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Emerging Microbes & Infections Dec 2024Histoplasmosis is an endemic mycosis in North America frequently reported along the Ohio and Mississippi River Valleys, although autochthonous cases occur in non-endemic...
Histoplasmosis 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 , (Nam1) and (Nam2). To bridge the molecular epidemiological gap, we genotyped 93 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, and ; with sporadic cases caused by in California and Texas. While is prevalent in eastern states, 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 thrives in places with warmer and drier conditions, while 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.
Topics: Histoplasmosis; Histoplasma; Genotype; Genomics; Texas
PubMed: 38465644
DOI: 10.1080/22221751.2024.2315960 -
Biodiverse Species Elicit Distinct Patterns of Pulmonary Inflammation following Sublethal Infection.MSphere Aug 2020is an endemic dimorphic fungus that can cause disease in healthy and immunocompromised individuals after the transition of inhaled spores into the facultative...
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 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 (strain G186A) and (strain G217B) using comprehensive flow cytometric and single-cell analyses. Within 8 days after inoculation, induced a significantly higher infiltration of neutrophils and inflammatory monocytes into the lung compared to 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 -expressing strains indicated that , but not , 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 but not infection, despite similar intracellular growth dynamics within AMJ2-C11 alveolar macrophages Together, our data suggest that , but not , 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. Acute pulmonary histoplasmosis in healthy individuals comprises most of the disease burden caused by the fungal pathogen 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 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 and imply that the progression of clinical disease is considerably more heterogeneous than previously recognized.
Topics: Animals; Disease Models, Animal; Flow Cytometry; Genetic Variation; Histoplasma; Histoplasmosis; Lung; Macrophages, Alveolar; Male; Mice; Mice, Inbred C57BL; Pneumonia
PubMed: 32848006
DOI: 10.1128/mSphere.00742-20 -
Analytical and Bioanalytical Chemistry Oct 2017This work sought to assess optimal extraction conditions in the study of the metalloproteome of the dimorphic fungus Histoplasma capsulatum. One of the body's responses...
This work sought to assess optimal extraction conditions in the study of the metalloproteome of the dimorphic fungus Histoplasma capsulatum. One of the body's responses to H. capsulatum infection is sequestration of zinc within host macrophage (MØ), as reported by Vignesh et al. (Immunity 39:697-710, 2013) and Vignesh et al. (PLOS Pathog 9:E1003815, 2013). Thus, metalloproteins containing zinc were of greatest interest as it plays a critical role in survival of the fungus. One challenge in metalloproteomics is the preservation of the native structure of proteins to retain non-covalently bound metals. Many of the conventional cell lysis, separation, and identification techniques in proteomics are carried out under conditions that could lead to protein denaturation. Various cell lysis techniques were investigated in an effort to both maintain the metalloproteins during lysis and subsequent analysis while, at the same time, serving to be strong enough to break the cell wall, allowing access to cytosolic metalloproteins. The addition of 1% Triton x-100, a non-ionic detergent, to the lysis buffer was also studied. Seven lysis methods were considered and these included: Glass Homogenizer (H), Bead Beater (BB), Sonication Probe (SP), Vortex with 1% Triton x-100 (V, T), Vortex with no Triton x-100 (V, NT), Sonication Bath, Vortex, and 1% Triton x-100 (SB, V, T) and Sonication Bath, Vortex, and no Triton x-100 (SB, V, NT). A Qubit® Assay was used to compare total protein concentration and inductively coupled plasma-mass spectrometry (ICP-MS) was utilized for total metal analysis of cell lysates. Size exclusion chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS) was used for separation of the metalloproteins in the cell lysate and the concentration of Zn over a wide molecular weight range was examined. Additional factors such as potential contamination sources were also considered. A cell lysis method involving vortexing H. capsulatum yeast cells with 500 μm glass beads in a 1% Triton x-100 lysis buffer (V, T) was found to be most advantageous to extract intact zinc metalloproteins as demonstrated by the highest Zn to protein ratio, 1.030 ng Zn/μg protein, and Zn distribution among high, mid, and low molecular weights suggesting the least amount of protein denaturation. Graphical abstract In this work, several cell lysis techniques and two lysis buffers were investigated to evaluate the preservation of the zinc metalloproteome of H. capsulatum while maintaining compatibility with the analytical techniques employed.
Topics: Detergents; Fungal Proteins; Histoplasma; Histoplasmosis; Humans; Metalloproteins; Octoxynol; Proteomics; Sonication; Zinc
PubMed: 28801743
DOI: 10.1007/s00216-017-0556-7 -
PLoS Neglected Tropical Diseases Aug 2019Histoplasmosis is a neglected disease that affects mainly immunocompromised patients, presenting a progressive dissemination pattern and a high mortality rate, mainly...
BACKGROUND
Histoplasmosis is a neglected disease that affects mainly immunocompromised patients, presenting a progressive dissemination pattern and a high mortality rate, mainly due to delayed diagnosis, caused by slow fungal growth in culture. Therefore, a fast, suitable and cost-effective assay is required for the diagnosis of histoplasmosis in resource-limited laboratories. This study aimed to develop and evaluate two new molecular approaches for a more cost-effective diagnosis of histoplasmosis.
METHODOLOGY
Seeking a fast, suitable, sensitive, specific and low-cost molecular detection technique, we developed a new Loop-mediated Isothermal Amplification (LAMP) assay and nested PCR, both targeting the Internal Transcribed Spacer (ITS) multicopy region of Histoplasma capsulatum. The sensitivity was evaluated using 26 bone marrow and 1 whole blood specimens from patients suspected to have histoplasmosis and 5 whole blood samples from healthy subjects. All specimens were evaluated in culture, as a reference standard test, and Hcp100 nPCR, as a molecular reference test. A heparin-containing whole blood sample from a heathy subject was spiked with H. capsulatum cells and directly assayed with no previous DNA extraction.
RESULTS
Both assays were able to detect down to 1 fg/μL of H. capsulatum DNA, and ITS LAMP results could also be revealed to the naked-eye by adding SYBR green to the reaction tube. In addition, both assays were able to detect all clades of Histoplasma capsulatum cryptic species complex. No cross-reaction with other fungal pathogens was presented. In comparison with Hcp100 nPCR, both assays reached 83% sensitivity and 92% specificity. Furthermore, ITS LAMP assay showed no need for DNA extraction, since it could be directly applied to crude whole blood specimens, with a limit of detection of 10 yeasts/μL.
CONCLUSION
ITS LAMP and nPCR assays have the potential to be used in conjunction with culture for early diagnosis of progressive disseminated histoplasmosis, allowing earlier, appropriate treatment of the patient. The possibility of applying ITS LAMP, as a direct assay, with no DNA extraction and purification steps, makes it suitable for resource-limited laboratories. However, more studies are necessary to validate ITS LAMP and nPCR as direct assay in other types of clinical specimens.
Topics: Blood; Bone Marrow; DNA, Ribosomal Spacer; Histoplasma; Histoplasmosis; Humans; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Prospective Studies; Sensitivity and Specificity
PubMed: 31449526
DOI: 10.1371/journal.pntd.0007692 -
Infection and Immunity Jun 1987The sulfhydryl blocking agent p-chloromercuriphenylsulfonic acid (PCMS) irreversibly inhibited the mycelium-to-yeast transitions of two virulent strains of Histoplasma...
The sulfhydryl blocking agent p-chloromercuriphenylsulfonic acid (PCMS) irreversibly inhibited the mycelium-to-yeast transitions of two virulent strains of Histoplasma capsulatum, G184A and G222B, when the temperature of incubation was raised to 37 degrees C, and the block persisted even after the cultures were washed free of PCMS. Instead of transforming to yeast cells, PCMS-treated mycelia continued to grow as mycelia at the elevated temperatures. A less virulent strain (Downs) was more temperature sensitive, but it showed a similar irreversible effect at 34 degrees C. Therefore, the mycelium-to-yeast transition of H. capsulatum is not required for the adaptation of mycelia to elevated temperatures but probably results from the temperature-dependent activation of yeast-specific genes. The transition to yeast is inferred to be obligate for pathogenicity in mice because PCMS-treated mycelia failed to cause infection, and no fungi were seen in tissues after PCMS-treated mycelia were injected into mice.
Topics: 4-Chloromercuribenzenesulfonate; Animals; Female; Histoplasma; Histoplasmosis; Hot Temperature; Mice; Oxygen Consumption
PubMed: 3032799
DOI: 10.1128/iai.55.6.1355-1358.1987 -
MSphere Aug 2023The rapid development of CRISPR/CRISPR-associated (Cas) systems has revolutionized the ability to produce genetic mutations in a desired locus, particularly in organisms...
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. 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 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 species and applicable for multiple genes. The optimized system shows promise for accelerating reverse genetic studies in spp. IMPORTANCE The ability to eliminate gene product functions is central to understanding molecular mechanisms. In the fungal pathogen , methods to inactivate or deplete gene products are inefficient, which hampers progress in defining 's virulence mechanisms. We describe an efficient CRISPR/Cas-based system for generating gene deletions in and show its validation on multiple genes with selectable and non-selectable phenotypes.
Topics: CRISPR-Cas Systems; Gene Editing; Histoplasma; Gene Deletion; Plasmids
PubMed: 37389430
DOI: 10.1128/msphere.00178-23 -
BMC Infectious Diseases Dec 2018Histoplasmosis is a fungal infection highly endemic in the American continent. The disease can be severe in immunocompromised subjects. In immunocompetent subjects the...
BACKGROUND
Histoplasmosis is a fungal infection highly endemic in the American continent. The disease can be severe in immunocompromised subjects. In immunocompetent subjects the clinical manifestations are variable. Aim of this work was to review the cases of acute histoplasmosis in immunocompetent travelers reported in literature.
METHODS
A systematic review of literature was conducted. Electronic search was performed in Pubmed and LILACS. Two reviewers independently extracted data on demographic, clinical and radiological features, and treatment. Cases were classified according to Wheat's definitions.
RESULTS
Seventy-one studies were included in the analysis, comprising a total of 814 patients. Twenty-one patients diagnosed at the Centre of Tropical Diseases, Negrar (VR), Italy were also included. The most common travel destination was Central America (168 people, 29.8%); the most common way of exposure to histoplasma was the exploration of caves and/or contact with bat guano (349 people, 60.9%). The multivariate logistic regression model showed association between the development of disseminated histoplasmosis (DH) and activities that involved the exploration of caves and/or the contact with bats' guano (adjusted OR: 34.20 95% CI: 5.29 to 220.93) or other outdoor activities (adjusted OR: 4.61 95% CI: 1.09 to 19.56). No significant difference in the attack rate between countries of destination was observed (p-value: 0.8906, Kruskal-Wallis test).
CONCLUSIONS
Histoplasmosis often causes no or mild symptoms in immunocompetent individuals, although a severe syndrome may occur. The infection can mimic other diseases, and the epidemiological risk of exposure is an important clue to raise the index of suspicion.
Topics: Acute Disease; Animals; Histoplasma; Histoplasmosis; Humans; Immunocompetence; Travel
PubMed: 30563472
DOI: 10.1186/s12879-018-3476-z -
Journal of Clinical Microbiology Oct 2018The diagnosis of central nervous system (CNS) histoplasmosis is often difficult. Although cerebrospinal fluid (CSF) (1,3)-β-d-glucan (BDG) is available as a biological...
The diagnosis of central nervous system (CNS) histoplasmosis is often difficult. Although cerebrospinal fluid (CSF) (1,3)-β-d-glucan (BDG) is available as a biological marker for the diagnosis of fungal meningitis, there are limited data on its use for the diagnosis of meningitis. We evaluated CSF BDG detection, using the Fungitell assay, in patients with CNS histoplasmosis and controls. A total of 47 cases and 153 controls were identified. The control group included 13 patients with a CNS fungal infection other than histoplasmosis. Forty-nine percent of patients with CNS histoplasmosis and 43.8% of controls were immunocompromised. The median CSF BDG level was 85 pg/ml for cases, compared to <31 pg/ml for all controls ( < 0.05) and 82 pg/ml for controls with other causes of fungal meningitis ( = 0.27). The sensitivity for detection of BDG in CSF was 53.2%, whereas the specificity was 86.9% versus all controls and 46% versus other CNS fungal infections. CSF BDG levels of ≥80 pg/ml are neither sensitive nor specific to support a diagnosis of meningitis.
Topics: Adult; Biomarkers; Clinical Laboratory Techniques; Histoplasma; Histoplasmosis; Humans; Meningitis, Fungal; Proteoglycans; ROC Curve; Reagent Kits, Diagnostic; beta-Glucans
PubMed: 30021828
DOI: 10.1128/JCM.00663-18 -
Fungal Genetics and Biology : FG & B Jan 2022Histoplasma, a genus of dimorphic fungi, is the etiological agent of histoplasmosis, a pulmonary disease widespread across the globe. Whole genome sequencing has...
Histoplasma, a genus of dimorphic fungi, is the etiological agent of histoplasmosis, a pulmonary disease widespread across the globe. Whole genome sequencing has revealed that the genus harbors a previously unrecognized diversity of cryptic species. To date, studies have focused on Histoplasma isolates collected in the Americas with little knowledge of the genomic variation from other localities. In this report, we report the existence of a well-differentiated lineage of Histoplasma occurring in the Indian subcontinent. The group is differentiated enough to satisfy the requirements of a phylogenetic species, as it shows extensive genetic differentiation along the whole genome and has little evidence of gene exchange with other Histoplasma species. Next, we leverage this genetic differentiation to identify genetic changes that are unique to this group and that have putatively evolved through rapid positive selection. We found that none of the previously known virulence factors have evolved rapidly in the Indian lineage but find evidence of strong signatures of selection on other alleles potentially involved in clinically-important phenotypes. Our work serves as an example of the importance of correctly identifying species boundaries to understand the extent of selection in the evolution of pathogenic lineages. IMPORTANCE: Whole genome sequencing has revolutionized our understanding of microbial diversity, including human pathogens. In the case of fungal pathogens, a limiting factor in understanding the extent of their genetic diversity has been the lack of systematic sampling. In this piece, we show the results of a collection in the Indian subcontinent of the pathogenic fungus Histoplasma, the causal agent of a systemic mycosis. We find that Indian samples of Histoplasma form a distinct clade which is highly differentiated from other Histoplasma species. We also show that the genome of this lineage shows unique signals of natural selection. This work exemplifies how the combination of a robust sampling along with population genetics, and phylogenetics can reveal the precise genetic changes that differentiate lineages of fungal pathogens.
Topics: Genomics; Histoplasma; Histoplasmosis; Humans; Phylogeny; Whole Genome Sequencing
PubMed: 34942368
DOI: 10.1016/j.fgb.2021.103654 -
Einstein (Sao Paulo, Brazil) 2021Histoplasmosis is an infection caused by the dimorphic fungus Histoplasma capsulatum. The disease is endemic in several regions of tropical and temperate climate. The... (Review)
Review
Histoplasmosis is an infection caused by the dimorphic fungus Histoplasma capsulatum. The disease is endemic in several regions of tropical and temperate climate. The fungus presents opportunistic behavior, causing widespread infection in immunocompromised patients, resulting from complication of primary pulmonary infection, due to exogenous reinfection or reactivation of a quiescent source. In immunocompetent individuals, approximately 95% of pulmonary infections are asymptomatic. However, prolonged exposure to high amount spores may lead to acute or chronic lung infection. Due to the low amount of inoculum, primary cutaneous histoplasmosis caused by traumatic implantation is extremely rare and effectively treated with triazoles. Thus, the present study aims to report a case of primary cutaneous histoplasmosis that is difficult to treat in an immunocompetent patient, and to review the literature on the incidence of drug-resistant Histoplasma capsulatum strains in clinical practice.
Topics: Histoplasma; Histoplasmosis; Humans
PubMed: 34037087
DOI: 10.31744/einstein_journal/2021RC5488