Epidemiology of coccidioidomycosis in Argentina, an update.

The National Reference Laboratory in Clinical Mycology of Argentina conducted a retrospective review of human coccidioidomycosis cases diagnosed by the National Mycology Laboratory Network of Argentina between 2010 and 2022 to determine the burden of the disease in the country. A total of 100 human coccidioidomycosis cases were documented, with a higher prevalence in male patients (male-to-female ratio of 1.9:1), with a median age of 41 years. Comparing the number of cases between two 10-year periods (2000-2009 and 2010-2019), the increase was 36.51% (from 63 to 86 cases). Among the 100 recorded cases, 79 tested positive using the double immunodiffusion test. Spherules were observed in 19 cases through histopathology or direct microscopic examination and the fungus was isolated in 39 cases. Thirty-six isolates were identified as Coccidioides posadasii through partial sequencing of the Ag2/PRA gene. Catamarca province had the highest number of cases, comprising 64% of the total, with an incidence rate above 1.0-2.5/100,000 inhabitants until 2018. However, there has been a recent downward trend in the region from 2018 to 2022. It is concerning that more than half of diagnosed cases were chronic pulmonary or disseminated forms, indicating a lack of early disease detection. To rectify this issue, it is imperative to conduct targeted training programs for healthcare personnel and enhance public awareness within the endemic area. This will contribute to a better understanding of the true burden of coccidioidomycosis and enable the implementation of appropriate sanitary control measures.

Diagnóstico micológico de paracoccidioidomicosis en un hospital de área no endémica: metodología clásica y molecular / Mycological diagnosis of paracoccidioidomycosis in a hospital from a nonendemic area: classical and molecular methods

Introducción. La paracoccidioidomicosis es una micosis sistémica y endémica en Latinoamérica. El cambio climático y el movimiento migratorio del huésped enfatizan la necesidad de optimizar el diagnóstico de esta infección. Objetivo. Evaluar la implementación de la detección de ADN de Paracoccidioides spp. al diagnóstico micológico de pacientes con sospecha de paracoccidioidomicosis. Materiales y métodos. Estudio retrospectivo con datos de laboratorio de pacientes con sospecha de paracoccidioidomicosis en un hospital de área no endémica. Resultados. Se analizaron los resultados de las muestras de 19 pacientes con sospecha clínica de paracoccidioidomicosis. El 90 % de los pacientes había nacido o visitado un área endémica de esta micosis en Latinoamérica. En 14 pacientes varones adultos se confirmó paracoccidioidomicosis por diagnóstico convencional. El examen directo fue positivo en 12 pacientes con enfermedad comprobada y en 4 de ellos se obtuvo crecimiento del hongo. Se detectaron anticuerpos contra Paracoccidioides spp. en ocho pacientes con la enfermedad. Se realizó PCR anidada con muestras de 14 pacientes para detectar ADN de Paracoccidioides spp. En 9 de los 10 pacientes con diagnóstico convencional de paracoccidioidomicosis se obtuvo una prueba de PCR positiva. Conclusiones. La implementación de técnicas moleculares para detectar ADN de Paracoccidioides spp. complementa el diagnóstico convencional de paracoccidioidomicosis y permite instaurar el tratamiento antifúngico, sobre todo en los casos clínicos donde no se observa la presencia del hongo en las muestras clínicas. La migración actual de poblaciones humanas dificulta el diagnóstico de paracoccidioidiomicosis y otras infecciones endémicas, por lo que se requiere optimizar el diagnostico micológico en los laboratorios clínicos para tratar pacientes con este tipo micosis desatendida.

Introduction. Paracoccidioidomycosis is a systemic mycosis endemic in Latin America. Climate change and host migration emphasize the need to optimize this infection diagnosis. Objective. To evaluate the implementation of Paracoccidioides spp. DNA detection in the mycological diagnosis of patients with suspected paracoccidioidomycosis. Materials and methods. It is a retrospective study with laboratory data from patients with clinical suspicion of paracoccidioidomycosis, who consulted a university hospital from a non-endemic area. Results. We analyzed the laboratory results of samples from 19 patients with suspected paracoccidioidomycosis. Seventeen out of 19 patients were born in or had visited an endemic area in Latin America. Fourteen adult male patients were confirmed to have paracoccidioidomycosis by conventional diagnosis: the direct examination was positive in 12 samples while fungal growth was found only in 4. Anti-Paracoccidioides spp. antibodies were detected in 10 patients, 8 of them with proven paracoccidioidomycosis. Nested PCR for Paracoccidioides spp. detection was performed on clinical samples from 14 patients, and positive results were obtained for 9 out of 10 patients with the conventional diagnosis of paracoccidioidomycosis. Conclusions. The incorporation of molecular techniques to detect Paracoccidioides spp. DNA complements the conventional diagnosis of paracoccidioidomycosis. This tool allows the prescription of antifungal treatment in those cases where the fungus is not observed in the clinical samples. Current human migrations difficult the mycological diagnosis of paracoccidioidomycosis and other fungal infections. For this reason, it is necessary to improve mycological diagnosis in clinical laboratories to adequately treat patients with this neglected mycosis.

Evaluation of the CHROMagar Candida Plus medium for presumptive identification of yeasts and MALDI-TOF MS identification.

Rapid and accurate yeasts species identification in clinical laboratories is important for appropriate and timely antifungal treatment. We evaluate the performance of the new medium CHROMagar™ Candida Plus for presumptive identification of yeasts species and MALDI-TOF identification. We identify 303 strains belonging to 60 clinically relevant yeasts species by using the new medium. Presumptive identification was correct at the Candida albicans complex, Candida tropicalis and Pichia kudriavzevii (Candida krusei) species. However, although this medium was able to identify all Candida auris and Candida glabrata strains, other species were misidentified as C. auris or C. glabrata. A total of 215 strains were identified by using MALDI-TOF and evaluated two incubation temperatures (30°C and 37°C) and two incubation times (24 h and 72 h). Most strains (94%; 202/215) were correctly identified at the species (n:190) or complex level (n:12) at both temperatures and incubation times. However, we observed that the time of incubation (24 h vs. 72 h) affects the score values when yeasts are incubated at 37°C, but does not affect score values when yeasts are incubated at 30°C. In conclusion, the new medium has a good performance in the presumptive identification of the C. albicans complex, C. tropicalis and P. kudriavzevii (C. krusei). In addition, this medium is useful for the screening of C. auris and C. glabrata isolates, but identification should be confirmed by other more specific techniques, like MALDI-TOF.

Genotypic Diversity and Antifungal Susceptibility of Clinical Isolates of Cryptococcus Gattii Species Complex from Argentina.

The aim of this study was to determine the genotypic diversity of 22 Cryptococcus gattii species complex clinical isolates from Argentina and to place these genotypes within the diversity of clinical, veterinary and environmental isolates from Latin America. Mating type and antifungal susceptibility of the isolates were also determined. By URA5-RFLP, nine isolates were identified as molecular type VGI, 10 as VGII, one as VGIII and two as VGIV. Multilocus sequence typing (MSLT), following the International Society for Human and Animal Mycology (ISHAM) consensus MLST scheme, was used to determine the genotypic diversity. Our results suggest that, in Argentina, VGI isolates have low genetic diversity, while VGII isolates have high genetic diversity. Both isolates identified as VGIV by URA5-RFLP were genotyped by MLST as belonging to the currently named VGVI clade. From all isolates, eight sequence types (STs) were unique for Argentina, while five STs have been reported already in other countries, being of high interest the genotypes ST20 and ST7 since they belong to the subtypes VGIIa and VGIIb, respectively, which are associated with hypervirulent strains responsible for outbreaks in North America. To note, geographical analysis showed that some genotypes may be associated with some regions in Argentina. Most isolates were MATα, but we are reporting one isolate MATa for the first time in the country. Antifungal susceptibility tests showed that itraconazole, voriconazole and posaconazole had high activity against all isolates, while amphotericin B, fluconazole and 5-fluorocytosine were the least active drugs against all studied isolates.

COVID-19 associated with disseminated histoplasmosis in a kidney transplant patient / COVID-19 asociado a histoplasmosis diseminada en un paciente trasplantado renal

Abstract We report a case of disseminated histoplasmosis and COVID-19 infection in a renal transplant recipient in Argentina. The patient exhibited respiratory symptoms, and a chest computed tomography scan (CT) showed multiple bilateral centrilobular opacities with a tree-in-bud pattern in both lobes. The patient was initially treated as having bacterial community-acquired pneumonia, and then tuberculosis. A month later, histoplasmosis was diagnosed, and Histoplasma capsulatum LAmB clade was isolated from sputum, skin and oral lesions. The patient was hospitalized and treatment was started with intravenous liposomal amphotericin B. During the course of the antifungal therapy the respiratory symptoms worsened, a new chest CT showed a unilateral lesion with a ground glass appearance and SARS-CoV-2 was detected in a new nasopharyngeal sample. In addition, plasma therapy was administered, and the immunosuppressive regimen was adjusted (everolimus was interrupted, mycophenolate mofetil reduced, and meprednisone increased). Finally, the patient's progress was favorable and was discharged after five days on oral itraconazole treatment for histoplasmosis.

Resumen Se presenta un caso de histoplasmosis diseminada e infección por COVID-19 en un paciente trasplantado renal en Argentina. El paciente presentó un cuadro clínico respiratorio, y la tomografía computarizada (TC) de tórax mostró múltiples opacidades centrolobulillares bilaterales con patrón de árbol en brote. El paciente fue tratado inicialmente con antibióticos para agentes causantes de neumonía bacteriana adquirida en la comunidad y luego como tuberculosis. Un mes después se le diagnosticó una histoplasmosis diseminada y el hongo fue aislado del esputo, la piel y la mucosa oral. El hongo fue tipificado molecularmente como Histoplasma capsulatum clado LAmB. El paciente fue hospitalizado y se inició tratamiento con anfoteric-ina B liposomal vía intravenosa. Durante el transcurso de la terapia antifúngica los síntomas respiratorios del paciente empeoraron, una nueva TC de tórax mostró una lesión unilateral con apariencia de vidrio esmerilado y se detectó SARS-CoV-2 en el hisopado nasofaríngeo. El paciente fue tratado con plasmoterapia y se modificó el régimen de inmunosupresión (se interrumpió everolimus, se redujo micofenolato de mofetilo y se incrementó la meprednisona). La evolución del paciente fue favorable y fue dado de alta con tratamiento oral con itraconazol.

COVID-19 associated with disseminated histoplasmosis in a kidney transplant patient.

We report a case of disseminated histoplasmosis and COVID-19 infection in a renal transplant recipient in Argentina. The patient exhibited respiratory symptoms, and a chest computed tomography scan (CT) showed multiple bilateral centrilobular opacities with a tree-in-bud pattern in both lobes. The patient was initially treated as having bacterial community-acquired pneumonia, and then tuberculosis. A month later, histoplasmosis was diagnosed, and Histoplasma capsulatum LAmB clade was isolated from sputum, skin and oral lesions. The patient was hospitalized and treatment was started with intravenous liposomal amphotericin B. During the course of the antifungal therapy the respiratory symptoms worsened, a new chest CT showed a unilateral lesion with a ground glass appearance and SARS-CoV-2 was detected in a new nasopharyngeal sample. In addition, plasma therapy was administered, and the immunosuppressive regimen was adjusted (everolimus was interrupted, mycophenolate mofetil reduced, and meprednisone increased). Finally, the patient's progress was favorable and was discharged after five days on oral itraconazole treatment for histoplasmosis.

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.

Frequency and geographical distribution of genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii species complexes in Argentina / Frecuencia y distribución geográfica de genotipos y tipos sexuales de los complejos de especies Cryptococcus neoformans y Cryptococcus gattii en Argentina

Abstract The aim of this work was to know the frequency and geographical distribution of genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii species complexes isolated from human infections in Argentina during the period from April 2009 to April 2011. A multicenter study was conducted, in which 372 isolates were obtained from 61 laboratories throughout the country. Of those, 98.8% of the isolates belonged to the C. neoformans species complex and 1.1% to the C. gattii species complex. Genotype VNI (MATa) was the most frequently isolated (n = 326, 87.6%), followed by VNII (MATa) (n = 22, 5.9%), the recently described VNII-VNIV (aADa) hybrid (n = 14, 3.8%), VNIV (MATa) (n=4, 1.1%), VNIII (aADa) hybrid (n = 1, 0.3%), and VNIII (aADa) hybrid (n = 1, 0.3%). The Argentine Central region showed the greatest number of cases and genotype diversity. Interestingly, a relative high frequency was observed in genotype VNII (MATa) in the Cuyo, Northeast and Northwest regions and, also in VNII-VNIV (aADa) hybrids in the Northwest region. C. gattii species complex was isolated at a low rate; 3 VGI (MATa) and 1 VGII (MATa) isolates were obtained from the Northwest and Central regions. In conclusion, this study shows that genotype frequencies seem to vary among regions in Argentina and reveals a relatively high frequency of rare hybrids in the Northwest region. Further regional clinical and environmental studies may help to elucidate if those varia-tions in frequencies are associated with the existence of regional ecological niches or any other regional factors.

Resumen El objetivo de! trabajo fue conocer la frecuencia y la distribución geográfica de genotipos y tipos sexuales de aislados pertenecientes a los complejos de especies Cryptococcus neoformansy Cryptococcus gattii obtenidos de infecciones humanas en Argentina. Entre abril de 2009 y abril de 2011 se realizó un estudio multicéntrico del que se obtuvieron 372 aislados de 61 laboratorios de diferentes zonas del país. El 98,8% de los aislados pertenecieron al complejo C. neoformansy el 1,1% al complejo C. gattii. El genotipo VNI (MATa) fue el más frecuente (n = 326; 87,6%), le siguieron VNII (MATa) (n = 22; 5,9%), el híbrido VNII-VNIV (aADa) (n = 14; 3,8%), VNIV (MATa) (n =4; 1,1%) y los híbridos VNIII (aADa) (n = 1; 0,3%) y VNIII (aADa) (n = 1; 0,3%). La región Centro mostró el mayor número de casos y la mayor diversidad de genotipos. Cabe destacar que el genotipo VNII (MATa) tuvo una frecuencia relativamente alta en las regiones de Cuyo, Noreste y Noroeste. En esta última región, también fue alta la frecuencia del híbrido VNII-VNIV (aADa). La frecuencia de aislamiento de miembros del complejo C. gattii fue baja: se obtuvieron 3 aislados VGI (MATa) y 1 VGII (MATa) de las regiones Centro y Noroeste. En conclusión, este estudio muestra que las frecuencias de genotipos varían entre las distintas regiones de Argentina y señala la presencia de híbridos poco comunes en una frecuencia relativamente alta dentro de la región Noroeste. Contar con mayor número de estudios clínicos y ambientales regionales podría ayudar a elucidar si tales variaciones están asociadas a la existencia de nichos ecológicos particulares o a algún otro factor regional.

Frequency and geographical distribution of genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii species complexes in Argentina.

The aim of this work was to know the frequency and geographical distribution of genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii species complexes isolated from human infections in Argentina during the period from April 2009 to April 2011. A multicenter study was conducted, in which 372 isolates were obtained from 61 laboratories throughout the country. Of those, 98.8% of the isolates belonged to the C. neoformans species complex and 1.1% to the C. gattii species complex. Genotype VNI (MATα) was the most frequently isolated (n=326, 87.6%), followed by VNII (MATα) (n=22, 5.9%), the recently described VNII-VNIV (aADα) hybrid (n=14, 3.8%), VNIV (MATα) (n=4, 1.1%), VNIII (αADa) hybrid (n=1, 0.3%), and VNIII (αADα) hybrid (n=1, 0.3%). The Argentine Central region showed the greatest number of cases and genotype diversity. Interestingly, a relative high frequency was observed in genotype VNII (MATα) in the Cuyo, Northeast and Northwest regions and, also in VNII-VNIV (aADα) hybrids in the Northwest region. C. gattii species complex was isolated at a low rate; 3 VGI (MATα) and 1 VGII (MATα) isolates were obtained from the Northwest and Central regions. In conclusion, this study shows that genotype frequencies seem to vary among regions in Argentina and reveals a relatively high frequency of rare hybrids in the Northwest region. Further regional clinical and environmental studies may help to elucidate if those variations in frequencies are associated with the existence of regional ecological niches or any other regional factors.

Epidemiology of dermatophytoses in 31 municipalities of the province of Buenos Aires, Argentina: A 6-year study / Epidemiología de las dermatofitosis en 31 municipios de la provincia de Buenos Aires, Argentina: estudio de 6 años

Background: No reliable data are available in the province of Buenos Aires regarding the frequency of dermatophytoses and other fungal diseases. The distribution of the clinical forms and the species involved are also unknown. Aims: To present the data collected by the laboratories participating in the Mycology Network of the province of Buenos Aires (MNPBA) from a retrospective epidemiological survey on dermatophytoses. Methods: A descriptive and exploratory analysis was performed on the cases of dermatophytoses gathered between 2002 and 2007 by the Mycology Network of the province of Buenos Aires. Results: Of the 3966 dermatophytosis cases reported by 41 laboratories in 31 municipalities, more than a half occurred in three highly populated urban municipalities. The male:female ratio was 1:1.5. The most frequent clinical form was tinea unguium, diagnosed in 904 cases (51.83%), followed by tinea capitis (19.32%), tinea corporis (15.19%), tinea pedis (6.77%), tinea cruris (3.73%), and tinea manuum (2.18%). The species involved was identified in 1368 (33.49%) cases. Trichophyton rubrum was the most common species, with a frequency of 42.03%. An association was found between urban municipalities and T. rubrum or the Trichophyton mentagrophytes complex. Conclusions: Results from the MNPBA survey provide valuable information that should enable further interventions to be designed in order to prevent and control the disease

Antecedentes: No existen datos fiables acerca de la frecuencia de las dermatofitosis y otras enfermedades fúngicas en la provincia de Buenos Aires. Por otra parte, la distribución en la provincia de las formas clínicas y las especies involucradas no es conocida. Objetivos: El objetivo de este estudio fue reportar los datos recogidos por los laboratorios que participan en la Red de Micología de la Provincia de Buenos Aires (MNPBA) a través del análisis de encuestas epidemiológicas retrospectivas sobre casos notificados de dermatofitosis. Métodos: Se realizó un análisis descriptivo y exploratorio de los casos de dermatofitosis recogidos por los laboratorios de la Red de Micología de la provincia de Buenos Aires durante un período de 6 años (2002-2007). Resultados: Se registraron 3.966 casos procedentes de 41 laboratorios distribuidos en 31 municipios. Más de la mitad de los casos tuvieron lugar en tres municipios urbanos muy poblados. La proporción varón:mujer fue de 1:1,5. La forma clínica más frecuente fue tinea unguium, diagnosticada en 904 casos (51,83%), seguida de tinea capitis (19,32%), tinea corporis (15,19%), tinea pedis (6,77%), tinea cruris (3,73%) y tinea manuum (2,18%). La identificación de las especies de dermatofitos se realizó en 1.368 casos (33,49%). La especie predominante fue Trichophyton rubrum (42,03%). Se observó asociación entre los municipios de alta densidad poblacional y la presencia de T. rubrum y del complejo de especies Trichophyton mentagrophytes. Conclusiones: Los resultados de las encuestas de MNPBA generan información de calidad que permite el diseño de nuevas intervenciones para la prevención y control de estas micosis

Epidemiology of dermatophytoses in 31 municipalities of the province of Buenos Aires, Argentina: A 6-year study.

BACKGROUND: No reliable data are available in the province of Buenos Aires regarding the frequency of dermatophytoses and other fungal diseases. The distribution of the clinical forms and the species involved are also unknown. AIMS: To present the data collected by the laboratories participating in the Mycology Network of the province of Buenos Aires (MNPBA) from a retrospective epidemiological survey on dermatophytoses. METHODS: A descriptive and exploratory analysis was performed on the cases of dermatophytoses gathered between 2002 and 2007 by the Mycology Network of the province of Buenos Aires. RESULTS: Of the 3966 dermatophytosis cases reported by 41 laboratories in 31 municipalities, more than a half occurred in three highly populated urban municipalities. The male:female ratio was 1:1.5. The most frequent clinical form was tinea unguium, diagnosed in 904 cases (51.83%), followed by tinea capitis (19.32%), tinea corporis (15.19%), tinea pedis (6.77%), tinea cruris (3.73%), and tinea manuum (2.18%). The species involved was identified in 1368 (33.49%) cases. Trichophyton rubrum was the most common species, with a frequency of 42.03%. An association was found between urban municipalities and T. rubrum or the Trichophyton mentagrophytes complex. CONCLUSIONS: Results from the MNPBA survey provide valuable information that should enable further interventions to be designed in order to prevent and control the disease.

Comparison of PCR protocols for detecting Histoplasma capsulatum DNA through a multicenter study

Background. A multicenter study was conducted. A panel containing DNA from Histoplasma capsulatum, as well as negative and cross-reaction controls, was sent to five different laboratories, members of the MICOMOL network from CYTED Program. Aims. The objective was to assess the accuracy of different PCR protocols to detect H. capsulatum DNA. Methods. Seven different PCR protocols were tested. They were based on PCR techniques and used unicopy and multicopy targets. Results. Most of these protocols (4/7) were able to detect the smallest amounts of fungal DNA (102 fg/ml). Overall sensitivity was 86% and specificity was 100%. The protocol based on a unicopy target (SCAR220) presented lower sensitivity (43%) but 100% specificity. The real-time protocols tested were highly reproducible, sensitive, and specific. Neither false positives nor cross-reactions were detected in any protocol. Conclusions. All laboratories were able to amplify H. capsulatum DNA, and real-time PCR seems to be a promising tool to efficiently detect this pathogen in clinical samples (AU)

Brote de histoplasmosis en la provincia de Neuquén, Patagonia Argentina / Outbreak of histoplasmosis in province of Neuquén, Patagonia Argentina

Antecedentes. La República Argentina no registra casos autóctonos de histoplasmosis en regiones australes del país. Objetivo. Informar de un brote de histoplasmosis ocurrido en Zapala, Provincia de Neuquén, Patagonia Argentina. Métodos. Se evaluó el cuadro clínico y las características epidemiológicas de 5 pacientes involucrados en el brote. Se realizaron estudios ambientales para conocer la fuente de infección. Se analizó el perfil genético generado por RAPD-PCR con los primers 1281-1283 de los aislamientos de Histoplasma capsulatum del caso índice ( CI ) y se comparó con cepas de origen clínico no relacionadas con el brote. Resultados. Los pacientes residían en Zapala y no habían visitado otras áreas geográficas. Todos presentaron un síndrome gripal con imágenes radiológicas micronodulillares diseminadas. El CI necesitó tratamiento específico por la gravedad de su cuadro clínico, y los 4 pacientes restantes presentaron una sintomatología leve y no fueron tratados. La evolución clínica de todos fue favorable. Las cepas de H. capsulatum aisladas de hemocultivo y biopsia pulmonar del CI presentaron un perfil genético diferente al resto de las cepas analizadas. La presencia del hongo en el ambiente pudo inferirse mediante la detección de anticuerpos anti-Histoplasma en suero de ratones BALB/c inoculados con tierra de una alcantarilla que los trabajadores removieron después de un aluvión. Conclusiones. Este brote extiende el área endémica de histoplasmosis por debajo del paralelo 38° de latitud sur. El diagnóstico de histoplasmosis debe considerarse en pacientes de Neuquén, con síntomas compatibles con esta micosis, aun sin existir antecedentes epidemiológicos de viajes a áreas endémicas (AU)

Background: In Argentina, there are no reports of autochthonous cases of histoplasmosis in the southern regions of the country. Aim: To report a histoplasmosis outbreak in Zapala town, Province of Neuquén, Patagonia Argentina. Methods: We evaluated the clinical and epidemiological characteristics of 5 patients involved in the outbreak. Environmental studies were conducted to determine the source of infection. The genetic profile of Histoplasma capsulatum strains isolated from the index case (IC) were compared with clinical isolates from Argentinean patients not related to the outbreak, using RAPD-PCR with primers 1281-1283. Results: The patients were residents of Zapala, and had not visited other geographical areas before. All patients had an influenza-like syndrome, and X-ray revealed disseminated micronodular images throughout the lung parenchyma. The IC needed specific antifungal therapy; the remaining 4 patients had mild symptoms, and did not require therapy. All of them had a good clinical outcome. Strains of H. capsulatum isolated from blood culture and lung biopsy of the IC showed a genetic profile different from other strains analyzed. The presence of the fungus in the environment was demonstrated by the detection of anti- Histoplasma antibodies in BALB/c mice inoculated with soil obtained in a culvert where workers had dug up earth after a landslide. Conclusions: This outbreak suggests the histoplasmosis endemic area is under the 38◦ S parallel. Patients from Neuquén, Patagonia Argentina, with compatible symptoms of histoplasmosis should be tested, regardless of their travel or exposure history (AU)

Comparative analyses of classical phenotypic method and ribosomal RNA gene sequencing for identification of medically relevant Candida species.

As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequencing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as targets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, genotypic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirmation of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species.

Comparison of PCR protocols for detecting Histoplasma capsulatum DNA through a multicenter study.

BACKGROUND: A multicenter study was conducted. A panel containing DNA from Histoplasma capsulatum, as well as negative and cross-reaction controls, was sent to five different laboratories, members of the MICOMOL network from CYTED Program. AIMS: The objective was to assess the accuracy of different PCR protocols to detect H. capsulatum DNA. METHODS: Seven different PCR protocols were tested. They were based on PCR techniques and used unicopy and multicopy targets. RESULTS: Most of these protocols (4/7) were able to detect the smallest amounts of fungal DNA (10(2)fg/µl). Overall sensitivity was 86% and specificity was 100%. The protocol based on a unicopy target (SCAR220) presented lower sensitivity (43%) but 100% specificity. The real-time protocols tested were highly reproducible, sensitive, and specific. Neither false positives nor cross-reactions were detected in any protocol. CONCLUSIONS: All laboratories were able to amplify H. capsulatum DNA, and real-time PCR seems to be a promising tool to efficiently detect this pathogen in clinical samples.

Comparative analyses of classical phenotypic method and ribosomal RNA gene sequencing for identification of medically relevant Candida species

As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequencing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as targets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, genotypic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirmation of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species.

[Outbreak of histoplasmosis in province of Neuquén, Patagonia Argentina]. / Brote de histoplasmosis en la provincia de Neuquén, Patagonia Argentina.

BACKGROUND: In Argentina, there are no reports of autochthonous cases of histoplasmosis in the southern regions of the country. AIM: To report a histoplasmosis outbreak in Zapala town, Province of Neuquén, Patagonia Argentina. METHODS: We evaluated the clinical and epidemiological characteristics of 5 patients involved in the outbreak. Environmental studies were conducted to determine the source of infection. The genetic profile of Histoplasma capsulatum strains isolated from the index case (IC) were compared with clinical isolates from Argentinean patients not related to the outbreak, using RAPD-PCR with primers 1281-1283. RESULTS: The patients were residents of Zapala, and had not visited other geographical areas before. All patients had an influenza-like syndrome, and X-ray revealed disseminated micronodular images throughout the lung parenchyma. The IC needed specific antifungal therapy; the remaining 4 patients had mild symptoms, and did not require therapy. All of them had a good clinical outcome. Strains of H. capsulatum isolated from blood culture and lung biopsy of the IC showed a genetic profile different from other strains analyzed. The presence of the fungus in the environment was demonstrated by the detection of anti-Histoplasma antibodies in BALB/c mice inoculated with soil obtained in a culvert where workers had dug up earth after a landslide. CONCLUSIONS: This outbreak suggests the histoplasmosis endemic area is under the 38° S parallel. Patients from Neuquén, Patagonia Argentina, with compatible symptoms of histoplasmosis should be tested, regardless of their travel or exposure history.

Identificación rápida de Histoplasma capsulatum en lisados de cultivo / Rapid identification of Histoplasma capsulatum in culture lysates

Antecedentes. Histoplasma capsulatum es el agente causal de la histoplasmosis, micosis asociada principalmente a pacientes inmunocomprometidos. La rápida identificación del hongo a partir del cultivo permite el tratamiento temprano. Objetivo. Evaluar un sistema de PCR para dos dianas específicas de H. capsulatum en lisados acuosos de cultivos. Métodos. Se utilizaron dos técnicas de PCR previamente descritas que, en reacciones independientes, amplifican fragmentos específicos de 111 y 279 pb del gen AgM de H. capsulatum. Se analizaron 248 cepas de H. capsulatum y 68 de otras especies fúngicas.Para la amplificación se partió de un lisado acuoso (que contenía el ADN), obtenido por tres ciclos de hervido/enfriamiento rápido a 0°C. En casos particulares se obtuvo ADN purificado y/o se secuenció el producto la amplificación. Resultados. Las técnicas de PCR amplificaron las dos bandas a partir del lisado acuoso de 239 cepas de H. capsulatum; las 9 restantes sólo mostraron bandas de amplificación a partir de ADN purificado. No se observó amplificación específica a partir de lisado acuoso ni de ADN purificado de 66 cepas de especies distintas de H. capsulatum. Dos cepas de Emmonsia crescens presentaron ambas bandas de amplificación cuyas secuencias resultaron tener una homología superior al 97% con secuencias de H. capsulatum. El tiempo total de la prueba no superó las 7h con un 96% de sensibilidad, 97% de especificidad y un valor predictivo positivo de 99%. Conclusiones. El método es rápido, económico y puede ser utilizado como una alternativa para identificar presuntivamente H. capsulatum en lisados de cultivo sin purificar(AU)

Background. Histoplasma capsulatum is the agent of histoplasmosis, a deep mycosis mainly afflicting immunocompromised patients. Rapid identification of the fungus isolated from clinical specimens allows timely administration of specific treatment. Aim. To assess the ability of a dual PCR system targeting specific H. capsulatum DNA sites to identify fungal species in simple aqueous lysates from cultured fungi. Methods. We analysed the performance of two independent PCR reactions designed to amplify fragments of 111 and 279bp included in H. capsulatum-specific gene AgM. We used 248 H. capsulatum strains and 68 isolates of other fungal species. Reaction templates consisted of aqueous lysates of cultured fungi (either in mycelial or yeast phase) obtained after three cycles of boiling and immediate cooling at 0°C. Selected strains were submitted to conventional DNA extraction and/or sequencing. Results. Both PCR systems performed identically. Amplification from aqueous lysates was achieved from 239 H. capsulatum strains; the remaining 9 strains only showed specific bands when purified DNA was used as template. Of all other fungal species tested, only 2 Emmonsia crescens strains amplified H. capsulatum-specific bands and sequences of their amplified PCR products matched > 97% with H. capsulatum sequences. Total test time did not exceed 7h with 96% sensitivity, 97% specificity and 99% positive predictive value. Conclusions. The assay is fast, accurate and economical, and can be an alternative method for presumptive identification of H. capsulatum in simple culture lysates(AU)

[Rapid identification of Histoplasma capsulatum in culture lysates]. / Identificación rápida de Histoplasma capsulatum en lisados de cultivo.

BACKGROUND: Histoplasma capsulatum is the agent of histoplasmosis, a deep mycosis mainly afflicting immunocompromised patients. Rapid identification of the fungus isolated from clinical specimens allows timely administration of specific treatment. AIM: To assess the ability of a dual PCR system targeting specific H. capsulatum DNA sites to identify fungal species in simple aqueous lysates from cultured fungi. METHODS: We analysed the performance of two independent PCR reactions designed to amplify fragments of 111 and 279 bp included in H. capsulatum-specific gene AgM. We used 248 H. capsulatum strains and 68 isolates of other fungal species. Reaction templates consisted of aqueous lysates of cultured fungi (either in mycelial or yeast phase) obtained after three cycles of boiling and immediate cooling at 0°C. Selected strains were submitted to conventional DNA extraction and/or sequencing. RESULTS: Both PCR systems performed identically. Amplification from aqueous lysates was achieved from 239 H. capsulatum strains; the remaining 9 strains only showed specific bands when purified DNA was used as template. Of all other fungal species tested, only 2 Emmonsia crescens strains amplified H. capsulatum-specific bands and sequences of their amplified PCR products matched > 97% with H. capsulatum sequences. Total test time did not exceed 7h with 96% sensitivity, 97% specificity and 99% positive predictive value. CONCLUSIONS: The assay is fast, accurate and economical, and can be an alternative method for presumptive identification of H. capsulatum in simple culture lysates.