Phenotypic characterization of cryptic species in the fungal pathogen Histoplasma.

Histoplasmosis is an endemic mycosis that often presents as a respiratory infection in immunocompromised patients. Hundreds of thousands of new infections are reported annually around the world. The etiological agent of the disease, Histoplasma, is a dimorphic fungus commonly found in the soil where it grows as mycelia. Humans can become infected by Histoplasma through inhalation of its spores (conidia) or mycelial particles. The fungi transition into the yeast phase in the lungs at 37°C. Once in the lungs, yeast cells reside and proliferate inside alveolar macrophages. Genomic work has revealed that Histoplasma is composed of at least five cryptic phylogenetic species that differ genetically. Three of those lineages have received new names. Here, we evaluated multiple phenotypic characteristics (colony morphology, secreted proteolytic activity, yeast size, and growth rate) of strains from five of the phylogenetic species of Histoplasma to identify phenotypic traits that differentiate between these species: Histoplasma capsulatum sensu stricto, Histoplasma ohiense, Histoplasma mississippiense, Histoplasma suramericanum, and an African lineage. We report diagnostic traits for three species. The other two species can be identified by a combination of traits. Our results suggest that (i) there are significant phenotypic differences among the cryptic species of Histoplasma and (ii) those differences can be used to positively distinguish those species in a clinical setting and for further study of the evolution of this fungal pathogen.IMPORTANCEIdentifying species boundaries is a critical component of evolutionary biology. Genome sequencing and the use of molecular markers have advanced our understanding of the evolutionary history of fungal pathogens, including Histoplasma, and have allowed for the identification of new species. This is especially important in organisms where morphological characteristics have not been detected. In this study, we revised the taxonomic status of the four named species of the genus Histoplasma, H. capsulatum sensu stricto (ss), H. ohiense, H. mississippiense, and H. suramericanum, and propose the use of species-specific phenotypic traits to aid their identification when genome sequencing is not available. These results have implications not only for evolutionary study of Histoplasma but also for clinicians, as the Histoplasma species could determine the outcome of disease and treatment needed.

Histoplasmosis in the Republic of Congo dominated by African histoplasmosis, Histoplasma capsulatum var. duboisii.

The Republic of Congo (RoC) is one of the African countries with the most histoplasmosis cases reported. This review summarizes the current status regarding epidemiology, diagnostic tools, and treatment of histoplasmosis in the RoC. A computerized search was performed from online databases Medline, PubMed, HINARI, and Google Scholar to collect literature on histoplasmosis in the RoC. We found 57 cases of histoplasmosis diagnosed between 1954 and 2019, corresponding to an incidence rate of 1-3 cases each year without significant impact of the AIDS epidemic in the country. Of the 57 cases, 54 (94.7%) were cases of Histoplasma capsulatum var. duboisii (Hcd) infection, African histoplasmosis. Three cases (5.3%) of Histoplasma capsulatum var. capsulatum infection were recorded, but all were acquired outside in the RoC. The patients' ages ranged between 13 months to 60 years. An equal number of cases were observed in adults in the third or fourth decades (n = 14; 24.6%) and in children aged ≤15 years. Skin lesions (46.3%), lymph nodes (37%), and bone lesions (26%) were the most frequent clinical presentations. Most diagnoses were based on histopathology and distinctive large yeast forms seen in tissue. Amphotericin B (AmB) was first line therapy in 65% of the cases and itraconazole (25%) for maintenance therapy. The occurrence of African histoplasmosis in apparently normal children raises the possibility that African histoplasmosis is linked to environmental fungal exposure.

High prevalence and mortality due to Histoplasma capsulatum in the Brazilian Amazon: An autopsy study.

BACKGROUND: Histoplasmosis is acquired by inhalation of spores of the dimorphic fungus Histoplasma spp. Although this pathogen is distributed worldwide, it is more prevalent in the Americas. However, the real burden of histoplasmosis remains undefined in many endemic regions. METHODOLOGY: We conducted a series of 61 autopsies to individuals who died in a hospital in the Brazilian Amazon focused on infectious diseases. We performed a detailed histological and microbiological evaluation with genetic characterization of Histoplasma strains with the aim to evaluate the contribution of histoplasmosis to morbidity and mortality. Additionally, we assessed the clinicopathological correlation. PRINCIPAL FINDINGS: Evidence of Histoplasma infection was detected in 21 patients (34%). Eight cases were disseminated infections, all of them occurred in HIV-positive patients. Six cases were localized histoplasmosis, limited to the lungs. In seven patients Histoplasma DNA was detected by PCR in patients with no histological lesions. Histoplasma infection was detected in 38% of HIV-positive patients and was a major contributor to death in 22% of them. Lungs, liver and spleen were affected in all cases of disseminated histoplasmosis. Phylogenetic analysis of the strains suggested a high diversity of Histoplasma species circulating in the Brazilian Amazon. Histoplasmosis was clinically missed in 75% of the disseminated infections. CONCLUSIONS: The high incidence of histoplasmosis, the low index of clinical suspicion, and the severity of the disseminated disease highlight the need of proactively implementing sensitive routine screening methods for this pathogen in endemic areas. Antifungal prophylaxis against Histoplasma should be encouraged in the severely immunocompromised HIV patients in these areas. In conclusion, substantial mortality is associated with disseminated histoplasmosis among HIV-positive patients in the Brazilian Amazon.

Biodiverse Histoplasma Species Elicit Distinct Patterns of Pulmonary Inflammation following Sublethal Infection.

Histoplasma is an endemic dimorphic fungus that can cause disease in healthy and immunocompromised individuals after the transition of inhaled spores into the facultative intracellular yeast form. There is substantial diversity among Histoplasma species, but it is not clear how this heterogeneity impacts the progression of pathology and cellular immune responses during acute respiratory infection, which represents the vast majority of histoplasmosis disease burden. After inoculating mice intranasally with a sublethal inoculum, we characterized the immune response to Histoplasma capsulatum (strain G186A) and Histoplasma ohiense (strain G217B) using comprehensive flow cytometric and single-cell analyses. Within 8 days after inoculation, H. ohiense induced a significantly higher infiltration of neutrophils and inflammatory monocytes into the lung compared to H. capsulatum Microscopic analysis of infected lung tissue revealed that although the total number of fungi was similar within inflamed lung lesions, we observed different species-dependent intracellular yeast distribution patterns. Inoculation with gfp-expressing strains indicated that H. ohiense, but not H. capsulatum, was associated primarily with alveolar macrophages early after infection. Interestingly, we observed a significant reduction in the total number of alveolar macrophages 12 to 16 days after H. ohiense, but not H. capsulatum infection, despite similar intracellular growth dynamics within AMJ2-C11 alveolar macrophages in vitro Together, our data suggest that H. ohiense, but not H. capsulatum, preferentially interacts with alveolar macrophages early after infection, which may lead to a different course of inflammation and resolution despite similar rates of fungal clearance.IMPORTANCE Acute pulmonary histoplasmosis in healthy individuals comprises most of the disease burden caused by the fungal pathogen Histoplasma Fungal pneumonia is frequently delayed in diagnosis and treatment due to a prolonged period of quiescence early during infection. In this study, we used a murine respiratory model of histoplasmosis to investigate how different Histoplasma species modulate lung inflammation throughout the complete course of infection. We propose that a relatively low, sublethal inoculum is ideal to model acute pulmonary histoplasmosis in humans, primarily due to the quiescent stage of fungal growth that occurs in the lungs of mice prior to the initiation of inflammation. Our results reveal the unique course of lung immunity associated with divergent species of Histoplasma and imply that the progression of clinical disease is considerably more heterogeneous than previously recognized.

Acute histoplasmosis in travelers: a retrospective study in an Italian referral center for tropical diseases.

Purpose: Histoplasmosis is a fungal infection acquired through inhalation of Histoplasma capsulatum microconidia, mostly present in the Americas. Both immunocompetent and immunocompromised patients can present a wide spectrum of signs/symptoms, ranging from mild disease to a severe, disseminated infection. The aim of this observational study is to describe histoplasmosis cases diagnosed in travelers and their clinical/radiological and therapeutic pattern.Methods: Retrospective study at the Department of Infectious - Tropical Diseases and Microbiology (DITM) of Negrar, Verona, Italy, between January 2005 and December 2015.Results: Twenty-three cases of acute histoplasmosis were diagnosed, 17 of which belong to the same cluster. Seven of the 23 patients (30.4%) were admitted to hospital, four of whom underwent invasive diagnostic procedures. Thirteen patients (56.5%) received oral itraconazole. All patients recovered, although nine (39.1%) had radiological persisting lung nodules at 12 month follow up.Conclusions: Clinical, laboratory and radiological features of histoplasmosis can mimic other conditions, resulting in unnecessary invasive diagnostic procedures. However, a history of travel to endemic areas and of exposure to risk factors (such as visits to caves and presence of bats) should trigger the clinical suspicion of histoplasmosis. Treatment may be indicated in severe or prolonged disease.

Histoplasmosis Outbreaks in Brazil: Lessons to Learn About Preventing Exposure.

Histoplasmosis is considered the most common invasive opportunistic fungal disease in the Americas, with outbreaks and micro-epidemics reported for over 80 years. In Brazil, this disease has been described since 1946, reaching a remarkable incidence in the population, especially during the HIV-AIDS pandemic. In this study, published and unpublished outbreaks and micro-epidemics of histoplasmosis in Brazil were revisited by accessing different database sources and evaluating epidemiological and clinical features. We have found reports spanning 1946-2017, across 10 Brazilian states and with involvement of 370 humans and 2 dogs, and 13 disseminated cases and 3 deaths were reported. Rio de Janeiro had the largest number of outbreaks (n = 20/40; 50%) reported in this study. The majority of outbreaks and micro-epidemics was reported in caves (n = 21/40; 52.5%), followed by reports in abandoned/deactivated sites (n = 6/40; 15%), mines (n = 5/40; 12.5%), chicken coops (n = 4/40; 10%). Histoplasmosis is a serious health issue in Brazil considering the attractive and growing market of ecotourism throughout more than 7000 caves, and all levels of poultry farming activity are important to raise awareness about how dangerous this neglected disease can be and establish ways to decrease exposure to contaminated environmental sources through adequate preventive measures.

Disseminated histoplasmosis in an immunocompetent individual diagnosed with gastrointestinal endoscopy: a case report.

BACKGROUND: Histoplasmosis is one of the invasive fungal infections and presents with symptoms mainly in the lungs. Disseminated histoplasmosis (DH) is rare and its lesions in the gastrointestinal tract are even uncommon. The concomitant involvement of the upper and lower gastrointestinal tract has never been described in the immunocompetent individuals. CASE PRESENTATION: A 44-year-old immunocompetent Chinese man presented with fever, hepatosplenomegaly, fungal esophagitis and protuberant lesions with central depression and erosion along the mucous membrane of the colon. The patient was diagnosed as disseminated histoplasmosis by gastrointestinal endoscopy. CONCLUSIONS: Histoplasmosis should be taken caution in patients with fever and hepatosplenomegaly. Actions should be taken to avoid its disseminated infection associated high mortality.

Analyses of the genetic diversity and population structures of Histoplasma capsulatum clinical isolates from Mexico, Guatemala, Colombia and Argentina, using a randomly amplified polymorphic DNA-PCR assay.

We studied the genetic diversity and the population structure of human isolates of Histoplasma capsulatum, the causative agent of histoplasmosis, using a randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) assay to identify associations with the geographic distribution of isolates from Mexico, Guatemala, Colombia and Argentina. The RAPD-PCR pattern analyses revealed the genetic diversity by estimating the percentage of polymorphic loci, effective number of alleles, Shannon's index and heterozygosity. Population structure was identified by the index of association (IA) test. Thirty-seven isolates were studied and clustered into three groups by the unweighted pair-group method with arithmetic mean (UPGMA). Group I contained five subgroups based on geographic origin. The consistency of the UPGMA dendrogram was estimated by the cophenetic correlation coefficient (CCCr = 0.94, P = 0.001). Isolates from Mexico and Colombia presented higher genetic diversity than isolates from Argentina. Isolates from Guatemala grouped together with the reference strains from the United States of America and Panama. The IA values suggest the presence of a clonal population structure in the Argentinian H. capsulatum isolates and also validate the presence of recombining populations in the Colombian and Mexican isolates. These data contribute to the knowledge on the molecular epidemiology of histoplasmosis in Latin America.

Phylogenetic analysis of Histoplasma capsulatum var duboisii in baboons from archived formalin-fixed, paraffin embedded tissues.

Histoplasma capsulatum var. duboisii (Hcd) infections have been well documented to cause chronic granulomatous disease, mainly involving the skin of baboons and humans in African countries primarily. This retrospective study classified the subspecies of Histoplasma and developed a phylogenetic tree utilizing DNA sequences extracted from formalin-fixed, paraffin embedded (FFPE) tissues from 9 baboons from a research colony in Texas histologically diagnosed with Hcd. Based on sequence analysis of ITS-2, Tub-1, and ARF, Hcd isolated from the archived samples closely aligns with the African clade and has 88% sequence homology with a sample isolated from an individual in Senegal.

Mapping Histoplasma capsulatum Exposure, United States.

Maps of Histoplasma capsulatum infection prevalence were created 50 years ago; since then, the environment, climate, and anthropogenic land use have changed drastically. Recent outbreaks of acute disease in Montana and Nebraska, USA, suggest shifts in geographic distribution, necessitating updated prevalence maps. To create a weighted overlay geographic suitability model for Histoplasma, we used a geographic information system to combine satellite imagery integrating land cover use (70%), distance to water (20%), and soil pH (10%). We used logistic regression modeling to compare our map with state-level histoplasmosis incidence data from a 5% sample from the Centers for Medicare and Medicaid Services. When compared with the state-based Centers data, the predictive accuracy of the suitability score-predicted states with high and mid-to-high histoplasmosis incidence was moderate. Preferred soil environments for Histoplasma have migrated into the upper Missouri River basin. Suitability score mapping may be applicable to other geographically specific infectious vectors.

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.

Histoplasmosis in Africa: An emerging or a neglected disease?

Histoplasmosis in Africa has markedly increased since the advent of the HIV/AIDS epidemic but is under-recognised. Pulmonary histoplasmosis may be misdiagnosed as tuberculosis (TB). In the last six decades (1952-2017), 470 cases of histoplasmosis have been reported. HIV-infected patients accounted for 38% (178) of the cases. West Africa had the highest number of recorded cases with 179; the majority (162 cases) were caused by Histoplasma capsulatum var. dubuosii (Hcd). From the Southern African region, 150 cases have been reported, and the majority (119) were caused by H. capsulatum var. capsulatum (Hcc). There have been 12 histoplasmin skin test surveys with rates of 0% to 35% positivity. Most cases of Hcd presented as localised lesions in immunocompetent persons; however, it was disseminated in AIDS patients. Rapid diagnosis of histoplasmosis in Africa is only currently possible using microscopy; antigen testing and PCR are not available in most of Africa. Treatment requires amphotericin B and itraconazole, both of which are not licensed or available in several parts of Africa.

A matrix-assisted laser desorption/ionization time of flight mass spectrometry reference database for the identification of Histoplasma capsulatum.

The isolation of the pathogenic fungus Histoplasma capsulatum from cultures together with the visualization of typical intracellular yeast in tissues are the gold standard methods for diagnosis of histoplasmosis. However, cultures are time-consuming, require level 3 containment and experienced personnel, and usually call for an additional confirmation test. Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-ToF MS) has been established as a suitable tool for microbial identification in several clinical laboratories. A reference database has been constructed for the identification of H. capsulatum by MALDI-ToF MS by using six H. capsulatum strains previously identified by molecular methods. For validation, 63 fungal strains belonging to the Collection of the Spanish National Centre for Microbiology were tested against the new reference database combined with other commercial and in-house databases. In a blind assay, all H. capsulatum strains (n = 30) were correctly identified by the database and 86.6% had scores above 1.7. Considering both phases of the fungus for the same strain, the most reliable results were obtained with the mycelial phase, with only 13.3% of isolates having scores below 1.7. The new database was able to identify both morphological phases of the fungus. MALDI-ToF technology yields a prompt and simple identification from H. capsulatum yeast forms and early mycelial cultures. It allows for reducing response time and decreasing risk in fungus manipulation.

African histoplasmosis: new clinical and microbiological insights.

African histoplasmosis is defined as the fungal infection caused by Histoplasma capsulatum var. duboisii (Hcd). Studies focused on distinguishing Hcd and H. capsulatum var. capsulatum (Hcc), which coexist in Africa, are scarce or outdated, and African strains are continuously underrepresented. In this work, 13 cases of African patients with histoplasmosis diagnosed in the Spanish Mycology Reference Laboratory have been reviewed showing that 77% had disseminated disease and AIDS as underlying disease although Hcd infection has been classically considered a rare presentation in AIDS patients. Strains isolated from these patients and other clinical and reference strains were studied by assessing classical identification methods and performing a three-loci multi-locus sequence analysis (MLSA). Classical identification methods based on biochemical tests and measurement of yeast size proved to be useless in distinguishing both varieties. The MLSA defined an African cluster, with a strong statistical support, that included all strains with African origin. Finally, mating type was also determined by using molecular methods revealing an unequal mating type distribution in African strains. In conclusion, historical statements and classical identification methods were useless to distinguish between varieties, whereas molecular analyses revealed that all strains with African origin grouped together suggesting that traditional classification should be revised. Further investigation is required in order to unravel traditional concepts about Hcd infection and support results obtained in this work.

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.

Genome Sequences Reveal Cryptic Speciation in the Human Pathogen Histoplasma capsulatum.

Histoplasma capsulatum is a pathogenic fungus that causes life-threatening lung infections. About 500,000 people are exposed to H. capsulatum each year in the United States, and over 60% of the U.S. population has been exposed to the fungus at some point in their life. We performed genome-wide population genetics and phylogenetic analyses with 30 Histoplasma isolates representing four recognized areas where histoplasmosis is endemic and show that the Histoplasma genus is composed of at least four species that are genetically isolated and rarely interbreed. Therefore, we propose a taxonomic rearrangement of the genus.IMPORTANCE The evolutionary processes that give rise to new pathogen lineages are critical to our understanding of how they adapt to new environments and how frequently they exchange genes with each other. The fungal pathogen Histoplasma capsulatum provides opportunities to precisely test hypotheses about the origin of new genetic variation. We find that H. capsulatum is composed of at least four different cryptic species that differ genetically and also in virulence. These results have implications for the epidemiology of histoplasmosis because not all Histoplasma species are equivalent in their geographic range and ability to cause disease.

Development and Evaluation of a Molecular Diagnostic Method for Rapid Detection of Histoplasma capsulatum var. farciminosum, the Causative Agent of Epizootic Lymphangitis, in Equine Clinical Samples.

Histoplasma capsulatum var. farciminosum, the causative agent of epizootic lymphangitis (EZL), is endemic in parts of Africa. Diagnosis based on clinical signs and microscopy lacks specificity and is a barrier to further understanding this neglected disease. Here, a nested PCR method targeting the internal transcribed spacer (ITS) region of the rRNA operon was validated for application to equine clinical samples. Twenty-nine horses with signs of EZL from different climatic regions of Ethiopia were clinically examined. Blood samples and aspirates of pus from cutaneous nodules were taken, along with blood from a further 20 horses with no cutaneous EZL lesions. Among the 29 horses with suspected cases of EZL, H. capsulatum var. farciminosum was confirmed by extraction of DNA from pus and blood samples from 25 and 17 horses, respectively. Positive PCR results were also obtained with heat-inactivated pus (24 horses) and blood (23 horses) spotted onto Whatman FTA cards. Two positive results were obtained among blood samples from 20 horses that did not exhibit clinical signs of EZL. These are the first reports of the direct detection of H. capsulatum var. farciminosum in equine blood and at high frequency among horses exhibiting cutaneous lesions. The nested PCR outperformed conventional microscopic diagnosis, as characteristic yeast cells could be observed only in 14 pus samples. The presence of H. capsulatum var. farciminosum DNA was confirmed by sequencing the cloned PCR products, and while alignment of the ITS amplicons showed very little sequence variation, there was preliminary single nucleotide polymorphism-based evidence for the existence of two subgroups of H. capsulatum var. farciminosum This molecular diagnostic method now permits investigation of the epidemiology of EZL.

Filamentous Fungi.

Filamentous mycoses are often associated with significant morbidity and mortality. Prompt diagnosis and aggressive treatment are essential for good clinical outcomes in immunocompromised patients. The host immune response plays an essential role in determining the course of exposure to potential fungal pathogens. Depending on the effectiveness of immune response and the burden of organism exposure, fungi can either be cleared or infection can occur and progress to a potentially fatal invasive disease. Nonspecific cellular immunity (i.e., neutrophils, natural killer [NK] cells, and macrophages) combined with T-cell responses are the main immunologic mechanisms of protection. The most common potential mold pathogens include certain hyaline hyphomycetes, endemic fungi, the Mucorales, and some dematiaceous fungi. Laboratory diagnostics aimed at detecting and differentiating these organisms are crucial to helping clinicians make informed decisions about treatment. The purpose of this chapter is to provide an overview of the medically important fungal pathogens, as well as to discuss the patient characteristics, antifungal-therapy considerations, and laboratory tests used in current clinical practice for the immunocompromised host.

Histoplasma capsulatum Infection with Extensive Lytic Bone Lesions Mimicking LCH.

Multiple lytic bone lesions in a child can be a manifestation of various diseases like Langerhans cell histiocytosis, metastatic neuroblastoma, leukemia, hyperparathyroidism, multifocal osteomyelitis and histoplasmosis. Disseminated histoplasmosis caused by Histoplasma capsulatum var. duboisii is well known to present with multiple osteolytic lesions in immunocompromised adults and is mostly restricted to the African subcontinent. Histoplasmosis seen in American and Asian countries is caused by Histoplasma capsulatum var. capsulatum, which presents with pulmonary and systemic manifestations and rarely bone involvement. We report a case of histoplasmosis, caused by H. capsulatum var. capsulatum with extensive lytic bone lesions in a 13 year old immunocompetent boy who presented with prolonged fever, weight loss and multiple boggy swellings. He responded to amphotericin and is currently on Itraconazole. This case is unique for extensive osteolytic lesions with H. capsulatum var. capsulatum infection in an immunocompetent child.

Molecular identification of Histoplasma capsulatum using rolling circle amplification.

Histoplasmosis is a systemic fungal disease that occurs worldwide, causing symptomatic infection mostly in immunocompromised hosts. Etiological agent is the dimorphic fungus, Histoplasma capsulatum, which occurs in soil contaminated with bird or bat droppings. Major limitation in recognition of H. capsulatum infections is the low awareness, since other diseases may have similar symptomatology. The molecular methods have gained importance because of unambiguous diagnostic ability and efficiency. The aim of this study was to develop and evaluate a padlock probe in view of rolling circle amplification (RCA) detection method which targets ITS (Internal Transcribed Spacer) rDNA of H. capsulatum enabling rapid and specific detection of the fungus in clinical samples. Two padlock probes were designed and one of these (HcPL2) allowed specific amplification of H. capsulatum DNA while no cross-reactivity was observed with fungi used as negative controls. This method proved to be effective for H. capsulatum specific identification and demonstrated to be faster than the traditional method of microbiological identification.