Fabio Scordino

New Insight into Molecular Phylogeny and Epidemiology of Sporothrix schenckii Species Complex Based on Calmodulin-Encoding Gene Analysis of Italian Isolates

In this study, we investigated phylogenetic relationships among Italian Sporothrix schenckii isolates, by comparing their partial calmodulin sequences. In this analysis, we used 26 environmental strains of S. schenckii, plus two autochthonous clinical isolates. The results showed that our clinical strains grouped with S. schenckii sensu stricto isolates, whereas all 26 environmental isolates co-clustered with Sporothrix albicans (now regarded as a synonym of Sporothrix pallida), a non-pathogenic species closely related to S. schenckii. Furthermore, the group of environmental strains was found to be quite heterogeneous and further subdivided into two subgroups. The data reported here also showed that molecular methods, for specific identification of S. schenckii, developed before the description of its closely related species should be used with caution because of the possibility of false positive results, which could lead to inappropriate antifungal therapy. This study improves our understanding of the distribution of these new closely related Sporothrix species which also showed significant differences in antifungal susceptibilities.

A multiplex PCR protocol for rapid identification of Candida glabrata and its phylogenetically related species Candida nivariensis and Candida bracarensis

We have developed a multiplex PCR protocol for the detection of Candida glabrata and its closely related species Candida nivariensis and Candida bracarensis. The method uses four PCR primers, targeting the ITS1 region and the 5.8S ribosomal RNA gene. The combination of these primers yielded unique results to all Candida species tested. The PCR assay we developed was found to be a rapid, specific and easy to perform method and it will be useful for characterizing large numbers of isolates for epidemiological studies.

Current methods for identifying clinically important cryptic Candida species

In recent years, the taxonomy of the most important pathogenic Candida species (Candida albicans, Candida parapsilosis and Candida glabrata) has undergone profound changes due to the description of new closely-related species. This has resulted in the establishment of cryptic species complexes difficult to recognize in clinical diagnostic laboratories. The identification of these novel Candida species seems to be clinically relevant because it is likely that they differ in virulence and drug resistance. Nevertheless, current phenotypic methods are not suitable to accurately distinguish all the species belonging to a specific cryptic complex and therefore their recognition still requires molecular methods. Since traditional mycological techniques have not been useful, a number of molecular based methods have recently been developed. These range from simple PCR-based methods to more sophisticated real-time PCR and/or MALDI-TOF methods. In this article, we review the current methods designed for discriminating among closely related Candida species by highlighting, in particular, the limits of the existing phenotypic tests and the development of rapid and specific molecular tools for their proper identification.

Isolation and molecular characterization of Candida africana from Jos, Nigeria

During a survey of the prevalence of Candida spp. in Jos, Plateau State, Nigeria, two atypical C. albicans isolates were recovered. These two yeasts were germ tube positive, chlamydospore-negative and gave a green color on CHROMagar Candida. Molecular analysis performed by amplification of the hwp1 gene showed that these two isolates belonged to C. africana, a newly proposed Candida species closely related to C. albicans. Based on the presence or absence of an intron in DNA sequences encoding rRNA, the two C. africana, including all C. albicans isolates examined, were found to belong to genotype A and no other genotypes or species such as C. dubliniensis were found. To our knowledge, this is the first isolation of C. africana in Nigeria.

Cryptococcus neoformans / Cryptococcus gattii Species Complex in Southern Italy: An Overview on the Environmental Diffusion of Serotypes, Genotypes and Mating-Types

Given the lack of comprehensive molecular epidemiology studies in Reggio Calabria and Messina, Italy, we decided to perform an extensive environmental sampling to describe the current molecular epidemiology of C. neoformans/C. gattii species complex in southern Italy. In this study, we report the occurrence of serotypes, genotypes and mating-types of isolates of the C. neoformans/C. gattii species complex recovered from environmental sources. In addition, a number of environmental C. neoformans var. grubii strains, isolated in 1997 by our laboratory, were also retrospectively examined in order to compare their genotypes with those recently found and to infer the possible epidemiological changes in our country. One hundred and twenty-two isolates were identified as being C. neoformans, whereas only one was found to belong to C. gattii serotype B, genotype VGI and mating-type alpha. Our data revealed that all environmental isolates of C. neoformans recovered here as well as those previously isolated in 1997 belong to serotype A and genotype VNI and posses a mating-type alpha allele.

Genotyping and fluconazole susceptibility of Candida albicans strains from patients with vulvovaginal candidiasis in Jos, Nigeria

Abstract Objective To investigate the fluconazole susceptibility of the genotypes of Candida albicans ( C. albicans ) strains isolated from patients with vulvovaginal candidiasis (VVC) in Jos, Plateau State, Nigeria. Methods A total of one hundred and seventy seven (177) Candida isolates were examined. The strains were obtained from 320 female patients with VVC and were identified using both phenotypic and molecular methods. Their genotypes were determined, based on the presence or absence of a transposable intron in the 25S rDNA. Results Eighty four (84) strains were recognized as being C. albicans and all the 84 C. albicans strains resulted to be genotype A. Antifungal susceptibility testing showed that 13 of those isolates (15.48%) were resistant to fluconazole. Conclusions Based on these data, we concluded that C. albicans genotype A was common among VVC patients in Jos, and resistance to fluconazole is quite high. To our knowledge, this is the first study that reports C. albicans genotypes in Jos and its resistance to fluconazole.

Draft Genome Sequence of the Dimorphic Fungus Sporothrix pallida, a Nonpathogenic Species Belonging to Sporothrix, a Genus Containing Agents of Human and Feline Sporotrichosis

ABSTRACT Sporothrix pallida is considered to be a mostly avirulent environmental fungus, phylogenetically closely related to the well-known pathogen Sporothrix schenckii. Here, we present the first assembly of its genome, which provides a valuable resource for future comparative genomic studies between nonpathogenic and pathogenic Sporothrix spp.

Whole-genome sequencing and in silico analysis of two strains of Sporothrix globosa

Sporothrix globosa is a thermo-dimorphic fungus belonging to a pathogenic clade that also includes Sporothrix schenckii, which causes human and animal sporotrichosis. Here, we present the first genome assemblies of two S. globosa strains providing data for future comparative genomic studies in pathogenic Sporothrix species.

Molecular Characterization of the N-Acetylglucosamine Catabolic Genes in Candida africana, a Natural N-Acetylglucosamine Kinase (HXK1) Mutant.

Background In this study we report the genetic characterization, including expression analysis, of the genes involved in the uptake (NGT1) and catabolism (HXK1/NAG5, DAC1/NAG2, NAG1) of the aminosugar N-acetylglucosamine (GlcNAc) in Candida africana, a pathogenic biovariant of Candida albicans that is naturally unable to assimilate the GlcNAc. Results DNA sequence analysis of these genes revealed a number of characteristic nucleotide substitutions including a unique and distinctive guanine insertion that shifts the reading frame and generates a premature stop codon (TGA) 154 bp downstream of the ATG start codon of the HXK1 gene encoding the GlcNAc-kinase, a key enzyme of the GlcNAc catabolic pathway. However, all examined genes produced transcripts even though different levels of expression were observed among the Candida isolates examined. In particular, we found an HXK1-idependent relationship of the NGT1 gene and a considerable influence of the GlcNAc-kinase functionality on the transcription of the DAC1 and NAG1 genes. Additional phenotypic analysis revealed that C. africana isolates are hyperfilamentous in the first 24-48h of growth on filament-inducing media and revert to the yeast morphological form after 72h of incubation on these media. Conclusions Our results show that C. africana is a natural HXK1 mutant, displaying a number of phenotypic characteristics distinct from typical C. albicans isolates.

Molecular characterization of patulin producing and non-producing Penicillium species in apples from Morocco

The isolation of patulin-producing Penicillia in apples collected in different markets in four localities in Morocco is reported. Fungi were identified by β-tubulin sequencing and further characterized using a specific PCR-based method targeting the isoepoxydon dehydrogenase (IDH) gene to discriminate between patulin-producing and non-producing strains. Production of patulin was also evaluated using standard cultural and biochemical methods. Results showed that 79.5% of contaminant fungi belonged to the genus Penicillium and that Penicillium expansum was the most isolated species (83.9%) followed by Penicillium chrysogenum (~9.7%) and Penicillium crustosum (~6.4%). Molecular analysis revealed that 64.5% of the Penicillium species produced the expected IDH-amplicon denoting patulin production in these strains. However, patulin production was not chemically confirmed in all P. expansum strains. The isolation of IDH(-)/patulin(+) strains poses the hypothesis that gentisylaldehyde is not a direct patulin precursor, supporting previous observations that highlighted the importance of the gentisyl alcohol in the production of this mycotoxin. Total agreement between IDH-gene detection and cultural/chemical methods employed was observed in 58% of P. expansum strains and for 100% of the other species isolated. Overall the data reported here showed a substantial genetic variability within P. expansum population from Morocco.