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Background: Disseminated Kaposi sarcoma (DKS) is present in patients with advanced HIV infection in whom co-infection with other opportunistic pathogens can occur. Bone marrow (BM) aspirate and biopsy comprise a robust diagnostic tool in patients with fever, cytopenias, and abnormal liver tests. However, the yield in patients with DKS has not been determined., Objective: The aim of this study was to evaluate the utility of BM aspirate and biopsy in patients with DKS., Methods: We included 40 male patients with a recent diagnosis of DKS. BM aspirate and biopsy was performed as part of the workup to rule out co-infections., Results: In four patients, Mycobacterium avium complex (MAC) was recovered from culture. In other four patients, intracellular yeasts were observed in the Grocott stain, diagnosed as Histoplasma. The yield of BM was calculated in 20%. Only 12 patients (30%) had fever and 11 (27.5%) had pancytopenia. Alkaline phosphatase (ALP) above normal values and C-reactive protein (CRP) were higher in patients with positive results for BM than in those with negative results (63% vs. 21.9%, and 3.0 vs. 1.2 mg/L; p = 0.03 in both comparisons). No differences were found when complete blood-count abnormalities were compared., Conclusion: We recommend performing a BM aspirate for stains, culture, and biopsy in all HIV patients with DKS, as this will permit the early diagnosis of co-infections and prevent further complications in those who receive chemotherapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Epidemiological cut-off values (ECVs) have been used as a tool to detect the acquisition of resistance mechanisms to antifungal drugs. In this context, the objective of this study was to determine the ECVs for classic antifungals against Histoplasma capsulatum var. capsulatum isolates from human immunodeficiency virus (HIV)-infected patients with a diagnosis of disseminated histoplasmosis. First, minimum inhibitory concentrations (MICs) for amphotericin B (AmB), itraconazole (ITR), fluconazole (FLU), voriconazole (VCZ) and caspofungin (CAS) were determined against 138 H. capsulatum isolates in the filamentous form by the broth microdilution method; antifungal ECVs were then calculated. MIC ranges were 0.0078-1 µg/mL for AmB, 0.0005-0.0625 µg/mL for ITR, 2 to ≥256 µg/mL for FLU, 0.0078-1 µg/mL for VCZ and ≤0.0156 to ≥32 µg/mL for CAS. The obtained ECVs were 0.5, 0.0313, 128, 0.5 and 16 µg/mL for AmB, ITR, FLU, VCZ and CAS, respectively. The percentage of wild-type isolates was 96.4% for AmB, 98.6% for ITR and 99.3% for FLU, VCZ and CAS. Although these results do not cover all phylogenetic species of H. capsulatum, they bring important information on strains from Brazil. In addition, the assessed isolates were from HIV-positive patients, which may not reflect the antifungal ECVs against isolates from immunocompetent individuals or from other sources. Finally, this study pioneers the initiative of establishing ECVs for five antifungal agents against H. capsulatum var. capsulatum, providing a criterion for the interpretation of susceptibility results as well as a monitoring strategy for the emergence of antifungal resistance., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Mammalian body temperature triggers differentiation of the fungal pathogen Histoplasma capsulatum into yeast cells. The Drk1 regulatory kinase and an interdependent network of Ryp transcription factors establish the yeast state. Beyond morphology, the differentiation-dependent expression program equips yeasts for invasion and survival within phagosomes. Yeast cells produce α-glucan and the Eng1 endoglucanase which hide yeasts from immune detection. Secretion of yeast phase-specific Sod3 and CatB detoxify phagocyte-derived reactive oxygen molecules. Histoplasma cells adapt to iron and zinc limitation in activated macrophages by production of siderophores and the Zrt2 transporter, respectively. Yeasts also respond to inflammation-associated hypoxia. Histoplasma pathogenicity thus relies on factors controlled by yeast differentiation as well as environment-dependent responses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Histoplasma capsulatum (Hc) exists in the soil and is capable of adapting to the shift in environment during infection to ensure survival. Yeast encounter a restrictive host environment low in nutrients such as zinc. In this study we functionally analyzed a putative zinc regulated transporter, HcZrt2, in zinc limiting conditions by complementation of HcZrt2 and gene knockdown through RNA interference (RNAi). Complementation analysis demonstrated HcZrt2's ability to functionally replace the characterized Saccharomyces cerevisiae zinc plasma membrane transporters Zrt1 and Zrt2 in zinc deficient medium. Gene silencing revealed that HcZrt2 is essential for growth in zinc deficient medium and plays a role in zinc accumulation. Fungal burden was reduced in mice infected with HcZrt2 silenced strains compared to a control strain. Sixty-seven percent of mice infected with a lethal dose of HcZrt2-RNAi#1 survived, and 100% of mice infected with HcZrt2-RNAi#2 withstood lethal infection. Our data suggest that HcZrt2 is a vital part of zinc homeostasis and essential for the pathogenesis of histoplasmosis., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Microbial pathogens induce or inhibit death of host cells during infection, with significant consequences for virulence and disease progression. Death of an infected host cell can either facilitate release and dissemination of intracellular pathogens or promote pathogen clearance. Histoplasma capsulatum is an intracellular fungal pathogen that replicates robustly within macrophages and triggers macrophage lysis by unknown means. To identify H. capsulatum effectors of macrophage lysis, we performed a genetic screen and discovered three mutants that grew to wild-type levels within macrophages but failed to elicit host-cell death. Each mutant was defective in production of the previously identified secreted protein Cbp1 (calcium-binding protein 1), whose role in intracellular growth had not been fully investigated. We found that Cbp1 was dispensable for high levels of intracellular growth but required to elicit a unique transcriptional signature in macrophages, including genes whose induction was previously associated with endoplasmic reticulum stress and host-cell death. Additionally, Cbp1 was required for activation of cell-death caspases-3/7, and macrophage death during H. capsulatum infection was dependent on the pro-apoptotic proteins Bax and Bak. Taken together, these findings strongly suggest that the ability of Cbp1 to actively program host-cell death is an essential step in H. capsulatum pathogenesis., (© 2015 John Wiley & Sons Ltd.)

The host pulmonary response to the fungus Histoplasma capsulatum was evaluated, through the profile of cytokines detected by the MagPix magnetic beads platform in lung homogenates and by lung-granulomas formation, from mice intra-nasally infected with mycelial propagules (M-phase) of two virulent H. capsulatum strains, EH-46 and G-217B. Results highlight that mice lung inflammatory response depends on the H. capsulatum strain used, during the first step of the fungal infection. IL-1β and TNF-α increased their concentrations in mice infected with both strains. The highest levels of IL-6, IL-17, and IL-23 were found in EH-46-infected mice, whereas levels of IL-22 were variable at all post-infection times for both strains. Significant increases of IL-12, IFN-γ, IL-4, and IL-10 were associated to EH-46-infected mice. Histological lung findings from EH-46-infected mice revealed incipient and numerous well-developed granulomas, distributed in lung-lobes at the 14th and the 21st days after infection, according to cytokine profiles., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The present paper is an overview of the primary events that are associated with the histoplasmosis immune response in the murine model. Valuable data that have been recorded in the scientific literature have contributed to an improved understanding of the clinical course of this systemic mycosis, which is caused by the dimorphic fungus Histoplasma capsulatum. Data must be analyzed carefully, given that misinterpretation could be generated because most of the available information is based on experimental host-parasite interactions that used inappropriate proceedings, i.e., the non-natural route of infection with the parasitic and virulent fungal yeast-phase, which is not the usual infective phase of the etiological agent of this mycosis. Thus, due to their versatility, complexity, and similarities with humans, several murine models have played a fundamental role in exploring the host-parasite interaction during H. capsulatum infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Human immunodeficiency virus (HIV)-associated disseminated Histoplasma capsulatum capsulatum infection often mimics tuberculosis. This disease is well know in the United States but is dramatically underdiagnosed in Central and South America. In the Amazon region, given the available incidence data and the regional HIV prevalence, it is expected that, every year, 1,500 cases of histoplasmosis affect HIV patients in that region alone. Given the mortality in undiagnosed patients, at least 600 patients would be expected to die from an undiagnosed but treatable disease. The lack of a simple diagnostic tool and the lack of awareness by clinicians spiral in a vicious cycle and made a major problem invisible for 30 years. The HIV/acquired immunodeficiency syndrome community should tackle this problem now to prevent numerous avoidable deaths from HIV-associated histoplasmosis in the region and elsewhere.

The multiplex PCR developed from a suspension of the yeast fungi correctly identified fifty-one clinical of H. capsulatum var. capsulatum strains isolated from clinical samples and soil specimens. The multiplex PCR was developed by combining two pairs of primers, one of them was specific to the H. capsulatum and the other one, universal for fungi, turned out to be specific to H. capsulatum, regardless of the fungus isolate studied. Primers designed to amplify a region of about 390-bp (Hc I-Hc II) and a region of approximately 600-bp (ITS1-ITS4) were used to identify a yeast isolated as H. capsulatum when both regions could be amplified. Absolute agreement (100 % sensitivity) could be shown between this assay and the cultures of H. capsulatum according to their morphological characteristics. Failure to amplify the target DNA sequence by PCR with primers Hc I-Hc II in the presence of the ITS1-ITS4 amplicon in isolates of P. brasiliensis, Cryptococcus neoformans, Trichosporon spp, Candida glabrata, C. albicans, C. tropicalis, C. parapsilosis, C. krusei, or Penicillium marneffei was an unequivocal sign of the high specificity of this assay. The assay specificity was also found to be 100 %. Incipient yeast forms obtained from clinical samples were identified as H. capsulatum by the PCR assay described before the morphological characteristics were registered shortening the time of diagnosis.

The objective of this study was to develop an image-based cytometric methodology for the quantification of viable pathogenic yeasts, which can offer increased sensitivity and efficiency when compared to the traditional colony forming unit (CFU) assay. Live/dead yeast quantification by flow cytometry has been previously demonstrated, however, adoption of flow cytometric detection of pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome. Here, we quantitatively assess the growth of H. capsulatum yeasts within RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with Cellometer image-based cytometry; this method faithfully recapitulates growth trends as measured by traditional CFU enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of bone marrow-derived macrophages with a GFP-expressing strain of C. albicans. To demonstrate that image-based cytometry can be used as a tool to assess the susceptibility of fungi to antifungal drugs, we perform dose response experiments with the antifungal drugs amphotericin B and itraconazole and show that image-based cytometry allows rapid assessment of the kinetics of cytotoxicity induced by these antifungals. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens, either alone or in association with host cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We report a case of Histoplasma capsulatum endocarditis in which Histoplasma antigen assay and fungal blood cultures were negative. The diagnosis was made by microscopic examination and culture of the excised valve. Histoplasma capsulatum should be considered in the differential diagnosis of culture-negative endocarditis in regions where it is endemic and in travelers.

A histoplasmosis outbreak affecting 6 previously healthy Air Force cadets is herein presented. The patients suffered from fever and respiratory symptoms after having cleaned an abandoned hangar soiled with pigeons and bat droppings. They all presented fever, myalgia, tachypnea, and nonproductive cough. Chest X-ray and CT scan studies showed disseminated reticulonodular images affecting both lungs. Hiliar adenomegalies were also observed. All patients achieved a favourable outcome without antifungal treatment. Both serologic tests searching for specificic antibodies (immunodiffusion and counterimmunoelectrophoresis) and histoplasmin skin tests were positive in all cases. Five soil samples mixed with pigeons and bat droppings were collected from the hangar. Suspensions of these samples were inoculated into 20 hamsters by intraperitoneal injection; mycelial phase of H. capsulatum was isolated from liver and spleen cultures. The genetic profile of this strain was compared with 12 isolates obtained from Argentinean patients, and a great degree of homogeneity was observed (> 96% similarity). Although histoplasmosis is endemic in the wet Pampas, this is the first epidemic outbreak reported south of the 34th parallel.

Monoclonal antibodies to Histoplasma capsulatum can modify pathogenesis. We now show that monoclonal antibody H1C to a 70-kDa antigen increases intracellular fungal growth and reduces macrophage nitric oxide release but has no effect on fungal burden or survival in murine infection. This further demonstrates the complexities of host-pathogen interactions.

The conventional means of diagnosis of histoplasmosis presents difficulties because of the delay to the time that the diagnosis is made, indicating the need for the implementation of molecular assays. We evaluated 146 clinical samples from 135 patients suspected of having histoplasmosis using a previously reported nested PCR assay for the Histoplasma capsulatum-specific 100-kDa protein (the Hc100 PCR). In order to determine the specificity of this molecular test, we also used samples from healthy individuals (n = 20), patients suspected of having respiratory disease with negative fungal cultures (n = 29), and patients with other proven infections (n = 60). Additionally, a sizable collection of DNA from cultures of H. capsulatum and other medically relevant pathogens was studied. A panfungal PCR assay that amplified the internal transcribed spacer 2 region was also used to identify all fungal DNAs. All PCR-amplified products were sequenced. Of the 146 clinical samples, 67 (45.9%) were positive by culture and PCR, while 9 samples negative by culture were positive by PCR. All the sequences corresponding to the 76 amplified products presented > or =98% identity with H. capsulatum. The Hc100 PCR exhibited a sensitivity of 100% and specificities of 92.4% and 95.2% when the results were compared to those for the negative controls and samples from other proven clinical entities, respectively; the positive predictive value was 83% and the negative predictive value was 100%; the positive and negative likelihood rates were 25 and 0, respectively. These results suggest that the Hc100 nested PCR assay for the detection of H. capsulatum DNA is a useful test in areas where mycosis caused by this organism is endemic.

Therapeutic effects of Sodium Iodide (NaI), Potassium Iodide (KI), ground berries of "Endod" (Phytolacca dodecandra) and Penstrip were evaluated on 70 cases of equine hitoplasmosis (EH). Response to each treatment was assessed using clinical examination of the lesions. Statistically significant difference (P = 0.0036) in therapeutic effect was observed among the different remedies. Cases treated either with a combination of NaI and Penstrip (F = 6.34, P = 0.004) or "Endod" and Penstrip (F = 3.64, P = 0.031) demonstrated significant response. The difference in response to treatment between early and advanced cases of EH was statistically significant (t = 2.22, P = 0.0148).

The dimorphism of Histoplasma reflects a developmental switch in morphology and lifestyle that is necessary for virulence. The dimorphism regulating kinase DRK1 and the Histoplasma WOR1 homolog RYP1 mediate the thermally induced transition to the pathogenic yeast-phase program. The genes expressed as part of this regulon influence the host-pathogen interaction to favor Histoplasma virulence. While surface localized HSP60 supports yeast attachment to host macrophages, yeast alpha-glucan polysaccharides conceal immunostimulatory cell wall beta-glucans from detection by macrophage receptors. Intramacrophage growth of yeast cells is facilitated by CBP a secreted, protease-resistant calcium-binding protein tailored to function within the phagolysosomal environment. In some Histoplasma strains, YPS3 promotes dissemination of yeast from pulmonary infection sites. The Histoplasma yeast-phase program includes additional cell surface and extracellular molecules that potentially function in further aspects of Histoplasma virulence.

Monoclonal antibodies (MAbs) to a cell surface histone on Histoplasma capsulatum modify murine infection and decrease the growth of H. capsulatum within macrophages. Without the MAbs, H. capsulatum survives within macrophages by modifying the intraphagosomal environment. In the present study, we aimed to analyze the affects of a MAb on macrophage phagosomes. Using transmission electron and fluorescence microscopy, we showed that phagosome activation and maturation are significantly greater when H. capsulatum yeast are opsonized with MAb. The MAb reduced the ability of the organism to regulate the phagosomal pH. Additionally, increased antigen processing and reduced negative costimulation occur in macrophages that phagocytose yeast cells opsonized with MAb, resulting in more-efficient T-cell activation. The MAb alters the intracellular fate of H. capsulatum by affecting the ability of the fungus to regulate the milieu of the phagosome.

The pathogenic fungus Histoplasma capsulatum causes disease ranging from mild to fatal in healthy and immunocompromised humans. Infection rates reach 80% in endemic areas, including the Midwestern United States. We used inbred mice to identify a 300-fold difference in fungal burden. A/J mice showed lower fungal burden and morbidity than C57BL/6J mice, a reversal of the trend observed for many bacterial pathogens. We mapped the differences in fungal burden to discrete locations on chromosomes 1, 6, 15 and 17 with high significance. Substitution of a single resistant chromosome 17 onto the susceptible background was sufficient to lower fungal burden. These loci will allow dissection of the fungal-specific immune program.

Cutaneous infection by histoplasmosis in Cuban HIV patients was researched. In a case series study, all HIV patients admitted to "Pedro Kouri" Institute from January 1st, 1992 to June 30th, 2003, who had been diagnosed with cutaneous histoplasmosis, were included. Of 44 patients with histoplasmosis, 52% (23 cases) developed the progressive disseminated form of histoplasmosis, which behaved as a subacute weakening disease. Young adults represented 56.5% and 82.6% were males mainly Caucasian (91.3%). Most of cases came from the Western provinces. Histoplasmosis was a marker disease in 39.1% of cases. CD4+ T-lymphocyte counting under 200 cell/mm3 was present in 78.9% of patients. Histoplasmosis seems to behave as an important marker disease for AIDs in seropositive patients. Serology was not the diagnosing method of choice for this cutaneous disease in AIDS patients.

Visceral leishmaniasis, progressive disseminated histoplasmosis and penicilliosis marneffei are infections found in both human and animals. Their clinical manifestations, morphology of the pathogens under microscope are similar. Misdiagnosis was common and prognosis was poor when wrong therapy was given. This article is to introduce the epidemiology, clinical manifestation, laboratory findings and the treatment of these infections.

Epizootic lymphangitis (EL) was experimentally reproduced in four horses that had been purchased from an EL-free district. Two horses were injected with either 0.2 mL of the yeast form of Histoplasma capsulatum var. farciminosum (HCF) in pus (Horse 1), or 0.2 mL (ca. 20 mg) of a suspension in saline of the mycelial form (Horse 2), both into the pre-scapular and pre-femoral lymph nodes, with scarification of the skin of the left hind limb, conjunctiva of the right eye and the nasal membrane of the right nostril. The two other horses served as controls. Nodular lesions of EL appeared during the fourth week of infection at all sites in the horse infected with the yeast form. Lesions only appeared in the lymph nodes and skin scratches of the horse infected with the mycelial suspension after three months. The control horses showed no clinical signs. The yeast form was recovered from the lesions of both infected horses. Similarly, the mycelial form was isolated from both horses on Sabouraud's Dextrose Agar. This experiment showed the reproducibility of clinical EL through experimental infection, and has laid the groundwork for the evaluation of the potency of a vaccine against EL using a vaccination and challenge experiment.

Dimorphism and pathogenesis of Hisdistoplasma capsulatum is a dimorphic fungal pathogen with worldwide significance, which causes a broad spectrum of disease. In the saprophytic stage, it lives as a mycelial form consisting of hyphae bearing both macro and microconidia. Infection with H. capsulatum occurs by inhalation of microconidia (1-4 x 2-6 microm) or small mycelia fragments (5-8 microm) in the terminal bronchioles and alveoli of the lung. Inhaled conidia then convert into the yeast form that is responsible for the pathogenesis of histoplasmosis. As a soil fungus with no known requirements for interacting with a mammalian host as a necessary stage of its life cycle, the number of its strategies for successful pathogenesis is particularly remarkable. They include dimorphic mould-yeast transition, entry into host macrophages, subcellular localization, intracellular survival and proliferation during clinically unapparent infection with capacity for reactivation. H. capsulatum became the subject of increasing studies concurrently with the rising prevalence of human immunodeficiency. This paper presents an overall view of advances in the investigation of H. capsulatum dimorphic transition and pathogenesis.

A 3.5-year-old Thoroughbred mare presented at necropsy with a large mass at the root of the mesentery and multiple smaller mesenteric masses. The mucosa of the small intestine contained numerous raised nodules. Histologic examination revealed severe granulomatous mesenteric lymphadenitis and enteritis. Epithelioid macrophages and multinucleated giant cells frequently contained numerous intracytoplasmic yeast organisms, which were strongly positive on immunohistochemical staining when using a polyclonal antibody against Histoplasma spp. A diagnosis of abdominal histoplasmosis was made based on the gross, microscopic, and immunohistochemical findings.

The fungus, Histoplasma capsulatum, produces a persistent infection. Reactivation histoplasmosis is largely a result of impaired immunity, but the perturbations associated with escape of the fungus from host defenses remain ill-defined. We analyzed a murine model of reactivation to elucidate the host defects that permit reactivation. C57BL/6 mice were infected intranasally and, 42 days later, they were depleted of CD4(+) and CD8(+) cells. Elimination of these cells, but not either alone, produced a persistent infection over several weeks. Neutralization of IFN-gamma, TNF-alpha, or both did not induce reactivation. Endogenous IL-10 exacerbated reactivation. Depletion of T cells in B cell(-/-) mice induced a markedly higher burden in organs when compared with wild type. However, the infection remained persistent. Elimination of CD4(+) cells alone or neutralization of cytokines increased the fungal load. The persistent infection was not dependent on gammadelta T cells or NK cells. Elimination of Thy-1.2(+) cells in mice given mAb to CD4 and CD8 transformed reactivation into a progressive, lethal infection in B cell(-/-) and wild-type mice, but the tempo of progression was accelerated in the former. The data reveal the complex control by the host to prevent reactivation of this fungus.

Histoplasmosis is a fungal infection caused by the Histoplasma capsulatum. Mucosal manifestations are uncommon, but, when present, they are usually associated with pulmonary or chronic disseminated infection. The course of the disease is often related to the host immune response. The purpose of this article is to describe the clinical and microscopic findings of unusual involvement of nasal cartilage and septal destruction, and oral lesions of histoplasmosis in an immunosuppressed patient who presented an unusual form of the disease.

Histofarcin, a skin test antigen for the diagnosis of epizootic lymphangitis, was locally produced from the mycelial form of Histoplasma capsulatum var. farciminosum (HCF) in disease-endemic districts of Ethiopia and tested for its application in the field between April 2002 and May 2003. The test was evaluated using 108 mules, 84 in endemic and 24 in disease-free districts. Microscopic and mycological examinations of clinical lesions were used as the "gold standard" for the validation of the test. The concentration of histofarcin that caused an optimum reaction was 0.2-0.4 mg/mL in a 0.1 mL dose and this was attained 24-48 h post-injection. The sensitivity and specificity of the histofarcin test were 90.3% (CI = 73.1, 97.5) and 69% (95%, CI = 48.1, 84.9%) in disease-endemic districts. On the other hand, specificity was 100% (CI=94.8, 100) in disease-free districts. Positive and negative predictive values of the histofarcin test were 77.78% (95% CI = 60.4, 89.3) and 85.71% (95% CI = 62.6, 96.2), respectively. A marginal substantial agreement (kappa = 0.61, P = 0.0000) was observed between the clinical status and the result of the histofarcin test. A large proportion (31%) of 'false positives' was recorded in endemic districts, which could be due to the pre-clinical stage of the disease. The latter ended in lower specificity and positive predictive value of the test since the true infection status of a 'false positive' could not be known on the basis of clinical features. Therefore, standard test validation procedures including slaughtering and isolation of HCF is required. After proper validation, we conclude that the histofarcin test could play a significant role in detecting early infection, and differentiating of EL from glanders, ulcerative lymphangitis, and sporotrichosis.

Between October 1998 and April 1999, 51 persons belonging to two separate groups developed acute pulmonary histoplasmosis after visiting a cave in Costa Rica. The first group consisted of 61 children and 14 adults from San Jose, Costa Rica; 44 (72%) were diagnosed with acute histoplasmosis. The second group comprised 14 tourists from the United States and Canada; 9 (64%) were diagnosed with histoplasmosis. After a median incubation time of 14 days, the most common symptoms were headache, fever, cough, and myalgias. Risk factors for developing histoplasmosis included crawling (odds ratio [OR] = 17.5, 95% confidence interval [CI] = 2.3-802) and visiting one specific room (OR = 3.4, 95% CI = 1.0-12.3) in the cave. Washing hands (OR = 0.1, 95% CI = 0.01-0.6) after exiting the cave was associated with a decreased risk of developing histoplasmosis. Histoplasma capsulatum was isolated from bat guano collected from inside the cave. Persons who explore caves, whether for recreation or science, should be aware of the risk bat-inhabited caves pose for developing histoplasmosis, especially if they are immunocompromised in any way.

Leukotrienes are classical mediators of inflammatory response. New aspects of leukotriene function have recently been described. We examine here the previously unreported role that leukotrienes play in the regulation of cytokines in a murine model of histoplasmosis. We demonstrate that administration of MK 886, a leukotriene synthesis inhibitor, caused Histoplasma capsulatum-infected mice to die by the day 15 of infection, whereas the correlating death rate in untreated infected mice was 0%. Treating infected animals with MK 886 inhibited leukotriene synthesis but increased leukocyte recruitment to the lungs. Subsequent to this phenomenon, levels of tumor necrosis factor alpha, interleukin-1 (IL-1), IL-6, and KC chemoattractant cytokines and fungi in the lung parenchyma increased, as did inflammatory response. In contrast, IL-2, IL-5, IL-12, and gamma interferon cytokine levels actually decreased. Thus, murine response to pulmonary histoplasmosis may be leukotriene modulated. This finding may enable us to alter the course of the immune response and inflammation caused by histoplasmosis. The data from the present study suggest an important new strategy for immunologic or drug intervention in human patients.

We determined whether the absence of IL-10 in mice influenced protective and memory immunity to Histoplasma capsulatum. IL-10(-/-) mice cleared primary and secondary infection more rapidly than wild-type controls. Administration of mAb to TNF-alpha or IFN-gamma, but not GM-CSF, abrogated protection in naive IL-10(-/-) mice; mAb to TNF-alpha, but not IFN-gamma or GM-CSF, subverted protective immunity in secondary histoplasmosis. The inflammatory cell composition in IL-10(-/-) mice was altered in those given mAb to IFN-gamma or TNF-alpha. More Gr-1(+) and Mac-3(+) cells were present in lungs of IL-10(-/-) mice given mAb to IFN-gamma, and treatment with mAb to TNF-alpha sharply reduced the number of CD8(+) cells in lungs of IL-10(-/-) mice. We ascertained whether the lack of IL-10 modulated memory T cell generation or the protective function of cells. The percentage of CD3(+), CD44(high), CD62(low), and IFN-gamma(+) cells in IL-10(-/-) mice was higher than that of wild-type at day 7 but not day 21 or 49 after immunization. Fewer splenocytes from immunized IL-10(-/-) mice were required to mediate protection upon adoptive transfer into infected TCR alphabeta(-/-) mice. Hence, deficiency of IL-10 confers a salutary effect on the course of histoplasmosis, and the beneficial effects of IL-10 deficiency require endogenous TNF-alpha and/or IFN-gamma. Memory cell generation was transiently increased in IL-10(-/-) mice, but the protective function conferred by cells from these mice following immunization is strikingly more vigorous than that of wild-type.

Histoplasma capsulatum is a successful intracellular pathogen of mammalian macrophages. As such, this fungus must survive and/or subvert hostile environmental onslaughts in a professionally antimicrobial host cell. H. capsulatum uses different host receptors for binding to macrophages (beta 2 integrins) than it uses for binding to dendritic cells (the fibronectin receptor); the fungus experiences different degrees of success in survival in these two cells. Surface expression of HSP60 as the specific adhesin for macrophage beta 2 integrins represents a novel mechanism for binding. Long considered a resident of the phagolysosome, H. capsulatum may also reside in a modified phagosome without experiencing phagolysosomal fusion. H. capsulatum must compete with the host to acquire the essential nutrient iron, and has several potential mechanisms for accomplishing this necessary feat. Finally, H. capsulatum displays morphotype-specific expression of several genes, and a calcium-binding protein expressed only by the pathogenic yeast phase has been demonstrated as essential for full virulence. An organism's environment is of great importance to its success or failure, and H. capsulatum is good at finding or making the right environment in the host.

Research in medical mycology has traditionally been a mix of exciting biology and frustrating genetics, although the excitement has been steadily increasing as genetic obstacles have been successfully overcome. Now, a variety of fungal pathogens can be studied using molecular techniques derived from classical bacterial and yeast genetics, but with selective and strategic adaptations. Histoplasma capsulatum is the best-studied of the primary pathogens known as 'dimorphic' fungi, and tailored molecular genetic strategies are beginning to reveal a repertoire of genes and gene products intimately associated with pathogenesis.

Histoplasma capsulatum is an effective intracellular parasite of macrophages and causes the most prevalent fungal respiratory disease in the United States. A "dimorphic" fungus, H. capsulatum exists as a saprophytic mold in soil and converts to the parasitic yeast form after inhalation. Only the yeasts secrete a calcium-binding protein (CBP) and can grow in calcium-limiting conditions. To probe the relation between calcium limitation and intracellular parasitism, we designed a strategy to disrupt CBP1 in H. capsulatum using a telomeric linear plasmid and a two-step genetic selection. The resultingcbp1 yeasts no longer grew when deprived of calcium, and they were also unable to destroy macrophages in vitro or proliferate in a mouse model of pulmonary infection.

Histoplasma capsulatum, the causative agent of the most common systemic fungal infection, histoplasmosis, has become subject to increasing study in parallel with rising prevalence of human immunodeficiency. This review presents a summary of the advances made in the investigation of H. capsulatum genomics, molecular epidemiology, pathogenesis, and molecular genetics.

The presence of functional T cells is often required for successful resolution of infections with intracellular pathogens, yet the mechanisms by which they contribute to elimination of the invading pathogen in primary and secondary immunity are only partly understood. We report that increased mortality of naive alpha/beta TCR+ or CD4+ T cell-depleted mice infected with the fungus Histoplasma capsulatum is associated with impairment of IFN-gamma production. Upon secondary infection, mice concomitantly depleted of CD4+ and CD8+ cells exhibit decreased survival beyond day 25 of rechallenge, whereas elimination of either T cell subset or B cell deficiency does not result in accelerated mortality compared with controls. Remarkably, despite a decrease of H. capsulatum CFU in lungs of CD4+ plus CD8+-deficient mice, a progressive increase in spleen CFU is observed. The ability to control fungus growth in lungs is associated with vigorous TNF-alpha, but not IFN-gamma, production by bronchoalveolar lavage cells. In contrast, spleen cells from CD4+ plus CD8+-deficient mice are unable to produce TNF-alpha. Thus, the cellular and molecular requirements for protective immunity vary between primary and secondary infection. Furthermore, in secondary histoplasmosis, a sharp contrast can be drawn between lungs and spleens in their reliance upon T cells to control fungal replication. The opposing activities of these organs can be ascribed in part to differential production of TNF-alpha.

The new synthetic antifungal agent, L-Lysyl-L-Norvalyl-FMDP, inhibits growth of the yeast form of Histoplasma capsulatum. The compound is transported into the fungal cells by peptide permeases, cleaved intracellularly to constitutive amino acids, and the released C-terminal amino acid inhibits glucosamine-6-phosphate synthase. Promising antihistoplasmal in vivo activity of the FMDP-peptide was observed in an organ load test in mice.

To determine the optimum incubation period for recovery of fungi from clinical specimens in an area where Histoplasma capsulatum is endemic, we reviewed the results of 4259 consecutive fungal cultures. Of the total 1306 fungi isolated, 92.6% were detected by day 7, and 97.9% were detected by day 14. For the 1027 yeast isolates, only 1.4% were detected after day 7, none of which was considered clinically significant. Of the 281 mould isolates, 91.1% were detected by day 14; and of those detected after day 14, only isolates of H. capsulatum were considered clinically significant. An incubation protocol based on type of specimen is suggested.

Human monocyte/macrophages (Mphi) were adhered to extracellular matrix proteins, and the intracellular growth of Histoplasma capsulatum (Hc) yeasts were quantified and compared with their growth in Mphi adhered to plastic. Freshly isolated monocytes and cultured monocyte/derived Mphi adhered to type 1 collagen gels, but not to nongelled collagen-, fibronectin-, laminin-, or vitronectin-coated surfaces, demonstrated significant fungistatic activity against Hc yeasts. Activation of Mphi developed immediately upon adherence to the collagen matrices (1 h) and did not require additional time in culture. In addition, many of the yeasts were digested by 24 h postinfection. Mphi adhered to collagen maintained their fungistatic activity for up to 4 days, during which time monolayers cultured on plastic were destroyed. Culture of Mphi in the presence of IFN-gamma or TNF-alpha for 24 h before infection did not augment the fungistatic activity of collagen-adherent Mphi. Likewise, culture of monocytes on collagen gels with IL-3, granulocyte-Mphi CSF (GM-CSF) or Mphi CSF (M-CSF) for 7 days did not enhance Mphi fungistatic activity above that obtained by monocytes cultured on collagen alone. The mechanism(s) of Mphi-mediated fungistasis was not associated with production of toxic oxygen radicals, nitric oxide, or the restriction of intracellular iron. However, experiments with horseradish peroxidase-labeled gold colloids and immunoelectron microscopy demonstrated that phagolysosomal fusion, which is minimal in Hc-infected Mphi adhered to plastic, is enhanced significantly at both 1 h and 24 h postinfection in Mphi adhered to collagen matrices. These data suggest that in vivo, matrix-bound Mphi may express a previously unrecognized antifungal activity that proceeds in the absence of exogenous cytokines and is mediated, in part, by overcoming the capacity of Hc yeasts to inhibit Mphi phagolysosomal fusion.

The natural reservoir of Histoplasma capsulatum var. duboisii, the etiological agent of histoplasmosis duboisii (African histoplasmosis) is not yet known. We report the isolation of H. capsulatum var. duboisii from soil admixed with bat guano and from the intestinal contents of a bat in a sandstone cave in a rural area, Ogbunike in Anambra State of Nigeria. Eight of 45 samples of soil admixed with bat guano yielded H. capsulatum var. duboisii. Of the 35 bats belonging to the species Nycteris hispida and Tadirida pumila examined, only one (N. hispida) yielded this fungus from its intestinal contents. Identification of the isolates as Histoplasma was confirmed by exoantigen tests and by mating with tester strains of H. capsulatum. In vitro conversion to large yeast from suggestive of H. capsulatum var. duboisii was obtained on brain heart infusion agar supplemented with sheep blood and glutamine or cysteine. Pathogenicity tests with mice for all the isolates confirmed their identity by the demonstration of large yeast forms (8-15 microns in diameter) within giant cells in the infected tissues. Investigations on the possible occurrence of human infections in the area are in progress.

We investigated the role of intracellular iron on the capacity of Histoplasma capsulatum (Hc) yeasts to multiply within human macrophages (Mphi). Coculture of Hc-infected Mphi with the iron chelator deferoxamine suppressed the growth of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by iron-free transferrin (apotransferrin). Chloroquine, which prevents release of iron from transferrin by raising endocytic and lysosomal pH, induced human Mphi to kill Hc. The effect of chloroquine was reversed by iron nitriloacetate, an iron compound that is soluble at neutral to alkaline pH, but not by holotransferrin, which releases iron only in an acidic environment. Chloroquine (40-120 mg/kg) given intraperitoneally for 6 d to Hc-infected C57BL/6 mice significantly reduced the growth of Hc in a dose-dependent manner. At 120 mg/kg there was a 17- and 15-fold reduction (P < 0.01) in CFU in spleens and livers, respectively. The therapeutic effect of chloroquine also correlated with the length of treatment. As little as 2 d of chloroquine therapy (120 mg/kg), when started at day 5 after infection, reduced CFU in the spleen by 50%. Treatment with chloroquine for 10 d after a lethal inoculum of Hc protected six of nine mice; all control mice were dead by day 11 (P = 0.009). This study demonstrates that: (a) iron is of critical importance to the survival and multiplication of Hc yeasts in human Mphi; (b) in vitro, chloroquine induces Mphi killing of Hc yeasts by restricting the availability of intracellular iron; and (c) in vivo, chloroquine significantly reduces the number of organisms in the spleens and livers of Hc-infected mice and can protect mice from a lethal inoculum of Hc yeasts. Thus, chloroquine may be effective in the treatment of active histoplasmosis and also may be useful in preventing relapse of histoplasmosis in patients with acquired immunodeficiency syndromes.

A cottony, light tan, filamentous fungus with pear-shaped microconidia and lacking tuberculated macroconidia was isolated from a bronchial lavage specimen. Subculture on several media at 37 degrees C failed to convert the fungus to a yeast form after several weeks; attempts at in vivo conversion in mice were also unsuccessful. Sera obtained several months apart showed M bands with Histoplasma capsulatum (HC) antigen by immunodiffusion and an increase in complement fixation titers with mycelial and yeast phase antigens of HC. Parallel identity was obtained on two occasions with exoantigen culture confirmation reagents for HC from Immuno-Mycologics as well as one of identity with Nolan reagents. Extracts from four Chrysosporium spp. strains had no identity reactions with HC with either kit. The fungus was identified as HC by the Accuprobe Histoplasma chemiluminescence-labeled DNA probe directed at ribosomal RNA, whereas all four Chrysosporium spp. isolates tested negative. DNA probes are a fast and accurate method to confirm the identity of aberrant fungal isolates.

Recombinant cytokines and colony-stimulating factors (CSFs) were tested for their abilities to activate human monocytes/macrophages (M phi) to inhibit the intracellular growth of or kill Histoplasma capsulatum yeasts. None of the cytokines or CSFs or combinations of cytokines and CSFs activated M phi fungistatic activity when they were added to M phi monolayers concurrently with yeasts. In contrast, culture of monocytes for 7 days in the presence of interleukin 3, granulocyte-M phi CSF, or M phi CSF stimulated M phi fungistatic (but not fungicidal) activity against H. capsulatum yeasts in a concentration-dependent manner. Optimal activation of M phi by CSFs required 5 days of coculture, and the cultures had to be initiated with freshly isolated peripheral blood monocytes. Culture of monocytes with combinations of CSFs or addition of CSFs during the 24 h of coculture with the yeasts did not further enhance M phi fungistatic activity for H. capsulatum. Addition of gamma interferon or tumor necrosis factor alpha to CSF-activated M phi also did not enhance M phi fungistatic activity. These results suggest that interleukin 3, granulocyte-M phi CSF, and M phi CSF may play a role in the cell-mediated immune response to H. capsulatum by enhancing monocyte/M phi fungistatic activity.

We report the recovery of Histoplasma capsulatum from blood specimens cultured for Mycobacterium sp. in BACTEC 13A radiometric medium. H. capsulatum was recovered from six of eight blood specimens submitted for mycobacterial cultures from five human immunodeficiency virus-positive individuals. Initial positive metabolic signals occurred at a mean of 11 days, but no organisms were detected with acid-fast stains. The bottles remained positive, and after an additional incubation (mean, 8 days), yeast cells morphologically compatible with H. capsulatum were detected when aliquots were stained with acridine orange. Therefore, when radiometric mycobacterial blood cultures with persistent positive metabolic signals and negative acid-fast stains are encountered, acridine orange staining and subculturing for a variety of microorganisms, including fungi, e.g., H. capsulatum, should be considered.