gDNA extraction from Candida albicans and Candida dubliniensis in subgingival samples in Argentina. Evaluation of different methods / Evaluación de diferentes métodos de extracción de gADN en Candida albicans y Candida dubliniensis en muestras subgingivales en Argentina

ABSTRACT The oral cavity constitutes a unique ecosystem with highly variable ecological niches that harbor a great variety of microorganisms, including yeasts. Molecular methods are currently considered the gold standard for identifying species, although they involve limitations associated with the disruption of yeast cell walls to release the genomic DNA (gDNA) for amplification. Aim: The aim of this study was to compare the performance of different methods for extracting gDNA from Candida albicans and Candida dubliniensis, subsequently amplifying DNA by PCR. Materials and Method: Fifty-two isolates (16 C. albicans and 36 C. dubliniensis) were obtained from subgingival biofilm of HIV+ patients with clinical signs of periodontal disease. The study evaluated 6 gDNA extraction methods and two PCR amplification methods. Furthermore, the presence of alleles of HWP1 gene was determined in C. albicans. Results: Comparisons of six methods show statistically significant differences (p<0.001) except for C. albicans in two of them. For C. dubliniensis, statistical differences were observed in all comparisons. Commercial methods were more efficient for concentrating gDNA than in-house methods, and both PCRs were effective. Ten heterozygous C. albicans isolates for this allele were positive for the HWP1-1 / HWP1-2 allele, one was homozygous for Wild Type HWP1-1 allele, and 5 were homozygous for novel/rare HWP1-2 allele. Conclusions: This study aims to provide simple, inexpensive strategies for phenotypic identification and molecular confirmation of Candida albicans and Candida dubliniensis for non-reference laboratories with low complexity and/or low budgets.

RESUMEN La cavidad oral constituye un ecosistema único con nichos ecológicos muy variables, capaz de albergar una gran variedad de microorganismos, incluidas las levaduras. Los métodos moleculares son considerados actualmente los métodos de identificación definitivos ya que a diferencia de los anteriores, nos brindan una correcta sensibilidad y especificidad. Sin embargo, existen limitaciones asociadas con la ruptura de las paredes celulares de estas levaduras para liberar el ADN genómico (gADN) necesario para la amplificación. Objetivo: El objetivo de este estudio fue comparar el rendimiento de diferentes métodos de extracción de gADN de Candida albicans y Candida dubliniensis, amplificando posteriormente por PCR. Materiales y Método: Se estudiaron 52 aislamientos, 16/52 de Candida albicans y 36/52 de Candida dubliniensis obtenidos de biofilm subgingival de pacientes VIH+ con signos clínicos de enfermedad periodontal. Se evaluaron seis métodos de extracción de gADN y la posterior amplificación se realizó por dos técnicas de PCR. Además en C. albicans se determinó la presencia de alelos para el gen HWP1. Resultados: Las comparaciones de seis métodos son estadísticamente significativas (p<0,001) excepto para C. albicans en dos de ellos. Para C. dubliniensis se observaron diferencias estadísticas en todas las comparaciones. Los métodos comerciales mostraron una mayor eficiencia en la concentración de gADN que los métodos caseros y ambos fueron efectivos en las dos PCR. 10 aislados de C. albicans resultaron positivos para el alelo HWP1-1/HWP1-2, siendo heterocigotos para este alelo. Solo un aislamiento fue homocigoto para el alelo HWP1-1 de tipo salvaje y 5 eran homocigotos para el alelo HWP1-2 nuevo/raro. Conclusiones: Este estudio tiene como objetivo proporcionar estrategias simples y económicas para la identificación fenotípica y confirmación molecular de Candida albicans y Candida dubliniensis para laboratorios de no referencia con baja complejidad y/o bajo presupuesto económico.

Antifungal activity of synthetic antiseptics and natural compounds against Candida dubliniensis before and after in vitro fluconazole exposure

ABSTRACT INTRODUCTION: This study evaluated the susceptibilities of oral candidiasis-derived Candida albicans, fluconazole-resistant (FR) Candida dubliniensis, and fluconazole-susceptible (FS) C. dubliniensis to synthetic antiseptics [chlorhexidine gluconate (CHX), cetylpyridinium chloride (CPC), and triclosan (TRC)] and natural compounds (carvacrol, eugenol and thymol). METHODS: Susceptibility tests were performed based on the M27-A3 reference method. The fluconazole-resistant C. dubliniensis strains were obtained after prolonged in vitro exposure to increasing fluconazole concentrations. The geometric mean values for minimum inhibitory concentrations and minimum fungicidal concentrations were compared among the groups. RESULTS: Fluconazole-susceptible C. dubliniensis was more sensitive to CPC and TRC than FR C. dubliniensis and C. albicans were. However, eugenol and thymol were more active against FR C. dubliniensis. The fungicidal activities of CHX and TRC were similar for the three groups, and FR C. dubliniensis and C. albicans had similar sensitivities to CPC. CONCLUSIONS: The resistance of C. dubliniensis to fluconazole affects its sensitivity the synthetic antiseptics and natural compounds that were tested.

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.

Differentiation of Candida albicans and Candida dubliniensis using Latex Agglutination Test / 대한의진균학회지

BACKGROUND: Candida dubliniensis is phenotypically similar to Candida albicans that may be underdiagnosed in clinical laboratory. In 2010, C. dubliniensis was first recovered from blood of a candidemia patient in Seoul, Korea. Also, a simple commercial latex agglutination (LA) test is available. OBJECTIVE: The aim of the present study was to investigate the prevalence of C. dubliniensis among isolates in our stocks during 2-years period (2010-2011) and to evaluate the ability of LA test (Bichro-Dubli Fumouze®) for the differentiation of C. albicans and C. dubliniensis. METHODS: A total 509 isolates including 504 C. albicans and 5 C. dubliniensis were examined for LA test, the presence of “spiking” on blood agar plate, and the germ tube test. Also all isolates were tested using the VITEK 2 ID-YST system. RESULTS: No C. dubliniensis was found in 504 isolates of initially identified as C. albicans. The LA test was positive only in 5 clinical isolates and 2 type strains of C. dubliniensis. CONCLUSIONS: The data show that the prevalence of C. dubliniensis in Korea is still expected to be extremely low and LA test is very rapid, simple, and reliable tool for the differentiation of C. albicans and C. dubliniensis.

Fluconazole and amphotericin-B resistance are associated with increased catalase and superoxide dismutase activity in Candida albicans and Candida dubliniensis

Introduction Candida dubliniensis, a new species of Candida that has been recovered from several sites in healthy people, has been associated with recurrent episodes of oral candidiasis in AIDS and HIV-positive patients. This species is closely related to C. albicans. The enzymatic activity of C. dubliniensis in response to oxidative stress is of interest for the development of drugs to combat C. dubliniensis. Methods Fluconazole- and amphotericin B-resistant strains were generated as described by Fekete-Forgács et al. (2000). Superoxide dismutase (SOD) and catalase assays were performed as described by McCord and Fridovich (1969) and Aebi (1984), respectively. Results We demonstrated that superoxide dismutase (SOD) and catalase activities were significantly higher (p<0.05) in the fluconazole- and amphotericin B-resistant strains of C. dubliniensis and C. albicans than in the sensitive strains. The catalase and SOD activities were also significantly (p<0.01) higher in the sensitive and resistant C. albicans strains than in the respective C. dubliniensis strains. Conclusions These data suggest that C. albicans is better protected from oxidative stress than C. dubliniensis and that fluconazole, like amphotericin B, can induce oxidative stress in Candida; oxidative stress induces an adaptive response that results in a coordinated increase in catalase and SOD activities. .

Utilização da microespectroscopia infravermelha (FT-IR) para teste de algoritmos estatísticos na diferenciação dos micro-organismos Candida albicans, Candida dubliniensis e Candida parapsilosis / Using FT-IR microspectroscopy for testing statistical algorithms for differentiation of Candida albicans, Candida dubliniensis and Candida parapsilosis

As espécies do gênero Candida são causadoras de diversas infecções fúngicas e, nos últimos anos, tem sido desenvolvidas novas tecnologias para auxiliar nos diagnósticos microbiológicos. Dentre as técnicas está a espectroscopia infravermelha junto com a análise estatística multivariada. O objetivo deste trabalho é comparar dois métodos: estatístico (análise multivariada) e não-estatístico (ajuste de curva), utilizando os espectros infravermelhos de Candida albicans, Candida dubliniensis e Candida parapsilosis para testar o potencial do uso de Análise Estatística Multivariada para discriminação de espectros de micro-organismos. Para isso foram obtidos, utilizando o Spectrum Spotlight 400 da PerkinElmer, 54 espectros infravermelhos, sendo 18 de cada espécie, na faixa de 4000 a 1000 cm-1, com resolução de 4 cm-1, no modo de transmissão, a 20 ºC. A análise dos espectros foi realizada através de três métodos: (1) inspeção visual direta dos espectros; (2) análise estatística multivariada; (3) ajuste de curva para a determinação de estruturas secundárias de proteínas. Na região de 1200 a 1000 cm-1, os espectros apresentam diferenças que podem ser percebidas numa inspeção visual direta. Uma banda próxima de 1070 cm-1 e outra próxima de 1045 cm-1 apresentam intensidades relativas diferentes para os três espectros. Por outro lado, as bandas da amida I, na região de 1710 a 1590 cm-1, apresentam aspectos visuais semelhantes com máximo em 1651 cm-1 para os espectros dos três micro-organismos. Esse fato torna possível submeter a análise estatística multivariada a um teste de sua capacidade de diferenciar três espectros de Candida. A análise estatística multivariada foi aplicada aos 54 espectros para investigar as regiões de 4000 a 1000 cm-1 com exceção da região de 2600 a 2300 cm-1 e de 1710 a 1590 cm-1 que corresponde a das bandas da amida I. A técnica selecionada foi a análise por componentes principais (PCA, Principal Componente Analysis), utilizando os primeiros quatro componentes principais, em conjunto com a técnica hierárquica de análise de agrupamento (HCA, Hierarchical Clustering Analysis) segundo o método de Ward. Foi utilizado para esta análise o software MINITAB 15 e o resultado mostra uma clara discriminação dos espectros dos três micro-organismos nas duas regiões consideradas. Adicionalmente foi obtido o espectro médio de cada micro-organismo nas bandas da amida I na região de 1710 a 1590 cm-1. Os três espectros médios assim obtidos foram analisados pelo método de ajuste de curva que não é estatístico para determinar as estruturas secundárias de proteínas. Para esta análise o software ORIGIN 7.5 foi utilizado e os resultados obtidos mostram estruturas conformacionais diferentes nos três micro-organismos. Esses resultados confirmam a discriminação obtida através da análise estatística multivariada e visual. Pode-se concluir que as análises estatísticas multivariadas baseadas em análise por componentes principais e análise de agrupamento com uso do algoritmo Ward é potencialmente útil para discriminar micro-organismos através de seus espectros infravermelhos. Além disso, as análises mostram que as bandas da amida I dos espectros infravermelhos de Candida albicans, Candida dubliniensis e Candida parapsilosis fornecem um conjunto de dados cuja estrutura de agrupamento é conhecida e que pode ser útil para testar e validar algoritmos estatísticos de análise de agrupamento.

Films of Candida albicans, Candida dubliniensis and Candida parapsilosis were prepared and the infrared spectra of these films were obtained in the region 4000 to 1000 cm-1, with resolution of 4 cm-1, in the transmission mode, at 20 ºC. Fifty four spectra were obtained, 18 of each microorganism, with the PerkinElmer Spotlight 400 FT-IR, which has a microscope attached to a FT-IR spectrophotometer. The spectra were analyzed through three methods: (1) mere visual inspection; (2) multivariate statistical analysis; (3) curve-fitting for determining secondary structures of proteins. In the region 1200 to 1000 cm-1, the spectral bands show differences that can be seen by a mere visual inspection. On the other hand, the amide I bands, in the region 1710 to 1590 cm-1, have the same visual aspect for the three microorganisms. Multivariate statistical analysis was applied to analyze these amide I bands of all the 54 spectra. Principal component analysis (PCA) and techniques of hierarchical cluster analysis (HCA, Hierarchical Clustering Analysis) according to Ward's method were applied using the software MINITAB 15. The results show a clear discrimination of the three microorganisms. The average spectrum of each microorganism was obtained in the amide I band. Each average spectrum was analyzed by curve-fitting for the determination of secondary structures of proteins. The software used was the ORIGIN 7.5 and the results confirm the discrimination obtained through multivariate statistical analysis. This result shows that multivariate statistical analysis can be useful to discriminate infrared spectra of different microorganisms. Furthermore, this work shows that the amide I bands of the infrared spectra of Candida albicans, Candida dubliniensis, and Candida parapsilosis provide a set of data of known group structure that can be useful to test statistical algorithms of cluster analysis.

A simple and reliable PCR-restriction fragment length polymorphism assay to identify Candida albicans and its closely related Candida dubliniensis

Candida dubliniensis is an emerging pathogen capable of causing superficial as well as systemic infections. Due to its close similarity to C. albcians, conventional methods based on phenotypic traits are not always reliable in identification of C. dubliniensis. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay to identify and discriminate between the two closely related species. The D1/D2 region of 28S rDNA was amplified by PCR and enzymatically digested by ApaI and BsiEI respectively. PCR products of both species were digested into two fragments by ApaI, but those of other yeast species were undigested. BsiEI cut the PCR products of C. albicans into two fragments but not those of C. dubliniensis. Thus two species were differentiated. We evaluated 10 reference strains representing 10 yeast species, among which C. albicans and C. dubliniensis were successfully identified. A total of 56 phenotypically characterized clinical isolates (42 C. albicans isolates and 14 C. dubliniensis isolates) were also investigated for intra-species variability. All tested isolates produced identical RFLP patterns to their respective reference strains except one initially misidentified isolate. Our method offers a simple, rapid and reliable molecular method for the identification of C. albicans and C. dubliniensis.

Detección e identificación de Candida dubliniensis por métodos fenotípicos en estomatitis sub-protésica: reporte de un caso / Detection and identification of methods for phenotypic Candida dubliniensis in denture stomatitis: report of case

La Estomatitis Sub-Protésica es una afección comúnmente observada en sujetos portadores de prótesis dentales removibles. Son numerosos los reportes publicados en los que se asocia esta patología con la presencia de hongos del Género Candida, y particularmente con Candida albicans. No obstante, algunas cepas identificadas como C. albicans, pudieran no tratarse en realidad de este microorganismo sino de Candida dubliniensis, más aún si se toma en cuenta que ambas especies producen clamidoconidias. Es por ello que resulta necesario comparar distintos métodos fenotípicos que permitan diferenciar en buena medida a ambas especies. En este artículo se describe el caso de una paciente portadora de prótesis removible superior con diagnóstico clínico de Estomatitis Sub-Protésica, a quien se le tomó muestras del paladar afectado y de la prótesis implicada. Se emplearon diferentes métodos fenotípicos de identificación diferencial de ambas especies, entre estos: CHROMagar Candida, Agar tabaco, Agar Pal´s, Agar Tween 80, medio de tomate y zanahoria y de Agar Sabouraud con NaCl al 6,5% (P/V). También se realizaron la pruebas de termotolerancia a 45ºC, aglutinación en latex, así como la identificación a través del sistema ID 32C (bioMerieux). Una vez empleados los métodos antes mencionados, la especie encontrada en el paladar de la paciente fue C. dubliniensis, en tanto que la que se halló en la prótesis fue C. albicans, siendo esta la primera vez que se detecta e identifica en nuestro país a C.dubliniensis en pacientes que presentan esta patología bucal.

Denture stomatitis is a condition commonly observed in subjects with removable dentures. There are numerous reports published in which this disease is associated with the presence of fungi of the genus Candida, particularly Candida albicans. However, some strains identified as C. albicans, may not actually be this organism but Candida dubliniensis, especially if one takes into account that both species produce clamidoconidias. That is why it is necessary to compare different phenotypic methods to differentiate largely to both species. This article describes the case of a patient with upper removable prosthesis with a clinical diagnosis of Denture Stomatitis, whom he took samples of the palate is affected and the prosthesis involved. Different methods were used phenotypic differential identification of both species, among them: CHROMagar Candida Agar snuff, Pal's Agar, Tween 80 Agar, tomato and carrot medium, and Sabouraud Agar with 6.5% NaCl (P / V ). Also thermotolerance tests performed at 45 ° C, latex agglutination, and identification by ID 32C system (bioMerieux) were used. Once employed the above methods, the species found in the mouth of the patient was C. dubliniensis, whereas that was found in the prosthesis was C. albicans, marking the first time it is detected and identified in our country C.dubliniensis in patients with this oral pathology.

The First Korean Case of Candidemia due to Candida dubliniensis

Candidemia due to uncommon Candida spp. appears to be increasing in incidence. C. dubliniensis has been increasingly recovered from individuals not infected with HIV. Identification of C. dubliniensis can be problematic in routine clinical practice due to its phenotypic resemblance to C. albicans. We report the first case of C. dubliniensis candidemia in Korea, which occurred in a 64-yr-old woman who presented with partial seizure, drowsiness, and recurrent fever. Germ-tube positive yeast that was isolated from blood and central venous catheter tip cultures formed smooth, white colonies on sheep blood agar and Sabouraud agar plates, indicative of Candida spp. C. dubliniensis was identified using the Vitek 2 system (bioMerieux, USA), latex agglutination, chromogenic agar, and multiplex PCR. The blood isolate was susceptible to flucytosine, fluconazole, voriconazole, and amphotericin B. After removal of the central venous catheter and initiation of fluconazole treatment, the patient's condition gradually improved, and she was cleared for discharge from our hospital. Both clinicians and microbiologists should be aware of predisposing factors to C. dubliniensis candidemia in order to promote early diagnosis and appropriate treatment.

Differentiation of Candida dubliniensis from Candida albicans with the use of killer toxins / Avaliação das toxinas killer na diferenciação entre Candida albicans e Candida dubliniensis

The aim of this study was to report the ability of killer toxins, previously used as biotyping techniques, as a new tool to differentiate C. albicans from C. dubliniensis. The susceptibility of C. albicans and C. dubliniensis to killer toxins ranged from 33.9 to 93.3 percent and from 6.67 to 93.3 percent, respectively.

Avaliou-se a capacidade das toxinas killer, previamente utilizadas na biotipagem de C. albicans, como método para diferenciar C. albicans de C. dubliniensis. A susceptibilidade de C. albicans e C. dubliniensis às toxinas killer variou de 33,9 por cento a 93,3 por cento para C. albicans e de 6,67 por cento a 93,3 por cento para C. dubliniensis.

Candida dubliniensis does not show phospholipase activity: true or false? / Candida dubliniensis não evidencia atividade fosfolipásica: verdadeiro ou falso?

INTRODUCTION: The phospholipase activity in Candida albicans and Candida dubliniensis isolated from oral candidiasis cases were studied. METHODS: The phospholipase activity was evaluated in egg yolk agar. RESULTS: All the C. albicans isolates (n = 48) showed phospholipase activity (mean Pz = 0.66). However, none of the C. dubliniensis isolates (n = 24) showed this activity. CONCLUSIONS: The authors discuss whether these findings are a true characteristic of C. dubliniensis or a consequence of the methodology employed, which includes the possibility that NaCl may have inhibited the enzymatic activity of C. dubliniensis.

INTRODUÇÃO: Avaliou-se a atividade fosfolipásica em Candida albicans e Candida dubliniensis isoladas de casos de candidíase oral. MÉTODOS: A atividade de fosfolipase foi avaliada em ágar gema de ovo. RESULTADOS: Todos os isolados de C. albicans (nº = 48) evidenciaram atividade fosfolipásica (média Pz = 0.66). Todavia, nenhum isolado de C. dubliniensis (nº= 24) demonstrou esta atividade. CONCLUSÕES: Os autores discutem se estes achados são uma característica verdadeira de C. dubliniensis ou uma conseqüência da metodologia empregada, a qual inclui a possibilidade de que o NaCl seja inibidor da atividade enzimática de C. dubliniensis.

Screening of Candida dubliniensis from Respiratory Samples in Korea / 대한의진균학회지

BACKGROUND: Candida dubliniensis is newly described yeast that is a close phylogenetic relative of C. albicans and isolates mainly from the oral cavity. OBJECTIVE: The aim of the present study was to screen for C. dubliniensis using the 'spiking' appearance on a blood agar plate (BAP), germ tube test with human pooled serum (HPS) and fetal bovine serum (FBS) and to investigate the prevalence of C. dubliniensis from respiratory samples in Korea. METHODS: A total 434 isolates of Candida spp. were examined for the presence of 'spiking' on BAP and the germ tube test with HPS and FBS. Also all isolates were tested using the VITEK 2 ID-YST system. RESULTS: No C. dubliniensis was found in the study population. C. albicans was the most frequently isolated species (74.9%). CONCLUSIONS: No C. dubliniensis was identified in our study. Further large-scale studies are needed to isolate and to confirm the prevalence of C. dubliniensis.

Appropriate Condition of Germ Tube Formation as Presumptive Identification Test for Candida albicans / 대한의진균학회지

BACKGROUND: Candida albicans is the most prevalent species found in human yeast infections. The germ tube test is still frequently used for its rapid presumptive identification. Recently Candida dubliniensis as well as C. albicans has been reported to form germ tubes. OBJECTIVE: The purpose of this study was to evaluate the germ tube test at various conditions for rapid presumptive identification of C. albicans. METHODS: C. albicans ATCC 14053, C. albicans ATCC 18804, C. dubliniensis ATCC MYA 646, and C. dubliniensis KCTC 17427 were tested. Human pooled serum (HPS), HBV, HCV infected patient serum, fetal bovine serum (FBS) and rabbit serum (RS) were used for germ tube test. The germ tube formation was evaluated at different keeping condition of various sera, after mixing with 5 different bacterial suspensions and at various incubation conditions. RESULTS: The germ tube formation of C. albicans was more in the RS or FBS than in the HPS. For the various sera fresh sample was always the best expression of germ-tube forming ability. In the HCV infected patient serum and mixing with Pseudomonas aeruginosa germ tube formation was suppressed. C. dubliniensis did not form germ tube in the HPS, only formed in the FBS or RS. CONCLUSION: For rapid presumptive identification of C. albicans not C. dubliniensis the best selection of serum is the fresh HPS. We recommend the examination with isolated colony free from bacteria after incubation for 2 to 3 hours.

Enzymatic and hemolytic activities of Candida dubliniensis strains

Candida dubliniensis is an opportunistic yeast that has been recovered from several body sites in many populations; it is most often recovered from the oral cavities of human immunodeficiency virus-infected patients. Although extensive studies on epidemiology and phylogeny of C. dubliniensis have been performed, little is known about virulence factors such as exoenzymatic and hemolytic activities. In this study we compared proteinase, hyaluronidase, chondroitin sulphatase and hemolytic activities in 18 C. dubliniensis and 30 C. albicans strains isolated from AIDS patients. C. albicans isolates produced higher amounts of proteinase than C. dubliniensis (p < 0.05). All the tested C. dubliniensis strains expressed hyaluronidase and chondroitin sulphatase activities, but none of them were significantly different from those observed with C. albicans (p > 0.05). Hemolytic activity was affected by CaCl2; when this component was absent, we did not notice any significant difference between C. albicans and C. dubliniensis hemolytic activities. On the contrary, when we added 2.5 g percent CaCl2, the hemolytic activity was reduced on C. dubliniensis and stimulated on C. albicans tested strains (p < 0.05).

C. dubliniensis é uma levedura oportunista que, embora já tenha sido isolada de vários sítios anatômicos é, com maior frequência, encontrada na boca de pacientes infectados pelo HIV. Embora tenham sido realizados numerosos estudos sobre a epidemiologia e filogenia, seus fatores de virulência como atividade exoenzimática e atividade hemolítica, são, ainda, pouco conhecidos. Neste estudo comparou-se a atividade in vitro de proteinase, hialuronidase, condroitin sulfatase e atividade hemolítica de 18 cultivos de C. dubliniensis com 30 cultivos de C. albicans, todos isolados de pacientes com SIDA. Foi evidenciada maior atividade de proteinase em C. albicans em relação a C. dubliniensis (p < 0,05). Todos os isolados de C. dubliniensis evidenciaram atividade de hialuronidase e condroitin-sulfatase de forma similar ao observado com C. albicans (p > 0,05). Constatou-se que a atividade hemolítica foi influenciada pelo CaCl2; em sua ausência não foram observadas diferenças na atividade hemolítica das duas espécies; todavia, ao se agregar 2,5 por cento de CaCl2, a atividade hemolítica de C. dubliniensis foi reduzida enquanto a de C. albicans, estimulada (p < 0,05).

PCR fingerprinting discriminating between Candida albicans and Candida dubliniensis / 中华传染病杂志

Objective To develop a rapid PCR fingerprinting for discriminating between Candida albicans and Candida dubliniensis isolates. Methods Genomic DNA purified from the two species was amplified by single primer PCR. Oligonucleotide of the minisatellite-specific core sequence of the wild-type phage M13 (5′-GAGGGTGGCGGTTCT-3′) was used as primer and the amplified products were analyzed by gel electrophoresis and microfluidic DNA chip assays. Results Of 17 candida isolates, the PCR fingerprinting generated five strain-specific bands for C. albicans and C. dubliniensis respectively, allowing identification to species level between them. The other bands were minor different in their species. By microfluidic DNA chip, the DNA fragments in size of amplified products for the C. dubliniensis were 960,1177,1297,1495,1797 bp and for the C. albicans 653,1323,1531,2021,2875 bp. Conclusions C. albicans and C. dubliniensis have distinguishable pattern by PCR fingerprinting using the single primer. The microfluidic DNA chip is proposed here as a simple, rapid and highly reproducible tool, especially for the epidemiological investigation.

Rapid Identification of Candida albicans and Candida dubliniensis Isolates With Two Specific Probes Si-multaneously / 中华皮肤科杂志

Objective To develop a microtitration plate en zyme immunoassay to differentiate Candida albicans from Candida dubliniensis isolates using two specific probes s imultaneously.Methods The fun-gus-specific universal primers derived from the internal transcribed s pacer region of fungal rDNA were labeled with biotin,while the C.albicans or C.dubliniensis specific capture probes were coated on the microplates.Genomic DNA purified from the two species was amplified by PCR.The biotinylated p roducts were captured by the probes coated on the microplates.The A 405 value was finally determined by the c olorimetric assay.Results The two species of Candida could be detected specifically.Out of 108clinical isolates originally identified as C.albicans on the basis of germtube formation,two isolates were positive for C.dubliniensis and negative for C.albicans.The other106isolates were positive for C.albicans and negative for C.dubliniensis.Conclusions Two-specific-probe hybridization method is rapid and re liable for differentiating C.albicans from C.dubliniensis.