Cross-reactivity of a Histoplasma capsulatum antigen enzyme immunoassay in urine specimens from persons with emergomycosis in South Africa.

Histoplasma antigen detection in urine is a rapid diagnostic method for disseminated histoplasmosis, although cross-reactivity has been reported in specimens from patients with other thermally dimorphic fungal infections. We tested urine specimens, from persons with suspected invasive fungal infections, using a commercial monoclonal antibody Histoplasma enzyme immunoassay (EIA) at a South African national mycology reference laboratory from August 2014 through December 2018. Corresponding fungal culture and histopathology results were obtained from an electronic laboratory information system. In some cases, cultured fungal isolates were sent with the urine specimen for species-level identification by phenotypic and molecular methods. Cross-reactivity was confirmed using culture filtrates of several fungal pathogens. Of 212 referred cases, 41 (19%) were excluded since they had no recorded clinical history (n = 1), alternative diagnoses were confirmed (n = 2), or no fungal culture or histopathology results (n = 38). Eighty-seven of 212 (41%) had laboratory evidence of an invasive fungal disease, while 84 (40%) did not. Of the 87 cases, 37 (43%) were culture-confirmed mycoses: emergomycosis (n = 18), histoplasmosis (n = 8), sporotrichosis (n = 6), cryptococcosis (n = 2), talaromycosis (n = 1), and other fungi isolated (n = 2). The sensitivity and specificity of the EIA were calculated for two groups: culture-confirmed (n = 37) and histology-confirmed invasive fungal disease (n = 50). The sensitivity and specificity of the EIA for diagnosis of histoplasmosis compared to culture were 88% (7/8, 95%CI 47-100%) and 72% (21/29, 95%CI 53-87%), respectively, and for diagnosis of emergomycosis/histoplasmosis compared to histology was 83% (29/35, 95%CI 66-93%) and 93% (14/15, 95%CI 68-100%), respectively. Cross-reactions occurred in urine specimens of patients with Emergomyces africanus infection and in culture filtrates of E. africanus, T. marneffei and Blastomyces species. A commercial Histoplasma EIA had satisfactory accuracy for diagnosis of culture-confirmed histoplasmosis, but cross-reacted in urine specimens from patients with invasive disease caused by the closely-related pathogen, E. africanus and in culture filtrates of E. africanus and other related fungi. LAY SUMMARY: Emergomyces africanus and Histoplasma capsulatum are fungi that cause a multi-system disease among HIV-seropositive persons with a low CD4 cell count. Handling live cultures of these fungi to confirm a diagnosis requires specialized laboratory equipment and infrastructure which is infrequently accessible in low-resource settings. The features of the two diseases (i.e., disseminated histoplasmosis and emergomycosis) may be indistinguishable when infected tissue is prepared, stained, and examined under a microscope. Enzyme immunoassays (EIA) have been developed as rapid diagnostic tools for the detection of a cell wall component of H. capsulatum in urine specimens, although cross-reactions have been reported in specimens from patients with other fungal infections. We evaluated the accuracy of a commercial Histoplasma EIA to diagnose histoplasmosis and to assess cross-reactions in urine specimens from persons with emergomycosis and in cultures of E. africanus and related fungi. We report a sensitivity and specificity of 88% (95%CI 47-100%) and 72% (95%CI 53-87%) for diagnosis of histoplasmosis compared to culture and 83% (95%CI 66-93%) and 93% (95%CI 68-100%) for diagnosis of either histoplasmosis/emergomycosis compared to a diagnosis made by microscopic examination of infected tissue. The assay cross-reacted in urine specimens from patients with emergomycosis and in culture filtrates of related fungi. Although the EIA cross-reacted with other related fungi, this test can decrease the time to diagnosis and facilitate early treatment of emergomycosis and histoplasmosis in South Africa.

IL-22 Promotes IFN-γ-Mediated Immunity against Histoplasma capsulatum Infection.

Histoplasma capsulatum is the agent of histoplasmosis, one of the most frequent mycoses in the world. The infection initiates with fungal spore inhalation, transformation into yeasts in the lungs and establishment of a granulomatous disease, which is characterized by a Th1 response. The production of Th1 signature cytokines, such as IFN-γ, is crucial for yeast clearance from the lungs, and to prevent dissemination. Recently, it was demonstrated that IL-17, a Th17 signature cytokine, is also important for fungal control, particularly in the absence of Th1 response. IL-22 is another cytokine with multiple functions on host response and disease progression. However, little is known about the role of IL-22 during histoplasmosis. In this study, we demonstrated that absence of IL-22 affected the clearance of yeasts from the lungs and increased the spreading to the spleen. In addition, IL-22 deficient mice (Il22-/-) succumbed to infection, which correlated with reductions in the numbers of CD4+ IFN-γ+ T cells, reduced IFN-γ levels, and diminished nitric oxide synthase type 2 (NOS2) expression in the lungs. Importantly, treatment with rIFN-γ mitigated the susceptibility of Il22-/- mice to H. capsulatum infection. These data indicate that IL-22 is crucial for IFN-γ/NO production and resistance to experimental histoplasmosis.

Cholestasis and disseminated histoplasmosis in a psoriatic patient on infliximab: case report and review of literature.

BACKGROUND: Histoplasma capsulatum is the most common endemic mycosis in the United States and frequently presents as an opportunistic infection in immunocompromised hosts. Though liver involvement is common in disseminated histoplasmosis, primary gastrointestinal histoplasmosis of the liver in absence of lung involvement is rare. Similarly, cholestatic granulomatous hepatitis in liver histoplasmosis is rarely seen. CASE PRESENTATION: We present a rare case of primary gastrointestinal histoplasmosis manifesting with acute granulomatous hepatitis and cholestasis in a 48-year-old female with psoriatic arthritis, receiving methotrexate and infliximab. The epidemiology, risk factors, clinical presentation, diagnosis, and treatment of histoplasmosis is discussed. Furthermore, we review the published cases of biopsy-proven disseminated histoplasmosis with cholestatic jaundice to highlight histoplasmosis involvement in the liver. CONCLUSION: Histoplasmosis should be considered in immunosuppressed patients with fever, chills, abdominal pain and cholestasis with progressive jaundice, particularly in subjects without evidence of biliary obstruction. Future studies are needed to accurately assess the risk of this fungal infection, specifically in patients on immunomodulatory therapy for autoimmune disease.

Colonic Infection by Histoplasma capsulatum in a Liver Transplant Patient: A Case Report.

Histoplasmosis is an infection caused by the dimorphic fungus Histoplasma capsulatum. While the lungs are the most common site of infection, disseminated disease affecting multiple organs can occur, particularly in immunocompromised patients. Gastrointestinal histoplasmosis is usually diagnosed in the context of disseminated disease and can present in any part of the digestive system, the ileum being the most frequently affected. We report the case of a 60-year-old female patient who underwent liver transplant for alcoholic liver cirrhosis. The patient had a 10 mm polypoid lesion in the sigmoid colon diagnosed in a screening colonoscopy performed 8 months prior to the transplant, but biopsy was not done for fear of bleeding due to extensive anorectal varices. There were no other lesions in the rest of the colon at that time. Four months after the transplant, the patient was asymptomatic and was submitted to a control colonoscopy, which showed 8 polypoid lesions in different parts of the colon, all of which were biopsied. Histologic results showed extensive infiltration of the colonic mucosa by Histoplasma capsulatum. Imaging and laboratorial screening for other sites of infection was negative, and the patient was treated with itraconazole for 12 months. A marked reduction in the dose of tacrolimus was necessary to maintain therapeutic levels during itraconazole treatment. Asymptomatic isolated colonic histoplasmosis is an uncommon manifestation of infection by Histoplasma capsulatum, with no previous reports in the literature of this condition affecting liver transplant recipients. This manuscript is compliant with the Helsinki Congress and the Istanbul Declaration.

Evaluation of a Histoplasma antigen lateral flow assay for the rapid diagnosis of progressive disseminated histoplasmosis in Colombian patients with AIDS.

BACKGROUND: Progressive disseminated histoplasmosis (PDH) is an important cause of mortality in persons living with HIV (PLHIV), especially in countries where patients have limited access to antiretroviral therapies and diagnostic testing. OBJECTIVE: A lateral flow assay (LFA) to detect Histoplasma capsulatum antigen in serum developed by MiraVista® was evaluated. METHODS: We tested 75 serum samples: 24 from PLHIV and culture-proven PDH and 51 from PLHIV with other fungal and bacterial infections as well as people without HIV. LFA devices were read manually (read by eye) and by an automated reader. RESULTS: When the LFA was read manually, sensitivity was 96% and specificity was 90%. When an automated reader was used, sensitivity was 92% and specificity was 94%. The Kappa index comparing manual and automated reader was 0.90. Cross-reactions were observed principally in samples from patients with proven diagnosis of paracoccidioidomycosis. CONCLUSIONS: The MiraVista® Diagnostics Histoplasma antigen LFA had high analytical performance and good agreement between manual and automated reader. This LFA allows Histoplasma antigen testing with minimal laboratory equipment and infrastructure requirements.

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.

The HIF-1α/LC3-II Axis Impacts Fungal Immunity in Human Macrophages.

The fungal pathogen Histoplasma capsulatum causes a spectrum of disease, ranging from local pulmonary infection to disseminated disease. The organism seeks residence in macrophages, which are permissive for its survival. Hypoxia-inducible factor 1α (HIF-1α), a principal regulator of innate immunity to pathogens, is necessary for macrophage-mediated immunity to H. capsulatum in mice. In the present study, we analyzed the effect of HIF-1α in human macrophages infected with this fungus. HIF-1α stabilization was detected in peripheral blood monocyte-derived macrophages at 2 to 24 h after infection with viable yeast cells. Further, host mitochondrial respiration and glycolysis were enhanced. In contrast, heat-killed yeasts induced early, but not later, stabilization of HIF-1α. Since the absence of HIF-1α is detrimental to host control of infection, we asked if large amounts of HIF-1α protein, exceeding those induced by H. capsulatum, altered macrophage responses to this pathogen. Exposure of infected macrophages to an HIF-1α stabilizer significantly reduced recovery of H. capsulatum from macrophages and produced a decrement in mitochondrial respiration and glycolysis compared to those of controls. We observed recruitment of the autophagy-related protein LC3-II to the phagosome, whereas enhancing HIF-1α reduced phagosomal decoration. This finding suggested that H. capsulatum exploited an autophagic process to survive. In support of this assertion, inhibition of autophagy activated macrophages to limit intracellular growth of H. capsulatum Thus, enhancement of HIF-1α creates a hostile environment for yeast cells in human macrophages by interrupting the ability of the pathogen to provoke host cell autophagy.

Histoplasma panniculitis in a patient with systemic lupus erythematosus.

Histoplasmosis usually presents primarily as lung infection. Occasionally, mainly in immunocompromised hosts, it can spread and cause systemic manifestations. Skin lesions have been reported in 10 to 15 percent of cases of disseminated histoplasmosis, and panniculitis has been described as an unusual form of presentation in affected patients. We present the case of a patient with systemic lupus erythematosus who presented cellulitis due to disseminated histoplasmosis.

Oropharyngeal histoplasmosis: a report of 10 cases.

A wide differential diagnosis must be entertained in patients with unusual oral and pharyngeal ulcerations. A mucosal biopsy is essential. We retrospectively reviewed 10 cases from the Infectious Diseases Division at Mayo Clinic Rochester (MN, USA), in which the diagnosis proved to be Histoplasma capsulatum infection. Between 1995 and 2016, 10 patients were diagnosed with oropharyngeal histoplasmosis. Common presenting symptoms included weight loss, weakness and oropharyngeal pain with ulcerations. Despite specialty evaluation at other facilities, diagnostic delay occurred in six patients due to lack of biopsy or fungal staining. Yeast forms consistent with H. capsulatum were identified in the biopsy specimens of all our patients. Treatment included intravenous amphotericin B and prolonged courses of azoles. Oral histoplasmosis occurred in both immunocompetent and immunosuppressed patients, and was a manifestation of disseminated infection. Severe pain involving all areas of the mouth was typical. Diagnostic delay may be avoided by early biopsy using fungal stains.

Histoplasma panniculitis in a patient with systemic lupus erythematosus

Abstract: Histoplasmosis usually presents primarily as lung infection. Occasionally, mainly in immunocompromised hosts, it can spread and cause systemic manifestations. Skin lesions have been reported in 10 to 15 percent of cases of disseminated histoplasmosis, and panniculitis has been described as an unusual form of presentation in affected patients. We present the case of a patient with systemic lupus erythematosus who presented cellulitis due to disseminated histoplasmosis.

Flying under the radar: Histoplasma capsulatum avoidance of innate immune recognition.

The dimorphic fungal pathogen Histoplasma capsulatum takes advantage of the innate immune system, utilizing host macrophages as a proliferative niche while largely avoiding stimulation of signaling host receptors. As a result, innate immune cells are unable to control H. capsulatum on their own. Not all host phagocytes respond to H. capsulatum in the same way, with neutrophils and dendritic cells playing important roles in impeding fungal growth and initiating a protective TH1 response, respectively. Dendritic cells prime T-cell differentiation after internalization of yeasts via VLA-5 receptors and subsequent degradation of the yeasts. Dendritic cell-expressed TLR7 and TLR9 promote a type I interferon response for TH1 polarization. In contrast to dendritic cells, macrophages provide a hospitable intracellular environment. H. capsulatum yeasts enter macrophages via binding to phagocytic receptors. Simultaneously, α-glucan masks immunostimulatory cell wall ß-glucans and a secreted endoglucanase removes exposed ß-glucans to minimize recognition of yeasts by Dectin-1. This review highlights how phagocytes interact with H. capsulatum yeasts and the mechanisms H. capsulatum uses to limit the innate immune response.

Accuracy of a Novel Histoplasmosis Enzyme Immunoassay to Evaluate Suspicious Lung Nodules.

BACKGROUND: Granulomas caused by infectious lung diseases present as indeterminate pulmonary nodules (IPNs) on radiography. Newly available serum enzyme immunoassay (EIA) for histoplasmosis has not been studied for the evaluation of IPNs. We investigated serum biomarkers of histoplasmosis antibodies as an indication of benign disease in IPNs from a highly endemic region. METHODS: A total of 152 serum samples from patients presenting with pulmonary nodules ≤30 mm in maximum diameter were analyzed for histoplasmosis antibodies by immunodiffusion and EIA IgG and IgM tests. Serology and FDG-PET/CT scan diagnostic test characteristics were estimated and compared. RESULTS: Cancer prevalence was 55% (n = 83). Thirty-nine (26%) individuals were positive for IgG histoplasmosis antibodies. Twelve samples were IgM antibody positive. Immunodiffusion serology was similar to IgM antibody results with 13 positive tests. Diagnostic likelihood ratios for benign disease were 0.62, 0.33, and 0.28 for FDG-PET/CT, IgG, and IgM antibodies, respectively. When both IgG and IgM were positive (n = 8), no nodules were cancerous and six were FDG-PET/CT avid. CONCLUSIONS: A positive EIA test for both IgM and IgG strongly suggested histoplasmosis etiology and benign granuloma for 12% of benign nodules arising from a highly endemic region. Presence of either IgG or IgM histoplasma antibodies was associated with benign disease. The EIA test was more sensitive in assessing histoplasma exposure than immunodiffusion serology. IMPACT: A new CLIA-certified histoplasmosis antibody EIA test measures histoplasmosis exposure, offers a possible alternative clinical diagnosis for benign IPNs, and may improve IPN evaluation while avoiding harmful invasive biopsies.

NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages.

LC3-associated phagocytosis (LAP) is an emerging non-canonical autophagy process that bridges signaling from pattern-recognition receptors (PRRs) to autophagic machinery. LAP formation results in incorporation of lipidated LC3 into phagosomal membrane (termed LAPosome). Increasing evidence reveals that LAP functions as an innate defense mechanism against fungal pathogens. However, the molecular mechanism involved and the consequence of LAP in regulating anti-fungal immune response remain largely unexplored. Here we show that Histoplasma capsulatum is taken into LAPosome upon phagocytosis by macrophages. Interaction of H. capsulatum with Dectin-1 activates Syk and triggers subsequent NADPH oxidase-mediated reactive oxygen species (ROS) response that is involved in LAP induction. Inhibiting LAP induction by silencing LC3α/ß or treatment with ROS inhibitor impairs the activation of MAPKs-AP-1 pathway, thereby reduces macrophage proinflammatory cytokine response to H. capsulatum. Additionally, we unravel the importance of NLRX1 in fungus-induced LAP. NLRX1 facilitates LAP by interacting with TUFM which associates with autophagic proteins ATG5-ATG12 for LAPosome formation. Macrophages from Nlrx1-/- mice or TUFM-silenced cells exhibit reduced LAP induction and LAP-mediated MAPKs-AP-1 activation for cytokine response to H. capsulatum. Furthermore, inhibiting ROS production in Nlrx1-/- macrophages almost completely abolishes H. capsulatum-induced LC3 conversion, indicating that both Dectin-1/Syk/ROS-dependent pathway and NLRX1-TUFM complex-dependent pathway collaboratively contribute to LAP induction. Our findings reveal new pathways underlying LAP induction by H. capsulatum for macrophage cytokine response.

Histoplasmosis of the head and neck in the immunocompetent patient: Report of 2 cases.

Histoplasmosis of the head and neck is rarely seen in immunocompetent patients. We report 2 new cases of histoplasmosis of the head and neck in immunocompetent patients, one an 80-year-old man and the other a 57-year-old man. The older man presented with oral cavity histoplasmosis; his symptoms included pain, dysphagia, and ulcerative lesions. The younger man had laryngeal histoplasmosis, which resulted in hoarseness and dyspnea. We discuss the methods of diagnosis and the classic findings in histoplasmosis, including the microscopic appearance of caseating granulomas, the results of periodic acid-Schiff staining and Gomori staining, and antibody detection of histoplasmosis. We also review the treatment options with antifungals, including amphotericin B and the oral conazole drugs. With an accurate diagnosis and proper treatment, both of our patients recovered well and their symptoms resolved. Because their symptoms overlapped with those of other, more common disease processes, an accurate diagnosis of these patients was essential to treating their infection.

Concentration-dependent protein loading of extracellular vesicles released by Histoplasma capsulatum after antibody treatment and its modulatory action upon macrophages.

Diverse pathogenic fungi secrete extracellular vesicles (EV) that contain macromolecules, including virulence factors that can modulate the host immune response. We recently demonstrated that the binding of monoclonal antibodies (mAb) modulates how Histoplasma capsulatum load and releases its extracellular vesicles (EV). In the present paper, we addressed a concentration-dependent impact on the fungus' EV loading and release with different mAb, as well as the pathophysiological role of these EV during the host-pathogen interaction. We found that the mAbs differentially regulate EV content in concentration-dependent and independent manners. Enzymatic assays demonstrated that laccase activity in EV from H. capsulatum opsonized with 6B7 was reduced, but urease activity was not altered. The uptake of H. capsulatum by macrophages pre-treated with EV, presented an antibody concentration-dependent phenotype. The intracellular killing of yeast cells was potently inhibited in macrophages pre-treated with EV from 7B6 (non-protective) mAb-opsonized H. capsulatum and this inhibition was associated with a decrease in the reactive-oxygen species generated by these macrophages. In summary, our findings show that opsonization quantitatively and qualitatively modifies H. capsulatum EV load and secretion leading to distinct effects on the host's immune effector mechanisms, supporting the hypothesis that EV sorting and secretion are dynamic mechanisms for a fine-tuned response by fungal cells.

Tumor Necrosis Factor Alpha Antagonism Reveals a Gut/Lung Axis That Amplifies Regulatory T Cells in a Pulmonary Fungal Infection.

Tumor necrosis factor (TNF) antagonists are popular therapies for inflammatory diseases. These agents enhance the numbers and function of regulatory T cells (Tregs), which are important in controlling inflammatory diseases. However, elevated Treg levels increase susceptibility to infections, including histoplasmosis. We determined the mechanism by which Tregs expand in TNF-neutralized mice infected with Histoplasma capsulatum Lung CD11c+ CD11b+ dendritic cells (DCs), but not alveolar macrophages, from H. capsulatum-infected mice treated with anti-TNF induced a higher percentage of Tregs than control DCs in vitro CD11b+ CD103+ DCs, understood to be unique to the intestines, were augmented in lungs with anti-TNF treatment. In the absence of this subset, DCs from anti-TNF-treated mice failed to amplify Tregs in vitro CD11b+ CD103+ DCs from TNF-neutralized mice displayed higher retinaldehyde dehydrogenase 2 (RALDH2) gene expression, and CD11b+ CD103+ RALDH+ DCs exhibited greater enzyme activity. To determine if CD11b+ CD103+ DCs migrated from gut to lung, fluorescent beads were delivered to the gut via oral gavage, and the lungs were assessed for bead-containing DCs. Anti-TNF induced migration of CD11b+ CD103+ DCs from the gut to the lung that enhanced the generation of Tregs in H. capsulatum-infected mice. Therefore, TNF neutralization promotes susceptibility to pulmonary H. capsulatum infection by promoting a gut/lung migration of DCs that enhances Tregs.

Mucocutaneous Manifestations of Infection by Histoplasma capsulatum in HIV-Negative Immunosuppressed Patients. / Manifestaciones mucocutáneas de la infección por Histoplasma capsulatum en pacientes inmunosuprimidos.

Histoplasmosis is a systemic mycosis caused by the dimorphous fungus Histoplasma capsulatum (H. capsulatum). The fungus enters the body through the respiratory tract in the form of microconidia, which are transformed into intracellular yeast-like structures in the lungs before disseminating hematogenously. Primary infection is usually asymptomatic and self-resolving. Some patients develop severe disease with acute or chronic respiratory involvement. Immunosuppressed patients, mainly those with altered cellular immunity, may have disseminated disease with variable mucocutaneous involvement characterized by papules, nodules, gummas, or ulcers with a granulomatous base. We report the case of 3 HIV-negative patients infected by H capsulatum in whom diagnosis based on the skin lesions proved essential for early initiation of treatment.