Thermotolerance of Histoplasma capsulatum at 40 °C predominates among clinical isolates from different Latin American regions

ABSTRACT The yeast phase of 22 Histoplasma capsulatum clinical isolates from Mexico, Argentina, Colombia, and Guatemala and three reference strains, one from Panama and two from the United States of America (USA), were screened for thermosensitivity characteristics using different analyses. Growth curves at 0, 3, 6, 12, 24, and 30 h of incubation at 37 and 40 °C, the growth inhibition percentage at 40 °C, and the doubling time at 37 and 40 °C were determined for all yeasts studied. Most of the isolates examined exhibited thermotolerant phenotypes at 40 °C, whereas a thermosensitive phenotype at 40 °C was only detected in the Downs reference strain from the USA. Growth inhibition values lower than 33.8% supported the predominance of the thermotolerant phenotype at 40 °C. The doubling time means found for the different isolates were 5.14 h ± 1.47 h at 37 °C and 5.55 h ± 1.87 h at 40 °C. This is the first report to underscore the predominance of thermotolerant and delayed doubling time phenotypes in H. capsulatum clinical isolates from different regions of Latin America.

Postpartum histoplasmosis in an HIV-negative woman: a case report and phylogenetic characterization by internal transcribed spacer region analysis

Abstract The present report describes the first case of postpartum disseminated histoplasmosis in a 24-year-old HIV-negative woman. On the tenth day after vaginal delivery, the patient presented with dyspnea, fever, hypotension, tachycardia, and painful hepatomegaly. Yeast-like Histoplasma capsulatum features were isolated in the buffy coat. The phylogenetic analysis demonstrated that the fungal isolate was similar to other H. capsulatum isolates identified in HIV patients from Ceará and Latin America. Thus, histoplasmosis development in individuals with transitory immunosuppression or during the period of immunological recovery should be carefully examined.

Multiplex polymerase chain reaction as an improved method for screening Histoplasma capsulatum mating types

Histoplasmosis is a systemic mycosis infection caused by Histoplasma capsulatum, a heterothallic ascomycete. The sexual reproduction of this fungus is regulated by the mating type (MAT1) locus that contains MAT1-1 and MAT1-2 idiomorphs, which were identified by uniplex polymerase chain reaction (PCR). This study aimed to optimise single-step multiplex PCR for the accurate detection of the distinct mating types of H. capsulatum. Among the 26 isolates tested, 20 had MAT1-1 genotype, while six showed MAT1-2 genotype, in agreement with the uniplex PCR results. These results suggest that multiplex PCR is a fast and specific tool for screening H. capsulatum mating types.

The use of nested Polymerase Chain Reaction (nested PCR) for the early diagnosis of Histoplasma capsulatum infection in serum and whole blood of HIV-positive patients* / Uso da Reação em Cadeia da Polimerase aninhada (PCR aninhada) para o diagnóstico precoce da infecção pelo Histoplasma capsulatum no soro e no sangue total de pacientes HIV positivos

The aim of the study was to detect the rDNA sequences and their regions in Histoplasma capsulatum, which could be considered species-specific and used as a molecular method for this diagnosis by the technique of nested polymerase chain reaction (nested PCR), employing specific sequences (primers) for H. capsulatum: 18S rDNA region (HC18), 100 kDa (HC100) and the sequence 5.8 S-ITS rDNA (HC5.8). The PCR sequences HC18, HC100 and HC5.8 resulted in a specificity of 100%. The molecular assays may increase the specificity, sensitivity and speed in the diagnosis of Histoplasmosis.

O objetivo do estudo consistiu em detectar seqüências no ADNr e as suas regiões no Histoplasma capsulatum, que pudessem ser consideradas espécie-específicas e usadas como método molecular para o diagnóstico pela técnica da reação em cadeia da polimerase aninhada ("nested PCR") com seqüências específicas ("primers") para H. capsulatum: regiões 18S ADNr (HC18), 100kDa (HC100) e a seqüência 5.8 S ADNr-ITS (HC5.8). A "nested PCR" com as seqüências HC18, HC100 e HC5.8 resultaram em 100% de especificidade. Os ensaios moleculares podem aumentar a especificidade, sensibilidade e rapidez na diagnose da Histoplasmose.

Brote de histoplasmosis en la Escuela de Cadetes de la Base Aérea de Morón, Provincia de Buenos Aires, República Argentina / Histoplasmosis outbreak in Morón, Buenos Aires Province, Argentina

Se describe un brote de histoplasmosis que afectó a 6 cadetes de la Fuerza Aérea Argentina, sin antecedentes patológicos previos. Todos consultaron por problemas respiratorios después de haber limpiado un hangar. En ese recinto se encontraron abundantes deyecciones de animales, presuntamente de palomas y murciélagos. Los pacientes sufrieron fiebre, mialgias, taquipnea y tos no productiva. Las radiografías y tomografías de tórax mostraron imágenes pulmonares micronodulares, engrosamiento de los tabiques interalveolares y adenopatías hiliares. Todos tuvieron una evolución favorable y no requirieron tratamiento antifúngico. Las pruebas de inmunodifusión y contrainmunoelectroforesis con antígenos de Histoplasma capsulatum fueron positivas, al igual que las intradermorreacciones con histoplasmina. Se recogieron 5 muestras de tierra del lugar, las que fueron inoculadas por vía intraperitoneal a 20 hámsteres. De los cultivos de hígado y bazo de dichos animales se consiguió aislar la fase micelial de H. capsulatum. La cepa aislada se comparó con las obtenidas de 12 pacientes argentinos utilizando perfiles genéticos y se observó un clado único con más de 96% de similitud, lo que confirma la homogeneidad de las cepas argentinas. Si bien la histoplasmosis es endémica en la Pampa húmeda, este es el primer brote totalmente documentado al sur del paralelo 34°.

An histoplasmosis outbreak affecting 6 previously healthy Air Force cadets is herein presented. The patients suffered from fever and respiratory symptoms after having cleaned an abandoned hangar soiled with pigeons and bat droppings. They all presented fever, myalgia, tachypnea, and nonproductive cough. Chest X-ray and CT scan studies showed disseminated reticulonodular images affecting both lungs. Hiliar adenomegalies were also observed. All patients achieved a favourable outcome without antifungal treatment. Both serologic tests searching for specificic antibodies (immunodiffusion and counterimmunoelectrophoresis) and histoplasmin skin tests were positive in all cases. Five soil samples mixed with pigeons and bat droppings were collected from the hangar. Suspensions of these samples were inoculated into 20 hamsters by intraperitoneal injection; mycelial phase of H. capsulatum was isolated from liver and spleen cultures. The genetic profile of this strain was compared with 12 isolates obtained from Argentinean patients, and a great degree of homogeneity was observed (> 96% similarity). Although histoplasmosis is endemic in the wet Pampas, this is the first epidemic outbreak reported south of the 34th parallel.

Identification of fungemia agents using the polymerase chain reaction and restriction fragment length polymorphism analysis

Prompt and specific identification of fungemia agents is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time-consuming and not without errors. The aim of the present study was to identify the following fungemia agents: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Cryptococcus gattii, and Histoplasma capsulatum using the polymerase chain reaction and restriction fragment length polymorphism analysis (PCR/RFLP). More specifically: a) to evaluate 3 different amplification regions, b) to investigate 3 different restriction enzymes, and c) to use the best PCR/RFLP procedure to indentify 60 fungemia agents from a culture collection. All 3 pairs of primers (ITS1/ITS4, NL4/ITS5 and Primer1/Primer2) were able to amplify DNA from the reference strains. However, the size of these PCR products did not permit the identification of all the species studied. Three restriction enzymes were used to digest the PCR products: HaeIII, Ddel and Bfal. Among the combinations of pairs of primers and restriction enzymes, only one (primer pair NL4/ITS5 and restriction enzyme Ddel) produced a specific RFLP pattern for each microorganism studied. Sixty cultures of fungemia agents (selected from the culture collection of Fundação de Medicina Tropical do Amazonas - FMTAM) were correctly identified by PCR/RFLP using the prime pair NL4/ITS5 and Ddel. We conclude that the method proved to be both simple and reproducible, and may offer potential advantages over phenotyping methods.

Inteins in pathogenic fungi: a phylogenetic tool and perspectives for therapeutic applications

Inteins or "internal proteins" are coding sequences that are transcribed and translated with flanking sequences (exteins). After translation, the inteins are excised by an autocatalytic process and the host protein assumes its normal conformation and develops its expected function. These parasitic genetic elements have been found in important, conserved proteins in all three domains of life. Most of the eukaryotic inteins are present in the fungi kingdom and the PRP8 intein is one of the most widespread inteins, occurring in important pathogens such as Cryptococcus neoformans (varieties grubii and neoformans), Cryptococcus gattii, Histoplasma capsulatum and Paracoccidioides brasiliensis. The knowledge of conserved and non-conserved domains in inteins have opened up new opportunities for the study of population variability in pathogenic fungi, including their phylogenetic relationships and recognition or diagnoses of species. Furthermore, inteins in pathogenic fungi should also be considered a promising therapeutic drug target, since once the autocatalytic splicing is inhibited, the host protein, which is typically vital, will not be able to perform its normal function and the fungal cell will not survive or reproduce.

Diagnóstico molecular de histoplasmosis humana en muestras de sangre entera / Molecular diagnosis of human histoplasmosis in whole blood samples

Se evaluó el uso de sangre entera para el diagnóstico molecular de histoplasmosis utilizando un método artesanal de extracción de ADN fúngico y una PCR anidada que amplifica una porción del gen HcP100 específica de Histoplasma capsulatum. La sangre entera se trató con liticasa, enzima lisante de Trichoderma harzianum y proteinasa K, seguido de una extracción fenólica. Este tratamiento permitió una lisis completa de las células, mostró buen rendimiento en la obtención de ADN y posibilitó la detección de la banda de 210 pb específica de H. capsulatum en la PCR anidada. El límite de detección fue de 0,25-1 levaduras/ml de sangre. El método se evaluó en 31 muestras de sangre de 19 pacientes con diagnóstico microbiológico de histoplasmosis, en 21 muestras de pacientes con otras micosis o infecciones por micobacterias y en 30 controles sanos. La PCR fue positiva en sangre para 17/19 pacientes con histoplasmosis (14/15 inmunocomprometidos y 3/4 sin inmunocompromiso aparente). Las muestras de sangre de los 30 controles sanos y de 20 pacientes con otras patologías fueron negativas, sólo hubo un falso positivo correspondiente a un paciente con infección por Mycobacterium avium-intracellulare. El método presentó 89% de sensibilidad y 96% de especificidad para el diagnóstico de histoplasmosis en sangre entera.

To assess the value of using whole blood samples for the molecular diagnosis of histoplasmosis, we applied an in-house DNA extraction method and a nested PCR targeting a 210 bp specific segment of the Histoplasma capsulatum HcP100 gene. A whole blood volume of 2.5-3 milliliters was centrifuged and the cellular pellet was treated with Trichoderma harzianum lyticase and proteinase K prior to applying a conventional phenol DNA extraction. This procedure allowed complete cell lysis, high DNA yield and specific amplification. The PCR detection limit was 0.25-1 yeast cells/ml of blood sample. The method was assessed on 31 blood samples from 19 patients with microbiological diagnosis of histoplasmosis, 30 healthy persons and 21 patients with other mycoses or mycobacterial diseases. Positive results were obtained in samples from 17/19 patients with histoplasmosis (14/15 immunocompromised and 3/4 without known immunological disorder). Blood samples from the 30 healthy controls and 20 patients with other conditions proved negative; the only false positive result was obtained from a patient with Mycobacterium avium-intracellulare infection. With 89% sensitivity and 98% specificity, this molecular method for detection of the agent in blood shows promising for the rapid diagnosis of human histoplasmosis.

Primer aislamiento de Histoplasma capsulatum de murciélago urbano Eumops bonariensis / First isolation of Histoplasma capsulatum from the urban bat Eumops bonariensis

Se comunica el primer aislamiento de Histoplasma capsulatum var. capsulatum de un murciélago macho de la especie Eumops bonariensis, capturado en la ciudad de Buenos Aires en 2003. Los aislamientos fueron recuperados de bazo e hígado e identificados fenotípicamente. Se los comparó por PCR, con 17 aislamientos clínicos, 12 de pacientes residentes en la ciudad de Buenos Aires y cinco de otros países de América, usando los iniciadores 1283, (GTG)5, (GACA)4 y M13. Con los cuatro iniciadores, los perfiles de los aislamientos de murciélago resultaron idénticos entre sí y más relacionados a los de pacientes de Buenos Aires que a los de otros países (porcentaje de similitud: 91-100% y 55-87%, respectivamente). La alta relación genética entre los aislamientos obtenidos del murciélago y de los humanos residentes en Buenos Aires sugiere una fuente común de infección. Este es el primer registro de E. bonariensis infectado con H. capsulatum en el mundo, y el primer aislamiento del hongo en la población de quirópteros de la Argentina. Así como estos mamíferos actúan como reservorio y dispersan el hongo en la naturaleza, la infección en murciélagos urbanos podría asociarse al elevado número de casos de histoplasmosis entre pacientes inmunodeprimidos en la ciudad de Buenos Aires.

We report the first isolation of Histoplasma capsulatum var. capsulatum from a male bat Eumops bonariensis captured in Buenos Aires city in 2003. The pathogen was recovered from spleen and liver specimens, and was identified by its phenotypic characteristics. PCR with primers 1283, (GTG)5, (GACA)4 and M13 was used to compare both bat isolates with 17 human isolates, 12 from patients residing in Buenos Aires city, and 5 from other countries of the Americas. The profiles obtained with the four primers showed that both bat isolates were identical to each other and closer to Buenos Aires patients than to the other isolates (similarity percentage: 91-100% and 55-97%, respectively). The high genetic relationship between bat isolates and those from patients living in Buenos Aires suggests a common source of infection. This is the first record of E. bonariensis infected with H. capsulatum in the world, and the first isolation of the fungus in the Argentinean Chiroptera population. In the same way as these wild mammals act as reservoir and spread the fungus in the natural environment, infection in urban bats could well be associated with the increase in histoplasmosis clinical cases among immunosuppressed hosts in Buenos Aires city.

I. El murciélago como reservorio y responsable de la dispersión de Histoplasma capsulatum en la naturaleza. II. Papel de los marcadores moleculares del hongo aislado de murciélagos infectados / I. Bats as reservoirs and responsible of histoplasma capsulatum spreading in nature. II. the role of molecular markers of the fungus isolated from infected bats

Objetivo: El presente trabajo refiere datos acerca de los aislamientos positivos y caracterización de cepas de Histoplasma capsulatum en murciélagos infectados y capturados en los estados de Guerrero, Morelos y Oaxaca: Pteronotus parnellii; P. dauyi; Myotis californicus; Mormoops megalophylla; Natalus stramineus; Artibeus hirsutus; Leptonycteris nivalis y L. Curasoae. Aportaciones sobre el aislamiento del hongo en murciélagos infectados: Para el estado de Oaxaca se aportan nuevos registros de infección de murciélagos por H. capsulatum en P. parnellii, P. dauyi y L. curasoae, siendo las dos últimas especies nuevos registros para el mundo. Los datos indican un alto riesgo de infección en murciélagos que utilizan cuevas con bóvedas bajas, relieve accidentado y abundantes depósitos de guano. Polimorfismo genético de H. capsulatum aislado de murciélagos infectados: Se analizaron los patrones polimórficos del DNA del hongo aislado de doce mulciélagos infectados, capaturados en Guerrero y Morelos, utilizando el polimirfismo del DNA amplificado al azar por la reacción en cadena de la polimerasa (RAPD-PCR), método conocido por su alta sensibilidad. Se encontraron dos patrones polimórficos diferentes en los doce aislados de H. capsulatum obtenidos de murciélagos infectados, que podrían representar marcadores moleculares del hongo para las áreas geografícas estudiadas, además de asociarse con los desplazamientos de los murciélagos en estas áreas. Conclusiones: Se sugiere el papel de los murciélagos como reservorios y responsables de la dispersión de H. capsulatum en la naturaleza, en relación al uso de refugios permanentes (cuevas), a su hábitos alimentarios, y sus desplazamientos y migraciones