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.

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.

Genotypic diversity, virulence, and molecular genetic tools in Histoplasma.

SUMMARYHistoplasmosis is arguably the most common fungal respiratory infection worldwide, with hundreds of thousands of new infections occurring annually in the United States alone. The infection can progress in the lung or disseminate to visceral organs and can be difficult to treat with antifungal drugs. Histoplasma, the causative agent of the disease, is a pathogenic fungus that causes life-threatening lung infections and is globally distributed. The fungus has the ability to germinate from conidia into either hyphal (mold) or yeast form, depending on the environmental temperature. This transition also regulates virulence. Histoplasma and histoplasmosis have been classified as being of emergent importance, and in 2022, the World Health Organization included Histoplasma as 1 of the 19 most concerning human fungal pathogens. In this review, we synthesize the current understanding of the ecological niche, evolutionary history, and virulence strategies of Histoplasma. We also describe general patterns of the symptomatology and epidemiology of histoplasmosis. We underscore areas where research is sorely needed and highlight research avenues that have been productive.

Tracing histoplasmosis genomic epidemiology and species occurrence across the USA.

ABSTRACTHistoplasmosis is an endemic mycosis in North America frequently reported along the Ohio and Mississippi River Valleys, although autochthonous cases occur in non-endemic areas. In the United States, the disease is provoked by two genetically distinct clades of Histoplasma capsulatum sensu lato, Histoplasma mississippiense (Nam1) and H. ohiense (Nam2). To bridge the molecular epidemiological gap, we genotyped 93 Histoplasma isolates (62 novel genomes) including clinical, environmental, and veterinarian samples from a broader geographical range by whole-genome sequencing, followed by evolutionary and species niche modelling analyses. We show that histoplasmosis is caused by two major lineages, H. ohiense and H. mississippiense; with sporadic cases caused by H. suramericanum in California and Texas. While H. ohiense is prevalent in eastern states, H. mississipiense was found to be prevalent in the central and western portions of the United States, but also geographically overlapping in some areas suggesting that these species might co-occur. Species Niche Modelling revealed that H. ohiense thrives in places with warmer and drier conditions, while H. mississippiense is endemic to areas with cooler temperatures and more precipitation. In addition, we predicted multiple areas of secondary contact zones where the two species co-occur, potentially facilitating gene exchange and hybridization. This study provides the most comprehensive understanding of the genomic epidemiology of histoplasmosis in the USA and lays a blueprint for the study of invasive fungal diseases.

Histoplasma antigens as novel players for the development of new enzyme immunoassays for the serodiagnosis of histoplasmosis: A comparative study of their analytical performance.

Definitive diagnosis of histoplasmosis relies on culture and/or cytology/histopathology; however, these procedures have limited sensitivity and cultures are time-consuming. Antibodies detection by immunodiffusion has low sensitivity in immunocompromised individuals and uses histoplasmin (HMN), a crude antigenic extract, as reagent. Novel protein antigen candidates have been recently identified and produced by DNA-recombinant techniques to obtain standardized and specific reagents for diagnosing histoplasmosis. To compare the analytical performance of novel enzyme-linked immunosorbent assays (ELISAs) for antibodies testing for diagnosing histoplasmosis using different Histoplasma capsulatum antigens as reagents. The H. capsulatum 100 kDa protein (Hcp100), the M antigen and its immunoreactive fragment F1 were produced by DNA-recombinant techniques. Galactomannan was purified from both the yeast and mycelial cell walls (yGM and mGM, respectively). The analytical performance of the ELISA tests for the serological detection of antibodies against these antigens was evaluated and compared with those obtained using HMN as reagent. Antibodies detection by the Hcp100 ELISA demonstrated 90.0% sensitivity and 92.0% specificity, versus 43.3% sensitivity and 95.0% specificity of the M ELISA, 33.3% sensitivity and 84.0% specificity of the F1 ELISA, 96.7% sensitivity and 94.0% specificity of the yGM ELISA, 83.3% sensitivity and 88.0% specificity of the mGM ELISA, and 70.0% sensitivity and 86.0% specificity for the HMN ELISA. In summary, Hcp100 is proposed as the most promising candidate for the serodiagnosis of histoplasmosis. The primary immunoreactive element in HMN proved to be GM rather than the M antigen. Nevertheless, a higher incidence of cross-reactions was noted with GM compared to M.

Hcp100 is a promising serodiagnostic candidate for histoplasmosis, boasting high sensitivity and specificity. Notably, GM, rather than M antigen, emerged as the primary immunoreactive element in HMN, despite a higher incidence of cross-reactions with GM compared to M.

Validation of a real-time polymerase chain reaction for the detection and quantification of the nucleic acid of Histoplasma from equine clinical samples.

Histoplasma capsulatum var. farciminosum (HCF) is a dimorphic fungus that causes epizootic lymphangitis in equids. Current diagnostic approaches, including culture, microscopy, and clinical presentation, lack speed, sensitivity, and specificity when diagnosing clinical cases. In this study, equine blood and pus samples on Whatman FTA cards from Senegal (n = 3), The Gambia (n = 19), Ethiopia (n = 16), and Mali (n = 13) were tested using a real-time PCR (qPCR) protocol. The assay was optimized and tested for its suitability to detect and quantify HCF in blood and pus loaded onto Whatman FTA cards at sampling. Whatman FTA cards were tested for their suitability for use with qPCR and were found to recover DNA more efficiently than from direct extraction. Using TaqMan fluorescent probes and specific primers, the assay demonstrated 100% analytical specificity when detecting multiple strains of Histoplasma and no false positives with off-target organisms. The assay's diagnostic performance was measured against an existing nested internal transcribed spacer PCR protocol using a receiver operating characteristic curve. The test was found to have a diagnostic specificity and sensitivity of 100% and 71.4%, respectively, when analyzing pus samples using a cycle threshold (Ct) cutoff determined by Youden's index (27.75). Blood sample cutoff Ct value was proposed at 34.55. Further optimization is required to improve the performance of the protocol when applied to blood samples. This study has, for the first time, demonstrated the ability to detect and quantify the DNA of Histoplasma spp. in equine blood and pus samples with a high degree of accuracy, providing a platform to further investigate the pathogenesis and epidemiology of this disease. IMPORTANCE: Histoplasmosis is a neglected yet major cause of morbidity and mortality in both equids and people in resource-scarce settings. One of the major hindrances to the control of histoplasmosis is a lack of readily available diagnostic tests. Tests are needed to support clinical decision-making and to be applied in population-based research to further understand this disease in situ. This paper reports, for the first time, the validation and application of a qPCR to detect Histoplasma directly from equine clinical samples, bypassing the need to culture this notoriously difficult organism. We report and comment on the performance of the qPCR in comparison with our previously developed nested PCR.

Histoplasmosis in HIV/AIDS patients in Amazonas, Northern Brazil.

Histoplasmosis is commonly observed in AIDS patients as a neglected opportunistic disease that has an important relationship with environmental factors. The present study described the clinical characteristics of HIV/AIDS patients diagnosed with disseminated histoplasmosis in a tertiary healthcare facility in Manaus, Amazonas, Brazil, and evaluated the patients' homes and urban environmental samples as a source of exposure to Histoplasma capsulatum. A review of medical records from 2017 to 2019 of patients with HIV/AIDS associated with histoplasmosis was carried out, as well as the collection of environmental samples in the homes of these patients. These samples were subjected to DNA extraction and then subjected to qPCR. A total of 62 patients diagnosed with HIV/AIDS and histoplasmosis were identified, which corresponds to 4.5% (n = 62/1372) of the HIV/AIDS cases detected in the period. Of these, 68% (n = 42/62) were male, with a mean age of 36 years and low education. In 47% (n = 29/62) of the cases, the diagnosis of HIV/AIDS and histoplasmosis occurred simultaneously. Mortality was 45% (n = 28/62), and 68% (n = 42/62) of these patients did not regularly use highly active antiretroviral therapy. The main symptoms found were respiratory, gastrointestinal, and weight loss, and in 81% (n = 50/62), the place of residence was in an urban area. A total of 57 environmental samples were analyzed, and the presence of Histoplasma capsulatum was not detected in any of the analyzed samples. There was a high mortality rate in the studied group of patients with AIDS and histoplasmosis. Most patients reported residing in urban areas of Manaus, with no history of travel to other areas previously known as being high risk for histoplasmosis.

Utilization of a Histoplasma capsulatum zinc reporter reveals the complexities of fungal sensing of metal deprivation.

Histoplasma capsulatum is a dimorphic fungal pathogen acquired via inhalation of soil-resident spores. Upon exposure to mammalian body temperatures, these fungal elements transform into yeasts that reside primarily within phagocytes. Macrophages (MΦ) provide a permissive environment for fungal replication until T cell-dependent immunity is engaged. MΦ activated by granulocyte macrophage colony stimulating factor (GM-CSF) induces metallothioneins (MTs) that bind zinc (Zn) and deprive yeast cells of labile Zn, thereby disabling fungal growth. Prior work demonstrated that the zinc transporter, ZRT2, was important for fungal survival in vivo. Hence, we constructed a yeast cell reporter strain that expresses green fluorescent protein (GFP) under control of the ZRT2 zinc-regulated promoter. This reporter accurately responds to a medium devoid of Zn. ZRT2 expression increased in GM-CSF, but not interferon-γ, stimulated MΦ. To examine the in vivo response, we infected mice with a reporter yeast strain and assessed ZRT2 expression at 0, 3, 7, and 14 days post-infection (dpi). ZRT2 expression minimally increased at 3 dpi and peaked at 7 dpi, corresponding with the onset of adaptive immunity. We discovered that the major MΦ populations that restrict Zn from the fungus are interstitial MΦ and exudate MΦ. Neutralizing GM-CSF blunted the control of infection but unexpectedly increased ZRT2 expression. This increase was dependent on another cytokine that activates MΦ to control H. capsulatum replication, M-CSF. These findings illustrate the reporter's ability to sense Zn in vitro and in vivo and correlate ZRT2 expression with GM-CSF and M-CSF activation of MΦ.IMPORTANCEPhagocytes use an arsenal of defenses to control the replication of Histoplasma yeasts, one of which is the limitation of trace metals. On the other hand, H. capsulatum combats metal restriction by upregulating metal importers such as the Zn importer ZRT2. This transporter contributes to H. capsulatum pathogenesis upon activation of adaptive immunity. We constructed a fluorescent ZRT2 transcriptional reporter to probe H. capsulatum Zn sensing during infection and exposed the role for M-CSF activation of macrophages when GM-CSF is absent. These data highlight the ways in which fungal pathogens sense metal deprivation in vivo and reveal the potential of metal-sensing reporters. The work adds a new dimension to study how intracellular pathogens sense and respond to the changing environments of the host.

The influence of a copper efflux pump in Histoplasma capsulatum virulence.

During the infectious process, pathogenic microorganisms must obtain nutrients from the host in order to survive and proliferate. These nutritional sources include the metallic nutrient copper. Despite its essentiality, copper in large amounts is toxic. Host defense mechanisms use high copper poisoning as a fungicidal strategy to control infection. Transcriptional analyses showed that yeast cultured in the presence of copper or inside macrophages (24 h) had elevated expression of CRP1, a copper efflux pump, suggesting that Histoplasma capsulatum could be exposed to a high copper environment in macrophages during the innate immune stage of infection. Accordingly, macrophages cultured in high copper are more efficient in controlling H. capsulatum growth. Also, silencing of ATP7a, a copper pump that promotes the copper influx in phagosomes, increases fungal survival in macrophages. The rich copper environment faced by the fungus is not dependent on IFN-γ, since fungal CRP1 expression is induced in untreated macrophages. Appropriately, CRP1 knockdown fungal strains are more susceptible to macrophage control than wild-type yeasts. Additionally, CRP1 silencing decreases fungal burden in mice during the phase of innate immune response (4-day postinfection) and CRP1 is required for full virulence in a macrophage cell lines (J774 A.1 and RAW 264.7), as well as primary cells (BMDM). Thus, induction of fungal copper detoxifying genes during innate immunity and the attenuated virulence of CRP1-knockdown yeasts suggest that H. capsulatum is exposed to a copper-rich environment at early infection, but circumvents this condition to establish infection.

Recyclable CRISPR/Cas9-mediated gene disruption and deletions in Histoplasma.

IMPORTANCE: Histoplasma is a primary fungal pathogen with the ability to infect otherwise healthy mammalian hosts, causing systemic and sometimes life-threatening disease. Thus far, molecular genetic manipulation of this organism has utilized RNA interference, random insertional mutagenesis, and a homologous recombination protocol that is highly variable and often inefficient. Targeted gene manipulations have been challenging due to poor rates of homologous recombination events in Histoplasma. Interrogation of the virulence strategies of this organism would be highly accelerated by a means of efficiently generating targeted mutations. We have developed a recyclable CRISPR/Cas9 system that can be used to introduce gene disruptions in Histoplasma with high efficiency, thereby allowing disruption of multiple genes.

Targeted gene deletions in the dimorphic fungal pathogen Histoplasma using an optimized episomal CRISPR/Cas9 system.

The rapid development of CRISPR/CRISPR-associated (Cas) systems has revolutionized the ability to produce genetic mutations in a desired locus, particularly in organisms with low rates of homologous recombination. Histoplasma is an important respiratory and systemic fungal pathogen that has few reverse genetic options. We describe an optimized CRISPR/Cas system for the efficient generation of mutations in desired genes. The limited requirements for CRISPR/Cas, namely a gene-targeting guide RNA (gRNA) and expression of a Cas endonuclease, enabled both the gRNA and the Streptococcus pyogenes Cas9 gene to be expressed from a single episomal vector. The gRNAs are expressed from a strong Pol(II) promoter, a critical parameter for increasing the recovery of mutated genes, and processed into the mature gRNA by ribozymes in the mRNA. Expression of dual-tandem gRNAs facilitates the generation of gene deletions at a good frequency which can be detected by PCR-based screening of pooled isolates resulting in the isolation of marker-less deletion mutants. The CRISPR/Cas system is encoded on an episomal telomeric vector facilitating curing strains of the CRISPR/Cas vector upon generation of the mutant. We demonstrate the successful application of this CRISPR/Cas system in diverse Histoplasma species and applicable for multiple genes. The optimized system shows promise for accelerating reverse genetic studies in Histoplasma spp. IMPORTANCE The ability to eliminate gene product functions is central to understanding molecular mechanisms. In the fungal pathogen Histoplasma, methods to inactivate or deplete gene products are inefficient, which hampers progress in defining Histoplasma's virulence mechanisms. We describe an efficient CRISPR/Cas-based system for generating gene deletions in Histoplasma and show its validation on multiple genes with selectable and non-selectable phenotypes.

A multicentre external quality assessment: A first step to standardise PCR protocols for the diagnosis of histoplasmosis and coccidioidomycosis.

BACKGROUND: In-house real-time PCR (qPCR) is increasingly used to diagnose the so-called endemic mycoses as commercial assays are not widely available. OBJECTIVES: To compare the performance of different molecular diagnostic assays for detecting Histoplasma capsulatum and Coccidioides spp. in five European reference laboratories. METHODS: Two blinded external quality assessment (EQA) panels were sent to each laboratory that performed the analysis with their in-house assays. Both panels included a range of concentrations of H. capsulatum (n = 7) and Coccidioides spp. (n = 6), negative control and DNA from other fungi. Four laboratories used specific qPCRs, and one laboratory a broad-range fungal conventional PCR (cPCR) and a specific cPCR for H. capsulatum with subsequent sequencing. RESULTS: qPCR assays were the most sensitive for the detection of H. capsulatum DNA. The lowest amount of H. capsulatum DNA detected was 1-4 fg, 0.1 pg and 10 pg for qPCRs, specific cPCR and broad-range cPCR, respectively. False positive results occurred with high concentrations of Blastomyces dermatitidis DNA in two laboratories and with Emergomyces spp. in one laboratory. For the Coccidioides panel, the lowest amount of DNA detected was 1-16 fg by qPCRs and 10 pg with the broad-range cPCR. One laboratory reported a false positive result by qPCR with high load of Uncinocarpus DNA. CONCLUSION: All five laboratories were able to correctly detect H. capsulatum and Coccidioides spp. DNA and qPCRs had a better performance than specific cPCR and broad-range cPCR. EQAs may help standardise in-house molecular tests for the so-called endemic mycoses improving patient management.

In silico-chemogenomic repurposing of new chemical scaffolds for histoplasmosis treatment.

BACKGROUND: Histoplasmosis is a systemic form of endemic mycosis to the American continent and may be lethal to people living with HIV/AIDS. The drugs available for treating histoplasmosis are limited, costly, and highly toxic. New drug development is time-consuming and costly; hence, drug repositioning is an advantageous strategy for discovering new therapeutic options. OBJECTIVE: This study was conducted to identify drugs that can be repositioned for treating histoplasmosis in immunocompromised patients. METHODS: Homologous proteins among Histoplasma capsulatum strains were selected and used to search for homologous targets in the DrugBank and Therapeutic Target Database. Essential genes were selected using Saccharomyces cerevisiae as a model, and functional regions of the therapeutic targets were analyzed. The antifungal activity of the selected drugs was verified, and homology modeling and molecular docking were performed to verify the interactions between the drugs with low inhibitory concentration values and their corresponding targets. RESULTS: We selected 149 approved drugs with potential activity against histoplasmosis, among which eight were selected for evaluating their in vitro activity. For drugs with low minimum inhibitory concentration values, such as mebendazole, everolimus, butenafine, and bifonazole, molecular docking studies were performed. A chemogenomic framework revealed lanosterol 14-α-demethylase, squalene monooxygenase, serine/threonine-protein kinase mTOR, and the ß-4B tubulin chain of H. capsulatum, respectively, as the protein targets of the drugs. CONCLUSIONS: Our strategy can be used to identify promising antifungal targets, and drugs with repositioning potential for treating H. capsulatum.

Pathogenicity & virulence of Histoplasma capsulatum - A multifaceted organism adapted to intracellular environments.

Histoplasmosis is a systemic mycosis caused by the thermally dimorphic fungus Histoplasma capsulatum. Although healthy individuals can develop histoplasmosis, the disease is particularly life-threatening in immunocompromised patients, with a wide range of clinical manifestations depending on the inoculum and virulence of the infecting strain. In this review, we discuss the established virulence factors and pathogenesis traits that make H. capsulatum highly adapted to a wide variety of hosts, including mammals. Understanding and integrating these mechanisms is a key step toward devising new preventative and therapeutic interventions.

Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages.

The fungal pathogen Histoplasma capsulatum (Hc) invades, replicates within, and destroys macrophages. To interrogate the molecular mechanisms underlying this interaction, we conducted a host-directed CRISPR-Cas9 screen and identified 361 genes that modify macrophage susceptibility to Hc infection, greatly expanding our understanding of host gene networks targeted by Hc. We identified pathways that have not been previously implicated in Hc interaction with macrophages, including the ragulator complex (involved in nutrient stress sensing), glycosylation enzymes, protein degradation machinery, mitochondrial respiration genes, solute transporters, and the ER membrane complex (EMC). The highest scoring protective hits included the complement C3a receptor (C3aR), a G-protein coupled receptor (GPCR) that recognizes the complement fragment C3a. Although it is known that complement components react with the fungal surface, leading to opsonization and release of small peptide fragments such as C3a, a role for C3aR in macrophage interactions with fungi has not been elucidated. We demonstrated that whereas C3aR is dispensable for macrophage phagocytosis of bacteria and latex beads, it is critical for optimal macrophage capture of pathogenic fungi, including Hc, the ubiquitous fungal pathogen Candida albicans, and the causative agent of Valley Fever Coccidioides posadasii. We showed that C3aR localizes to the early phagosome during Hc infection where it coordinates the formation of actin-rich membrane protrusions that promote Hc capture. We also showed that the EMC promotes surface expression of C3aR, likely explaining its identification in our screen. Taken together, our results provide new insight into host processes that affect Hc-macrophage interactions and uncover a novel and specific role for C3aR in macrophage recognition of fungi.

Molecular Detection of Histoplasma capsulatum in Antarctica.

We detected Histoplasma capsulatum in soil and penguin excreta in the Antarctic Peninsula by sequencing after performing species-specific PCR, confirming previous observations that this pathogen occurs more broadly than suspected. This finding highlights the need for surveillance of emerging agents of systemic mycoses and their transmission among regions, animals, and humans in Antarctica.

Equine Histoplasmosis in Ethiopia: Phylogenetic Analysis by Sequencing of the Internal Transcribed Spacer Region of rRNA Genes.

Equine histoplasmosis commonly known as epizootic lymphangitis (EL) is a neglected granulomatous disease of equine that is endemic to Ethiopia. It is caused by Histoplasma capsulatum variety farciminosum, a dimorphic fungus that is closely related to H. capsulatum variety capsulatum. The objective of this study was to undertake a phylogenetic analysis of H. capsulatum isolated from EL cases of horses in central Ethiopia and evaluate their relationship with H. capsulatum isolates in other countries and/or clades using the internal transcribed spacer (ITS) region of rRNA genes. Clinical and mycological examinations, DNA extraction, polymerase chain reaction (PCR), Sanger sequencing, and phylogenetic analysis were used for undertaking this study. Additionally, sequence data of Histoplasma isolates were retrieved from GenBank and included for a comprehensive phylogenetic analysis. A total of 390 horses were screened for EL and 97 were positive clinically while H. capsulatum was isolated from 60 horses and further confirmed with PCR, of which 54 were sequenced. BLAST analysis of these 54 isolates identified 29 H. capsulatum isolates and 14 isolates from other fungal genera while the remaining 11 samples were deemed insufficient for further downstream analysis. The phylogenetic analysis identified five clades, namely, African, Eurasian, North American 1 and 2, and Latin American A and B. The Ethiopian isolates were closely aggregated with isolates of the Latin American A and Eurasian clades, whereas being distantly related to isolates from North American 1 and 2 clades as well as Latin American B clade. This study highlights the possible origins and transmission routes of Histoplasmosis in Ethiopia.

Mac1-Dependent Copper Sensing Promotes Histoplasma Adaptation to the Phagosome during Adaptive Immunity.

Intracellular pathogens residing within macrophage phagosomes are challenged with recognizing the phagosomal environment and appropriately responding to changing host defense strategies imposed in this organelle. One such phagocyte defense is the restriction of available copper as a form of nutritional immunity during the adaptive immune response to fungal pathogens. The intracellular fungal pathogen Histoplasma capsulatum adapts to this decreased copper through upregulation of the Ctr3 copper transporter. In this study, we show that Histoplasma recognizes the characteristic low-copper phagosomal environment of activated macrophages through the copper-dependent transcriptional regulator Mac1. Multiple cis-acting regulatory sequences in the CTR3 promoter control upregulation of CTR3 transcription under low-copper conditions, and the loss of Mac1 function prevents induction of Ctr3 under low-copper conditions. During adaptive immunity, this loss of copper sensing by Mac1 attenuates Histoplasma virulence more severely than loss of Ctr3 alone, indicating that Mac1 controls the expression of additional mechanisms important for pathogenesis. Transcriptional profiling of Histoplasma yeasts identified a small regulon of Mac1-dependent genes, with the most strongly regulated genes encoding proteins linked to copper, iron, and zinc homeostasis and defenses against reactive oxygen (iron-requiring catalase [CatB] and superoxide dismutase [Sod4]). Accordingly, the loss of Mac1 function increased sensitivity to copper and iron restriction and blocked low-copper-induced expression of extracellular catalase activity. Thus, Mac1-mediated sensing of low-copper signals to Histoplasma yeasts their residence within the activated macrophage phagosome, and in response, Histoplasma yeasts produce factors, including non-copper-dependent factors, to combat the enhanced defenses of activated macrophages. IMPORTANCE Histoplasma capsulatum is a fungal pathogen that survives and grows within host macrophages. For successful infection, Histoplasma must sense and adapt to a dynamic intracellular environment over the course of an infection. We demonstrate that the copper-dependent transcription factor, Mac1, enables Histoplasma sensing of low copper that characterizes the phagosome environment of activated macrophages. Histoplasma recognition of this state leads not only to upregulation of copper acquisition mechanisms but also to other non-copper-related pathogenesis strategies, including scavenging of other metals and detoxification of reactive oxygen produced by host cells. The limited set of genes regulated by Histoplasma Mac1 compared to those of other fungal pathogens suggests a response that has been tailored specifically for Histoplasma's life inside the phagosome. Thus, low levels of phagosomal copper serve as a signal to Histoplasma, enabling responses to the enhanced antimicrobial defenses resulting from immune activation of macrophages.

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.

Histoplasmosis in Africa: Current perspectives, knowledge gaps, and research priorities.

BACKGROUND: Histoplasmosis is a chronic granulomatous disease caused by the thermally dimorphic fungus Histoplasma capsulatum. The 2 variants Histoplasma capsulatum var. capsulatum (Hcc) and Histoplasma capsulatum var. duboisii (Hcd) causes infection in humans and commonly termed classical or American histoplasmosis and African histoplasmosis, respectively. Histoplasma capsulatum var. farciminosum (Hcf) affects equines. In recent times, there have been heightened sensitization on fungal infections such as histoplasmosis in Africa, aimed at improving awareness among relevant stakeholders, particularly healthcare workers. This effort is expected to be paralleled with increased detection of both classical and African histoplasmosis, which has remained underdiagnosed over the years. In this narrative review, we describe the current perspectives of histoplasmosis in Africa, identify knowledge gaps, and suggest research priorities. METHODS: A PubMed, Google Scholar, and Africa Journal Online (AJOL) literature search was conducted for studies on histoplasmosis in Africa between 2000 and 2020. Histoplasmosis essays in medical mycology textbooks were also consulted. This narrative review was prepared from the data gathered. FINDINGS: In the past 2 decades, histoplasmosis in general has seen a relative increase in case detection in some Africa countries, probably attributable to the gradually increasing medical mycology advocacy efforts in Africa. Histoplasmosis cases are dominated by African histoplasmosis mostly in Western and Central Africa, while classical histoplasmosis is more common in Southern and Northern Africa. Although both classical and African histoplasmosis are common in Africa, the latter is more restricted to Africa, and cases outside the continent usually have a travel history to the continent. Despite the clinical and laboratory difference between African histoplasmosis and classical histoplasmosis, it is not straightforward to distinguish them. The typical manifestation of African histoplasmosis is the appearance of lesions affecting the skin, bones, and lymph nodes and unusually linked to human immunodeficiency virus (HIV)/AIDS. By contrast, classical histoplasmosis mostly affects the lungs and is often associated with immunosuppression, mainly HIV/AIDS. The present perspectives of histoplasmosis in Africa highlight unclear details on the true burden, strain diversity, infection route and genetic basis of African histoplasmosis, availability of specie-specific diagnostic tools, and compliance with recommended antifungal therapy. These knowledge gaps represent research questions that require scientific exploration. CONCLUSIONS: Despite a subtle increase in identifying histoplasmosis cases in Africa, it remains underdiagnosed and neglected in some parts of the continent. Increasing awareness and training among healthcare workers, bridging diagnostic and therapeutic gaps, and encouraging more research in Africa are crucial to improve the current perspectives of histoplasmosis in Africa.