Molecular detection of Histoplasma capsulatum in organ samples from bats randomly captured in urban areas of Araraquara, São Paulo state, Brazil.

The mycosis histoplasmosis is also considered a zoonosis that affects humans and other mammalian species worldwide. Among the wild mammals predisposed to be infected with the etiologic agent of histoplasmosis, bats are relevant because they are reservoir of Histoplasma species, and they play a fundamental role in maintaining and spreading fungal propagules in the environments since the infective mycelial phase of Histoplasma grows in their accumulated guano. In this study, we detected the fungal presence in organ samples of bats randomly captured in urban areas of Araraquara City, São Paulo, Brazil. Fungal detection was performed using a nested polymerase chain reaction to amplify a molecular marker (Hcp100) unique to H. capsulatum, which revealed the pathogen presence in organ samples from 15 out of 37 captured bats, indicating 40.5% of infection. Out of 22 Hcp100-amplicons generated, 41% corresponded to lung and trachea samples and 59% to spleen, liver, and kidney samples. Data from these last three organs suggest that bats develop disseminated infections. Considering that infected bats create environments with a high risk of infection, it is important to register the percentage of infected bats living in urban areas to avoid risks of infection to humans, domestic animals, and wildlife.

Unusual Differences in the Pulmonary Histopathology of Mice after Intranasal Infection with Mycelial Propagules of Histoplasma capsulatum Strains Classified as LAm A2 and NAm 2 Phylogenetic Species.

The ascomycete Histoplasma capsulatum is the causative agent of systemic respiratory mycosis histoplasmosis, which sometimes develops acute disseminated or chronic clinical forms, with the latter usually associated with granuloma formation. The present report shows differential histopathological changes in the pulmonary inflammatory response of mice infected intranasally with the mycelial morphotype of H. capsulatum strains with distinct genotypes, EH-46 and G-217B, classified as LAm A2 and NAm 2 phylogenetic species, respectively. Infected male BALB/c mice were sacrificed at different postinfection times, and their serial lung sections were stained with periodic acid-Schiff and analyzed via microscopy. In mice infected with the LAm A2 strain, the results showed progressive changes in the inflammatory infiltrate of the lung parenchyma during the first hours and days postinfection as well as granulomas with macrophages containing intracellular yeast cells, which prevailed at 14 and 21 days postinfection. Bronchiolar-associated lymphoid tissue was induced in mice infected with both strains, primarily in mice infected with the NAm 2 strain. Several lung sections from mice infected with the LAm A2 strain showed PAS-positive yeast cells aggregated in a perinuclear crown-like arrangement in macrophages from 3 h to 21 days postinfection. These findings highlight differences in the host pulmonary inflammatory response associated with distinct H. capsulatum species.

Central Nervous System Histoplasmosis: An Updated Insight.

Histoplasmosis is one of the systemic mycoses that can involve the Central Nervous System (CNS), and it is caused by the dimorphic ascomycete species of the Histoplasma capsulatum complex. Once in the CNS, this pathogen causes life-threatening injuries that are associated with clinical manifestations of meningitis, focal lesions (abscesses, histoplasmomas), and spinal cord injuries. The present review provides updated data and highlights a particular vision regarding this mycosis and its causative agent, as well as its epidemiology, clinical forms, pathogenesis, diagnosis, and therapy, focusing on the CNS.

Considerations about the Geographic Distribution of Histoplasma Species.

Histoplasmosis is a mycotic infection principally affecting pulmonary tissue; sometimes, histoplasmosis can progress into a systemic disease. This infection involves immunocompetent and immunosuppressed human and other mammalian hosts, depending on particular circumstances. Histoplasmosis infection has been documented worldwide. The infection is acquired by inhaling infective mycelial propagules of the dimorphic fungus Histoplasma capsulatum. New reports of clinical cases of histoplasmosis in extreme latitudes could be related to human social adaptations and climate changes in the world, which are creating new favorable environments for this fungus and for bats, its major natural reservoirs and dispersers. Histoplasma has been isolated from most continents, and it is considered a complex of cryptic species, consisting of various groups of isolates that differ genetically and correlate with a particular geographic distribution. Based on updated studies, Histoplasma taxonomy is adjusting to new genetic data. Here, we have suggested that Histoplasma has at least 14 phylogenetic species distributed worldwide and new genotypes that could be under deliberation. Histoplasma's geographic radiation began in South America millions of years ago when the continents were joined and the climate was favorable. For fungal spreading, the role of bats and some birds is crucial, although other natural factors could also participate.

Detection of Cytokines and Collectins in Bronchoalveolar Fluid Samples of Patients Infected with Histoplasma capsulatum and Pneumocystis jirovecii.

Histoplasmosis and pneumocystosis co-infections have been reported mainly in immunocompromised humans and in wild animals. The immunological response to each fungal infection has been described primarily using animal models; however, the host response to concomitant infection is unknown. The present work aimed to evaluate the pulmonary immunological response of patients with pneumonia caused either by Histoplasma capsulatum, Pneumocystis jirovecii, or their co-infection. We analyzed the pulmonary collectin and cytokine patterns of 131 bronchoalveolar lavage samples, which included HIV and non-HIV patients infected with H. capsulatum, P. jirovecii, or both fungi, as well as healthy volunteers and HIV patients without the studied fungal infections. Our results showed an increased production of the surfactant protein-A (SP-A) in non-HIV patients with H. capsulatum infection, contrasting with HIV patients (p < 0.05). Significant differences in median values of SP-A, IL-1ß, TNF-α, IFN-γ, IL-18, IL-17A, IL-33, IL-13, and CXCL8 were found among all the groups studied, suggesting that these cytokines play a role in the local inflammatory processes of histoplasmosis and pneumocystosis. Interestingly, non-HIV patients with co-infection and pneumocystosis alone showed lower levels of SP-A, IL-1ß, TNF-α, IFN-γ, IL-18, IL-17A, and IL-23 than histoplasmosis patients, suggesting an immunomodulatory ability of P. jirovecii over H. capsulatum response.

Histoplasma capsulatum Isolated from Tadarida brasiliensis Bats Captured in Mexico Form a Sister Group to North American Class 2 Clade.

Histoplasma capsulatum is a dimorphic fungus associated with respiratory and systemic infections in mammalian hosts that have inhaled infective mycelial propagules. A phylogenetic reconstruction of this pathogen, using partial sequences of arf, H-anti, ole1, and tub1 protein-coding genes, proposed that H. capsulatum has at least 11 phylogenetic species, highlighting a clade (BAC1) comprising three H. capsulatum isolates from infected bats captured in Mexico. Here, relationships for each individual locus and the concatenated coding regions of these genes were inferred using parsimony, maximum likelihood, and Bayesian inference methods. Coalescent-based analyses, a concatenated sequence-types (CSTs) network, and nucleotide diversities were also evaluated. The results suggest that six H. capsulatum isolates from the migratory bat Tadarida brasiliensis together with one isolate from a Mormoops megalophylla bat support a NAm 3 clade, replacing the formerly reported BAC1 clade. In addition, three H. capsulatum isolates from T. brasiliensis were classified as lineages. The concatenated sequence analyses and the CSTs network validate these findings, suggesting that NAm 3 is related to the North American class 2 clade and that both clades could share a recent common ancestor. Our results provide original information on the geographic distribution, genetic diversity, and host specificity of H. capsulatum.

Diagnostic accuracy of antigen detection in urine and molecular assays testing in different clinical samples for the diagnosis of progressive disseminated histoplasmosis in patients living with HIV/AIDS: A prospective multicenter study in Mexico.

BACKGROUND: The progressive disseminated histoplasmosis (PDH) has been associated with severe disease and high risk of death among people living with HIV (PLWHIV). Therefore, the purpose of this multicenter, prospective, double-blinded study done in ten Mexican hospitals was to determine the diagnostic accuracy of detecting Histoplasma capsulatum antigen in urine using the IMMY ALPHA Histoplasma EIA kit (IAHE), clarus Histoplasma GM Enzyme Immunoassay (cHGEI IMMY) and MiraVista Histoplasma Urine Antigen LFA (MVHUALFA); as well as the Hcp100 and 1281-1283220SCAR nested PCRs in blood, bone-marrow, tissue biopsies and urine. METHODOLOGY/PRINCIPAL FINDINGS: We included 415 PLWHIV older than 18 years of age with suspicion of PDH. Using as diagnostic standard recovery of H. capsulatum in blood, bone marrow or tissue cultures, or histopathological exam compatible, detected 108 patients (26%, [95%CI, 21.78-30.22]) with proven-PDH. We analyzed 391 urine samples by the IAHE, cHGEI IMMY and MVHUALFA; the sensitivity/specificity values obtained were 67.3% (95% CI, 57.4-76.2) / 96.2% (95% CI, 93.2-98.0) for IAHE, 91.3% (95% CI, 84.2-96.0) / 90.9% (95% CI, 87.0-94.0) for cHGEI IMMY and 90.4% (95% CI, 83.0-95.3) / 92.3% (95% CI, 88.6-95.1) for MVHUALFA. The Hcp100 nested PCR was performed on 393, 343, 75 and 297, blood, bone marrow, tissue and urine samples respectively; the sensitivity/specificity values obtained were 62.9% (95%CI, 53.3-72.5)/ 89.5% (95%CI, 86.0-93.0), 65.9% (95%CI, 56.0-75.8)/ 89.0% (95%CI, 85.2-92.9), 62.1% (95%CI, 44.4-79.7)/ 82.6% (95%CI, 71.7-93.6) and 34.9% (95%CI, 24.8-46.2)/ 67.3% (95%CI, 60.6-73.5) respectively; and 1281-1283220SCAR nested PCR was performed on 392, 344, 75 and 291, respectively; the sensitivity/specificity values obtained were 65.3% (95% CI, 55.9-74.7)/ 58.8% (95%CI, 53.2-64.5), 70.8% (95%CI, 61.3-80.2)/ 52.9% (95%CI, 46.8-59.1), 71.4% (95%CI, 54.7-88.2)/ 40.4% (95%CI, 26.4-54.5) and 18.1% (95%CI, 10.5-28.1)/ 90.4% (95%CI, 85.5-94.0), respectively. CONCLUSIONS/SIGNIFICANCE: The cHGEI IMMY and MVHUALFA tests showed excellent performance for the diagnosis of PDH in PLWHIV. The integration of these tests in clinical laboratories will certainly impact on early diagnosis and treatment.

Differential miRNA Expression in Human Macrophage-Like Cells Infected with Histoplasma capsulatum Yeasts Cultured in Planktonic and Biofilm Forms.

Histoplasma capsulatum affects healthy and immunocompromised individuals, sometimes causing a severe disease. This fungus has two morphotypes, the mycelial (infective) and the yeast (parasitic) phases. MicroRNAs (miRNAs) are small RNAs involved in the regulation of several cellular processes, and their differential expression has been associated with many disease states. To investigate miRNA expression in host cells during H. capsulatum infection, we studied the changes in the miRNA profiles of differentiated human macrophages infected with yeasts from two fungal strains with different virulence, EH-315 (high virulence) and 60I (low virulence) grown in planktonic cultures, and EH-315 grown in biofilm form. MiRNA profiles were evaluated by means of reverse transcription-quantitative polymerase chain reaction using a commercial human miRNome panel. The target genes of the differentially expressed miRNAs and their corresponding signaling pathways were predicted using bioinformatics analyses. Here, we confirmed biofilm structures were present in the EH-315 culture whose conditions facilitated producing insoluble exopolysaccharide and intracellular polysaccharides. In infected macrophages, bioinformatics analyses revealed especially increased (hsa-miR-99b-3p) or decreased (hsa-miR-342-3p) miRNAs expression levels in response to infection with biofilms or both growth forms of H. capsulatum yeasts, respectively. The results of miRNAs suggested that infection by H. capsulatum can affect important biological pathways of the host cell, targeting two genes: one encoding a protein that is important in the cortical cytoskeleton; the other, a protein involved in the formation of stress granules. Expressed miRNAs in the host's response could be proposed as new therapeutic and/or diagnostic tools for histoplasmosis.

Biofilm Formation by Histoplasma capsulatum in Different Culture Media and Oxygen Atmospheres.

Histoplasma capsulatum is a dimorphic fungus that causes an important systemic mycosis called histoplasmosis. It is an infectious disease with high prevalence and morbidity that affects the general population. Recently, the ability of these fungi to form biofilms, a phenotype that can induce resistance and enhance virulence, has been described. Despite some efforts, data regarding the impact of nutrients and culture media that affect the H. capsulatum biofilm development in vitro are not yet available. This work aimed to study H. capsulatum biofilms, by checking the influence of different culture media and oxygen atmospheres in the development of these communities. The biofilm formation by two strains (EH-315 and G186A) was characterized under different culture media: [Brain and Heart Infusion (BHI), Roswell Park Memorial Institute (RPMI) with 2% glucose, Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and nutrient medium HAM-F12 (HAM-F12) supplemented with glucose (18.2 g/L), glutamic acid (1 g/L), HEPES (6 g/L) and L-cysteine (8.4 mg/L)] and oxygen atmospheres (aerobiosis and microaerophilia), using the XTT reduction assay to quantify metabolic activities, crystal violet staining for biomass, safranin staining for the quantification of polysaccharide material and scanning electron microscopy (SEM) for the observation of topographies. Results indicated that although all culture mediums have stimulated the maturation of the communities, HAM-F12 provided the best development of biomass and polysaccharide material when compared to others. Regarding the oxygen atmospheres, both stimulated an excellent development of the communities, however in low oxygen conditions an exuberant amount of extracellular matrix was observed when compared to biofilms formed in aerobiosis, mainly in the HAM-F12 media. SEM images showed yeasts embedded by an extracellular matrix in several points, corroborating the colorimetric assays. However, biofilms formed in BHI, RPMI, and DMEM significantly induced yeast to hyphae reversal, requiring further investigation. The results obtained so far contribute to in vitro study of biofilms formed by these fungi and show that nutrition promoted by different media modifies the development of these communities. These data represent advances in the field of biofilms and contribute to future studies that can prove the role of these communities in the fungi-host interaction.

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.

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

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 ±â€¯1.47 h at 37 °C and 5.55 h ±â€¯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.

Heat Shock Protein 60, Insights to Its Importance in Histoplasma capsulatum: From Biofilm Formation to Host-Interaction.

Heat shock proteins (Hsps) are among the most widely distributed and evolutionary conserved proteins, acting as essential regulators of diverse constitutive metabolic processes. The Hsp60 of the dimorphic fungal Histoplasma capsulatum is the major surface adhesin to mammalian macrophages and studies of antibody-mediated protection against H. capsulatum have provided insight into the complexity involving Hsp60. However, nothing is known about the role of Hsp60 regarding biofilms, a mechanism of virulence exhibited by H. capsulatum. Considering this, the present study aimed to investigate the influence of the Hsp60 on biofilm features of H. capsulatum. Also, the non-conventional model Galleria mellonella was used to verify the effect of this protein during in vivo interaction. The use of invertebrate models such as G. mellonella is highly proposed for the evaluation of pathogenesis, immune response, virulence mechanisms, and antimicrobial compounds. For that purpose, we used a monoclonal antibody (7B6) against Hsp60 and characterized the biofilm of two H. capsulatum strains by metabolic activity, biomass content, and images from scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). We also evaluated the survival rate of G. mellonella infected with both strains under blockage of Hsp60. The results showed that mAb 7B6 was effective to reduce the metabolic activity and biomass of both H. capsulatum strains. Furthermore, the biofilms of cells treated with the antibody were thinner as well as presented a lower amount of cells and extracellular polymeric matrix compared to its non-treated controls. The blockage of Hsp60 before fungal infection of G. mellonella larvae also resulted in a significant increase of the larvae survival compared to controls. Our results highlight for the first time the importance of the Hsp60 protein to the establishment of the H. capsulatum biofilms and the G. mellonella larvae infection. Interestingly, the results with Hsp60 mAb 7B6 in this invertebrate model suggest a pattern of fungus-host interaction different from those previously found in a murine model, which can be due to the different features between insect and mammalian immune cells such as the absence of Fc receptors in hemocytes. However further studies are needed to support this hypothesis.

Novel clinical and dual infection by Histoplasma capsulatum genotypes in HIV patients from Northeastern, Brazil.

Histoplasmosis is a worldwide-distributed deep mycosis that affects healthy and immunocompromised hosts. Severe and disseminated disease is especially common in HIV-infected patients. At least 11 phylogenetic species are recognized and the majority of diversity is found in Latin America. The northeastern region of Brazil has one of the highest HIV/AIDS prevalence in Latin America and Ceará State has one of the highest death rates due to histoplasmosis in the world, where the mortality rate varies between 33-42%. The phylogenetic distribution and population genetic structure of 51 clinical isolates from Northeast Brazil was studied. For that morphological characteristics, exoantigens profile, and fungal mating types were evaluated. The genotypes were deduced by a MSLT in order to define local population structure of this fungal pathogen. In addition, the relationships of H. capsulatum genotypes with clinically relevant phenotypes and clinical aspects were investigated. The results suggest two cryptic species, herein named population Northeast BR1 and population Northeast BR2. These populations are recombining, exhibit a high level of haplotype diversity, and contain different ratios of mating types MAT1-1 and MAT1-2. However, differences in phenotypes or clinical aspects were not observed within these new cryptic species. A HIV patient can be co-infected by two or more genotypes from Northeast BR1 and/or Northeast BR2, which may have significant impact on disease progression due to the impaired immune response. We hypothesize that co-infections could be the result of multiple exposure events and may indicate higher risk of disseminated histoplasmosis, especially in HIV infected patients.

Molecular epidemiology of Colombian Histoplasma capsulatum isolates obtained from human and chicken manure samples.

The thermally dimorphic fungus Histoplasma capsulatum is the causative agent of histoplasmosis, one of the most prevalent endemic mycosis in the Americas. In tropical regions, agro-ecosystems require organic matter replacement, therefore, the use of organic fertilizers has increased disregarding the fact that certain number of such fertilizers might be contaminated with the fungus, and with their handling resulting in human cases and even outbreaks of histoplasmosis. Additionally, in Colombia, chicken manure is the most common raw material used in the production of organic fertilizers. In this work, we reported the isolation of this fungus from chicken manure, and genetically compared with 42 clinical isolates. The genetically compared environmental isolates grouped together with the clinical ones. Our result suggests that chicken manure may be one of H. capsulatum infection sources. Also, the phylogenetic analyses done with other H. capsulatum isolates indicate that the Colombian isolates are widely distributed in the relational tree thus reveling towards the great genetic diversity among the H. capsulatum Colombian isolates.

Dimorphism and Dissemination of Histoplasma capsulatum in the Upper Respiratory Tract after Intranasal Infection of Bats and Mice with Mycelial Propagules.

This article describes, for the first time, the role of the nasal mucosa (NM) as the initial site for the Histoplasma capsulatum mycelial-to-yeast transition. The results highlight that yeasts may arrive to the cervical lymph nodes (CLN) via phagocytes. Bats and mice were intranasally infected with H. capsulatum mycelial propagules and they were killed 10, 20, and 40 minutes and 1, 2, and 3 hours after infection. The NM and the CLN were monitored for fungal presence. Yeasts compatible with H. capsulatum were detected within the NM and the CLN dendritic cells (DCs) 2-3 hours postinfection, using immunohistochemistry. Histoplasma capsulatum was re-isolated by culturing at 28°C from the CLN of both mammalian hosts 2-3 hours postinfection. Reverse transcription-polymerase chain reaction assays were designed to identify fungal dimorphism, using mycelial-specific (MS8) and yeast-specific (YPS3) gene expression. This strategy supported fast fungal dimorphism in vivo, which began in the NM 1 hour postinfection (a time point when MS8 and YPS3 genes were expressed) and it was completed at 3 hours (a time point when only the YPS3 transcripts were detected) in both bats and mice. The presence of intracellular yeasts in the nasal-associated lymphoid tissue (NALT), in the NM nonassociated with the NALT, and within the interdigitating DCs of the CLN suggests early fungal dissemination via the lymph vessels.

Histoplasma capsulatum and Pneumocystis jirovecii coinfection in hospitalized HIV and non-HIV patients from a tertiary care hospital in Mexico.

BACKGROUND: Histoplasma capsulatum and Pneumocystis jirovecii are respiratory fungal pathogens that principally cause pulmonary disease. Coinfection with both pathogens is scarcely reported. This study detected this coinfection using specific molecular methods for each fungus in the bronchoalveolar lavage (BAL) of patients from a tertiary care hospital. MATERIALS AND METHODS: BAL samples from 289 hospitalized patients were screened by PCR with specific markers for H. capsulatum (Hcp100) and P. jirovecii (mtLSUrRNA and mtSSUrRNA). The presence of these pathogens was confirmed by the generated sequences for each marker. The clinical and laboratory data for the patients were analyzed using statistical software. RESULTS: The PCR findings separated three groups of patients, where the first was represented by 60 (20.8%) histoplasmosis patients, the second by 45 (15.6%) patients with pneumocystosis, and the last group by 12 (4.2%) patients with coinfection. High similarity among the generated sequences of each species was demonstrated by BLASTn and neighbor-joining algorithms. The estimated prevalence of H. capsulatum and P. jirovecii coinfection was higher in HIV patients.

Mixed infection by Histoplasma capsulatum isolates with different mating types in Brazilian AIDS-patients.

Mixed infection by Histoplasma capsulatum isolates with different mating types, in AIDS-patients are described in this study. Morphological, mating type-specific PCR assay and multilocus sequencing type analysis of H. capsulatum isolates recovered from two Brazilian AIDS-patients were performed. Five H. capsulatum isolates were recovered at different times from the two patients. Three isolates were obtained from bone marrow (day 1 - CE0411) and buffy coat cultures (day 1 - CE0311; day 2 - CE0511) of patient 1, and two isolates were isolated from buffy coat cultures (day 3 - CE2813; day 12 - CE2513) of patient 2. The mycelial colonies depicted different textures and pigmentation features. Dimorphic conversion to the yeast-phase in ML-Gema medium was achieved in all isolates. MAT1-1 idiomorph was identified in CE0311, CE0411 and CE2813 isolates; MAT1-2 idiomorph was found in CE0511 and CE2513 isolates. These H. capsulatum isolates were grouped within LAm A clade, highlighting that CE0311 and CE0411 isolates formed a subgroup supported by a high bootstrap value. The CE0511, CE2513, and CE2813 isolates clustered together with a Brazilian H151 isolate. This research reports mixed infections caused by H. capsulatum isolates with different mating types in Brazilian AIDS-patients for the first time in the literature.

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.

Detection of Histoplasma capsulatum in Organic Fertilizers by Hc100 Nested Polymerase Chain Reaction and Its Correlation with the Physicochemical and Microbiological Characteristics of the Samples.

Histoplasma capsulatum is the causative agent of histoplasmosis and this fungus inhabits soils rich in phosphorus and nitrogen that are enriched with bird and bat manure. The replacement of organic matter in agroecosystems is necessary in the tropics, and the use of organic fertilizers has increased. Cases and outbreaks due to the presence of the fungus in these components have been reported. The Instituto Colombiano Agropecuario resolution 150 of 2003 contains the parameters set by the Colombian Technical Standard (NTC 5167) on the physicochemical and microbiological features of fertilizers, but it does not regulate the search for H. capsulatum. The aim of this study was to demonstrate H. capsulatum presence in organic fertilizers by nested polymerase chain reaction (PCR). A total of 239 samples were collected: 201 (84.1%) corresponded to organic fertilizers, 30 (12.5%) to bird excrement, and 8 (3.4%) to cave soils. The Hc100 nested PCR had a detection limit of 0.1 pg/µL and a specificity of 100%. A total of 25 (10.5%) samples were positive and validated by sequencing. Seven of the positive samples represented locations where H. capsulatum was previously detected, suggesting the persistence of the fungus. No significant correlations were detected between the physicochemical and microbiological parameters with the presence of H. capsulatum by nested PCR, indicating the fungus existence in organic fertilizers that complied with the NTC 5167. The Hc100 nested PCR targeting H. capsulatum standardized in this work will improve the evaluation of organic fertilizers and ensure the prevention of outbreaks and cases due to manufacturing, marketing, and use of fertilizers contaminated with H. capsulatum.

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.