Orazio Romeo

First molecular method for discriminating between Candida africana, Candida albicans, and Candida dubliniensis by using hwp1 gene

The authors report here a polymerase chain reaction-based assay using a single primer pair (CR-f/CR-r), allowing discrimination between Candida africana and Candida albicans by using the hwp1 gene. The method also identifies Candida dubliniensis because it produces 3 different DNA fragments: approximately 700 bp for C. africana, 941 bp for C. albicans, and 569 for C. dubliniensis.

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.

Candida africana and its closest relatives

Candida africana is a recently described opportunistic yeast pathogen that has been linked to vaginal candidiasis. This yeast was first described, in 1995, as atypical chlamydospore‐negative Candida albicans strain, and subsequently proposed as a new Candida species on the basis of morphological, biochemical and physiological characteristics clearly different from those of typical C. albicans isolates. Phylogenetic studies based on the comparison of ribosomal DNA sequences demonstrated that C. africana and C. albicans isolates are too closely related to draw any conclusions regarding the status of a new species. Therefore, on the basis of these studies, some authors considered C. africana as a biovar of C. albicans even if genetic differences may be found if additional regions of genomic DNA are sequenced. The taxonomic situation of C. africana and its phylogenetic relationship with other Candida species is still controversial and remains, at present, a matter of debate. Our goal is to review the current knowledge about C. africana and highlight the development of rapid and accurate tests for its discrimination from C. albicans, Candida dubliniensis and Candida stellatoidea. Furthermore, through the analysis of literature data, we have found that C. africana has a worldwide distribution and a considerable number of features making its study particularly interesting.

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.

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.

Molecular Epidemiology of Candida albicans and Its Closely Related Yeasts Candida dubliniensis and Candida africana

ABSTRACT We performed a molecular study to determine the occurrence of Candida albicans, Candida africana, and Candida dubliniensis in different clinical samples. The study provides new insights into the epidemiology of candidiasis in hospitalized patients in three hospitals in southern Italy. It also reports the first detailed epidemiological data concerning the occurrence of C. africana in clinical samples.

High genetic variability in non-aflatoxigenic A. flavus strains by using Quadruplex PCR-based assay

Aflatoxigenic Aspergillus flavus isolates always show, by using a multiplex PCR-system, four DNA fragments specific for aflR, nor-1, ver-1, and omt-A genes. Non-aflatoxigenic A. flavus strains give variable DNA banding pattern lacking one, two, three or four of these genes. Recently, it has been found and reported that some aflatoxin non-producing A. flavus strains show a complete set of genes. Because less is known about the incidence of structural genes aflR, nor-1, ver-1 and omt-A in aflatoxin non-producing strains of A. flavus, we decided to study the frequencies of the aflatoxin structural genes in non-aflatoxigenic A. flavus strains isolated from food and feed commodities. The results can be summarized as following: 36.5% of the examined non-aflatoxigenic A. flavus strains showed DNA fragments that correspond to the complete set of genes (quadruplet pattern) as found in aflatoxigenic A. flavus. Forty three strains (32%) showed three DNA banding patterns grouped in four profiles where nor-1, ver-1 and omt-A was the most frequent profile. Twenty five (18.7%) of non-aflatoxigenic A. flavus strains yielded two DNA banding pattern whereas sixteen (12%) of the strains showed one DNA banding pattern. In one strain, isolated from poultry feed, no DNA bands were found. The nor-1 gene was the most representative between the four aflatoxin structural assayed genes. Lower incidence was found for aflR gene. Our data show a high level of genetic variability among non-aflatoxigenic A. flavus isolates that require greater attention in order to design molecular experiment to distinguish true aflatoxigenic from non-aflatoxigenic A. flavus strains.

Morphological, biochemical and molecular characterisation of the first Italian Candida africana isolate

One atypical isolate of the pathogenic yeast Candida albicans was isolated from an Italian patient with vulvovaginitis. The strain, germ tube positive and chlamydospore‐negative showed white‐thin turquoise colonies on Candida ID 2 medium. The yeast was identified as Candida africana by using morphological and biochemical tests. On the basis of the molecular results obtained in this study as well as in other studies, C. africana cannot be yet considered as a new species of Candida. It is possible that C. africana represents a new variant of C. albicans like the well‐known Candida stellatoidea. To our knowledge, this is the first isolation of C. africana in Italy.

Development and optimization of a new MALDI-TOF protocol for identification of the Sporothrix species complex

Accurate species identification of the Sporothrix schenckii complex is essential, since identification based only on phenotypic characteristics is often inconclusive due to phenotypic variability within the species. We used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species identification of 70 environmental and clinical isolates of the Sporothrix complex. A reference database was established for MALDI-TOF MS-based species identification according to minor adjustments in the manufacturers guidelines. The MALDI-TOF MS clearly distinguished strains of Sporothrix brasiliensis, Sporothrix globosa, Sporothrix mexicana, S. schenckii, Sporothrix luriei and Sporothrix pallida, enabling identification of all isolates at the species level, as confirmed by partial calmodulin gene sequence analyses. The present methodology is simple, reliable, rapid and highly suitable for routine identification in clinical mycology laboratories and culture collections, particularly for updating and reclassifying of deposited Sporothrix isolates.