Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.

Five new microsatellite loci were described and characterized for use as molecular markers for the identification and genetic differentiation of Candida albicans strains. Following the typing of 72 unrelated clinical isolates, the analysis revealed that they were all polymorphic, presenting from 5 to 30 alleles and 8 to 46 different genotypes. The discriminatory power obtained by combining the information generated by three microsatellites used in a multiplex PCR amplification strategy was 0.99, the highest ever reported. The multiplex PCR was later used to test a total of 114 C. albicans strains, including multiple isolates from the same patient collected from different body locations and along episodes of vulvovaginal infections. Three different scenarios for strain relatedness were identified: (i) different isolates that were revealed to be the same strain, (ii) isolates that were the same strain but that apparently underwent a process of microevolution, and (iii) isolates that corresponded to different strains. Analysis of the microevolutionary changes between isolates from recurrent infections indicated that the genotype alterations observed could be the result of events that lead to the loss of heterozygosity (LOH). In one case of recurrent infection, LOH was observed at the CAI locus, and this could have been related to exposure to fluconazole, since such strains were exposed to this antifungal during treatment. The analysis of microsatellites by a multiplex PCR strategy was found to be a highly efficient tool for the rapid and accurate differentiation of C. albicans strains and adequate for the identification of fine microevolutionary events that could be related to strain microevolution in response to environmental stress conditions. Candida albicans, the most common fungal pathogen, is a commensal yeast that belongs to the normal microbial population of the mouth, vagina, and gastrointestinal tract in humans. However, in people with a variety of transient or permanent immunocompromised conditions, including transplant recipients, chemotherapy patients, underweight neonates, and human immunodeficiency virus-infected individuals, it may become an invasive pathogen (30,56). Infections by opportunistic fungal agents are a major medical problem due to the growing number of immunocompromised patients with risk factors for such infections. Moreover, this problem is exacerbated by the fact that only a limited array of antifungal drugs is available and by the growing resistance among clinical isolates (39,46,55). The development of techniques and strategies that can accurately differentiate clinical isolates is of great relevance. These techniques should provide the ability to differentiate among strains responsible for clinical infections, as well as to trace their epidemiological pathways. Several molecular methods have been used to differentiate C. albicans strains, including electrophoretic karyotyping (2), the use of species-specific probes such as Ca3 or 27A in restriction enzyme analysis (28,35,36,43), P...

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The polymorphism of a new microsatellite locus (CAI) was investigated in a total of 114 Candida albicans strains, including 73 independent clinical isolates, multiple isolates from the same patient, isolates from several episodes of recurrent vulvovaginal infections, and two reference strains. PCR genotyping was performed automatically, using a fluorescence-labeled primer, and in the 73 independent isolates, 26 alleles and 44 different genotypes were identified, resulting in a discriminatory power of 0.97. CAI was revealed to be species specific and showed a low mutation rate, since no amplification product was obtained when testing other pathogenic Candida species and no genotype differences were observed when testing over 300 generations. When applying this microsatellite to the identification of strains isolated from recurrent vulvovaginal infections in eight patients, it was found that 13 out of 15 episodes were due to the same strain. When multiple isolates, obtained from the same patient and plated simultaneously, were typed for CAI, the same genotype was found in each case, confirming that the infecting population was clonal. Moreover, the same genotype appeared in isolates from the rectum and the vagina, revealing that the former could be a reservoir of potentially pathogenic strains. This new microsatellite proves to be a valuable tool to differentiate C. albicans strains. Furthermore, when compared to other molecular genotyping techniques, CAI proved to be very simple, highly efficient, and reproducible, being suitable for low-quantity and very-degraded samples and for application in large-scale epidemiological studies.It is known that opportunistic yeast pathogens are common residents of the mucosal surfaces of the gastrointestinal tract, genitourinary system, and oral cavity in warm-blooded animals. Although several yeast species can be associated with infection, the predominant causal agent of candidiasis is Candida albicans. This yeast causes several infections in humans, including a wide variety of life-threatening conditions triggered by bloodstream infections, especially in immunocompromised patients. Since pathogenicity and antifungal susceptibility often vary among strains, a rapid and accurate identification of the disease-causing strains of C. albicans is crucial for clinical treatment and epidemiological studies.Advances in molecular biology in the last 2 decades have allowed the development of rapid molecular genotyping techniques for clinical and epidemiological analysis. Several molecular typing methods have been developed to differentiate C. albicans strains, including electrophoretic karyotyping (2), the use of species-specific probes such as Ca3 or 27A in restriction enzyme analysis (20,23,27,29,32,33,35), and PCR-based methods (1,10,21,24,28,37). More recently, short tandem repeats (STRs) or microsatellites have assumed increasing importance as molecular markers in fields so diverse as oncogenetics, population genetics, and strain identification and characterization. They occur in se...

Candida albicans secreted aspartyl proteinases (Saps) are considered virulence-associated factors. Several members of the Sap family were claimed to play a significant role in the progression of candidiasis established by the hematogenous route. This assumption was based on the observed attenuated virulence of sap-null mutant strains. However, the exclusive contribution of SAP genes to their attenuated phenotype was not unequivocally confirmed, as the Ura status of these mutant strains could also have contributed to the attenuation. In this study, we have reassessed the importance of SAP1 to SAP6 in a murine model of hematogenously disseminated candidiasis using sap-null mutant strains not affected in their URA3 gene expression and compared their virulence phenotypes with those of Ura-blaster sap mutants. The median survival time of BALB/c mice intravenously infected with a mutant strain lacking SAP1 to SAP3 was equivalent to that of mice infected with wild-type strain SC5314, while those infected with mutant strains lacking SAP5 showed slightly extended survival times. Nevertheless, no differences could be observed between the wild type and a ⌬sap456 mutant in their abilities to invade mouse kidneys. Likewise, a deficiency in SAP4 to SAP6 had no noticeable impact on the immune response elicited in the spleens and kidneys of C. albicans-infected mice. These results contrast with the behavior of equivalent Ura-blaster mutants, which presented a significant reduction in virulence. Our results suggest that Sap1 to Sap6 do not play a significant role in C. albicans virulence in a murine model of hematogenously disseminated candidiasis and that, in this model, Sap1 to Sap3 are not necessary for successful C. albicans infection.The polymorphic yeast Candida albicans is an important opportunistic human pathogen causing infections that range from superficial mucosal lesions to life-threatening systemic disease. It is by far the most common cause of fungal invasive infections, which could be attributed to the little immunosuppression required to predispose an individual to invasive Candida infections (39). Host physical barriers and immune system integrity are crucial factors in controlling the establishment of infection. However, the high adaptability of C. albicans to different host niches, by the expression of appropriate sets of virulence-related genes, is also a determinant (19, 51). Several of these virulence attributes may participate in and influence the infective process, depending on the site and stage of invasion and on the nature of the host response (37). The secretion of hydrolytic enzymes during infection is required as a virulence attribute to aid adhesion, invasion, and the destruction of host immune factors, in addition to nutrient acquisition (21).Among these enzymes, secreted aspartyl proteinases (Sap), encoded by a 10-member gene family (SAP1 to SAP10) have been the most extensively studied (35). The 10 SAP genes that compose this family can be divided into subfamilies based on amino acid sequence ho...