Effectiveness of the National Quality Control Program in Mycology of Argentina and performance evaluation of participating laboratorios / Efectividad del Programa Nacional de Control de Calidad en Micología de Argentina y evaluación del desempeño de los laboratorios participantes

Abstract The National Quality Control Program in Mycology (PNCCM) of Argentina was establishedin 1996 to improve the quality of the mycological diagnosis, to help establish and to setup standardized procedures and continuous training of laboratory staff. The aim of this studywas to assess the effectiveness of the PNCCM in the 1996---2018 period. Data from the NationalMycology Laboratory Network (NMLN) and PNCCM database was used to estimate the increasein the number of controlled laboratories and jurisdictions, the percentage of participation, theimprovement in the quality of results and the adherence to the program. Satisfaction surveyswere performed to assess user satisfaction. The number of controlled laboratories increasedfrom 29 to 146; participation increased from 49% to 93% and general adherence was 72% inthe evaluated period (1996---2018). Improvement in the quality of the results was 15% for lowcomplexity samples; 7% for intermediate complexity samples and 14% for the identification ofhigh complexity strains. Up to 84% of the users consider the PNCCM to be ''very good'' and 16%''satisfactory''. These results show the importance of the PNCCM, which is widely accepted bymycological diagnostic laboratories from Argentina.

Resumen En 1996 se creó el Programa Nacional de Control de Calidad en Micología (PNCCM)de Argentina con el objetivo de mejorar la calidad del diagnóstico micológico, colaborar enel establecimiento de procedimientos estandarizados en aquellos laboratorios que carecen deellos y contribuir a la capacitación continua del personal.El objetivo de este estudio fue evaluar la efectividad del PNCCM en el período 1996-2018.Se utilizaron los datos de la base de la Red Nacional de Laboratorios de Micología (RNLM) ydel PNCCM para estimar el aumento en el número de laboratorios controlados y el porcentajede participación, la mejora de la calidad de los resultados y la adhesión al programa. Paraevaluar el grado de satisfacción de los usuarios, se analizaron las encuestas de satisfacción delos participantes. En el período evaluado, el número de laboratorios controlados aumentó de 29a 146, la participación aumentó de 49% a 93% y la adherencia general de los participantes fue del72%. La mejora de la calidad de los resultados de los laboratorios fue del 15% para muestras debaja complejidad, 7% para muestras de complejidad intermedia y 14% para la identificación decepas de alta complejidad. El 84% de los usuarios considera que el PNCCM es muy bueno y el 16%que es satisfactorio. Estos resultados evidencian la importancia del PNCCM, que es ampliamenteaceptado por los laboratorios que realizan diagnóstico micológico en nuestro país.

Effectiveness of the National Quality Control Program in Mycology of Argentina and performance evaluation of participating laboratories.

The National Quality Control Program in Mycology (PNCCM) of Argentina was established in 1996 to improve the quality of the mycological diagnosis, to help establish and to set up standardized procedures and continuous training of laboratory staff. The aim of this study was to assess the effectiveness of the PNCCM in the 1996-2018 period. Data from the National Mycology Laboratory Network (NMLN) and PNCCM database was used to estimate the increase in the number of controlled laboratories and jurisdictions, the percentage of participation, the improvement in the quality of results and the adherence to the program. Satisfaction surveys were performed to assess user satisfaction. The number of controlled laboratories increased from 29 to 146; participation increased from 49% to 93% and general adherence was 72% in the evaluated period (1996-2018). Improvement in the quality of the results was 15% for low complexity samples; 7% for intermediate complexity samples and 14% for the identification of high complexity strains. Up to 84% of the users consider the PNCCM to be "very good" and 16% "satisfactory". These results show the importance of the PNCCM, which is widely accepted by mycological diagnostic laboratories from Argentina.

Production of a latex agglutination reagent for the rapid diagnosis of cryptococcal meningitis / Producción de un reactivo de látex para el diagnóstico rápido de criptococosismeníngea

Abstract Cryptococcosis is a fungal disease affecting more than one million people per yearworldwide. Its main etiological agents are Cryptococcus neoformans species complex and Cryp-tococcus gattii species complex. Cryptococcal meningitis (CM) is considered an AIDS-definingcondition. Rapid diagnosis by cryptococcal antigen assays, either the latex agglutination test(LA) or the lateral flow assay, is key to decreasing mortality due to cryptococcal disease. Theaim of the study was to develop a latex agglutination reagent (LA-ANLIS) for the rapid and reliable diagnosis of cryptococcosis in Argentina. This reagent will be produced in order to supplythe NMLN (National Mycology Laboratory Network). The evaluation of LA-ANLIS performanceand its comparison with the Cryptococcus Antigen Latex Agglutination Test System (LA-IMMY)(Immuno-Mycologics, Inc., USA) were conducted in 94 samples of cerebrospinal fluid. LA-ANLISand LA-IMMY compared exhibited 100% positive agreement and 97% negative agreement. LA-ANLIS showed 94% sensitivity and 97% specificity with the positive and negative predictivevalues of 94% and 97%, respectively. The LA-ANLIS is a reliable, reproducible and cost-effectivereagent, especially useful in countries where the commercial kit is not generally available andmust be obtained at a high cost. National production of reagents is the best choice for a reliableaccess to the rapid diagnosis of CM in Argentina.

Resumen La criptococosis es una enfermedad fúngica que afecta a más de un millón de personas por año en todo el mundo. Los principales agentes etiológicos pertenecen a los complejos de especies Cryptococcus neoformans y Cryptococcus gattii. La criptococosis meníngea (CM) se considera una enfermedad marcadora de sida. El diagnóstico rápido de esta enfermedad a través de la detección del antígeno de Cryptococcus, ya sea por aglutinación en partículas de látex o por inmunocromatografía, es clave para disminuir la mortalidad. El objetivo del presente estudio fue desarrollar un reactivo de aglutinación en partículas de látex para el diagnóstico rápido y certero de la CM en Argentina. Este reactivo (denominado en adelante LA-ANLIS) será producido para abastecer a la Red Nacional de Laboratorios de Micología. Se evaluó el desempeno del reactivo LA-ANLIS, y se realizó una comparación con el reactivo comercial Immuno-Mycologics, Inc. (en adelante, LA-IMMY) utilizando 94 muestras de líquido cefalorraquídeo. Hubo un 100% de acuerdo positivo y un 97% de acuerdo negativo entre los resultados obtenidos con los reactivos LA-ANLIS y LA-IMMY. El reactivo LA-ANLIS mostró una sensibilidad del 94% y una especificidad del 97%; los valores predictivos positivo y negativo fueron del 94 y del 97%, respectivamente. Se concluye que el LA-ANLIS es un reactivo confiable y rentable, que arroja resultados reproducibles, por lo que es especialmente útil en países donde los reactivos comerciales generalmente no están disponibles o sus costos son elevados. La producción nacional de reactivos es la mejor opción para asegurar el acceso de todos los hospitales al diagnóstico rápido de la CM en Argentina.

Production of a latex agglutination reagent for the rapid diagnosis of cryptococcal meningitis.

Cryptococcosis is a fungal disease affecting more than one million people per year worldwide. Its main etiological agents are Cryptococcus neoformans species complex and Cryptococcus gattii species complex. Cryptococcal meningitis (CM) is considered an AIDS-defining condition. Rapid diagnosis by cryptococcal antigen assays, either the latex agglutination test (LA) or the lateral flow assay, is key to decreasing mortality due to cryptococcal disease. The aim of the study was to develop a latex agglutination reagent (LA-ANLIS) for the rapid and reliable diagnosis of cryptococcosis in Argentina. This reagent will be produced in order to supply the NMLN (National Mycology Laboratory Network). The evaluation of LA-ANLIS performance and its comparison with the Cryptococcus Antigen Latex Agglutination Test System (LA-IMMY) (Immuno-Mycologics, Inc., USA) were conducted in 94 samples of cerebrospinal fluid. LA-ANLIS and LA-IMMY compared exhibited 100% positive agreement and 97% negative agreement. LA-ANLIS showed 94% sensitivity and 97% specificity with the positive and negative predictive values of 94% and 97%, respectively. The LA-ANLIS is a reliable, reproducible and cost-effective reagent, especially useful in countries where the commercial kit is not generally available and must be obtained at a high cost. National production of reagents is the best choice for a reliable access to the rapid diagnosis of CM in Argentina.

Agentes de micosis endémicas en un área rural de Argentina: estudio seroepidemiológico en perros / Endemic fungal pathogens in a rural setting of Argentina: seroepidemiological study in dogs

AntecedentesLos hongos Paracoccidioides brasiliensis, Histoplasma capsulatum y Coccidioides sp. son los agentes etiológicos de paracoccidioidomicosis, histoplasmosis y coccidioidomicosis, respectivamente. Estas 3 micosis son endémicas en distintas áreas geográficas de Argentina. En Latinoamérica existen antecedentes de investigación de áreas endémicas que utilizan perros domésticos como indicadores epidemiológicos.ObjetivoDeterminar la presencia de P. brasiliensis, H. capsulatum y Coccidioides sp. en una zona rural del noreste de Argentina, denominada interfluvio Teuco-Bermejito, provincia del Chaco.MétodosSe evaluaron 89 sueros de igual número de perros domésticos que habitaban 10 comunidades del área. Mediante la técnica de western blot se buscaron anticuerpos específicos contra los antígenos extracelulares fúngicos específicos: anti-gP43 de P. brasiliensis, anti-H/M de H. capsulatum y anti-120, 82 y 48kDa de Coccidioides sp.ResultadosNueve de los 89 sueros (10%) mostraron anticuerpos específicos contra uno o más de los antígenos probados. Ocho sueros tuvieron anticuerpos anti-H/M y uno solamente tuvo anticuerpos anti-M. Uno de estos 9 sueros reveló, además, anti-gp43 y otro reaccionó contra los 3 antígenos probados.ConclusionesEste es el primer estudio realizado en Argentina que utiliza perros domésticos para detectar áreas endémicas de paracoccidioidomicosis, histoplasmosis y coccidioidomicosis. Nuestros resultados indican que H. capsulatum es el principal agente de micosis endémicas en el interfluvio Teuco-Bermejito. Probablemente, los humanos que habitan esta zona tengan una exposición similar a este hongo y, por tanto, el diagnóstico de histoplasmosis debe considerarse cuando los pacientes que habitan esta área geográfica presenten manifestaciones clínicas pulmonares o mucocutáneas compatibles con la enfermedad(AU)

BackgroundThree fungal species causing human disease, namely Paracoccidioides brasiliensis, Histoplasma capsulatum and Coccidioides sp., are endemic in different areas of Argentina. Rates of infection in domestic dogs have been used in other Latin American countries as indicators of the presence of these pathogens in a given area. We used such an approach to investigate the epidemiological relevance of paracoccidiodomycosis, histoplasmosis and coccidioidomycosis in our country.AimTo investigate the presence of P. brasiliensis, H. capsulatum and Coccidioides sp. in a rural area of Argentina called Interfluvio Teuco-Bermejito, located in Chaco province.MethodsWe applied Western Blotting to determine the presence of specific antibodies in sera from 89 domestic dogs inhabiting the area. Antibodies against the following extra-cellular fungal antigens were investigated: gP43 of P. brasiliensis, H/M of H. capsulatum and 120, 82 and 48kDa antigen bands of Coccidioides sp.ResultsSpecific antibodies against H. capsulatum were found in 9/89 (10%) sera: 8 reacted against both H and M antigens and 1 only reacted against antigen M. Of these 9 sera, one showed additional anti-gp43 activity and another reacted against all the fungal antigens tested.ConclusionsThis is the first study using dog infection to assess the presence of endemic fungal pathogens in Argentina. Our results suggest that H. capsulatum is the main dimorphic fungal pathogen in the Interfluvio Teuco-Bermejito area. Therefore, the diagnosis of histoplasmosis should be taken into account in patients living in this geographic region who show pulmonary or mucocutaneous symptoms compatible with the disease(AU)

[Endemic fungal pathogens in a rural setting of Argentina: seroepidemiological study in dogs]. / Agentes de micosis endémicas en un área rural de Argentina: estudio seroepidemiológico en perros.

BACKGROUND: Three fungal species causing human disease, namely Paracoccidioides brasiliensis, Histoplasma capsulatum and Coccidioides sp., are endemic in different areas of Argentina. Rates of infection in domestic dogs have been used in other Latin American countries as indicators of the presence of these pathogens in a given area. We used such an approach to investigate the epidemiological relevance of paracoccidiodomycosis, histoplasmosis and coccidioidomycosis in our country. AIM: To investigate the presence of P. brasiliensis, H. capsulatum and Coccidioides sp. in a rural area of Argentina called Interfluvio Teuco-Bermejito, located in Chaco province. METHODS: We applied Western Blotting to determine the presence of specific antibodies in sera from 89 domestic dogs inhabiting the area. Antibodies against the following extra-cellular fungal antigens were investigated: gP43 of P. brasiliensis, H/M of H. capsulatum and 120, 82 and 48kDa antigen bands of Coccidioides sp. RESULTS: Specific antibodies against H. capsulatum were found in 9/89 (10%) sera: 8 reacted against both H and M antigens and 1 only reacted against antigen M. Of these 9 sera, one showed additional anti-gp43 activity and another reacted against all the fungal antigens tested. CONCLUSIONS: This is the first study using dog infection to assess the presence of endemic fungal pathogens in Argentina. Our results suggest that H. capsulatum is the main dimorphic fungal pathogen in the Interfluvio Teuco-Bermejito area. Therefore, the diagnosis of histoplasmosis should be taken into account in patients living in this geographic region who show pulmonary or mucocutaneous symptoms compatible with the disease.

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

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

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

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

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