Frequency and geographical distribution of genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii species complexes in Argentina.

The aim of this work was to know the frequency and geographical distribution of genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii species complexes isolated from human infections in Argentina during the period from April 2009 to April 2011. A multicenter study was conducted, in which 372 isolates were obtained from 61 laboratories throughout the country. Of those, 98.8% of the isolates belonged to the C. neoformans species complex and 1.1% to the C. gattii species complex. Genotype VNI (MATα) was the most frequently isolated (n=326, 87.6%), followed by VNII (MATα) (n=22, 5.9%), the recently described VNII-VNIV (aADα) hybrid (n=14, 3.8%), VNIV (MATα) (n=4, 1.1%), VNIII (αADa) hybrid (n=1, 0.3%), and VNIII (αADα) hybrid (n=1, 0.3%). The Argentine Central region showed the greatest number of cases and genotype diversity. Interestingly, a relative high frequency was observed in genotype VNII (MATα) in the Cuyo, Northeast and Northwest regions and, also in VNII-VNIV (aADα) hybrids in the Northwest region. C. gattii species complex was isolated at a low rate; 3 VGI (MATα) and 1 VGII (MATα) isolates were obtained from the Northwest and Central regions. In conclusion, this study shows that genotype frequencies seem to vary among regions in Argentina and reveals a relatively high frequency of rare hybrids in the Northwest region. Further regional clinical and environmental studies may help to elucidate if those variations in frequencies are associated with the existence of regional ecological niches or any other regional factors.

Reidentification and antifungal susceptibility profile of Candida guilliermondii and Candida famata clinical isolates from a culture collection in Argentina.

The aim of this work was to reidentify strains previously identified as Candida guilliermondii and Candida famata by conventional phenotypic methods conserved in a culture collection from Argentina using ribosomal DNA sequencing, ACT1 gene sequencing, and matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI-TOF MS). In addition, we performed antifungal susceptibility tests of eight antifungal drugs commonly used in clinical treatment. We identified 68 isolates belonging to the Candida guilliermondii species complex (59 C. guilliermondii, 8 C. fermentati, and 1 Candida carpophila), 16 isolates belonging to the Candida famata species complex (8 C. famata, 6 Debaryomyces nepalensis, 1 Debaryomyces fabryi, and 1 Debaryomyces tyrocola). Although sequencing of ITS region was able to identify C. guilliermondii and D. nepalensis isolates, sequencing of ACT1 gene seems to be the most appropriate technique for differentiation between C. fermentati and C. carpophila and between members of the C. famata species complex others than D. nepalensis. MALDI-TOF MS has a good potential for the identification of these yeasts, particularly in clinical laboratories since is a rapid and easy to perform technique. Here, we report the first isolation of D. tyrocola from a human patient and the first isolation of D. nepalensis from lungs and blood of human patients. Finally, correct identification and determination of antifungal susceptibility of those closely related species could be a useful tool for clinicians to choose the most effective antifungal treatment.

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.

First Case of Fungemia Due to Pseudozyma aphidis in a Pediatric Patient with Osteosarcoma in Latin America.

We report the first case of blood infection due to Pseudozyma aphidis in Latin America. We contribute evidence showing this organism to be a potential human pathogen, and we provide new data about its identification, drug susceptibility, and treatment outcome.

Molecular identification, genotyping, and antifungal susceptibility testing of clinically relevant Trichosporon species from Argentina.

Trichosporon species are emerging causative agents of mycoses; most are documented in immunocompromised patients. Species identification is important for epidemiological purposes in order to better define species clinical associations and to improve antifungal treatment. Here, we studied a collection of 41 Trichosporon strains recovered from hospitalized patients in Argentina. All strains were identified by sequencing the D1/D2 domain of 26S, internal transcribed spacer (ITS) regions, and intergenic spacer 1 (IGS1) region. In addition, we determined the IGS1 region genotypes of the suspected T. asahii strains. Antifungal susceptibility of all strains was investigated. Thirty-eight of the 41 strains in this study were identified as follows: 29 T. asahii, 3 T. inkin, 3 T. montevideense, 2 T. faecale, and 1 T. dermatis. The identity of the three remaining strains could not be confirmed. Strain DMic 114126 (Culture collection of the Mycology Department (DMic), National Institute of Infectious Diseases "Dr. Carlos G. Malbrán".) may represent a T. asahii subspecies or a new Trichosporon species, strain DMic 94750 was identified as T. cf. guehoae and strain DMic 114132 as T. cf. akiyoshidainum. The distribution of T. asahii genotypes was as follows: 12 genotype 3, 9 genotype 1, 4 genotype 4, 2 genotype 5, and 2 genotype 7. Amphotericin B minimal inhibitory concentrations (MICs) were ≤1 mg/l for 78% (32/41) of the strains. Fluconazole MICs were ≥2 mg/l for 90% of the strains. However, itraconazole, voriconazole, ketoconazole, and posaconazole MICs were ≤1 mg/l for 100% of the strains. Terbinafine MICs were ≤1 mg/l for 98% 40/41 of the strains.

Cryptococcus gattii in urban trees from cities in North-eastern Argentina.

In the city of Buenos Aires, Argentina, Cryptococcus gattii genotype AFLP4/VGI was found to be associated with decaying wood in hollows of different tree species. The aim of this study was to investigate the presence of C. gattii in the environment of riverside cities of the river Paraná, and to describe its serotypes and molecular types. Five hundred samples were collected in 50 parks by swabbing tree hollows. The samples were inoculated on caffeic acid agar supplemented with chloramphenicol, and incubated at 28 °C for 1 week with a daily observation. The isolates were identified by conventional methods. The serotype was determined by slide agglutination with specific antisera. Molecular typing was carried out by PCR-RFLP of the URA5 gene. Four isolates of C. gattii were recovered: Cryptococcus gattii serotype B, genotype AFLP4/VGI, isolated from Eucalyptus sp. in the city of Rosario and from Grevillea robusta in the city of La Paz; and C. gattii serotype C, genotype AFLP5/VGIII, isolated from two different Tipuana tipu trees in the city of Resistencia. Here, we report for the first time the isolation of C. gattii serotype C, genotype AFLP5/VGIII, from environmental samples in Argentina.

Comparative analyses of classical phenotypic method and ribosomal RNA gene sequencing for identification of medically relevant Candida species.

As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequencing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as targets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, genotypic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirmation of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species.

Comparative analyses of classical phenotypic method and ribosomal RNA gene sequencing for identification of medically relevant Candida species

As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequencing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as targets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, genotypic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirmation of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species.

Primer caso de fungemia por una levadura relacionada con Candida pseudorugosa / First case of fungemia from a yeast related with Candida pseudorugosa

Se presenta el primer caso de fungemia por una especie de Candida relacionada con Candida pseudorugosa. La identificación de las especies de levaduras es de importancia a nivel epidemiológico y para el tratamiento de los pacientes que cursan una infección por levaduras.

Primer caso de fungemia por una levadura relacionada con Candida pseudorugosa / First case of fungemia from a yeast related with Candida pseudorugosa

Se presenta el primer caso de fungemia por una especie de Candida relacionada con Candida pseudorugosa. La identificación de las especies de levaduras es de importancia a nivel epidemiológico y para el tratamiento de los pacientes que cursan una infección por levaduras. (AU)

First case report of bloodstream infection due to a Candida species closely related to the novel species Candida pseudorugosa.

Candida pseudorugosa is a novel species closely related to Candida rugosa for which only one case has been reported. We report the first case of a bloodstream infection in humans caused by a Candida sp. closely related to C. pseudorugosa. We contribute evidence to show this organism as a potential human pathogen that may be misidentified by conventional methods, also pointing out its lower sensitivity to azoles and other antifungal agents.

Prevalence of Candida dubliniensis fungemia in Argentina: identification by a novel multiplex PCR and comparison of different phenotypic methods.

Candida dubliniensis is an emerging pathogen that can cause invasive disease in patients who have a variety of clinical conditions. C. dubliniensis is often misidentified as Candida albicans by clinical laboratories. In Argentina, incidence data are still scarce, and only one systemic infection has been reported. This study aims to determine the prevalence of C. dubliniensis in blood samples in Argentina, to evaluate a novel PCR multiplex as well as several phenotypic methods for the identification of this yeast, and to know the susceptibility profile of isolates against seven antifungal drugs. We have found that prevalence in Argentina appears to be lower than that reported in other countries, occurring only in 0.96% of the Candidemia cases recovered in 47 hospitals during a 1-year period. All C. dubliniensis clinical isolates included in this study were genetically identical when comparing ITS genes sequences. This is in agreement with the previous studies suggesting little genetic variation within this species. The novel multiplex PCR proved to be 100% sensitive and specific for the identification of C. dubliniensis. Therefore, we propose its use as a rapid and inexpensive method for laboratories having access to molecular techniques. Although no single phenotypic test has proved to be infallible, both colony morphology on tobacco agar, as well as abundant chlamydospore formation on both tobacco agar and on sunflower seed agar, may be used as a presumptive differentiation method in routine mycology laboratories. It has been suggested that C. dubliniensis may have higher propensity to develop azole antifungal drug resistance than C. albicans. In this study, one of the five clinical isolates of C. dubliniensis was resistant to fluconazole.

[Concordance between phenotypical features and PCR-REA for the identification of Malassezia spp]. / Concordancia entre características fenotípicas y PCR-REA en la identificación de especies de Malassezia.

The genus Malassezia has been recently revised and nowadays includes 11 species that cannot always be differentiated from each other by physiological and morphological tests. This study was aimed to evaluate the correlation between a molecular method and conventional phenotypic features in the identification of Malassezia spp. To achieve this aim, 92 Argentinean clinical strains isolated between 2001 and 2005 were analyzed along with three reference strains (Malassezia furfur CBS 7019, Malassezia sympodialis CBS 7222 and Malassezia slooffiae CBS 7956). By using PCR and restriction enzyme analysis with three different DNA endonucleases (PCR-REA), the molecular method consistently identified all three reference strains and all 92 clinical isolates as follows: 63 M. sympodialis, 18 M. furfur, 10 Malassezia globosa and one Malassezia obtusa. Phenotypic studies undentified 85 clinical isolates and two of the reference strains (total agreement > 91%). In particular for M. sympodialis, M. furfur and M. globosa, the species more frequently involved in human pathology, the agreement ranged between 84 and 96%. This result suggests that phenotypic studies are suitable for the presumptive identification of important Malassezia species in the clinical medical mycology laboratories where molecular methodologies are not available.

Concordancia entre características fenotípicas y PCR-REA en la identificación de especies de Malassezia / Concordance between phenotypical features and PCR-REA for the identification of Malassezia spp

El género Malassezia ha sido revisado recientemente y, hasta el momento,se acepta que incluye 11 especies, que no siempre pueden ser diferenciadasmediante pruebas fisiológicas y morfológicas. En este trabajo se investigó laconcordancia entre las pruebas fenotípicas y un método molecular rápido enla identificación de especies de ese género. Se analizaron 92 aislamientosclínicos de Malassezia spp. obtenidos de pacientes argentinos entre 2001 y2005 y tres cepas de referencia (Malassezia furfur CBS-7019, Malasseziasympodialis CBS-7222 y Malassezia slooffiae CBS-7956). El método molecular,basado en amplificación del ADN por PCR y su digestión con tres diferentesendonucleasas (PCR-REA), permitió identificar inequívocamente los 92aislamientos clínicos (63 M. sympodialis, 18 M. furfur, 10 Malassezia globosa yuna Malassezia obtusa) y las tres cepas de referencia. Mediante las pruebasfenotípicas se logró tipificar 85 aislamientos y dos de las cepas de referencia(concordancia total > 91%). Para las especies más frecuentementeinvolucradas en patología humana, tales como M. sympodialis, M. furfur yM. globosa, la concordancia osciló entre 84-96%, lo que indicaría que losestudios fenotípicos son útiles para la identificación presuntiva de estosmiembros del género en laboratorios que aún no tienen acceso a lametodología molecular(AU)

The genus Malassezia has been recently revised and nowadays includes11 species that cannot always be differentiated from each other byphysiological and morphological tests. This study was aimed to evaluate thecorrelation between a molecular method and conventional phenotypic featuresin the identification of Malassezia spp. To achieve this aim, 92 Argentineanclinical strains isolated between 2001 and 2005 were analyzed along withthree reference strains (Malassezia furfur CBS 7019, Malassezia sympodialisCBS 7222 and Malassezia slooffiae CBS 7956). By using PCR and restrictionenzyme analysis with three different DNA endonucleases (PCR-REA), themolecular method consistently identified all three reference strains and all92 clinical isolates as follows: 63 M. sympodialis, 18 M. furfur, 10 Malasseziaglobosa and one Malassezia obtusa. Phenotypic studies undentified 85 clinicalisolates and two of the reference strains (total agreement > 91%). In particularfor M. sympodialis, M. furfur and M. globosa, the species more frequentlyinvolved in human pathology, the agreement ranged between 84 and 96%.This result suggests that phenotypic studies are suitable for the presumptiveidentification of important Malassezia species in the clinical medical mycologylaboratories where molecular methodologies are not available(AU)