3 samples). Seven isolates had a MIC=2 mg/L of voriconazole. Seven of nine itraconazole and posaconazole resistant isolates harboured the same TR34/L98H mutation of cyp51A. These findings, together with the occurrence of resistant clinical isolates, suggest that azole resistance should be considered in primary patient care.

Cryptococcus gattii serotype-C strains isolated in Bangalore, Karnataka, India

During a retrospective study on cryptococcosis carried out in Bangalore, Karnataka, India, four Cryptococcus gattii strains were isolated from one HIV‐positive and three HIV‐negative patients, two of which had unknown predisposing conditions. Serotyping and genotyping showed that the isolates were C. gattii serotype C, mating‐type α and genotype VGIV. All the isolates were identical by multilocus sequence typing, but presented a low similarity compared with a set of 17 C. gattii global control strains. The comparison with a larger number of previously reported C. gattii strains, including African isolates, revealed a close relationship between Indian and African serotype‐C isolates.