Identificación de cepas del complejo Candida albicans aisladas de muestras clínicas en la región de Valparaíso, Chile / Identification of strains of the Candida albicans complex isolated clinical samples in the Valparaíso region, Chile

Introducción: C. albicans es reconocida como la especie más virulenta del género y representa la causa más frecuente de candidiasis en humanos. A nivel taxonómico, C.albicans se clasifica como un complejo de especies estrechamente relacionadas que incluye a C. albicans sensu stricto (s.s), C. dubliniensis y C. africana. Objetivo: identificar las especies del complejo C. albicans aisladas desde distintas muestras de pacientes de la quinta región de Valparaíso. Materiales y método: Se identificaron 103 cepas del complejo C. albicans, aisladas desde muestras superficiales y profundas durante el año 2020. La identificación se realizó en base a morfofisiología y la amplificación del gen HWP1. Resultados: Se identificaron 100 cepas como C. albicans s.s, 2 como C. dubliniensis y 1 como C. africana. Dentro de las cepas identificadas como C. albicans s.s se observaron cuatro patrones de tamaños de fragmentos genéticos. Conclusiones: C. albicans s.s fue la especie más frecuente y en base al genotipo de HPW1 se describen cuatro patrones ( H1 a H4). (AU)

Introduction: C. albicans is recognized as the most virulent species of the genus and represents the major cause of candidiasis in humans. At the taxonomic level, C. albicansis classified as a complex of closely related species that includes C. albicans sensu stricto (s.s), C. dubliniensis, and C. africana. Objective: to identify the species of the C. albicans complex isolated from different samples of patients from the fifth region of Valparaíso. Materials and method: 103 strains of the C. albicans complex were identified, isolated from superficial and deep samples during the year 2020. The identification was carried out based on morphophysiology and the amplification of the HWP1 gene. Results: 100 strains were identified as C. albicans s.s, 2 as C. dubliniensis and 1 as C. africana. Within the strains identified as C. albicans s.s, 4 patterns of fragment sizes were observed. Conclusions: C. albicans s.s was the most frequent species and based on the HPW1 genotype, four patterns are described (H1 to H4).(AU)

Rapid identification of microbial contaminants in pharmaceutical products using a PCA/LDA-based FTIR-ATR method

Microbiological quality of pharmaceuticals is fundamental in ensuring efficacy and safety of medicines. Conventional methods for microbial identification in non-sterile drugs are widely used; however they can be time-consuming and laborious. The aim of this paper was to develop a chemometric-based rapid microbiological method (RMM) for identifying contaminants in pharmaceutical products using Fourier transform infrared with attenuated total reflectance spectrometry (FTIR-ATR). Principal components analysis (PCA) and linear discriminant analysis (LDA) were used to obtain a predictive model capable of distinguishing Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), Enterococcus faecium (ATCC 8459), Escherichia coli (ATCC 8739), Micrococcus luteus (ATCC 10240), Pseudomonas aeruginosa (ATCC 9027), Salmonella typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 6538), and Staphylococcus epidermidis (ATCC 12228) microbial growth. FTIR-ATR spectra provide data on proteins, DNA/RNA, lipids, and carbohydrates constitution of microbial growth. Microbial identification provided by PCA/LDA based on FTIR-ATR method were compatible with those obtained using traditional microbiological methods. The chemometric-based FTIR-ATR method for rapid identification of microbial contaminants in pharmaceutical products was validated by assessing the sensitivity (93.5%), specificity (83.3%), and limit of detection (17-23 CFU/mL of sample). Therefore, we propose that FTIR-ATR spectroscopy may be used for rapid identification of microbial contaminants in pharmaceutical products and taking into account the samples studied

Development of amphotericin b Based organogels against mucocutaneous fungal infections

Amphotericin B is a broad spectrum antifungal agent used to treat fungal infections. Organogel is a semisolid preparation in which the apolar phase gets immobilized within spaces of the three-dimensional structure. The current study aimed at the formulation and comparative evaluation of sorbitan monostearate organogels and pluronic lecithin organogels (PLO). Different compositions of span 60 based organogels were prepared by varying the concentrations of the span 60 and PLO gels were prepared by varying the concentration of Pluronic F 127. The developed organogels were subjected to various characteristics such as pH, viscosity, spreadability, extrudability, and drug release studies. The optimized formulations were evaluated against Candida albicans and carried out ex vivo release study. The optimized formulation was selected from span 60 based organogels, and pluronic lecithin organogels were S1 and P1, respectively. The optimized formulation (S1) showed effective inhibition against Candida albicans. The skin irritation test was carried out on albino mice for optimized formulations and results showed that no irritation to the skin. Based on the results, organogels prepared by sorbitan monostearate showed better antifungal activity, and also all the formulations were found to be stable and safe throughout the study period.

Atividade antifúngica de metabólitos produzidos por Streptococcus mutans sobre Candida albicans / Antifungal activity of metabolites produced by Streptococcus mutans on Candida albicans

RESUMO A formação de biofilmes e produção de hifas por Candida albicans são importantes fatores de virulência, principalmente, para a aderência à mucosa e invasão tecidual. A busca por metabólitos secundários produzidos por S. mutans é de suma importância, pois poderá fornecer novas estratégias terapêuticas no combate às infecções por Candida, possibilitando o desenvolvimento de medicamentos capazes de inibir os mecanismos de patogenicidade das espécies desse gênero. Desse modo, o objetivo desse estudo foi obter o extrato bruto e frações a partir do sobrenadante da cultura de 4 h de S. mutans (UA159) e avaliar seus efeitos sobre os mecanismos de patogenicidade de C. albicans (ATCC18804) por meio de estudos in vitro. Após o cultivo de S. mutans, o extrato bruto foi obtido via partição líquido-líquido com acetato de etila (3x) e posteriormente fracionado em coluna de sílica derivatizada C-18 eluída com gradiente MeOH:H2O, fornecendo cinco frações (SM-F1, SM-F2, SM-F3, SM-F4 e SM-F5). A identificação das substâncias contidas no extrato bruto e frações foi realizada utilizando cromatografia gasosa acoplada a espectrometria de massas (CGEM), sendo encontradas as seguintes substâncias: ácido octanóico e uridina no extrato bruto, ácido propanóico, (3R)-3-Methyl-1,4-bis(trimethylsilyl)piperazine-2,5- dione, pirimidina, gulose e oleamida na SM-F1 e ácido nicotínico e triptofano na SM- F2. O extrato bruto e as frações foram submetidos aos ensaios de bioatividade sobre a formação de hifas de C. albicans e analisados por microscopia óptica e microscopia eletrônica de varredura (MEV). O extrato bruto e a fração SM-F2, que resultou em maior inibição das hifas, foram investigados na expressão de genes de C. albicans envolvidos no mecanismo de filamentação (CPH1, EFG1, HWP1, UME6 e YWP1) por PCR quantitativo em tempo real (RT-qPCR). Além disso, o potencial de inibição do extrato bruto, frações SM-F1 e SM-F2 foi avaliado sobre a formação de biofilmes de C. albicans, analisados pela quantificação de células viáveis (UFC/mL) e MEV Os resultados demonstraram inibição significativa na formação de hifas de C. albicans pelo extrato bruto e principalmente, pela SM-F2, com alterações significativas na expressão de todos os genes analisados. O extrato bruto e SM-F2 regularam negativamente a expressão dos genes CPH1, EFG1, HWP1 e UME6, em contrapartida, regularam positivamente a expressão do gene YWP1, envolvido no mecanismo de dispersão das leveduras. Nas análises de UFC/mL, os resultados demonstraram redução nas contagens de C. albicans nos biofilmes formados quando em contato com o extrato bruto, frações SM-F1 e SM-F2 em todas as concentrações testadas, sendo que o extrato bruto reduziu totalmente as células de C. albicans na concentração 15 mg/mL. Os resultados do nosso estudo demonstraram que o extrato bruto e frações de S. mutans apresentaram efeitos inibitórios sobre importantes mecanismos de virulência de C. albicans, fornecendo evidências que S. mutans produz substâncias com ação antifúngica, tornando-o promissor na busca de novos compostos antimicrobianos para prevenção e tratamento das candidoses humanas(AU)

The biofilm formation and hyphae production by Candida albicans are important virulence factors, mainly for mucosal adhesion and tissue invasion. The search for secondary metabolites produced by S. mutans is of great importance, since it may provide new therapeutic strategies against Candida infections, enabling the development of drugs that inhibit the mechanisms of pathogenicity of the species of this genus. Thus, the objective of this study was to obtain the crude extract and fractions from the supernatant of the 4 h culture of S. mutans (UA159) and evaluate their effects on the mechanisms of pathogenicity of C. albicans (ATCC18804) through in vitro studies. After culture of S. mutans, the crude extract was obtained via liquid-liquid partition with ethyl acetate (3x) and then fractionated on a C-18 derivatized silica column eluted with MeOH:H2O gradient, providing five fractions (SM-F1, SM-F2, SM-F3, SM-F4 and SM-F5). The identification of the substances contained in the crude extract and fractions was performed by gas chromatography coupled to mass spectrometry (GC-MS). The following substances were found: octanoic acid and uridine in crude extract, propanoic acid, (3R) -3-methyl -1,4-bis (trimethylsilyl) piperazine-2,5-dione, pyrimidine, gulose and oleamide in SM-F1 and nicotinic acid and tryptophan in SM-F2. The crude extract and fractions were submitted to bioactivity assays on hyphae formation by C. albicans and analyzed by optical microscopy and scanning electron microscopy (SEM). The crude extract and SM-F2 fraction, which resulted in highest inhibition of hyphae, were investigated in the expression of C. albicans genes involved in the filamentation mechanism (CPH1, EFG1, HWP1, UME6 and YWP1) by quantitative real-time PCR (RT-qPCR). In addition, the potential of inhibition of the crude extract, SM-F1 and SM-F2 fractions in biofilms of C. albicans were evaluated by the quantification of viable cells (CFU/mL) and SEM. The data obtained were statistically analyzed by Graph Pad Prism 5.0, with a significance level of 5%. The results demonstrated a significant inhibition on C. albicans hyphae formation by the crude extract and mainly by SMF2, with significant alterations in the expression of all genes analyzed. The crude extract and SM-F2 negatively regulated the expression of the CPH1, EFG1, HWP1 and UME6 genes, in contrast, positively regulated the expression of the YWP1 gene, involved in the mechanism of yeast dispersion. In the CFU/mL analyzes, the results showed a reduction in the counts of C. albicans in the biofilms formed when in contact with the crude extract, SM-F1 and SM-F2 fractions in all tested concentrations and the crude extract totally reduced C. albicans cells at 15 mg/mL concentration. The results of our study demonstrated that the crude extract and fractions of S. mutans presented inhibitory effects on important mechanisms of virulence of C. albicans, providing evidence that S. mutans produces substances with antifungal action, making it promising in the search for new compounds antimicrobials for the prevention and treatment of human candidoses(AU)

Factores relacionados a candidiasis oral en niños y adolescentes con VIH, caracterización de especies y susceptibilidad antifúngica / Factors related to oral candidiasis in HIV children and adolescents, species characterization and antifungal susceptibility

Resumen Introducción: Se desconocen los factores asociados a la candidiasis oral en población pediátrica con infección por VIH de los países en desarrollo. Objetivo: Identificar los factores asociados a la colonización por Candida, candidiasis oral y la susceptibilidad in vitro a antifúngicos, en niños y adolescentes con infección por VIH institucionalizados en la ciudad de Tijuana, México. Materiales y Métodos: Se examinó la cavidad oral de 30 niños y adolescentes con infección por VIH, se obtuvo una muestra de la mucosa oral para identificar las especies de Candida mediante cultivo y auxonograma. La susceptibilidad a los antifúngicos se determinó de acuerdo al CLSI. Los indicadores del estado inmunológico y falla virológica se clasificaron conforme a la OMS. Resultados: Se identificaron seis especies de Candida, 53% colonizantes y 47% causantes de candidiasis. Los factores asociados a candidiasis fueron alta carga viral (p = 0,001), menor recuento de LTCD4+ (p = 0,002) y esquema TARAA (p ≤ 0,014). La especie prevalente fue C. glabrata (33%); sin embargo, C. albicans (27%) fue más resistente a fluconazol (p = 0,001). Las especies resistentes a itraconazol se identificaron en esquemas que incluyen un INNTR (p = 0,041). Conclusiones: Los niños y adolescentes con infección por VIH institucionalizados mostraron una prevalencia elevada de Candida spp. colonizante y resistencia a los antifúngicos relacionada con los INNTR .

Background: Factors associated with candidiasis and colonization in HIV-positive children and adolescents in developing countries are not well understood. Aim: To identify the factors associated with oral Candida colonization and candidiasis in institutionalized HIV-positive children and adolescents in Tijuana, México, as well as the response of the isolates to antifungals. Materials and Methods: Sample of the oral mucosa of 30 HIV positive children and adolescents were obtained to isolate and identify Candida species by culture and metabolic profile. Antifungal drugs susceptibility was determined according to CLSI. Indicators of immunological and virologic failure were classified in accordance to WHO criteria. Results: Six Candida species were identified from oral mucosa, 53% colonizers and 47% in candidiasis. Factors associated with candidiasis and oral colonization were viral load (p = 0,001), CD4+ counts (p = 0,002) and HAART regimen (p ≤ 0,014). The most prevalent species was C. glabrata (33%), but C. albicans (27%) was more resistant to fluconazole (p = 0,001). Itraconazol resistant species were identified in regimens that include an NNRTI (p = 0,041). Conclusion: HIV-positive children and adolescents living in an orphanage showed high prevalence of colonizing Candida spp. and resistance to antifungals, related to NNRTI.

Historia sucinta de Candida albicans, blanca pero no tanto / Brief history of Candida, white but not so much

From the begin of clinical microbiology in the second half of the nineteenth century, the fungi were neglected as contaminants without relevance for health, belonging the major advances of their study to the fields of milk derivatives and beer industries. However, the seek for the etiological agent of thrush, a very common oral pathology affecting the newborn, put the yeasts on the table near 1840 with three capital papers - Berg, Gruby and Bennett - speaking about spores from vegetable as parasites of animal and human beings. The door was open, and very soon, in 1853, came the decisive description by Robin of the Oidium albicans as the causative agent of this painful disease. Seventy years after, in 1923, Christine Marie Berkhout, rejecting this name, defined the genus as Candida, leaving the specie with the iterative Latin name of Candida albicans, that means "White-white". Or, perhaps, with a fine sense of humor, she has made an oxymoron, because "candida" means a brilliant white and "albicans" a matt one, both opposite adjectives. Or, may be, Christine is still saying us: "White…but not so white".

Prevalencia y sensibilidad a los antifúngicos de Candida albicans y sus especies relacionadas, Candida dubliniensis y Candida africana aisladas de muestras vulvovaginales en un hospital de Argentina / Prevalence and antifungal susceptibility of Candida albicans and its related species Candida dubliniensis and Candida africana isolated from vulvovaginal samples in a hospital of Argentina

Candida africana taxonomical status is controversial. It was proposed as a separate species within the Candida albicans species complex; however, phylogenetic analyses suggested that it is an unusual variety of C. albicans. The prevalence of C. albicans-related species (Candida dubliniensis and C. africana) as vulvovaginal pathogens is not known in Argentina. Moreover, data on antifungal susceptibility of isolates causing vulvovaginal candidiasis is scarce. The aims of this study were to establish the prevalence of C. dubliniensis and C. africana in vaginal samples and to evaluate the antifungal susceptibilities of vaginal C. albicans species complex strains. We used a molecular-based method coupled with a new pooled DNA extraction methodology to differentiate C. dubliniensis and C. africana in a collection of 287 strains originally identified as C. albicans isolated from an Argentinian hospital during 2013. Antifungal susceptibilities to fluconazole, clotrimazole, itraconazole, voriconazole, nystatin, amphotericin B and terbinafine were evaluated by using the CLSI M27-A3 and M27-S4 documents. Of the 287 isolates, 4 C. dubliniensis and one C. africana strains (1.39% and 0.35% prevalence, respectively) were identified. This is the first description of C. africana in Argentina and its identification was confirmed by sequencing the ITS2 region and the hwp1 gene. C. dubliniensis and C. africana strains showed very low MIC values for all the tested antifungals. Fluconazole-reduced-susceptibility and azole cross-resistance were observed in 3.55% and 1.41% of the C. albicans isolates, respectively. These results demonstrate that antifungal resistance is still a rare phenomenon in this kind of isolates.

La clasificación taxonómica de Candida africana está en discusión, es considerada una nueva especie dentro del complejo C. albicans o una variedad inusual de C. albicans. La prevalencia de las especies relacionadas a C. albicans (C. dubliniensis y C. africana) como agentes de vulvovaginitis en Argentina se desconoce. Los objetivos de este trabajo fueron determinar la prevalencia de C. dubliniensis y C. africana en muestras vaginales y evaluar la sensibilidad a los antifúngicos de aislamientos vaginales de las especies del complejo C. albicans. Para diferenciar C. dubliniensis y C. africana utilizamos un método molecular asociado a un nuevo método de extracción de ADN. Se utilizó una colección de 287 cepas originalmente identificadas como C. albicans aisladas durante 2013 en un hospital de Argentina. Se evaluó la sensibilidad a fluconazol, clotrimazol, itraconazol, voriconazol, nistatina, anfotericina B y terbinafina utilizando los documentos M27-A3 y M27-S4 del CLSI. De los 287 aislamientos, se identificaron 4 C. dubliniensis y 1 C. africana (1,39 y 0,35% de prevalencia, respectivamente). Esta es la primera descripción de C. africana en Argentina. Su identificación fue confirmada por secuenciación de la región ITS2 y del gen hwp1. Las cepas identificadas como C. dubliniensis y C. africana mostraron valores de CIM muy bajos para todos los antifúngicos probados. En los aislamientos de C. albicans, la sensibilidad reducida al fluconazol y la resistencia cruzada a todos los azoles se observó en el 3,55% y el 1,41%, respectivamente. Estos resultados demuestran que la resistencia a los antifúngicos es todavía un fenómeno raro en este tipo de aislamientos.

Perfil fitoquímico e determinação da atividade antimicrobiana de Syzygium cumini (L. ) Skeels (Myrtaceae) frente a microrganismos bucais / Phytochemical profile and determination of antimicrobial activity of Syzygiumcumini (L. ) Skeels (Myrtaceae) against oral microorganisms

RESUMO Este estudo teve como objetivo determinar o perfil fitoquímico e avaliar a atividade antimicrobiana in vitro do extrato etanólico da casca do caule de Syzygium cumini(L.) Skeels frente a microrganismos bucais. O perfil fitoquímico do extrato foi traçado através da determinação espectrofotométrica quantitativa para verificar o teor de taninos, flavonóides, saponinas e polifenóis. A atividade antimicrobiana foi determinada através da Concentração Inibitória Mínima (CIM), por meio da técnica de microdiluição em caldo, utilizando-se as seguintes linhagens de microrganismos: Streptococcus mutans (25175), Streptococcus oralis (10557) e Candida albicans (10231). Uma quantidade apreciável de fitocontituintes foi observada, especialmente de taninos (100,58 ± 1,81). Os extratos apresentaram atividade antimicrobiana inibindo o crescimento das linhagens em estudo, destacando-se essa atividade sobre o crescimento de C. albicans (CIM=250 µg/mL). Já as CIMs para Streptococcus foram baixas. Diante dos resultados expostos, pode-se concluir que o perfil fitoquímico foi traçado e que, dentre os microrganismos testados, o extrato etanólico da casca de S. cumini apresentou forte potencial de inibição sobre o crescimento de C. albicans e fraca inibição frente aos Streptococcus testados. Este estudo sugere que mais pesquisas devem ser realizadas dando continuidade à bioprospecção, por meio de análises experimentais com essa espécie vegetal, objetivando, no futuro, que essa planta possa ser utilizada clinicamente para tratar candidose bucal.

ABSTRACT This study aimed to determine the phytochemical profile and to evaluate the in vitro antimicrobial activity of the ethanol extract of stem bark of Syzygium cumini (L.) Skeels against oral microorganisms. The phytochemicalprofile of the extract was traced through a quantitative spectrophotometric determination in order to check the tannin, flavonoids, saponins, and polyphenols content. The antimicrobial activity was determined through minimum inhibitory concentration (MIC) by the broth microdilution technique, using the following strains of microorganisms: Streptococcus mutans (25175), Streptococcus oralis (10557) and Candida albicans (10231). An appreciable amount of fitocontituintes was observed, particularly the tannin (100.58 ± 1.81). The extracts showed antimicrobial activity, inhibiting the growth of the strains under study, with this activity being more intense on the growth of C. albicans ( MIC = 250 mg / mL). On the other hand, the MICs of the Streptococcus were low. In face of the mentioned results, we can conclude that the phytochemical profile was traced and that, among the tested microorganisms, the ethanol extract of S. cumini bark showed strong potential to inhibit the growth of C. albicans and weak inhibition against the Streptococcus tested. This study suggests that more research should be done by proceeding with the bioprospecting, through experimental tests with this plant`s species, aiming that in the future this substance can be used clinically for the treatment of oral candidiasis.

Actividad antifúngica de melanina en cepas clínicas de Candida spp / Antifungal activity of melanin in clinical isolates of Candida spp

Background: Melanocytes are cells located in epidermis and mucous membranes that synthesize melanin and cytokines. It is known that melanin has antimicrobial activity and that melanocytes are melanized in presence of microbial molecules. Objective: To study the antifungal activity of melanin on Candida spp. Methodology: The minimum inhibitory concentration (MIC) to melanin was determined in 4 Candida ATCC strains (C. albicans SC5314, C. parapsilosis 22019, C. glabrata 2001, C. krusei 6258) and 56 clinical isolates of Candida spp. (33 C. albicans, 12 C. glabrata, 3 C. famata, 3 C. krusei, 3 C. parapsilosis, 2 C. tropicalis) using a broth microdilution method. In addition, the antifungal activity of melanocytes and mice melanoma cells was tested against C. albicans. Results: Melanin inhibited the tested isolates, including the susceptible dose-dependent and fluconazole-resistant strains; MIC range and MIC50 were 0.09-50 μg/mL and 6.25 μg/mL, respectively. Pigmented cells lysates inhibited C. albicans. Conclusions: Melanin is able to inhibit clinical isolates of Candida spp. Melanization could be an important protective mechanism of melanocytes.

Introducción: Los melanocitos son células presentes en piel y en mucosas que sintetizan melanina, además de citoquinas. Es sabido que melanina presenta actividad antimicrobiana y que los melanocitos se melanizan al ser expuestos a moléculas microbianas. Objetivo: Estudiar la actividad antifúngica de melanina en cepas clínicas de Candida spp. Metodología: Se midió la concentración inhibitoria mínima (CIM) a melanina, de 4 cepas de Candida ATCC (C. albicans SC5314, C. parapsilosis 22019, C. glabrata 2001 y C. krusei 6258) y 56 aislados clínicos de Candida spp. (33 C. albicans, 12 C. glabrata, 3 C. famata, 3 C. krusei, 3 C. parapsilosis, 2 C. tropicalis) mediante un método de microdilución en caldo. Además se estudió el efecto antifúngico de lisados de melanocitos y células de melanoma de ratón en C. albicans. Resultados: Melanina inhibió las cepas analizadas, incluso cepas susceptibles dosis-dependiente y resistentes a fluconazol, siendo los rangos de CIM y CIM50 de 0,09-50 μg/mL y 6,25 μg/ mL, respectivamente. Los lisados de células pigmentadas inhibieron C. albicans. Conclusiones: Melanina es capaz de inhibir cepas clínicas de Candida spp. La melanización podría ser un importante mecanismo protector de los melanocitos.

MOLECULAR TYPING OF Candida albicans ISOLATES FROM HOSPITALIZED PATIENTS / Tipagem molecular de Candida albicans isoladas de pacientes hospitalizados

SUMMARY Introduction: The majority of nosocomial fungal infections are caused by Candida spp. where C. albicans is the species most commonly identified. Molecular methods are important tools for assessing the origin of the yeasts isolated in hospitals. Methods: This is a study on the genetic profifiles of 39 nosocomial clinical isolates of C. albicans using two typing methods: random amplifified polymorphic DNA (RAPD) and microsatellite, two different primers for each technique were used. Results: RAPD provided 10 and 11 different profiles with values for SAB of 0.84 ± 0.126 and 0.88 ± 0.08 for primers M2 and P4, respectively. Microsatellite using two markers, CDC3 and HIS3, allowed the observation of six and seven different alleles, respectively, with combined discriminatory power of 0.91. Conclusions: Although genetic variability is clear, it was possible to identify high similarity, suggesting a common origin for at least a part of isolates. It is important to emphasize that common origin was proven from yeasts isolated from colonization (urine, catheter or endotracheal secretions) and blood culture from the same patient, indicating that the candidemia must have started from a site of colonization. The combination of RAPD and microsatellite provides a quick and efficient analysis for investigation of similarity among nosocomial isolates of C. albicans. .

RESUMO Introdução: A maioria das infecções fúngicas hospitalares são causadas por Candida spp. e C. albicans é a espécie mais comumente identificada. Métodos moleculares são ferramentas importantes para a avaliação da origem das leveduras isoladas em hospitais. Métodos: Este é um estudo sobre o perfil genético de 39 isolados clínicos nosocomiais de C. albicans através das técnicas de RAPD e microssatélite, foram usados dois diferentes iniciadores para cada técnica. Resultados: RAPD forneceu 10 e 11 diferentes perfis com valores de SAB 0,84 ± 0,126 e 0,88 ± 0,08 para os primers M2 e P4, respectivamente. A análise de microssatélites, usando os marcadores CDC3 e HIS3 permitiu a observação de seis e sete diferentes alelos respectivamente, com poder discriminatório combinado de 0,91. Conclusões: Embora seja clara a variabilidade genética, foi possível identificar alta similaridade, sugerindo origem comum para pelo menos parte deles. É importante enfatizar que foi comprovada origem comum de leveduras isoladas de colonização (urina, cateter ou secreção orotraqueal) e hemocultura do mesmo paciente, indicando que a candidemia deve ter iniciado a partir de um sítio de colonização. A combinação das técnicas RAPD e microssatélites fornece uma análise rápida e eficiente para investigação de similaridade entre isolados nosocomiais de C. albicans. .

Distribución de lesiones bucales producidas por Candida albicans en pacientes VIH/SIDA / Distribution of oral lesions produced by Candida albicans in HIV patients

Antecedentes: La candidiasis orofaríngea es la infección fúngica oportunista más frecuente en pacientes infectados por VIH. Clínicamente se puede presentar como queilitis angular, eritematosa, pseudomembranosa e hiperplásica. Objetivo. En el presente estudio se analizó la distribución de lesiones candidiásicas y su ubicación en la cavidad bucal en 29 pacientes VIH positivo. Método. Los pacientes fueron evaluados y controlados en el Servicio de Dermatología del Hospital Regional de Talca y se analizaron sus lesiones a nivel bucal y se tomaron muestras que permitieron aislar Candida albicans. Resultados y conclusiones. La candidiasis pseudomembranosa fue encontrada en mayor porcentaje (47.1 por ciento) y su principal ubicación fue en el paladar, la candidiasis eritematosa, la segunda más frecuente (45.1 por ciento) y su principal ubicación fue el dorso de la lengua. La queilitis angular se presentó el menor porcentaje (7.8 por ciento) y no se encontraron lesiones de tipo hiperplásica. Existe una fuerte asociación entre el tipo de variante clínica de candidiasis oral y su ubicación en la cavidad oral.

Background. The oropharyngeal candidiasis is the most common opportunistic fungal infection in HIV-infected patients. Clinically can be presented as angular cheilitis, erythematous, pseudomembranous and hyperplastic. Aims. The present study analyzed the clinical types of oral candidiasis and its location in the oral cavity in 29 HIV positive patients Methods. Patients were evaluated and controlled in a dermatology department of the Hospital Regional de Talca and analyzed their lesions at the oral level and samples for isolating Candida albicans. Results and conclusions. Pseudomembranous candidiasis was found in highest percentage (47.1 percent) and its main location was on the palate, erythematous candidiasis, the second most frequent (45.1 percent) and its principal location was the back of the tongue. Angular cheilitis was presented the lowest percentage (7.8 percent) and there were no hyperplastic type lesion. There is a strong association between the variant type of clinic oral candidiasis and its location in the oral cavity.

Presentación inusual de onicomicosis por candida albicans / Unusual presentation of onychomycosis caused by Candida albicans

Antecedentes: la onicomicosis es la infección de las uñas producida por hongos dermatofitos y no dermatofitos, como las levaduras y mohos. A los dermatofitos corresponde la inmensa mayoría de estas infecciones, pero informes recientes señalan el incremento de las infecciones producidas por los otros agentes referidos; dentro de éstos merecen especial atención el incremento reportado en diversos estudios de las especies del género Candida. La onicomicosis por Candida spp usualmente ocurre en inmunosupresos, siendo más frecuente en mujeres y en uñas de las manos. Caso clínico: masculino de 15 años con lesión en la uña del primer dedo de la mano derecha de un año de evolución, constituida por distrofia, cambios de coloración, opacidad y onicolisis, afecta el borde distal y medial; se consideró el diagnóstico clínico de onicomicosis, se tomó muestras para estudio microbiológico e indicó tratamiento con terbinafina oral mientras se esperaba el resultado del cultivo, sin obtener mejoría satisfactoria en 6 semanas. Se aisló Candida albicans, por lo que se indicó fluconazol vía oral 150mg/semana por 16 semanas, con lo que se logró la remisión completa. Conclusión: el paciente descrito es un masculino sin antecedentes personales patológicos u ocupacionales que favorescan la onicomicosis por Cándida, lo que nos demuestra la necesidad del aislamiento del agente etiológico de la onicomicosis para elegir la mejor opción terapéutica, y así obtener los mejores resultados. Dado que en nuestro medio los estudios de sensibilidad para antimicóticos son muy limitados, se necesita mejorar la disponibilidad de los mismos, lo cual nos ayudará a determinar la sensibilidad o resistencia a los agentes antimicóticos con que contamos en la actualidad...

Differentiation between Candida albicans and Candida dubliniensis using hypertonic Sabouraud broth and tobacco agar / Diferenciação entre Candida albicans e Candida dubliniensis usando caldo Sabouraud hipertônico e ágar Tabaco

INTRODUCTION: Opportunistic fungal infections in immunocompromised hosts are caused by Candida species, and the majority of such infections are due to Candida albicans. However, the emerging pathogen Candida dubliniensis demonstrates several phenotypic characteristics in common with C. albicans, such as production of germ tubes and chlamydospores, calling attention to the development of stable resistance to fluconazole in vitro. The aim of this study was to evaluate the performance of biochemistry identification in the differentiating between C. albicans and C. dubliniensis, by phenotyping of yeast identified as C. albicans. METHODS: Seventy-nine isolates identified as C. albicans by the API system ID 32C were grown on Sabouraud dextrose agar at 30°C for 24-48h and then inoculated on hypertonic Sabouraud broth and tobacco agar. RESULTS: Our results showed that 17 (21.5%) isolates were growth-inhibited on hypertonic Sabouraud broth, a phenotypic trait inconsistent with C. albicans in this medium. However, the results observed on tobacco agar showed that only 9 (11.4%) of the growth-inhibited isolates produced characteristic colonies of C. dubliniensis (rough colonies, yellowish-brown with abundant fragments of hyphae and chlamydospores). CONCLUSIONS: The results suggest that this method is a simple tool for screening C. albicans and non-albicans yeast and for verification of automated identification.

INTRODUÇÃO: Infecções fúngicas oportunistas em hospedeiros imunocomprometidos são causadas por espécies de Candida, cuja maioria das infecções se deve a Candida albicans. Entretanto, o patógeno emergente Candida dubliniensis demonstra várias características fenotípicas em comum com C. albicans, tais como produção de tubo germinativo e clamidósporos, solicitando atenção por desenvolver resistência in vitro estável ao fluconazol. O objetivo do presente estudo foi avaliar a performance da identificação bioquímica na diferenciação entre C. albicans e Candida dubliniensis, analisando fenotipicamente leveduras previamente identificadas como C. albicans. MÉTODOS: Setenta e oito isolados identificados como C. albicans pelo sistema API ID 32C foram cultivados em ágar Sabouraud dextrose a 30°C por 24-48h e em seguida inoculados em caldo hipertônico Sabouraud e agar tabaco. RESULTADOS: Nossos resultados mostraram que 17 (21,5%) isolados tiveram o crescimento inibido no caldo hipertônico Sabouraud, característica fenotípica inconsistente para C. albicans neste meio de cultura. Entretanto, os resultados observados em ágar tabaco mostraram que somente 9 (11,4%) dos isolados inibidos produziram colônias características de C. dubliniensis (colônias rugosas, marrom-amarelada com fragmentos de hifas e abundantes clamidósporos). CONCLUSÕES: Os resultados obtidos sugerem que este é um instrumento simples para triagem entre leveduras de C. albicans e não-albicans, bem como confirmação de identificação automatizada.

Multilocus Sequence Typing for Candida albicans Isolates from Candidemic Patients: Comparison with Southern Blot Hybridization and Pulsed-field Gel Electrophoresis Analysis / 대한진단검사의학회지

BACKGROUND: We evaluated the efficacy of multilocus sequence typing (MLST) for assessing the genetic relationship among Candida albicans isolates from patients with candidemia in a hospital setting. METHODS: A total of 45 C. albicans isolates from 21 patients with candidemia were analyzed. The MLST results were compared with results obtained by Southern blot hybridization (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE analysis included karyotyping and restriction endonuclease analysis of genomic DNAs using BssHII (REAG-B) and SfiI (REAG-S). RESULTS: The 45 isolates yielded 20 unique diploid sequence types (DSTs) by MLST, as well as 12 karyotypes, 15 REAG-B patterns, 13 REAG-S patterns, and 14 C1 fingerprinting types. Microevolution among intra-individual isolates was detected in 6, 5, 3, 5, and 7 sets of isolates by MLST (1 or 2 allelic differences), REAG-B, REAG-S, C1 fingerprinting, and a combination of all methods, respectively. Among 20 DSTs, 17 were unique, and 3 were found in more than 1 patient. The results of 2 DSTs obtained from 9 patient isolates were in agreement with REAG and C1 fingerprinting patterns. However, the remaining DST, which was shared by 2 patient isolates, showed 2 different PFGE and C1 fingerprinting patterns. In addition, 3 sets of isolates from different patients, which differed in only 1 or 2 alleles by MLST, also exhibited different PFGE or C1 fingerprinting patterns. CONCLUSIONS: MLST is highly discriminating among C. albicans isolates, but it may have some limitations in typing isolates from different patients, which may necessitate additional analysis using other techniques.

Molecular fingerprinting methods for studies involving oral Candida albicans: [review] / Métodos de caracterização molecular para estudos envolvendo Candida albicans de origem bucal: [revisão]

OBJECTIVE: This review was aimed to discuss the literature concerning the fingerprint methods for epidemiological studies of oral-borne Candida albicans. DISCUSSION: Interest in obtaining a better understanding of the pathogenesis, epidemiology, genetics and evolution of Candida albicans has led to the development of innumerable investigations. These studies have employed fingerprinting systems, such as multilocus enzyme electrophoresis, electrophoretic karyotyping, randomly amplified polymorphic DNA, and restriction length fragment polymorphism, with and without hybridization. The efficacy of these systems has been examined at different levels of discrimination. A validation strategy has been delineated which compares two or more unrelated methods. Moreover, the different fingerprinting patterns produced could be registered in database programs and submitted to comparison with parameters of the host and characteristics of the pathogen. These procedures permit urrent and retrospective comparison of a selection of clinical and epidemiologically important strains, which could show one or several characteristics of the host or pathogen. Additionally, the sum of this growing amount of information could contribute even more to the understanding of the dynamics of infectious organisms in human populations, the complex relationship between commensalism and infection, and genetic and evolutionary mechanisms. CONCLUSIONS: Multiple molecular systems are available for studies involving C. albicans. This growing amount of information contributes to the understanding of the dynamics of this fungus in human populations.

OBJETIVO: Esta revisão discute as informações existentes acerca dos métodos de caracterização para estudos epidemiológicos envolvendo Candida albicans de origem bucal. DISCUSSÃO: O interesse no melhor entendimento da patogênese, epidemiologia, genética e evolução de C. albicans tem levado os pesquisadores à condução de inúmeras investigações. Esses estudos empregam sistemas de caracterização molecular como eletroforese de enzimas multilocus, cariotipagem por eletroforese, amplificação do DNA polimórfico ao acaso e polimorfismo dos fragmentos de restrição com e sem hidridização. A eficácia desses sistemas tem sido avaliada nos seus diferentes níveis de discriminação. Uma estratégia de validação foi delineada, a qual compara dois ou mais métodos não relacionados. Ainda, os diferentes padrões de caracterização molecular produzem dados que podem ser avaliados por programas computacionais e permite a comparação comparâmetros do hospedeiro e características do patógeno. Tais procedimentos permitem comparações correntes e retrospectivas de cepas clínicas e epidemiologicamente importantes, que podem mostrar uma ou mais características do hospedeiro ou do patógeno. A somatória do montante de informação pode contribuir para o entendimento da dinâmica dos organismos infecciosos em populações humanas, as relações complexas entre comensalismo e infecção, e mecanismos genéticos e evolutivos. CONCLUSÕES: Vários sistemas de caracterização molecular estão disponíveis para estudos envolvendo C. albicans. Este aumento de informação contribui na compreensão da dinâmica deste fungo em populações humanas.

Comparison among tomato juice agar with other three media for differentiation of Candida dubliniensis from Candida albicans

O presente estudo teve como objetivo comparar o ágar suco de tomate, um tradicional meio utilizado para observação de ascósporos em leveduras, com o ágar semente de niger, ágar caseína e ágar semente de girassol, na diferenciação fenotípica entre C. albicans e C. dubliniensis. Após 48 h de incubação a 30 ºC, os 26 isolados de C. dublinienis (100%) evidenciaram a formação de clamidoconídios igualmente em todos os meios comparados. Entretanto, quando semeados com C. albicans, a formação de clamidoconídios foi raramente observada, resultando nos seguintes percentuais de ausência destas estruturas: ágar suco de tomate (92,47%), ágar niger (96,7%), ágar caseína (91,39%), ágar semente de girassol (96,7%). Estes resultados permitem-nos sugerir a utilização do ágar suco de tomate como mais um meio que, já no primo-isolamento, é capaz de, presuntivamente, diferenciar C. albicans de C. dubliniensis.

Value of morphotyping for the characterization of Candida albicans clinical isolates

Until recently, morphotyping, a method evaluating fringe and surface characteristics of streak colonies grown on malt agar, has been recommended as a simple and unexpensive typing method for Candida albicans isolates. The discriminatory power and reproducibility of Hunter's modified scheme of Phongpaichit's morphotyping has been evaluated on 28 C. albicans isolates recovered from the oral cavity of asymptomatic human immunodeficiency virus-positive subjects, and compared to two molecular typing methods: randomly amplified polymorphic DNA (RAPD) fingerprinting, and contour clamped homogeneous electric field (CHEF) electrophoretic karyotyping. Morphological features of streak colonies allowed to distinguish 11 different morphotypes while RAPD fingerprinting yielded 25 different patterns and CHEF electrophoresis recognized 9 karyotypes. The discriminatory power calculated with the formula of Hunter and Gaston was 0.780 for morphotyping, 0.984 for RAPD fingerprinting, and 0.630 for karyotyping. Reproducibility was tested using 43 serial isolates from 15 subjects (2 to 6 isolates per subject) and by repeating the test after one year storage of the isolates. While genetic methods generally recognized a single type for all serial isolates from each of the subjects studied, morphotyping detected strain variations in five subjects in the absence of genetic confirmation. Poor reproducibility was demonstrated repeating morphotyping after one year storage of the isolates since differences in at least one character were detected in 92.9 percent of the strains.

Identification of Candida dubliniensis in a diagnostic microbiology laboratory

Candida dubliniensis is an emerging yeast pathogen isolated mainly from immunocompromised patients. As molecular tests are currently unsuitable for use in routine diagnostic laboratories, we compared a variety of phenotypic techniques for differentiating C. albicans and C. dubliniensis. The tests included: colony colour on CHROMagar C and ida medium; growth at 37 degrees C and 45 degrees C; ability to produce germ tubes and chlamydospores; and the Auxacolor system. The organisms included 105 isolates previously identified as C. albicans, 10 reference strains of C. albicans, 2 reference strains of C. dubliniensis and 102 fresh clinical isolates identified as C. albicans. None of the tests alone was satisfactory but a combination of 3 tests may be suitable for presumptive identification of C. dubliniensis

Candida albicans morphotypes from oral cavity in several families

Se estudiaron 108 personas provenientes de 22 familias compuestas de 3 a 8 miembros cada una, para la búsqueda de morfotipos de candida albicans de la cavidad bucal con o sin piezas dentarias. Por la técnica de phongpaichit et. al., morfotipos idénticos o diferentes fueron detectados en 7 grupos familiares. En 10 familias, c. albicans fue aislada de la familia y de protesis dentarias removibles de un mismo individuo. La franja discontinua, considerada como probable indicador de virulencia fue detectada en 9 (16,7 porciento) de las cepas de c. albicans de personas con dentición completa y de saliva de usuarios y no usuarios de prótesis. La franja continua fue verificada en 5 (83,3 porciento) de las cepas de c. albicans aisladas de un mismo sitio. El morfotipo es simple, fácil de ejecutar, puede servir para detectar infección cruzada y como un posible indicador de riesgo de candidosis. C. albicans fue la especie prevalente (86,4 porciento), detectándose a lo menos en 2 miembros de 14 familias