Differentiation of Candida albicans, Candida glabrata, and Candida krusei by FT-IR and chemometrics by CHROMagar™ Candida.

Pathogenic Candida species are detected in clinical infections. CHROMagar™ is a phenotypical method used to identify Candida species, although it has limitations, which indicates the need for more sensitive and specific techniques. Infrared Spectroscopy (FT-IR) is an analytical vibrational technique used to identify patterns of metabolic fingerprint of biological matrixes, particularly whole microbial cell systems as Candida sp. in association of classificatory chemometrics algorithms. On the other hand, Soft Independent Modeling by Class Analogy (SIMCA) is one of the typical algorithms still little employed in microbiological classification. This study demonstrates the applicability of the FT-IR-technique by specular reflectance associated with SIMCA to discriminate Candida species isolated from vaginal discharges and grown on CHROMagar™. The differences in spectra of C. albicans, C. glabrata and C. krusei were suitable for use in the discrimination of these species, which was observed by PCA. Then, a SIMCA model was constructed with standard samples of three species and using the spectral region of 1792-1561cm-1. All samples (n=48) were properly classified based on the chromogenic method using CHROMagar™ Candida. In total, 93.4% (n=45) of the samples were correctly and unambiguously classified (Class I). Two samples of C. albicans were classified correctly, though these could have been C. glabrata (Class II). Also, one C. glabrata sample could have been classified as C. krusei (Class II). Concerning these three samples, one triplicate of each was included in Class II and two in Class I. Therefore, FT-IR associated with SIMCA can be used to identify samples of C. albicans, C. glabrata, and C. krusei grown in CHROMagar™ Candida aiming to improve clinical applications of this technique.

Multiplex PCR designed to differentiate species within the Candida glabrata complex.

BACKGROUND: No phenotypic methods are available to unequivocally differentiate species within the Candida glabrata complex. AIMS: To develop a new multiplex PCR method to differentiate between the three species of the C. glabrata species complex, as well as using it to study a C. glabrata collection to discover strains of the newly described species. METHODS: The method was developed based on the Internal Transcribed Spacer (ITS) sequence differences between the species. It was validated by using a blinded collection of strains and, finally, the new molecular method was used to study a collection of 192 C. glabrata species complex strains. The obtained results were compared with ITS sequencing. RESULTS: The proposed method showed 100% concordance with ITS sequencing and proved to be effective for clinical and epidemiological applications. Two Candida bracarensis and three Candida nivariensis were found out of the 192 studied strains (0.93% and 1.40% prevalence, respectively). CONCLUSIONS: A fast, inexpensive, robust and highly reproducible multiplex PCR method is presented. Its usefulness is demonstrated by studying a large collection of C. glabrata sensu lato strains.

Candida glabrata species complex prevalence and antifungal susceptibility testing in a culture collection: First description of Candida nivariensis in Argentina.

The presence of the cryptic species belonging to the Candida glabrata complex has not been studied in Argentina. We analyzed a collection of 117 clinical isolates of C. glabrata complex belonging to a National Culture Collection of Instituto Nacional de Microbiología "Dr. Carlos G. Malbrán" from Argentina (40 isolates from blood samples, 18 from other normally sterile sites, 20 from vagina, 14 from urine, 7 from oral cavity, 3 from catheter, 1 from a stool sample and 14 isolates whose clinical origin was not recorded). The aims of this work were to determine the prevalence of the cryptic species Candida nivariensis and Candida bracarensis and to evaluate the susceptibility profile of isolates against nine antifungal drugs. Identification was carried out by using classical phenotypic tests, CHROMagar™ Candida, PCR and MALDI-TOF. The minimal inhibitory concentrations of amphotericin B, 5-fluorocytosine, fluconazole, itraconazole, voriconazole, ketoconazole, posaconazole, caspofungin and anidulafungin were determined according to the EDef 7.3 (EUCAST) reference document. Of the 117 isolates, 114 were identified as C. glabrata and three as C. nivariensis by using PCR and MALDI-TOF. There were no major differences between C. nivariensis and C. glabrata susceptibility profiles. No resistant strains were found to echinocandins. We have found that the percentage of C. nivariensis in our culture collection was 2.56. This is the first description of C. nivariensis in Argentina, and data obtained could contribute to the knowledge of the epidemiology of this cryptic species.

Candida glabrata among Candida spp. from environmental health practitioners of a Brazilian Hospital

Abstract The incidence of the species Candida albicans and non-albicans Candida was evaluated in a Brazilian Tertiary Hospital from the environment and health practitioners. In a 12-month period we had a total positivity of 19.65% of Candida spp. The most recurring non-albicans Candida species was C. glabrata (37.62%), generally considered a species of low virulence, but with a higher mortality rate than C. albicans. Subsequently, C. parapsilosis (25.74%) and C. tropicalis (16.86%) were the second and third most commonly isolated species. Considering the total samples collected from the emergency room and from the inpatient and the pediatric sector, 19.10% were positive for Candida spp., with the predominance of non-albicans Candida species (89.42%). The high percentage of positivity occurred in the hands (24.32%) and the lab coats (21.88%) of the health care assistants. No sample of C. albicans presented a profile of resistance to the drugs. All the non-albicans Candida species presented a decreased susceptibility to miconazole and itraconazole, but they were susceptible to nystatin. Most of the isolates were susceptible to fluconazole and amphotericin B. As expected, a high resistance rate was observed in C. glabrata and C. krusei, which are intrinsically less susceptible to this antifungal agent. The contamination of environmental surfaces by Candida spp. through hand touching may facilitate the occurrence of Candida infections predominantly in immunocompromised patients. In addition to that, the antifungal agents used should be carefully evaluated considering local epidemiologic trends in Candida spp. infections, so that therapeutic choices may be better guided.

Candida glabrata among Candida spp. from environmental health practitioners of a Brazilian Hospital.

The incidence of the species Candida albicans and non-albicans Candida was evaluated in a Brazilian Tertiary Hospital from the environment and health practitioners. In a 12-month period we had a total positivity of 19.65% of Candida spp. The most recurring non-albicans Candida species was C. glabrata (37.62%), generally considered a species of low virulence, but with a higher mortality rate than C. albicans. Subsequently, C. parapsilosis (25.74%) and C. tropicalis (16.86%) were the second and third most commonly isolated species. Considering the total samples collected from the emergency room and from the inpatient and the pediatric sector, 19.10% were positive for Candida spp., with the predominance of non-albicans Candida species (89.42%). The high percentage of positivity occurred in the hands (24.32%) and the lab coats (21.88%) of the health care assistants. No sample of C. albicans presented a profile of resistance to the drugs. All the non-albicans Candida species presented a decreased susceptibility to miconazole and itraconazole, but they were susceptible to nystatin. Most of the isolates were susceptible to fluconazole and amphotericin B. As expected, a high resistance rate was observed in C. glabrata and C. krusei, which are intrinsically less susceptible to this antifungal agent. The contamination of environmental surfaces by Candida spp. through hand touching may facilitate the occurrence of Candida infections predominantly in immunocompromised patients. In addition to that, the antifungal agents used should be carefully evaluated considering local epidemiologic trends in Candida spp. infections, so that therapeutic choices may be better guided.

Biofilm microbial communities of denture stomatitis.

INTRODUCTION: Denture stomatitis is a common lesion that affects denture wearers. Its multifactorial etiology seems to depend on a complex and poorly characterized biofilm. The purpose of this study was to assess the composition of the microbial biofilm obtained from complete denture wearers with and without denture stomatitis using culture-independent methods. METHODS: Samples were collected from healthy denture wearers and from patients with denture stomatitis. Libraries comprising about 600 cloned 16S ribosomal DNA (rDNA) bacterial sequences and 192 cloned eukaryotic internal transcribed spacer (ITS) region sequences, obtained by polymerase chain reactions, were analyzed. RESULTS: The partial 16S rDNA sequences revealed a total of 82 bacterial species identified in healthy subjects and patients with denture stomatitis. Twenty-seven bacterial species were detected in both biofilms, 29 species were exclusively present in patients with denture stomatitis, and 26 were found only in healthy subjects. Analysis of the ITS region revealed the presence of Candida sp. in both biofilms. CONCLUSION: The results revealed the extent of the microbial flora, suggesting the existence of distinct biofilms in healthy subjects and in patients with denture stomatitis.

Genetic diversity among clinical isolates of Candida glabrata analyzed by randomly amplified polymorphic DNA and multilocus enzyme electrophoresis analyses.

The genetic diversity of 47 clinical and reference strains of Candida glabrata from several geographical origins and diverse clinical disorders, with different antifungal susceptibilities, as well as their genetic relationships were studied through multilocus enzyme electrophoresis (MLEE) and randomly amplified polymorphic DNA (RAPD) techniques. The genetic diversity estimated for 11 MLEE loci measured as average heterozygosity (h) was 0.055. A high level of genetic relatedness among isolates was established by cluster analysis. Forty-nine RAPD markers were analyzed, and the average genetic diversity among isolates, estimated by Shannon's index (Ho), was 0.372. The PhiST values estimated through an analysis of molecular variance to assess genetic differentiation among isolates revealed no genetic differentiation among them. Our results revealed very low genetic diversity among isolates, a lack of differentiation, and no association with their geographic origin and the clinical characteristics.