Enzyme production by Candida albicans and Candida dubliniensis in periodontal HIV-positive patients receiving and not receiving antiretroviral therapy / Producción enzimática de Candida albicans y Candida dubliniensis de pacientes con periodontitis VIH+ con y sin tratamiento antirretroviral

ABSTRACT Candida dubliniensis (Cd) and Candida albicans (Ca) are the most frequently isolated yeasts in HIV+ patients. Some of the enzymes produced by these yeasts are considered virulence factors since they contribute to pathogenicity of Candida spp. The aim of the present study was to compare production of enzymes such as phospholipase (Ph), proteinase (P), and hemolysin (H) by Cd and Ca strains isolated from periodontal HIV-positive patients receiving and not receiving highly active antiretroviral therapy (HAART). Subgingival biofilm samples were obtained using paper points, and a sample of oral mucosa was taken using a swab. Phenotypic and molecular methods were used to isolate 39 strains of Candida, including 25 strains of Cd and 14 strains of Ca, obtained from 33 periodontal pocket samples and 6 oral mucosa samples collected from 15 HIV+ patients (8 receiving and 7 not receiving HAART). Malt egg-yolk agar, albumin agar and blood agar were used to evaluate pH, P and H production respectively. The strains were inoculated in duplicate and incubated at 37 ºC. Colony and halo diameters were measured. A greater proportion of Ca was observed in patients not receiving HAART, and a higher proportion of Cd was observed in those under HAART, Chi2 p< 0.001. Phospholipase production was observed in 92.9% percent of isolated Ca strains but in none of the isolated Cd strains. Proteinase production was high in Ca and Cd strains isolated from patients not receiving HAART. Hemolysin production was observed in all the studied strains, though it was significantly higher (p=0.04) in Ca and Cd strains isolated from patients not receiving HAART. To sum up, the proportion of Candida dubliniensis strains was highest in the subgingival biofilm of patients receiving HAART, and Cd strains were found to express fewer virulence factors than Ca strains.

RESUMEN Las levaduras más aisladas en pacientes VIH+ son Candida dubliniensis (Cd) y Candida albicans (Ca). Algunas de sus enzimas constituyen factores de virulencia ya que favorecen la diseminación tisular. El objetivo fue comparar la producción de enzimas como fosfolipasa (F), proteinasa (P) y hemolisina (H) en cepas de Cd y Ca aisladas de pacientes VIH+ tratados y no tratados con antirretrovirales (TARGA). Se realizó la toma del biofilm de placa subgingival con conos de papel y la muestra de la mucosa bucal con hisopo. Se aislaron y tipificaron por métodos fenotípicos y moleculares 39 cepas: 25 de Cd y 14 Ca, obtenidas 33 de bolsas periodontales y 6 de mucosa bucal de 15 pacientes VIH+ (8 con y 7 sin tratamiento). Se utilizó agar malta con yema de huevo, agar albúmina y agar sangre para demostrar la producción de F, P y H, respectivamente. Se inocularon por duplicado e incubaron a 37°C. Se midieron los diámetros de las colonias y los de hidrólisis alrededor de las mismas. Se observó mayor proporción de Ca en los pacientes sin tratamiento y mayor proporción de Cd en los con tratamiento; Chi2 p< 0.001. El 92,9% de las Ca estudiadas, fueron productoras de fosfolipasa. En tanto que ninguna Cd produjo la enzima. En cuanto a la producción de proteinasa se observa una alta producción tanto en las cepas de Ca, como en las Cd aisladas en los pacientes no tratados. Todas las cepas estudiadas produjeron hemolisina, observándose una diferencia estadísticamente significativa (p=0,04) en ambas especies a favor de la alta producción de la enzima en las cepas obtenidas de pacientes no tratados. Podemos concluir que en el biofilm subgingival, en los pacientes bajo TARGA, se aíslan mayor proporción de Candida dubliniensis las cuales expresan menos factores de virulencia.

Primer aislamiento de Candida auris en Chile / First isolation de Candida auris in Chile

Resumen Candida auris es una levadura multi-resistente emergente con rápida diseminación mundial. Desde el primer reporte el 2009, varios aislados a través de los cinco continentes han sido identificados como agentes de infecciones asociadas a la atención en salud. Brotes independientes y simultáneos por C. auris se han vuelto prioridad para la comunidad hospitalaria y científica. Además, los errores en identificación y los perfiles de multi-resistencia, raramente observados para otras especies de Candida, resultan en una difícil erradicación y fallas terapéuticas frecuentes en infecciones por C. auris. Presentamos el primer aislamiento de una cepa de C. auris en un hospital en Santiago, en un paciente proveniente de la India, que fue admitido para tratamiento de su pie diabético. La cepa fue recuperada de un cultivo de tejido e identificada por VITEK® 2 Compact. La identificación de C. auris fue confirmada por MALDI-TOF MS y secuenciación. El aislado fue resistente a fluconazol y susceptible a anfotericina y caspofungina, según puntos de corte recomendados por el CDC. La emergencia de C. auris es alarmante debido a que el modo de transmisión dentro del ambiente hospitalario no es claro y probablemente es multifactorial.

Candida auris is an emerging multi-drug-resistant fungus that is rapidly spreading worldwide. Since the first reports in 2009, many isolates across five continents have been identified as agents of hospital-associated infections. Independent and simultaneous outbreaks of C. auris are becoming a major concern for healthcare and scientific community. Moreover, laboratory misidentification and multi-drug-resistant profiles, rarely observed for other non-albicans Candida species, result in difficult eradication and frequent therapeutic failures of C. auris infections. In this article we present the first case of isolation of a strain of C. auris at a hospital in Santiago, in a patient coming from India, who was admitted for treatment of diabetic foot complications. The strain was recovered from a tissue culture and identified by VITEK® 2 Compact. The accurate identification of C. auris was confirmed by means of MALDI-TOF MS and DNA sequence analysis. The isolate was resistant to fluconazole, retaining only susceptibility to amphotericin and caspofungin with MIC breakpoints recommended by CDC. The emergence of C. auris is alarming because the mode of transmission within the healthcare environment is not clear and is likely to be multifactorial.

Evaluación de MALDI-TOF MS para la identificación de levaduras patógenas oportunistas de muestras clínicas / Evaluation of MALDI-TOF MS for the identification of opportunistic pathogenic yeasts of clinical samples

Resumen La espectrometría de masas MALDI-TOF MS es una técnica rápida y sencilla para identificar microorganismos por análisis proteico. Se estudiaron 304 aislados de levaduras procedentes de micosis superficiales y profundas, con el objetivo de comparar tres métodos: convencional (bioquímico y morfológico), MALDI-TOF MS, y reacción en cadena de la polimerasa (RPC, método de referencia). Se estudiaron 24 especies con predominio de Candida spp y Cryptococcus spp. La identificación por método convencional fue de 258/304 cepas, mientras que por MALDI-TOF MS fue de: 277/304 cepas (84,8 versus 91,2%, p = no significativo). El coeficiente Kappa entre el MALDI-TOF MS y la RPC reportó una excelente concordancia (0,99). La sensibilidad y la especificidad de MALDI-TOF MS para la identificación de levaduras patógenas oportunistas de muestras clínicas fueron de 94,6% y 99%; respectivamente. MALDI-TOF MS demostró ser una herramienta de alta precisión para la identificación de levaduras patógenas.

MALDI-TOF MS mass spectrometry is a rapid and straightforward technique to identify microorganisms by protein analysis. The study was performed in 304 yeast isolates from superficial and deep mycoses, in order to compare three methods: conventional (biochemical and morphological), MALDI-TOF MS, and polymerase chain reaction (PCR, reference). We included 24 species with predominance of Candida spp and Cryptococcus spp. The identification by conventional methods was 258/304 strains, while by MALDI-TOF MS was: 277/304 strains (84.8% versus 91.2%, P = not significant). The Kappa coefficient comparing MALDI-TOF-MS with PCR reported excellent concordance (0.99). The sensitivity and specificity of MALDI-TOF MS for the diagnosis of opportunistic pathogenic yeasts of clinical samples were 94.6% and 99% respectively. MALDI-TOF MS is a simple, fast and reliable tool for pathogenic yeasts.

ERG11 gene polymorphisms and susceptibility to fluconazole in candida isolates from diabetic and kidney transplant patients

Abstract INTRODUCTION: Candidiasis is the most frequent opportunistic mycosis in humans and can cause mortality, particularly in immunodeficient patients. One major concern is the increasing number of infections caused by drug-resistant Candidas trains, as these cannot be efficiently treated with standard therapeutics. The most common mechanism of fluconazole resistance in Candida is mutation of ERG11, a gene involved in the biosynthesis of ergosterol, a compound essential for cell integrity and membrane function. METHODS: Based on this knowledge, we investigated polymorphisms in the ERG11 gene of 3 Candida species isolated from immunocompromised and immunocompetent patients. In addition, we correlated the genetic data with the fluconazole susceptibility profile of the Candida isolates. RESULTS: A total of 80 Candida albicans, 8 Candida tropicalis and 6 Candida glabrata isolates were obtained from the saliva of diabetic, kidney transplant and immunocompetent patients. Isolates were considered susceptible to fluconazole if the minimum inhibitory concentration was lower than 8 μg/mL. The amino acid mutations F105L, D116E, K119N, S137L, and K128T were observed in C. albicans isolates, and T224C and G263A were found in C. tropicalis isolates. CONCLUSIONS: Despite the high number of polymorphisms observed, the mutations occurred in regions that are not predicted to interfere with ergosterol synthesis, and therefore are not related to fluconazole resistance.

Identification and susceptibility of clinical isolates of Candida spp. to killer toxins / Identificação e susceptibilidade de isolados clínicos de Candida spp. para toxinas assassinas

Abstract Although invasive infections and mortality caused by Candida species are increasing among compromised patients, resistance to common antifungal agents is also an increasing problem. We analyzed 60 yeasts isolated from patients with invasive candidiasis using a PCR/RFLP strategy based on the internal transcribed spacer (ITS2) region to identify different Candida pathogenic species. PCR analysis was performed from genomic DNA with a primer pair of the ITS2-5.8S rDNA region. PCR-positive samples were characterized by RFLP. Restriction resulted in 23 isolates identified as C. albicans using AlwI, 24 isolates as C. parapsilosis using RsaI, and 13 as C. tropicalis using XmaI. Then, a group of all isolates were evaluated for their susceptibility to a panel of previously described killer yeasts, resulting in 75% being susceptible to at least one killer yeast while the remaining were not inhibited by any strain. C. albicans was the most susceptible group while C. tropicalis had the fewest inhibitions. No species-specific pattern of inhibition was obtained with this panel of killer yeasts. Metschnikowia pulcherrima, Pichia kluyveri and Wickerhamomyces anomalus were the strains that inhibited the most isolates of Candida spp.

Resumo Embora as infecções invasivas e a mortalidade causada por espécies de Candida estejam aumentando entre pacientes comprometidos, a resistência a agentes antifúngicos comuns também é um problema crescente. Analisamos 60 leveduras isoladas de pacientes com candidíase invasiva utilizando como estratégia PCR/RFLP baseada na região espaçadora transcrita interna (ITS2) para identificar diferentes espécies patogênicas de Candida. A análise por PCR foi realizada a partir de ADN genómico com um par de iniciadores da região ITS2-5.8S rDNA. As amostras PCR-positivas foram caracterizadas por RFLP. A restrição resultou em 23 isolados identificados como C. albicans usando AlwI, 24 isolados como C. parapsilosis usando RsaI e 13 como C. tropicalis usando XmaI. Em seguida, avaliou-se o grupo de todos os isolados quanto à sua susceptibilidade a um painel de leveduras killer previamente descritas, resultando em 75% sendo suscetíveis a pelo menos uma levedura killer, enquanto que as restantes não foram inibidas por qualquer cepa. C. albicans foi o grupo mais suscetível enquanto C. tropicalis teve o menor número de inibições. Não se obteve um padrão de inibição específico da espécie com este painel de leveduras killer. Metschnikowia pulcherrima, Pichia kluyveri e Wickerhamomyces anomalus foram as cepas que inibiram a maioria dos isolados de Candida spp.

Isolation of Candida spp. from denture-related stomatitis in Pará, Brazil

ABSTRACT The aim of this study was to isolate and identify Candida species from the oral cavity of denture wearers with denture-related stomatitis who were attended at the University Federal of Pará (Belém City, Pará State, Brazil). A total of 36 denture wearers with denture-related stomatitis were included, and type I (50%), type II (33%) and type III (17%) stomatitis were observed. Candida spp. were isolated from 89% of the cases and included five different Candida species. C. albicans was the most frequently recovered species (78% of the cases), followed by C. famata and C. tropicalis. We observed a significant association between Candida species isolation and unsatisfactory denture condition (p = 0.0017). Our results demonstrated the highly frequency of Candida species isolation in denture wearers with denture-related stomatitis and showed the relationship between these species and poor denture maintenance.

Variability in the clinical distributions of Candida species and the emergence of azole-resistant non-Candida albicans species in public hospitals in the Midwest region of Brazil

Abstract INTRODUCTION Incidence and antifungal susceptibility of Candida spp. from two teaching public hospitals are described. METHODS The minimum inhibitory concentrations of fluconazole, voriconazole, itraconazole, and amphotericin B were determined using Clinical Laboratory Standard Institute broth microdilution and genomic differentiation using PCR. RESULTS Of 221 Candida isolates, 50.2% were obtained from intensive care unit patients; 71.5% were recovered from urine and 9.1% from bloodstream samples. Candida parapsilosis sensu stricto was the most common candidemia agent. CONCLUSIONS We observed variations in Candida species distribution in hospitals in the same geographic region and documented the emergence of non-C. albicans species resistant to azoles.

Nueva reacción en cadena de la polimerasa múltiple para el diagnóstico específico de especies implicadas en la candidiasis humana / A new multiplex PCR for species-specific diagnosis of human candidiasis

RESUMEN Introducción. Las candidiasis son un grupo de infecciones oportunistas causadas por levaduras del género Candida. Candida albicans es la especie de mayor prevalencia en las infecciones superficiales y profundas. Sin embargo, en la última década, la frecuencia de especies diferentes a C. albicans ha aumentado y, por ende, su relevancia clínica, lo cual exige la utilización de técnicas diagnósticas que permitan su detección y el tratamiento adecuado de los pacientes afectados. Objetivo. Diseñar y optimizar una técnica de reacción en cadena de la polimerasa múltiple (PCR múltiple) considerando parámetros termodinámicos para la detección simultánea de cinco especies de Candida relevantes en la etiología de la candidiasis humana. Materiales y métodos. Para el diseño de los cebadores se consideraron restricciones físicas y termodinámicas que afectan la PCR múltiple, usando el programa Gene Runner y la herramienta Mult-PSOS. Como plantillas se utilizaron la región transcrita interna 2 (ITS2) (AJ249486.1) para C. albicans y la topoisomerasa II (TOPII) para C. parasilopsis (AB049144.1), C. krusei (AB049139.1), C. tropicalis (AB049141.1) y C. guillermondii (AB049145.1), y como moldes, extractos de ADN total obtenidos de cepas ATCC y de aislamientos clínicos de las especies de Candida. Resultados. Se diseñaron diez cebadores para la amplificación simultánea de las especies de Candida. Se obtuvo el siguiente patrón de bandas: C. albicans (206 pb), C. guillermondii (244 pb), C. tropicalis (474 pb), C. parasilopsis (558 pb) y C. krusei (419 pb). Conclusión. El ensayo diseñado de PCR múltiple permitió la amplificación simultánea y eficiente de todos los amplicones correspondientes a las especies estudiadas de Candida, así como su adecuada resolución en gel de agarosa al 1,3 %.

ABSTRACT Introduction: Candidiases is a group of opportunistic infections caused by yeasts belonging to the genus Candida. Candida albicans is the most prevalent species in both superficial and deep infections, however, the clinical importance of non-albicans Candida has increased during the last decade, driving an urgent need for diagnostic tests that allow for species-level resolution and selection of the optimum therapeutic approach. Objective: To design and to optimize a new multiplex PCR assay for the simultaneous identification of the five most relevant species of Candida involved in human candidiasis etiology. Materials and methods: For primers design, the physical and thermodynamic restrictions that affect multiplex PCR performance were analyzed using Gene Runner and Mult-PSOS. As templates, the internal transcribed region 2 (ITR2) was selected for C. albicans (AJ249486.1), and topoisomerase II (TOPII) for C. parasilopsis (AB049144.1), C. krusei (AB049139.1), C. tropicalis (AB049141.1), and C. guillermondii (AB049145.1). We used ATCC strains of all these five species and clinical isolates as templates. Results: We designed ten oligonucleotides for the simultaneous amplification of the Candida species. The electrophoresis band profile was: C. albicans (206 bp), C. guillermondii (244 bp), C. tropicalis (474 bp), C. parasilopsis (558 bp), and C. krusei (419 bp). Conclusion: The new multiplex PCR assay designed in this study allowed a simultaneous and efficient amplification of the amplicons corresponding to the five species of Candida under study, with an adequate resolution in standard agarose gel.

Comparison of four molecular approaches to identify Candida parapsilosis complex species

Since the description of Candida orthopsilosis and C. metapsilosis in 2005, several methods have been proposed to identify and differentiate these species from C. parapsilosis sensu stricto. Species-specific uniplex polymerase chain reaction (PCR) was performed and compared with sequencing of the D1/D2 region of the LSU 28S rDNA gene, microsatellite typing of C. parapsilosis sensu stricto, and PCR-restriction fragment length polymorphism patterns in the ITS1-5.8S-ITS2 region of the rDNA gene. There was agreement between results of testing of 98 clinical isolates with the four PCR-based methods, with 59 isolates identified as C. parapsilosis sensu stricto, 37 as C. orthopsilosis, and two as C. metapsilosis.

Phenotypic and genotypic detection of Candida albicans and Candida dubliniensis strains isolated from oral mucosa of AIDS pediatric patients

ABSTRACT The aim of this study was to assess a collection of yeasts to verify the presence of Candida dubliniensis among strains isolated from the oral mucosa of AIDS pediatric patients which were initially characterized as Candida albicans by the traditional phenotypic method, as well as to evaluate the main phenotypic methods used in the discrimination between the two species and confirm the identification through genotypic techniques, i.e., DNA sequencing. Twenty-nine samples of C. albicans isolated from this population and kept in a fungi collection were evaluated and re-characterized. In order to differentiate the two species, phenotypic tests (Thermotolerance tests, Chromogenic medium, Staib agar, Tobacco agar, Hypertonic medium) were performed and genotypic techniques using DNA sequencing were employed for confirmation of isolated species. Susceptibility and specificity were calculated for each test. No phenotypic test alone was sufficient to provide definitive identification of C. dubliniensis or C. albicans, as opposed to results of molecular tests. After amplification and sequencing of specific regions of the 29 studied strains, 93.1% of the isolates were identified as C. albicans and 6.9% as C. dubliniensis. The Staib agar assay showed a higher susceptibility (96.3%) in comparison with other phenotypic techniques. Therefore, genotypic methods are indispensable for the conclusive identification and differentiation between these species.

Analysis of the genetic diversity of Candida isolates obtained from diabetic patients and kidney transplant recipients

Yeasts of the genus Candida have high genetic variability and are the most common opportunistic pathogenic fungi in humans. In this study, we evaluated the genetic diversity among 120 isolates of Candida spp. obtained from diabetic patients, kidney transplant recipients and patients without any immune deficiencies from Paraná state, Brazil. The analysis was performed using the ITS1-5.8S-ITS2 region and a partial sequence of 28S rDNA. In the phylogenetic analysis, we observed a consistent separation of the species C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. parapsilosis, C. metapsilosis and C. orthopsilosis, however with low intraspecific variability. In the analysis of the C. albicans species, two clades were formed. Clade A included the largest number of isolates (91.2%) and the majority of isolates from GenBank (71.4%). The phylogenetic analysis showed low intraspecific genetic diversity, and the genetic polymorphisms between C. albicans isolates were similar to genetic divergence found in other studies performed with isolates from Brazil. This low genetic diversity of isolates can be explained by the geographic proximity of the patients evaluated. It was observed that yeast colonisation was highest in renal transplant recipients and diabetic patients and that C. albicans was the species most frequently isolated.

Susceptibility and molecular characterization of Candida species from patients with vulvovaginitis

Abstract Vulvovaginal candidiasis affects women of reproductive age, which represents approximately 15–25% of vaginitis cases. The present study aimed to isolate and characterize yeast from the patients irrespective of the presentation of clinical symptoms. The isolates were subjected to in vitro susceptibility profile and characterization by molecular markers, which intended to assess the distribution of species. A total of 40 isolates were obtained and identified through the CHROMagar, API20aux and by ITS and D1/D2 regions sequencing of DNAr gene. Candida albicans strains were genotyped by the ABC system and the isolates were divided into two genotypic groups. The identity of the C. albicans, C. glabrata, C. guilliermondii, C. kefyr and Saccharomyces cerevisiae isolates was confirmed by the multilocus analysis. The strains of Candida, isolated from patients with complications, were found to be resistant to nystatin but sensitive to fluconazole, amphotericin B and ketoconazole, as observed by in vitro sensitivity profile. The isolates from asymptomatic patients, i.e., the colonized group, showed a dose-dependent sensitivity to the anti-fungal agents, fluconazole and amphotericin B. However, the isolates of C. albicans that belong to distinct genotypic groups showed the same in vitro susceptibility profile.

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.

Rapid detection of Candida species in bronchoalveolar lavage fluid from patients with pulmonary symptoms

Abstract Candida species, especially C. albicans, are commensals on human mucosal surfaces, but are increasingly becoming one of the important invasive pathogens as seen by a rise in its prevalence in immunocompromised patients and in antibiotic consumption. Thus, an accurate identification of Candida species in patients with pulmonary symptoms can provide important information for effective treatment. A total of 75 clinical isolates of Candida species were obtained from the bronchoalveolar lavage fluid of both immunocompromised and immunocompetent patients with pulmonary symptoms. Candida cultures were identified based on nuclear ribosomal Internal Transcribed Spacer (ITS1-ITS2 rDNA) sequence analysis by polymerase chain reaction–restriction fragment length polymorphisms (PCR-RFLP). Molecular identification indicated that the isolates belonged predominantly to C. albicans (52%), followed by C. tropicalis (24%), C. glabrata (14.7%), C. krusei (5.3%), C. parapsilosis (1.3%), C. kefyr (1.3%) and C. guilliermondii (1.3%). Given the increasing complexity of disease profiles and their management regimens in diverse patients, rapid and accurate identification of Candida species can lead to timely and appropriate antifungal therapy.

Novel point mutations in the ERG11 gene in clinical isolates of azole resistant Candida species

The azoles are the class of medications most commonly used to fight infections caused by Candida sp. Typically, resistance can be attributed to mutations in ERG11 gene (CYP51) which encodes the cytochrome P450 14α-demethylase, the primary target for the activity of azoles. The objective of this study was to identify mutations in the coding region of theERG11 gene in clinical isolates of Candidaspecies known to be resistant to azoles. We identified three new synonymous mutations in the ERG11 gene in the isolates of Candida glabrata (C108G, C423T and A1581G) and two new nonsynonymous mutations in the isolates of Candida krusei - A497C (Y166S) and G1570A (G524R). The functional consequence of these nonsynonymous mutations was predicted using evolutionary conservation scores. The G524R mutation did not have effect on 14α-demethylase functionality, while the Y166S mutation was found to affect the enzyme. This observation suggests a possible link between the mutation and dose-dependent sensitivity to voriconazole in the clinical isolate of C. krusei. Although the presence of the Y166S in phenotype of reduced azole sensitivity observed in isolate C. kruseidemands investigation, it might contribute to the search of new therapeutic agents against resistant Candida isolates.

Analysis of the Vaginal Microbiome by Next-Generation Sequencing and Evaluation of its Performance as a Clinical Diagnostic Tool in Vaginitis

BACKGROUND: Next-generation sequencing (NGS) can detect many more microorganisms of a microbiome than traditional methods. This study aimed to analyze the vaginal microbiomes of Korean women by using NGS that included bacteria and other microorganisms. The NGS results were compared with the results of other assays, and NGS was evaluated for its feasibility for predicting vaginitis. METHODS: In total, 89 vaginal swab specimens were collected. Microscopic examinations of Gram staining and microbiological cultures were conducted on 67 specimens. NGS was performed with GS junior system on all of the vaginal specimens for the 16S rRNA, internal transcribed spacer (ITS), and Tvk genes to detect bacteria, fungi, and Trichomonas vaginalis. In addition, DNA probe assays of the Candida spp., Gardnerella vaginalis, and Trichomonas vaginalis were performed. Various predictors of diversity that were obtained from the NGS data were analyzed to predict vaginitis. RESULTS: ITS sequences were obtained in most of the specimens (56.2%). The compositions of the intermediate and vaginitis Nugent score groups were similar to each other but differed from the composition of the normal score group. The fraction of the Lactobacillus spp. showed the highest area under the curve value (0.8559) in ROC curve analysis. The NGS and DNA probe assay results showed good agreement (range, 86.2-89.7%). CONCLUSIONS: Fungi as well as bacteria should be considered for the investigation of vaginal microbiome. The intermediate and vaginitis Nugent score groups were indistinguishable in NGS. NGS is a promising diagnostic tool of the vaginal microbiome and vaginitis, although some problems need to be resolved.

FREQUENCY OF Candida SPECIES IN A TERTIARY CARE HOSPITAL IN TRIANGULO MINEIRO, MINAS GERAIS STATE, BRAZIL / Frequência de espécies de Candida em hospital terciário do Triângulo Mineiro, Minas Gerais, Brasil

Infections by Candida species are a high-impact problem in public health due to their wide incidence in hospitalized patients. The goal of this study was to evaluate frequency, susceptibility to antifungals, and genetic polymorphism of Candida species isolated from clinical specimens of hospitalized patients. The Candida isolates included in this study were obtained from blood cultures, abdominal fluids, and central venous catheters (CVC) of hospitalized patients at the Clinical Hospital of the Federal University of Uberlândia during the period of July 2010 - June 2011. Susceptibility tests were conducted by the broth microdilution method. The RAPD-PCR tests used employed initiator oligonucleotides OPA09, OPB11, and OPE06. Of the 63 Candida isolates, 18 (28.5%) were C. albicans, 20 (31.7%) were C. parapsilosis complex species, 14 (22.2%) C. tropicalis, four (6.4%) C. glabrata, four (6.4%) C. krusei, two (3.3%) C. kefyr, and one (1.6%) C. lusitaniae. In vitro resistance to amphotericin B was observed in 12.7% of isolates. In vitro resistance to azoles was not detected, except for C. krusei. The two primers, OPA09 and OPB11, were able to distinguish different species. Isolates of C. albicans and C. parapsilosis complex species presented six and five clusters, respectively, with the OPA09 marker by RAPD-PCR, showing the genetic variability of the isolates of those species. It was concluded that members of the C. parapsilosis complex were the most frequent species found, and most isolates were susceptible to the antifungals amphotericin B, flucozanole, and itraconazole. High genetic polymorphisms were observed for isolates of C. albicans and C. parapsilosis complex species, mainly with the OPA09 marker.

As infecções causadas por espécies de Candida são problema de grande impacto para a saúde pública, devido à alta incidência em pacientes hospitalizados e como causa de mortalidade. O presente estudo teve como objetivo avaliar a frequência de Candida spp. isoladas de pacientes hospitalizados, assim como a sensibilidade aos antifúngicos e o polimorfismo genético por RAPD-PCR. Os microrganismos incluíram isolados de hemocultura, líquido abdominal e ponta de cateter venoso central de pacientes internados no Hospital de Clínicas da Universidade Federal de Uberlândia, região do Triângulo Mineiro, Minas Gerais, Brasil, no período de julho de 2010-junho de 2011. Os testes de sensibilidade aos antifúngicos foram realizados por microdiluição em caldo e na análise por RAPD-PCR foram utilizados os oligonucleotídeos OPA09, OPB11, e OPE06. Dos 63 isolados, 18 (28,5%) foram C. albicans, 20 (31,7%) C. parapsilosis, 14 (22,2%) C. tropicalis, quatro (6,4%) C. glabrata, quatro (6,4%) C. krusei, dois (3,3%) C. kefyr, e um (1,6%) C. lusitaniae. Resistência in-vitro à anfotericina B foi observada em 12,7% dos isolados. Não foi observada resistência in-vitro aos azólicos, exceto para os isolados de C. krusei. Os oligonucleotídeos OPA09 e OPB11 possibilitaram distinguir diferentes espécies. Isolados de C. albicans apresentaram seis clusters e o complexo C. parapsilosis, cinco clusters, com o iniciador OPA09, por RAPD-PCR, mostrando a variabilidade genética daquelas espécies. Conclui-se que o complexo C. parapsilosis foi a espécie mais frequente, e a maioria dos isolados foi sensível in vitro aos antifúngicos testados. Alto polimorfismo genético foi observado para os isolados de C. albicans e complexo C. parapsilosis, principalmente com o oligonucleotídeo OPA09.

Species identification and antifungal susceptibility profile of candida isolates from ICU patients in Sohag University hospital, Upper Egypt

Background: Candida species are among the most common fungal pathogens in ICU patients. Candida albicans was the predominant species, but a shift toward non-albicans Candida species has been recently observed

Objectives: To detect the prevalence of different Candida species and determine their antifungal susceptibility profile in ICU patients using phenotypic methods, the Vitek 2 system compared with CHROMagar Candida agar and a genotypic method; PCR-RFLP

Methodology: Various clinical samples were collected from 248 ICU patients in Sohag University Hospital from the period between September 2014 and May 2015. Samples were cultured on CHROMagar Candida agar. Results were compared with those of Vitek 2 system and confirmed by PCR- RFLP method and antifungal susceptibility profiles were analyzed by disc diffusion and Vitek 2 antifungal susceptibility tests

Results: The study revealed an overall isolation rate of Candida species among ICU patients was 29% by PCR-RFLP. Candida albicans was the most frequent species isolated [40.3%]. Non- albicans Candida species including Candida tropicalis [22.2%], Candida glabrata [18%], Candida krusei [12.5%], C. parapsilosis [4.2%], C. dubliniensis [1.4%] and Candida guilliermondii [1.4%] were also isolated. The sensitivity of vitek 2 with regard to correct identification of Candida species was 96%; the specificity was 100%, also CHROMagar Candida agar enable the correct identification with sensitivity 89%, specificity 100%. Vitek 2 antifungal susceptibility tests results were found to be an accurate method as it was compared with the disc diffusion method for fluconazole, voriconazole and amphotracin B

Conclusion: CHROMagar Candida agar supported by Vitek 2 system is a valuable method for identification of common Candida species, these methods are easy to interpret and give rapid results in comparison with the expensive PCR-RFLP method. Although amphotericin B and fluconazole are widely used in clinical practice, there was no evidence of enhanced resistance. Moreover, voriconazole could be used in treatment of fluconazole-resistant Candida species

Three Cases of Candidiasis Misidentified as Candida famata by the Vitek 2 System

No abstract available.

Candida species biofilm and Candida albicans ALS3 polymorphisms in clinical isolates

Over the last decades, there have been important changes in the epidemiology of Candida infections. In recent years, Candida species have emerged as important causes of invasive infections mainly among immunocompromised patients. This study analyzed Candida spp. isolates and compared the frequency and biofilm production of different species among the different sources of isolation: blood, urine, vulvovaginal secretions and peritoneal dialysis fluid. Biofilm production was quantified in 327 Candida isolates obtained from patients attended at a Brazilian tertiary public hospital (Botucatu, Sao Paulo). C. albicans ALS3 gene polymorphism was also evaluated by determining the number of repeated motifs in the central domain. Of the 198 total biofilm-positive isolates, 72 and 126 were considered as low and high biofilm producers, respectively. Biofilm production by C. albicans was significantly lower than that by non-albicans isolates and was most frequently observed in C. tropicalis. Biofilm production was more frequent among bloodstream isolates than other clinical sources,in urine, the isolates displayed a peculiar distribution by presenting two distinct peaks, one containing biofilm-negative isolates and the other containing isolates with intense biofilm production. The numbers of tandem-repeat copies per allele were not associated with biofilm production, suggesting the evolvement of other genetic determinants.