[Molecular identification and in vitro antifungal susceptibility of blood isolates of the Candida parapsilosis species complex in Venezuela]. / Identificación molecular y sensibilidad a los antifúngicos de aislamientos de sangre del complejo Candida parapsilosis en Venezuela.

BACKGROUND: Candida parapsilosis is a species complex consisting of Candida parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis. Studies worldwide have described its epidemiology and susceptibility to antifungal agents. AIMS: The aims of this study were to carry out the molecular identification of blood isolates belonging to the Candida parapsilosis species complex, and to determine their in vitro susceptibility to antifungals of systemic use. METHODS: A study of 86 strains of C. parapsilosis species complex collected in 2008-2011 and obtained from the Candidaemia Surveillance Network of Mycology Department of the Rafael Rangel National Institute of Hygiene, was made. Secondary alcohol-dehydrogenase gene amplification was performed using polymerase chain reaction, and the products were analysed by restriction fragments length polymorphisms using the enzyme BanI. Susceptibility tests were performed using Etest®, following the manufacturer's instructions with modifications. RESULTS: Of the 86 isolates studied, 81 (94.2%) were C. parapsilosis sensu stricto, 4 (4.6%) C. orthopsilosis, and one (1.2%) C. metapsilosis. C. parapsilosis isolates were susceptible to amphotericin B and caspofungin, showing low rates of resistance to fluconazole and voriconazole. C. orthopsilosis and C. metapsilosis were susceptible to all the antifungals tested. CONCLUSIONS: The results obtained in Venezuela provide for the first time important information about the distribution of C. parapsilosis species complex in cases of candidaemia, and support the need for continuing surveillance programs, including molecular discrimination of species and antifungal susceptibility tests, which may guide specific therapy.

[Superficial mycoses: casuistry of the Mycology Department of the Instituto Nacional de Higiene "Rafael Rangel", Caracas, Venezuela (2001-2014)]. / Micosis superficiales: casuística del Departamento de Micología del Instituto Nacional de Higiene "Rafael Rangel", Caracas, Venezuela (2001-2014).

The superficial mycoses are very common infectious diseases and therefore are a frequent reason for medical consultation. The aim of this study was to determine the diagnostic frequency of superficial mycoses in the Mycology Department of the Instituto Nacional de Higiene "Rafael Rangel" during 14 years (2001-2014). A retrospective cross-sectional study was performed to review the mycological records of patients with presumptive diagnosis of superficial mycosis. Nails, hairs and epidermal scales were the processed samples. The identification of fungi was performed by macro and microscopic observation of colonies and biochemical and physiological tests, as required of the isolated agent. For the investigation of Malassezia spp. only direct examination was performed. Of the 3 228 samples processed, 1 098 (34%) were positive and their distribution according to the etiological agent was: dermatophytes 79.5%; 10.9% yeasts; non-dermatophytes fungi 5.1% and 4.5% Malassezia spp. The most frequently isolated dermatophyte was Trichophyton rubrum Complex (70.1%), followed by T mentagrophytes complex (15.1%), Microsporum canis (9.4%) and Epidermophyton floccosum (4%). The most frequent ringworms Were: Tinea unguium (66.8%), followed by Tineapedis (16.4%) and Tinea capitis (8.1%). Candida parapsilosis complex (37.5%) was the most frequently isolated yeast and Fusarium spp. (53.6%) was the most isolated among non-dermatophyte fungi, followed by Aspergillus spp. (19.6%) and Acremonium spp. (10.7%). The identification of the etiological agent is essential to guide appropriate treatment. This study constitutes an important contribution to the knowledge of the epidemiology of superficial mycoses in our country.

Micosis superficiales: casuística del Departamento de Micología del Instituto Nacional de Higiene “Rafael Rangel”, Caracas,Venezuela (2001-2014) / Superficial mycoses: casuistry of the Mycology Department of the Instituto Nacional de Higiene “Rafael Rangel”, Caracas, Venezuela (2001-2014)

Las micosis superficiales son muy comunes y por ello son motivo de consulta médica frecuente. El objetivo de este trabajo fue conocer la frecuencia de diagnóstico de las micosis superficiales en el Departamento de Micología del Instituto Nacional de Higiene “Rafael Rangel” en Caracas, Venezuela, durante 14 años (2001-2014). Se realizó un estudio transversal y retrospectivo de revisión de historias micológicas de pacientes con diagnóstico presuntivo de micosis superficial. Las muestras procesadas fueron uñas, pelos y escamas epidérmicas. La identificación de los hongos se realizó mediante observación macro y microscópica de las colonias y pruebas de identificación bioquímicas y fisiológicas, según requerimiento del agente aislado. Para la investigación de Malassezia spp. solo se realizó examen directo. De las 3228 muestras procesadas, 1098 (34%) resultaron positivas y su distribución según el agente etiológico fue: 79,5% dermatofitos; 10,9% levaduras; 5,1% hongos no dermatofitos y 4,5% Malassezia spp. El dermatofito más aislado fue el Complejo Trichophyton rubrum (70,1%), seguido del Complejo T. mentagrophytes (15,1%), Microsporum canis (9,4%) y Epidermophyton floccosum (4%). Las tiñas más frecuentes fueron: Tinea unguium (66,8%), seguida de Tinea pedis (16,4%) y Tinea capitis (8,1%). En el grupo de levaduras el Complejo Candida parapsilosis (37,5%) fue el más aislado y entre los hongos no dermatofitos el más frecuente fue Fusarium spp. (53,6%), seguido de Aspergillus spp. (19,6%) y Acremonium spp. (10,7%). La identificación del agente etiológico es fundamental para orientar un tratamiento adecuado. Esta casuística constituye un aporte importante para el conocimiento de la epidemiología de las micosis superficiales en nuestro país.

The superficial mycoses are very common infectious diseases and therefore are a frequent reason for medical consultation. The aim of this study was to determine the diagnostic frequency of superficial mycoses in the Mycology Department of the Instituto Nacional de Higiene “Rafael Rangel” during 14 years (2001-2014). A retrospective cross-sectional study was performed to review the mycological records of patients with presumptive diagnosis of superficial mycosis. Nails, hairs and epidermal scales were the processed samples. The identification of fungi was performed by macro and microscopic observation of colonies and biochemical and physiological tests, as required of the isolated agent. For the investigation of Malassezia spp. only direct examination was performed. Of the 3 228 samples processed, 1 098 (34%) were positive and their distribution according to the etiological agent was: dermatophytes 79.5%; 10.9% yeasts; non-dermatophytes fungi 5.1% and 4.5% Malassezia spp. The most frequently isolated dermatophyte was Trichophyton rubrum Complex (70.1%), followed by T. mentagrophytes complex (15.1%), Microsporum canis (9.4%) and Epidermophyton floccosum (4%). The most frequent ringworms were: Tinea unguium (66.8%), followed by Tinea pedis (16.4%) and Tinea capitis (8.1%). Candida parapsilosis complex (37.5%) was the most frequently isolated yeast and Fusarium spp. (53.6%) was the most isolated among non-dermatophyte fungi, followed by Aspergillus spp. (19.6%) and Acremonium spp. (10.7%). The identification of the etiological agent is essential to guide appropriate treatment. This study constitutes an important contribution to the knowledge of the epidemiology of superficial mycoses in our country.

Frecuencia y perfil de sensibilidad in vitro de aislamientos del Complejo Candida parapsilosis provenientes de pacientes con candidemias / Frequency and in vitro susceptibility profile of Candida parapsilosis Complex isolates from patients with candidemia

El objetivo de este trabajo fue conocer la frecuencia y el perfil de sensibilidad in vitro de aislamientos del Complejo Candida parapsilosis provenientes de casos de candidemias. Se estudiaron 754 cepas (Periodo 2008-2011), de la Red de Vigilancia de Candidemia del Instituto Nacional de Higiene “Rafael Rangel”. La identificación de las cepas se realizó por pruebas fenotípicas. La sensibilidad in vitro a los antifúngicos se evaluó por el método de Etest® y se determinó la concentración mínima inhibitoria a anfotericina B (AB), caspofungina (CS), fluconazol (FZ), y voriconazol (VZ). Se calcularon los puntos de corte epidemiológicos (PCE) y los rangos de cepas salvajes (PS) para cada antifúngico. El 43,6% de las cepas (n=328) fueron identificadas como Complejo C. parapsilosis; todas fueron sensibles a AB y presentaron bajos porcentajes de resistencia a FZ (4,3%), VZ (1,2%) y CS (0,6%). Los PCE y los rangos de PS (en µg/mL) fueron: FZ: 2/0,03-2; VZ y AB: 0,06/0,002-0,06 y CS: 0,5/0,002-0,5 respectivamente. Los resultados de este estudio aportaron información importante sobre el comportamiento del Complejo C. parapsilosis frente a los antifúngicos más utilizados en el tratamiento de las candidemias.

The aim of this study was to determine the frequency and in vitro susceptibility profile of Candida parapsilosis Complex isolates from patients with candidemia. Seven hundred and fifty four (754) strains (Period 2008-2011), from the Candidemia Surveillance Network of the Instituto Nacional de Higiene “Rafael Rangel” were studied. The strains identification was performed by phenotypic methods. In vitro antifungal susceptibility was evaluated by the Etest® method and minimum inhibitory concentration for amphotericin B (AB), caspofungin (CS), fluconazole (FZ), and voriconazole (VZ) was determined. Epidemiological cut off values (ECV) and ranges for wild type strains (WT) were also calculated for each antifungal. Forty three point six (43.6%) of the isolates (n=328) belonged to C. parapsilosis Complex; all of them were susceptible to AB and showed low resistance percentages to FZ (4.3%), VZ (1.2%) and CS (0.6%). The ECV and WT strains ranges (in mcg/mL) were: FZ: 2/0.03-2; VZ and AB: 0.06/0.002-0.06 and CS: 0.5/0.002-0.5 respectively. The results of this study provided important information about the behavior of the C. parapsilosis Complex against the most commonly antifungal agents used for the treatment of candidemias.

Micoteca del Instituto Nacional de Higiene “Rafael Rangel”: 60 años preservando la biodiversidad fúngica de interés médico / Fungal collection of the National Institute of Hygiene “Rafael Rangel”: 60 years preserving fungal biodiversity of medical interest in Venezuela

La Micoteca del Instituto Nacional de Higiene “Rafael Rangel” (INHRR) fue creada en el año 1955 y es la colección de hongos microscópicos autóctonos más grande y representativa del país. Cuenta con 2.500 cepas pertenecientes a 77 géneros y 165 especies de hongos y actinomicetos, de importancia médica, epidemiológica, industrial e histórica, preservados por duplicado bajo los métodos de agua por Castellani y aceite mineral. La colección tiene presencia a nivel internacional a través del catálogo y la página web del Centro Venezolano de Colecciones de Microorganismos (CVCM), que a su vez está afiliada a la Federación Mundial de Colecciones de Cultivos (WFCC). Además, a través de su membresía a la Federación Latinoamericana de Colecciones de Cultivos (FELACC), sus datos están disponibles en la página web de la Asociación Argentina de Microbiología (AAM). La conservación de hongos microscópicos es fundamental, debido a su importancia en el funcionamiento de los ecosistemas y a su impacto en la vida del hombre. Esta Micoteca garantiza la preservación ex situ de la biodiversidad fúngica. Sus características la consolidan como una unidad cónsona con las exigencias de los ámbitos científico, tecnológico y docente, para el desarrollo de investigaciones científicas, particularmente en el área de medicina.

The fungal collection (Mycothec) of the National Institute of Hygiene “Rafael Rangel” (INHRR) was created in 1,955 and is the largest and more representative collection of the country’s indigenous microscopic fungi. It has 2,500 strains belonging to 77 genera and 165 species of fungi and actinomycetes retaining medical, epidemiological, industrial and historical importance, preserved by duplicate under water by Castellani and mineral oil methods. The collection has international presence through the catalog and the website of the Venezuelan Center of Microorganism Collections (CVCM), which in turn belongs to the World Federation of Culture Collections (WFCC). In addition, through its membership to the Latin American Federation of Culture Collections (FELACC) the data are accessible on the website of the Argentinian Association of Microbiology (AAM). The conservation of microscopic fungi is essential, due to its importance in the ecosystems functioning and their impact on human life. This Mycothec guarantee the ex situ conservation of fungal biodiversity. Its characteristics consolidate it as a consonant unit with the requirements of scientific, technological, and educational areas for the development of scientific research, particularly in the ​​medicine area.

Estudio comparativo entre los sistemas automatizados Vitek YBC® y Microscan Walk Away RYID® con los métodos fenotípicos convencionales para la identificación de levaduras de interés clínico / A comparative study between the Vitek YBC® and Microscan Walk Away RYID® automated systems with conventional phenotypic methods for the identification of yeasts of clinical interest

El objetivo de este trabajo fue comparar la identificación de levaduras de interés clínico por los métodos automatizados Vitek YBC® y Microscan Walk Away RYID® con los métodos fenotípicos convencionales. Se utilizaron 193 aislamientos de levaduras provenientes de muestras clínicas y cinco cepas controles. Todas las levaduras fueron identificadas por los métodos automatizados antes nombrados y los métodos fenotípicos convencionales de asimilación de carbohidratos, visualización de la morfología microscópica con agar harina de maíz y el uso de agar cromogénico. Para evaluar las variables se utilizaron tablas de contingencia de 2×2, Chi cuadrado de Mc Nemar, el índice Kappa, y se calcularon los valores de concordancia, así como los errores mayores y menores de los métodos automatizados. Las levaduras se dividieron en dos grupos: 1) de aislamiento frecuente y 2) de aislamiento poco frecuente. Los sistemas Vitek YBC® y Microscan Walk Away RYID® fueron concordantes en un 88,4 y 85,9% respectivamente, cuando se compararon con los métodos fenotípicos convencionales. Aunque ambos sistemas automatizados se pueden utilizar para la identificación de levaduras, la presencia de errores mayores y menores indica la posibilidad de identificaciones incorrectas, por lo tanto, el operador de estos equipos debe utilizar paralelamente pruebas fenotípicas como la visualización de la morfología microscópica en agar harina de maíz y el agar cromogénico, sobre todo frente a levaduras de aislamiento poco frecuente. Los sistemas automatizados son una herramienta valiosa, sin embargo, la experiencia y el criterio del microbiólogo son una fortaleza importante para asegurar la calidad de los resultados.

The aim of this study was to compare the identification of clinically relevant yeasts by the Vitek YBC® and Microscan Walk Away RYID® automated methods with conventional phenotypic methods. One hundred and ninety three yeast strains isolated from clinical samples and five controls strains were used. All the yeasts were identified by the automated methods previously mentioned and conventional phenotypic methods such as carbohydrate assimilation, visualization of microscopic morphology on corn meal agar and the use of chromogenic agar. Variables were assessed by 2×2 contingency tables, McNemar’s Chi square, the Kappa index, and concordance values were calculated, as well as major and minor errors for the automated methods. Yeasts were divided into two groups: 1) frequent isolation and 2) rare isolation. The Vitek YBC® and Microscan Walk Away RYID® systems were concordant in 88.4 and 85.9% respectively, when compared to conventional phenotypic methods. Although both automated systems can be used for yeasts identification, the presence of major and minor errors indicates the possibility of misidentifications; therefore, the operator of this equipment must use in parallel, phenotypic tests such as visualization of microscopic morphology on corn meal agar and chromogenic agar, especially against infrequently isolated yeasts. Automated systems are a valuable tool; however, the expertise and judgment of the microbiologist are an important strength to ensure the quality of the results.

Susceptibilidad in vitro a cinco antifúngicos de aislamientos del Complejo Fusarium solani provenientes de úlceras corneales / In vitro susceptibility to five antifungals of Fusarium solani Complex isolates obtained from corneal ulcers

El Complejo Fusarium solani (CFS) se encuentra distribuido en la naturaleza, causando un amplio espectro de infecciones en los humanos, desde superficiales, como la queratitis, hasta infecciones fúngicas invasoras, caracterizándose por su resistencia a los antimicóticos. El objetivo de esta investigación fue determinar la susceptibilidad in vitro del CFS frente a cinco antifúngicos. Se utilizaron 30 aislados obtenidos de úlceras corneales provenientes de la colección de cultivos del Departamento de Micología del Instituto Nacional de Higiene “Rafael Rangel” y se siguió el protocolo descrito en el documento M38-A2 del Instituto de Estándares Clínicos y de Laboratorio (CLSI), determinando las Concentraciones Mínimas Inhibitorias (CMI) por microdilución en caldo para anfotericina B, itraconazol, posaconazol, voriconazol y fluconazol. En general, todas las drogas presentaron CMI elevadas, siendo voriconazol y anfotericina B los antifúngicos que exhibieron mejor actividad, mientras que itraconazol, posaconazol y fluconazol mostraron actividad nula. Los resultados de este estudio aportaron información importante sobre el comportamiento del CFS frente a los antifúngicos de uso común en la práctica clínica por primera vez en Venezuela. Es imprescindible que el médico conozca la actividad de estas drogas para poder tomar decisiones y orientar una conducta terapéutica adecuada.

The Fusarium solani Complex (FSC) is distributed in nature, producing a wide spectrum of infections in humans, from superficial ones such as keratitis, to invasive fungal infections, characterized by their resistance to antimycotics. The purpose of this investigation was to determine the in vitro susceptibility of the FSC to five antifungals. We used 30 isolates obtained from corneal ulcers kept at the culture collection of the Instituto Nacional de Higiene “Rafael Rangel” and we followed the protocol described in the M38-A2 document of the Clinical and Laboratory Standards Institute (CLSI) determining the Minimal Inhibitory Concentrations (MIC) by broth microdilution for amphotericin B, itraconazole, posaconazole, voriconazole and fluconazole. In general, all the drugs presented high MICs, voriconazole and amphotericin B being the antifungals which showed the best activity, while itraconazole, posaconazole and fluconazole showed a null activity. The results of this study provided, for the first time in Venezuela, important information about the behavior of the FSC towards commonly used antifungals. It is mandatory that physicians know the activity of these drugs in order to be able to take decisions and devise an guide an appropriate therapeutic management.

[A comparative study between the Vitek YBC and Microscan Walk Away RYID automated systems with conventional phenotypic methods for the identification of yeasts of clinical interest]. / Estudio comparativo entre los sistemas automatizados Vitek YBC y Microscan Walk Away RYID con los-métodos fenotípicos convencionales para la identificación de levaduras de interés clínico.

The aim of this study was to compare the identification of clin- ically relevant yeasts by the Vitek YBC and Microscan Walk Away RYID automated methods with conventional phenotypic methods. One hundred and ninety three yeast strains isolated from clinical samples and five controls strains were used. All the yeasts were identified by the automated methods previously mentioned and conventional phenotypic methods such as carbohydrate assimilation, visualization of microscopic morphology on corn meal agar and the use of chromogenic agar. Variables were assessed by 2 x 2 contingency tables, McNemar's Chi square, the Kappa index, and concordance values were calculated, as well as major and minor errors for the automated methods. Yeasts were divided into two groups: (1) frequent isolation and (2) rare isolation. The Vitek YBC and Microscan Walk Away RYID systems were concordant in 88.4 and 85.9% respectively, when compared to conventional phenotypic methods. Although both automated systems can be used for yeasts identification, the presence of major and minor errors indicates the possibility of misidentifications; therefore, the operator of this equipment must use in parallel, phenotypic tests such as visualization of microscopic morphology on corn meal agar and chromogenic agar, especially against infrequently isolated yeasts. Automated systems are a valuable tool; however, the expertise and judgment of the microbiologist are an important strength to ensure the quality of the results.

Validación del inóculo por densitometría para las pruebas de susceptibilidad a los antifúngicos en especies del género Fusarium / Validation of the inoculum by densitometry for antifungal susceptibility testing in Fusarium species

El objetivo de este trabajo fue validar la preparación del inóculo por densitometría para las pruebas de susceptibilidad a los antifúngicos en especies del género Fusarium. Se emplearon 15 aislamientos clínicos de Fusarium spp. para preparar los inóculos por espectrofotometría y contaje de unidades formadoras de colonias en cámara de Neubauer, siguiendo los protocolos establecidos por los documentos de referencia M38-A2 del Instituto de Estándares Clínicos y de Laboratorio (CLSI) y E.DEF 9.1 del Comité Europeo para Pruebas de Susceptibilidad a los Antimicrobianos (EUCAST), respectivamente. En paralelo se determinaron las lecturas por densitometría para ambos procedimientos. Se estableció un rango de 0,5-0,7 unidades McFarland para la preparación del inóculo por densitometría según el CLSI, y un rango de 0,2-0,8 unidades McFarland para la metodología descrita por el EUCAST. Con este estudio, se logró validar la preparación del inóculo para las pruebas de susceptibilidad en Fusarium spp., utilizando la densitometría como método alternativo de los procedimientos descritos internacionalmente, con considerables ventajas para ser implementado en los laboratorios de microbiología clínica. La variabilidad en cuanto a la capacidad de esporulación y tamaño de las conidias, sobre todo en especies poco frecuentes de Fusarium, sugiere la necesidad de validar el inóculo por especie.

The purpose of this work was to validate the preparation of the inoculum by densitometry for antifungal susceptibility testing in Fusarium species. Fifteen clinical isolates of Fusarium spp. were used to prepare the inocula by spectrophotometry and counting of colony forming units in a Neubauer chamber, according to the protocols established by the reference documents M38-A2 of the Clinical and Laboratory Standards Institute (CLSI), and E.DEF 9.1 of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), respectively. Densitometry readings were determined in parallel for both procedures. A range of 0.5-0.7 McFarland units was established for inocula preparation by densitometry according to the CLSI, and a range of 0.2-0.8 McFarland units was established for the methodology described by EUCAST. This study allowed validating the preparation of the inocula for antifungal susceptibility testing in Fusarium spp., using densitometry as an alternative method for other procedures described internationally, with considerable advantages that can be implemented at clinical microbiology laboratories. The variability regarding sporulation capacity and conidia size, especially in less frequent Fusarium species, suggests the need of validating inocula per species.

Diagnóstico de neumocistosis en pacientes oncológicos: por la técnica de inmunofluorescencia directa / Neumocistosis in oncological patients: diagnoses for immunofluorescense direct technique

El paciente con enfermedades oncológicas tiene un alto riesgo para desarrollar infecciones respiratorias, y neumonía por Pneumocystis jirovecii. En Venezuela existen pocos estudios sobre la neumocistosis en pacientes oncológicos. El objetivo de este trabajo fue detectar la presencia de Pneumocystis jirovecii en pacientes oncológicos a través de la técnica de inmunofluorescencia directa. Se recibieron, durante 10 meses, 31 muestras respiratorias (lavado broncoalveolar, esputo espontáneo e inducido, aspirados traqueales), de ellas 8 (25,5 por ciento) resultaron positivas. La distribución por tipo de cáncer fue la siguiente: 18 tumores sólidos y 13 leucemias y linfomas. La positividad entre los grupos estudiados no fue estadísticamente significativa (P>0,05). Los exámenes de laboratorio complementarios, relacionados tampoco fueron estadísticamente significativos (P>0,05). Es necesario incluir este diagnóstico en estudio microbiológico diferencial de infecciones del tracto respiratorio inferior en pacientes con cáncer, estos pacientes cursan con una sintomatología general inespecífica y tendrán una alta posibilidad de desarrollar neumocistosis.

The patient with malignancy disease has a high risk to develop respiratory infections for Pneumocystis jirovecii pneumonia. Investigations about pneumocystosis in oncological patients in Venezuela are scarce. The objective of this work was to detect Pneumocystis jirovecii in oncological patients by the method of direct immunofluorescence technique. Thirty one respiratory specimens (bronchoalveolar lavage, spontaneous and induced sputum, and tracheal aspirates) received in 10 months, 8 specimens of them (25.5) were positive the distribution by malignancy disease was the following: 18 solid tumors, and 13 leukemias, and lymphomas. No statistically significant differences were found between the studied groups and positive results (P>0.05). The complementary laboratory tests, related to the presence of Pneumocystis, were not statistically significant either P>0.05). Is necessary to include this diagnosis in the microbiological differential study of low respiratory tract infections in oncological patients, since these patients show unspecific symptoms, and have a high possibility to develop pneumocystosis.

Clasificación e identificación de especies de actinomicetos: Un estudio fenotípico comparativo / Classification and identification of Actinomyces species: A comparative phenotype study

Las pruebas fisiológicas y bioquímicas constituyen dos de las principales metodologías utilizadas, principalmente en los laboratorios de microbiología, para la identificación y diferenciación de los actinomicetos. La finalidad de este trabajo fue comparar y evaluar los métodos fenotípicos que son utilizados de manera rutinaria en la identificación de estos microorganismos. Se estudiaron setenta y tres cepas de actinomicetos provenientes de tres laboratorios de microbiología de Venezuela. El comportamiento fisiológico y bioquímico de las cepas en estudio fue evaluado mediante pruebas de descomposición de diferentes sustratos. Los resultados obtenidos permitieron observar diferencias en la identificación preliminar de las cepas realizada por estos laboratorios, conduciendo algunas veces a una nueva identificación de las mismas, gracias a la utilización de estos sustratos. Se pudo observar que la metodología empleada permitió la reclasificación taxonómica de casi todas las cepas estudiadas, lo que sugiere que es necesario estandarizar la metodología de identificación para los actinomicetos.

Physiologic and biochemical tests constitute two of the main methodologies, mainly used in microbiology laboratories, for the identification and differentiation of actinomyces. The purpose of this work was to compare and evaluate the phenotypic methods used routinely for the identification of these microorganisms. The study included seventy three actinomyces strains from three microbiology laboratories in Venezuela. The physiologic and biochemical behavior of the strains was evaluated through decomposition tests using different substrates. The results obtained showed differences in the preliminary identification of the strains done in the various laboratories, leading occasionally to a new identification arisen from the use of these substrates. It was seen that with the methods used it was possible to taxonomically reclassify almost all the strains studied, suggesting that it is necessary to standardize the methods for the identification of actinomyces.

Evaluación de la técnica de PCR anidada para el diagnóstico de Pneumocystis jirovecii / Evaluation of the nested PCR technique for Pneumocystis jirovecii diagnosis

Se realizó un estudio comparativo entre las técnicas de inmunofluorescencia directa (IFD) y la reacción en cadena de la polimerasa anidada (PCRa) en 62 muestras del tracto respiratorio inferior, con la finalidad de evaluar la técnica de PCRa para el diagnóstico de Pneumocystis jirovecii. Al comparar ambas técnicas, la PCRa obtuvo valores de sensibilidad 100%; especificidad 79,2%; valor predictivo positivo 58,3%; valor predictivo negativo 100% y una concordancia de 84% con la técnica de IFD. La PCRa para el diagnóstico de neumocistosis predice con éxito la ausencia de la enfermedad cuando el resultado es negativo. Ante un resultado positivo, debe tomarse en cuenta la condición clínica del paciente, ya que la prueba no es capaz de discriminar entre colonización e infección.

A comparative study between the direct immunofluorescency technique (DIF) and the nested polymerase chain reaction (nPCR) was carried out in 62 samples taken from the lower respiratory tract in order to evaluate the nPCR technique for Pneumocystis jirovecii diagnosis. When comparing both techniques, nPCR showed 100% sensitivity, 79.2% specificity, 58.3% positive predictive value, 100% negative predictive value, and 84% of agreement with the DIF technique. nPCR used for pneumocystosis diagnosis successfully predicts absence of disease when the result is negative. With a positive result, the clinical condition of the patient should be taken into account since the test is not capable of discriminating between colonization and infection.

MICs and minimum fungicidal concentrations of amphotericin B, itraconazole, posaconazole and terbinafine in Sporothrix schenckii.

The in vitro susceptibility of 62 isolates of Sporothrix schenckii in its mycelial form, from Latin-American countries (Peru, Venezuela, Brazil and Uruguay) and Spain, to amphotericin B (AB), itraconazole (IZ), posaconazole (PZ) and terbinafine (TB) was determined by measuring the MICs and minimum fungicidal concentrations (MFCs) using a standardized Clinical and Laboratory Standards Institute method. In general, TB was the most active drug, with the lowest geometric mean (GM) MIC and MFC values amongst isolates from the five countries tested. IZ and PZ showed almost the same activity against all strains tested, except for isolates from Uruguay where IZ gave the highest GM MIC (10.68 mg l(-1)). AB showed the widest MIC range (0.03-16.0 mg l(-1)); however, this drug was less active against 79 % of isolates (MICs above 1 mg l(-1)). MFCs were 5 to 20 times higher than the MICs, but the lowest GM MFC and range values were found for TB. IZ and PZ gave the highest GM MFC. MFC may be a better predictor of therapeutic response than MIC, especially in immunosuppressed patients, making the use of IZ and PZ an inappropriate treatment. There were some differences in susceptibility according to the geographical source of the isolates, with the MIC being lower for TB in Venezuelan strains (P=0.066) and the MFC higher for PZ in Peruvian strains (P=0.02). Thus, geographical origin may be important for appropriate treatment, and may relate to the identification of species of the S. schenckii complex.

Candida spp. in vitro susceptibility profile to four antifungal agents. Resistance surveillance study in Venezuelan strains.

The aim of this study was to determine in vitro susceptibility profiles of Venezuelan strains of Candida spp. to four antifungal agents. One hundred and forty five (145) isolates were recovered during a 1-year period (June 2006 to June 2007) from clinical specimens of patients with severe Candida spp. infections in 15 hospitals. In vitro susceptibilities to amphotericin B, fluconazole, itraconazole and voriconazole were determined by modified Etest. Non Candida albicans Candida spp. were the most frequently isolated yeasts (72.4%) in comparison with C. albicans (27.6%). Candida spp. strains showed MIC ranges between <0.002 and 0.5 mug/ml to amphotericin B. While none were found to be resistant to voriconazole, 5.5% and 27.6% of the test strains were resistant to fluconazole and itraconazole, respectively. C. albicans remains the most susceptible of the yeasts studied to fluconazole and itraconazole (P<0.05) when compared with non C. albicans Candida spp. C. krusei showed the greater cross-resistance to azoles, followed by C. glabrata, C. tropicalis and C. parapsilosis, while C. albicans isolates did not demonstrate this characteristic. It is very important to carry out the correct species identification of clinical yeast isolates because they show up variations in both distribution and susceptibility profiles according to the hospital, patient's underlying disease, clinical specimen analyzed, and the geographical region in which the studies were conducted. The Mycology Department of the INHRR is the national reference center responsible for antifungal resistance surveillance, performing the susceptibility tests with isolates recovered from hospitalized patients in public health centres which do not have mycological diagnosis laboratories.

[Pneumocystosis in Venezuelan patients: epidemiology and diagnosis (2001-2006)]. / Neumocistosis en pacientes venezolanos: diagnóstico y epidemiología (2001-2006).

The objective of this work was to investigate the epidemiology of pneumocystosis in Venezuelan patients utilizing a retrospective study during a six year period. One hundred and twenty nine clinical samples collected from patients with AIDS, cancer and non-AIDS-non-cancer low respiratory tract infection patients were processed by direct immunofluorescence technique. Pneumocystosis was diagnosed in 30 patients with a general frequency of 23.3%, which varied according to the patient's group: 36.6% in AIDS patients, 38% in cancer patients, and 10.4% in non-AIDS-non-cancer low respiratory tract infection patients. This study demonstrated the existence of differences in pneumocystosis frequency related to the patient's underlying disease, and that the illness is an important health problem in immunocompromised patients in Venezuela. Pneumocystosis must be suspected in non-immunocompromised patients with signs and symptoms of low respiratory tract infection, and the study of this illness must include COPD and cancer patients. Direct immunofluorescence is a useful technique for pneumocystosis diagnosis, however, it requires an optimal sample and skilled personnel in the laboratory.

[Distribution and antifungal susceptibility of Candida clinical isolations coming from six health care centers in the metropolitan area of Caracas, Venezuela (years 2003-2005)]. / Distribución y sensibilidad a los antifúngicos de aislamientos clínicos de Candida en seis centros de salud del área metropolitana de Caracas, Venezuela (años 2003-2005).

The aim of this study was to investigate the frequency and antifungal susceptibility of Candida clinical isolations coming from patients with candidiasis in six health care centers of Caracas, Venezuela metropolitan area. The laboratory reports were retrospectively revised from January 2003 through August 2005. The isolated yeasts identification was carried out by conventional methods and antifungal susceptibility was evaluated by ATB-fungus (bioMérieux, France) and Etest (AB Biodisk, Solna, Sweden). One thousand nine hundred seventy seven (1.977) yeasts were studied and their susceptibility testing were carried out only in 1,414 of them. C. albicans was the most isolated yeast (46.7%) and none-albicans Candida-species represented more than half of the isolations (53.4%). All the isolated yeasts evaluated presented CMIs<1 microg/ml to anfotericina B and showed variable susceptibility percentages to fluconazole (91.5%), itraconazole (80%) and voriconazole (98.6%).

Diagnóstico inmunológico de las micosis sistémicas durante cinco años 2002-2006 / Immunodiagnosis of systemic mycosis during a five-year period 2002-2006

Resumen El objetivo de este estudio fue determinar la frecuencia diagnóstica de las micosis sistémicas en el Departamento de Micología del Instituto Nacional de Higiene “Rafael Rangel” (INHRR) durante 5 años (2002-2006). Se revisaron retrospectivamente las historias micológicas en el período señalado y sólo se tomaron en cuenta los diagnósticos iniciales de las micosis detectadas, descartando los controles sucesivos. Las muestras (suero y líquido cefalorraquídeo) fueron procesadas por las técnicas de inmunodifusión doble y aglutinación con látex. De 7653 muestras, 409 (5,3%) resultaron positivas para el diagnóstico de micosis sistémicas. 39% (2990/7653) procedían de pacientes con VIH/SIDA, con 203/2990 (6,8%) casos positivos y 4663/7653 (61%) procedían de pacientes sin VIH/SIDA, con una positividad general de 4,4% (206/4663 casos). La histoplasmosis fue la micosis más frecuente en los pacientes con SIDA (67%), seguida de la criptococosis con 26,6%. La paracoccidioidomicosis se diagnosticó con mayor frecuencia en pacientes sin VIH/SIDA (54,4%), seguida de la histoplasmosis (32%) y la criptococosis (11,2%). La vigilancia epidemiológica de las micosis es esencial para fomentar los conocimientos sobre las mismas. El Departamento de Micología del INHRR funciona como centro de referencia para el diagnóstico micológico y realiza una parte importante de los estudios de estas enfermedades a escala nacional. La presentación de las revisiones de casuística en forma sistemática contribuye con el conocimiento de estas patologías en nuestro país.

Abstract The purpose of this study was to determine the diagnostic frequency of systemic mycoses at the Mycology Department of the Instituto Nacional de Higiene “Rafael Rangel” (INHRR) during a five-year period (2002-2006). Mycology case histories during the mentioned period were revised retrospectively and only the initial diagnoses of the mycoses detected were considered, discarding successive controls. Samples (serum and spinal fluid) were processed by double immunodiffusion and latex agglutination techniques. Of 7653 samples, 409 (5,3%) were positive for systemic mycoses. Thirty nine percent (2990/7653) came from HIV/AIDS patients, with 203/2990 (6,8%) positives, and 4663/7653 (61%) from non HIV/AIDS patients, with a 4,4% (206/4663) general positivity. Histoplasmosis was the most frequent mycosis in AIDS patients (67%), followed by cryptococcosis with 26,6%. Paracoccidioidomycosis was diagnosed more frequently in non HIV/AIDS patients (54,4%), followed by histoplasmosis (32%) and cryptococcosis (11,2%). Epidemiological surveillance of mycoses is essential to improve knowledge regarding them. The Mycology Department of the INHRR functions as a reference center for mycological diagnosis and performs an important part of these studies at a national scale. The systematic presentation of case revisions contributes to the knowledge of these pathologies in our country.

Epidemiología de la neumocistosis / Neumocystosis the epidemiology

La infección por Pneumocystis jirovecii ha sido considerada como parte del complejo SIDA. Conocer las siguientes variables: frecuencia de infección por Pneumocystis jirovecil en una población con factores de riesgo conocidos para la enfermedad y estado comórbidos de los pacientes estudiados, demostrar la infección por Pneumocystis jirovecii por inminoflurencia en muestras de esputo inducido o en material obtenido por broncofibroscopia y lavado bronquial y conocer las variables paraclínicas que se correlacionan con esta infección. Se diseño un estudio transversal, descriptivo, de pacientes con criterios de infección respiratoria baja, de cualquier edad o sexo, ingresados al Hospital General del Oeste desde mayo de 2004 a junio de 2005. Se calcularon: sensibilidad, especificidad y razones de verosimilitud de las variables en función del diagnóstico obtenido por inmunoflurescencia. Se identificaron 10 casos de neumocistosis, 40 por ciento presentaba SIDA y 60 por ciento otras condiciones comórbidas sin SIDA. El dolor pleurítico, hipoalbuminemia, LDH elevada, alteración de la auscultación pulmonar y alteraciones radiológicas compatibles con la infección, obtuvieron valores de moderada capacidad diagnóstica para la enfermedad. La neumocistosis debe ser sospechada en pacientes sin SIDA y/o con factores de riesgo para la enfermedad como neumopatías crónicas, contactos hospitalarios y viajes rurales o urbanos La inmunoflorescencia y el uso del esputo inducido son la técnica de elección para el diagnóstico de neumocistosis.