Histoplasma capsulatum 100-kilodalton antigen: recombinant production, characterization, and evaluation of its possible application in the diagnosis of histoplasmosis.

The goal of the present work was to develop a novel reagent with potential for histoplasmosis diagnosis. For this purpose, the genetic sequence of the 100 kDa protein of Histoplasma capsulatum (Hcp100) was cloned and expressed as a secretory protein in Pichia pastoris. After optimizing the culture conditions and purifying by immobilized metal ion affinity chromatography, the highest yield of Hcp100 reached approximately 1.3 mg/l with > 90% purity in shake flasks using basal salt medium supplemented with casamino acids after 72 h of methanol induction. To investigate its potential for diagnosis, its detection in urine samples using specific polyclonal antibodies as reagent was evaluated by dot blot in 6 patients with progressive disseminated histoplasmosis (PDH), of whom all had AIDS. Antigen was detected in urine from all 6 (100%) PDH patients. Urine samples from a pool of 20 healthy individuals did not react with the anti-Hcp100 antibodies. The dot blot assay performed in this study provides preliminary data of a simple technology that can be performed in medical institutions with limited resources to facilitate the rapid diagnosis of histoplasmosis, particularly the disseminated forms. Hence, use of these assays may provide a rapid diagnostic tool of PDH in endemic areas for histoplasmosis where PDH-related mortality is high, hastening treatment and improving patient survival. Finally, this novel antigen and its specific antibodies may provide an alternative diagnostic reagent to the largely unknown and poorly characterized polysaccharide antigens (HPA, galactomannan, histoplasmin) frequently used in the diagnostic tests. KEY POINTS: Few antigens are used as laboratory tools for the immunodiagnosis of histoplasmosis. P. pastoris was an excellent system for recombinant Hcp100 expression. Maximum expression levels of rHcp100 were achieved in BSM with 1% casamino acids. Dot blot assays with anti-rHcp100 antisera can be successfully used for diagnosing PHD.

[Disseminated histoplasmosis in patients with AIDS. Buenos Aires, 2009-2014]. / Histoplasmosis diseminada en pacientes HIV/Sida. Buenos Aires, 2009-2014.

A retrospective study was carried out on 171 cases of disseminated histoplasmosis diagnosed in HIV/AIDS patients during the period 2009-2014. Although HIV diagnosis rates remained stable over the study period, a sensible increase in the number of histoplasmosis cases was observed in the last three years. Disseminated histoplasmosis was prevalent in males with an average age of 37.8 years. At diagnosis, only 54/171 (31.6%) were receiving HAART, and CD4+ T-lymphocyte counts ranged from 4 to 264 cells/upsilon. Cutaneous lesions, including ulcerated papules or molluscoid plaques, were present in 110/171 (64.3%), with Histoplasma capsulatum being observed in all skin scraping specimens upon Giemsa staining. Respiratory manifestations were second in frequency with bronchoalveolar lavage showing a high diagnostic performance. Radiological findings included milliary patterns, interstitial infiltrates, and focalized condensations. Out of 141 blood cultures performed, H. capsulatum was isolated in 82 (58.2%). No significant difference in diagnostic performance was found between blood cultures and skin scraping (p = 0.6164). Other opportunistic infections were observed in 70/171 (40.9%) prior to or concomitantly with histoplasmosis. Association with Mycobacterium tuberculosis was recorded in 16/171 (9.4%) and one had a multi-drug resistant isolate. The severity of histoplasmosis determined the monotherapy with amphotericin B deoxycholate in 115 (67.3%), itraconazole in 42 (24.5%), and combined therapies in 14 (8.2%). Mortality was 19.9% (34/171). Finally, we emphasize that the higher prevalence in the last three years of the study should prompt the medical community to consider the diagnosis of histoplasmosis to reduce mortality of AIDS patients.

In vitro activities of amphotericin B, terbinafine, and azole drugs against clinical and environmental isolates of Aspergillus terreus sensu stricto.

The antifungal susceptibilities of 40 clinical and environmental isolates of A. terreus sensu stricto to amphotericin B, terbinafine, itraconazole, and voriconazole were determined in accordance with CLSI document M38-A2. All isolates had itraconazole and voriconazole MICs lower than epidemiologic cutoff values, and 5% of the isolates had amphotericin B MICs higher than epidemiologic cutoff values. Terbinafine showed the lowest MICs. No significant differences were found when MICs of clinical and environmental isolates were compared.

Genetic diversity of Histoplasma capsulatum strains isolated from Argentina based on nucleotide sequence variations in the internal transcribed spacer regions of rDNA.

The internal transcribed spacer (ITS) regions of rDNA genes of 49 Histoplasma capsulatum (48 from clinical samples and one from soil) isolates were examined. Nucleotide sequence heterogeneity within this region was useful for phylogenetic classification of H. capsulatum and species identification. Thus, in 45 of 49 isolates we observed higher percentages of identity in the nucleotide sequences of ITS regions when the isolates studied herein were compared with those reported in our country in the South America B clade. Phylogenetic analyses of rDNA sequences corresponding to the 537 bp of the ITS region obtained from H. capsulatum isolates assigned South America type B clade (45 isolates), North America type 1 and Asia clade (2 isolates each one). H. capsulatum strains isolated from soil and from patients living in Argentina (45 of 49) clustered together with the H. capsulatum isolates of the South America B clade. The high level of genetic similarity among our isolates suggests that almost one genetic population is present in the microenvironment. Isolates described as H. capsulatum var. capsulatum or var. farciminosum (2 isolates) did not form a monophyletic group and were found in the Asia clade. Subsequent studies are needed to properly identify these isolates.

Sporothrix Brasiliensis: A Review of an Emerging South American Fungal Pathogen, Its Related Disease, Presentation and Spread in Argentina.

Sporotrichosis, caused by Sporothrix schenckii and related species, is the most frequent implantation mycosis in Latin America. In Argentina, over the last 8 years, there have been 0.16 new cases per month of feline sporotrichosis in 2011, increasing to 0.75 cases per month in 2019 and involving zoonotic transmission to humans. Molecular identification by polymerase chain reaction (PCR) detected Sporothrix brasiliensis in these feline and zoonotic outbreaks. This study will focus on different feline and human sporotrichosis outbreaks caused by S. brasiliensis in Argentina during 2011-2019. We will address the sources of infection and environmental hotspots, as well as the application of several treatment strategies for improving the pharmacotherapy of the different clinical forms of the disease. Finally, we will provide a detailed summary of the clinical aspects and new advances in host-pathogen interactions, virulence factors and immune response, focusing on state-of-the-art diagnostic tools and potential vaccine candidates.

Phenotypic and genotypic characterization of Aspergillus lentulus and Aspergillus fumigatus isolates in a patient with probable invasive aspergillosis.

Characteristic morphological and phylogenetic analyses demonstrated the presence of Aspergillus fumigatus and Aspergillus lentulus as the aetiological agents in a case of probable invasive aspergillosis (IA). This is believed to be the first report of an A. lentulus strain isolated from a patient with probable IA in Argentina.

Genetic-relatedness of peri-implants and buccal Candida albicans isolates determined by RAPD-PCR.

Molecular techniques have been used in recent studies to identify a wide range of potential bacterial pathogens in periimplant pockets of the oral cavity. However, the prevalence and molecular epidemiology of yeasts and species distribution related to periimplantitis are as yet unknown. The aim of this study was to determine the prevalence and distribution of yeasts in periimplant biofilm and to study genetic relatedness of Candida albicans. Yeasts recovered from periimplant biofilm samples (n=89) and buccal samples (n=120) were studied in 40 immunocompetent nonsmoking patients who visited the dental clinic of the Asociación Implantodontológica Argentina, Buenos Aires, Argentina, and had received oral rehabilitation with implants for more than five years. Yeasts recovered from samples were studied by typing assays using RAPDPCR. The prevalence of yeasts in the periimplant sulcus was 73% (n=29). C. albicans was the most prevalent species identified in this study population. The RAPD analysis showed identical genotypes in most C. albicans spp. from the two different sampling sites: buccal and periimplant. These findings suggest that periimplant biofilm is an ecological niche that favors the growth of yeast species. Most C. albicans found in periimplant biofilm originate from the endogenous infection caused by commensal strains.

Las técnicas moleculares se han utilizado en estudios recientes para identificar una gran diversidad de patógenos bacterianos de surcos periimplantarios de cavidad bucal. Sin embargo, la prevalencia y epidemiología molecular de especies de levaduras en relación con la periimplantitis son aún desconocidas. El objetivo de este estudio fue determinar la prevalencia y distribución de las levaduras en la biopelícula periimplantaria y estudiar la relación genética de Candida albicans. Se estudiaron 40 pacientes inmunocompetentes no fumadores que se asistieron en la clínica dental de la Asociación Implantodontológica Argentina, Buenos Aires, Argentina, y que habían recibido rehabilitación oral con implantes durante más de cinco años. Las levaduras aisladas de las muestras de biopelícula periimplantaria (n = 89) y bucales (n = 120), fueron identificadas por métodos micológicos tradicionales y moleculares. Se obtuvo el ADN de C. albicans y se realizaron estudios moleculares por RAPD PCR. La prevalencia de levaduras en el surco alrededor del implante fue de 73 % (n = 29). C. albicans fue la especie más frecuente identificada en esta población de estudio. El análisis RAPD permitió identificar idénticos genotipos de C. albicans en ambos nichos ecológicos estudiados, periimplantar y bucal. Según los resultados obtenidos, el surco periiplantario es un nicho ecológico que favorece el crecimiento de especies de levaduras del género Candida. La mayoría de los aislamientos de C. albicans periimplantarios se originan a partir de la infección endógena causada por cepas comensales.

Histoplasmosis diseminada en pacientes HIV/SIDA: Buenos Aires, 2009-2014 / Disseminated histoplasmosis in patients with AIDS. Buenos Aires, 2009-2014

Durante 2009-2014, en nuestro hospital fueron diagnosticados 171 casos de histoplasmosis en pacientes con HIV/sida: 64 en 2009-2011, y 107 en 2012-2014. El mayor número de casos durante el segundo trienio no tuvo relación con la infección por HIV, cuya tasa de diagnóstico permaneció estable. La relación hombre/mujer fue 3.7:1 y la edad media 37.4 años. Al diagnóstico, solo 54/171 (31.6%) recibían TARGA y el recuento de linfocitos T CD4+ varió entre 4 y 264 células/μl. El 64.3% (n = 110) presentó lesiones cutáneas (pápulas ulceradas o "moluscoides") positivas para Histoplasma capsulatum al examen microscópico con tinción Giemsa. Siguieron en frecuencia las manifestaciones respiratorias y el lavado broncoalveolar fue positivo en 41/171 (24.0%) pacientes. Las imágenes radiológicas más frecuentes en orden decreciente fueron patrón miliar, infiltrados intersticiales y condensaciones focales. Se aisló H. capsulatum en 82 (58.2%) de los 141 hemocultivos realizados. No hubo diferencia significativa entre el rendimiento diagnóstico del hemocultivo y el de las escarificaciones cutáneas (p = 0.6164). Otras infecciones oportunistas previas o concomitantes con la histoplasmosis se observaron en 70/171 (40.9%). Se registró asociación con tuberculosis en 16/171 (9.4%) y un aislamiento de Mycobacterium tuberculosis resultó multirresistente. La gravedad del caso determinó el tratamiento con anfotericina B desoxicolato en 115 (67.3%), con itraconazol en 43 (25.1%), y con terapias combinadas en 14 (8.2%). La letalidad fue 19.9% (34/171). La mayor prevalencia en el segundo trienio debe alertar a la comunidad médica a considerar el diagnóstico de la histoplasmosis en pacientes con HIV/sida para disminuir la mortalidad.

A retrospective study was carried out on 171 cases of disseminated histoplasmosis diagnosed in HIV/AIDS patients during the period 2009-2014. Although HIV diagnosis rates remained stable over the study period, a sensible increase in the number of histoplasmosis cases was observed in the last three years. Disseminated histoplasmosis was prevalent in males with an average age of 37.8 years. At diagnosis, only 54/171 (31.6%) were receiving HAART, and CD4+ T-lymphocyte counts ranged from 4 to 264 cells/μl. Cutaneous lesions, including ulcerated papules or molluscoid plaques, were present in 110/171 (64.3%), with Histoplasma capsulatum being observed in all skin scraping specimens upon Giemsa staining. Respiratory manifestations were second in frequency with bronchoalveolar lavage showing a high diagnostic performance. Radiological findings included milliary patterns, interstitial infiltrates, and focalized condensations. Out of 141 blood cultures performed, H. capsulatum was isolated in 82 (58.2%). No significant difference in diagnostic performance was found between blood cultures and skin scraping (p = 0.6164). Other opportunistic infections were observed in 70/171 (40.9%) prior to or concomitantly with histoplasmosis. Association with Mycobacterium tuberculosis was recorded in 16/171 (9.4%) and one had a multi-drug resistant isolate. The severity of histoplasmosis determined the monotherapy with amphotericin B deoxycholate in 115 (67.3%), itraconazole in 42 (24.5%), and combined therapies in 14 (8.2%). Mortality was 19.9% (34/171). Finally, we emphasize that the higher prevalence in the last three years of the study should prompt the medical community to consider the diagnosis of histoplasmosis to reduce mortality of AIDS patients.

Subgingival distribution of yeast and their antifungal susceptibility in immunocompetent subjects with and without dental devices

Las levaduras del genero Candida colonizan el biofilm subgingival.Su hallazgo constituye un reservorio favorable para su multiplicación. El propósito de éste trabajo fue investigar la presencia de levaduras del género Candida en el biofilm subgingivalde individuos con enfermedad gingivoperiodontal a fin de establecer la prevalencia de especies y los perfiles desusceptibilidad a fluconazol y voriconazol de las mismas. Se obtuvieron muestras del biofilm subgingival en cien pacientes inmunocompetentes, no fumadores, con salud gingivoperiodontal, gingivitis y periodontitis, con y sin aparatologíabucal. Las muestras se sembraron en medio cromogénico diferencial y las levaduras aisladas se identificaronmediante micromorfología y pruebas bioquímicas. Los estudios de sensibilidad a fluconazol y voriconazol se realizaron según las normas CLSI M44-A. La prevalencia de levadurasen el biofilm subgingival fue del 40 por ciento IC95 por ciento (30.5-50.3) siendo el 10 por ciento y 30 por ciento la frecuencia de las mismas para los pacientes sin y con aparatología bucal respectivamente.C. albicans fue la levadura más frecuente. Encontramos otras especies como C. parapsilosis, C. dubliniensis, C. tropicalis, C. guilliermondii y C. sake. Solo se observó resistencia a los azoles en C. dubliniensis y C. guilliermondii. El tratamiento con aparatología bucal incrementó la prevalencia de levadurasen bolsa periodontal en forma significativa. Es importante destacar la presencia en el fluido subgingival de especies de Candida no albicans consideradas emergentes y con sensibilidad disminuida a los antifúngicos como C. dubliniensisy C. guilliermondii

Yeasts colonize the subgingival biofilm, which becomes a reservoir that favors their reproduction. The purpose of the present work was to determine the prevalence of yeasts of the Candida genus in the subgingival biofilm of gingivoperiodontal disease patients, including users and non-users of dental devices, and their susceptibility to fluconazole and voriconazole. Samples of subgingival pockets of immunocompetent nonsmokers showing gingivitis and periodontitis were inoculated in a differential chromogenic medium. Sixty three percent of subjects used dental devices. Yeasts were identified and susceptibility to fluconazole and voriconazole was tested following CLSI M44-A standards. The prevalence of yeasts in the subgingival biofilm was 40% CI 95% (30.5-50.3); 10% were patients who did not use dental appliances. The most frequently observed yeasts were C. albicans, and C. parapsilosis, C. dubliniensis, C. tropicalis and C. guilliermondii. Only C. dubliniensis and C. guilliermondii showed resistance to azoles. The use of dental devices significantly increased the prevalence of yeasts in periodontal pockets inpatients presenting gingivitis. It is noteworthy that non albicans Candida species, such as C. dubliniensis and C. guilliermondii, considered emerging species, which have a diminished susceptibility to antifungal agents were found in the crevicular fluid of immunocompetent patients.