Defining Galactomannan Positivity in the Updated EORTC/MSGERC Consensus Definitions of Invasive Fungal Diseases.

The consensus definitions of invasive fungal diseases from the EORTC/MSGERC were recently revised and updated. They now include consensus cutoff values for the galactomannan test that support the diagnosis of probable invasive aspergillosis. In this supplement article, we provide a rationale for these proposed thresholds based on the test's characteristics and performance in different patient populations and in different specimen types.

Efficacy of Cerebrospinal Fluid Beta-d-Glucan Diagnostic Testing for Fungal Meningitis: a Systematic Review.

Several case reports and cohort studies have examined the use of (1,3)-beta-d-glucan measurement with cerebrospinal fluid to diagnose fungal meningitis. This systematic review aims to characterize the evidence regarding cerebrospinal fluid (1,3)-beta-d-glucan measurement to detect fungal meningitis. We searched PubMed for (1,3)-beta-d-glucan and each of several distinct fungi, cerebrospinal fluid, and meningitis. Summary data including diagnostic performance (where applicable) were recorded. A total of 939 records were examined via a PubMed search. One hundred eighteen records remained after duplicates were removed, and 104 records were excluded, as they did not examine cerebrospinal fluid, included animals, or focused on nonfungal infections. Fourteen studies were included in this systematic review. A variety of fungi, including species of Candida, Aspergillus, Exserohilum, Cryptococcus, Histoplasma, and Coccidioides, were studied, although most were case reports. Diagnostic accuracy was examined in 5 studies. Cerebrospinal fluid (CSF) (1,3)-beta-d-glucan measurement showed >95% sensitivity in the corticosteroid injection-related outbreak of Exserohilum rostratum One study in Histoplasma meningitis found 53% (53/87) sensitivity and 87% (133/153) specificity, while another study of Cryptococcus meningitis found 89% (69/78) sensitivity and 85% (33/39) specificity. CSF (1,3)-beta-d-glucan testing may be useful, primarily as a nonspecific marker of fungal meningitis. Although the FDA black box warning states that Cryptococcus spp. do not make (1,3)-beta-d-glucan, the current evidence shows that (1,3)-beta-d-glucan is detectable in cryptococcal meningitis. Organism-specific testing should be used in conjunction with (1,3)-beta-d-glucan measurement.

Comparison of three anti-coccidioides antibody enzyme immunoassay kits for the diagnosis of coccidioidomycosis.

Coccidioidomycosis is a common cause of community-acquired pneumonia in endemic areas of the southwestern United States. Clinical presentations range from self-limited disease to severe, disseminated disease. As such, early and accurate diagnosis is essential to ensure appropriate treatment and monitoring. Currently available diagnostic testing has variable accuracy, particularly in certain patient populations, and new tests may offer improved accuracy for the diagnosis of coccidioidomycosis. Serum samples from patients with coccidioidomycosis and controls were tested for immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies using the MVista Coccidioides antibody detection EIA and two commonly used commercial enzyme immunoassay (EIA) kits: the IMMY Omega EIA and the Meridian Premier EIA. The sensitivity of the IgG antibody detection was 87.4% using the MVista test compared to 46.6% for IMMY and 70.9% for Meridian. The sensitivity for IgM antibody detection was 61.2% for the MVista test, 22.3% for IMMY and 29.1% for Meridian. For IgG antibody detection, specificity was 90% for the MVista EIA, 94.6% for IMMY, 96.4% for Meridian. For IgM antibody detection, specificity was 95.3% for the MVista test 98.2% for IMMY and 99.1% for Meridian. The MVista Coccidioides antibody EIA offers improved sensitivity, including among high-risk patient populations, for the detection of IgG and IgM antibodies in comparison to other currently available EIAs.

Current Concepts in the Epidemiology, Diagnosis, and Management of Histoplasmosis Syndromes.

Histoplasmosis is a global disease endemic to regions of all six inhabited continents. The areas of highest endemicity lie within the Mississippi and Ohio River Valleys of North America and parts of Central and South America. As a result of climate change and anthropogenic land utilization, the conditions suitable for Histoplasma capsulatum are changing, leading to a corresponding change in epidemiology. The clinical manifestations of histoplasmosis are protean, variably resembling other common conditions such as community-acquired pneumonia, tuberculosis, sarcoidosis, Crohn's disease, or malignancy. Making a successful diagnosis is contingent on a thorough understanding of epidemiology, common clinical presentations, and best testing practices for histoplasmosis. While most subclinical or self-limited diseases do not require treatment in immunocompetent patients, all immunocompromised patients and those with progressive disseminated disease or chronic pulmonary disease should be treated. Liposomal amphotericin B is the preferred agent for severe or disseminated disease, while itraconazole is adequate for milder cases and "step-down" therapy following response to amphotericin B. In this review, we discuss the current evidence-based approaches to the epidemiology, diagnosis, and management of histoplasmosis.

Novel canine anti-Coccidioides immunoglobulin G enzyme immunoassay aids in diagnosis of coccidioidomycosis in dogs.

The diagnosis of coccidioidomycosis (CM) in dogs is typically based on clinical presentation, serology, and (less frequently) spherule identification. Agar gel immunodiffusion (AGID) is the most commonly employed serological method, but AGID is slow (requiring up to a week for titer). A Coccidioides antigen enzyme immunoassay (EIA) is also available; however, sensitivity is low in CM dogs. An antibody EIA was developed to detect canine immunoglobulin G (IgG) reacting to Coccidioides antigens. Serum was evaluated from dogs with pathology proven CM and/or AGID positive CM, as well as dogs with histoplasmosis, blastomycosis, non-fungal infections, or healthy dogs. A standard curve was used to convert optical density (OD) values into EIA units (EU). Serum and urine samples from CM dogs were also tested in the antigen EIA. Sensitivity and specificity for IgG were 89.2% and 97.2%, respectively, upon evaluation of dogs with proven or probable CM and control dogs. Cross-reactivity was observed in 7.7% and in 6.4% of dogs with histoplasmosis or blastomycosis, respectively. The antigen EIA alone was insensitive (33.8%). Combined IgG and antigen testing increased sensitivity to 93.2%, as three dogs were IgG-negative but had detectable serum or urine antigen. In 22 dogs with proven CM, sensitivity was statistically similar for antibody EIA and AGID (86% and 73%; P = .487). The MiraVista® canine Coccidioides antibody IgG EIA may aid in the diagnosis of CM by improving turnaround time with comparable sensitivity to AGID. Serial or concurrent testing by antibody and antigen EIAs may be beneficial when screening dogs for CM.

Cryptococcal meningitis is a cause for cross-reactivity in cerebrospinal fluid assays for anti-Histoplasma, anti-Coccidioides and anti-Blastomyces antibodies.

BACKGROUND/OBJECTIVES: Antibody detection is commonly used for diagnosis of histoplasmosis, and cross-reactions have been recognised due to endemic mycoses but not cryptococcosis. We observed cross-reactions in an anti-Histoplasma antibody enzyme immunoassay (EIA) in the cerebrospinal fluid (CSF) from a patient with cryptococcal meningitis and sought to assess the risk of cross-reactive anti-Histoplasma antibodies in persons with cryptococcal meningitis. METHODS: An anti-cryptococcal antibody EIA was developed to measure CSF antibody response in HIV-infected subjects from Kampala, Uganda and previously healthy, HIV-negative subjects at the National Institutes of Health (NIH) with cryptococcal meningitis. Specimens were tested for cross-reactivity in assays for IgG anti-Histoplasma, anti-Blastomyces and anti-Coccidioides antibodies. RESULTS: Among 61 subjects with cryptococcal meningitis (44 Kampala cohort, 17 NIH cohort), elevated CSF anti-cryptococcal antibody levels existed in 38% (23/61). Of the 23 CSF specimens containing elevated anti-cryptococcal antibodies, falsely positive results were detected in antibody EIAs for histoplasmosis (8/23, 35%), coccidioidomycosis (6/23, 26%) and blastomycosis (1/23, 4%). Overall, 2% (2/81) of control CSF specimens had elevated anti-cryptococcal antibody detected, both from Indiana. CONCLUSIONS: Cryptococcal meningitis may cause false-positive results in the CSF for antibodies against Histoplasma, Blastomyces and Coccidioides. Fungal antigen testing should be performed to aid in differentiating true- and false-positive antibody results in the CSF.

Improvement in Diagnosis of Histoplasma Meningitis by Combined Testing for Histoplasma Antigen and Immunoglobulin G and Immunoglobulin M Anti-Histoplasma Antibody in Cerebrospinal Fluid.

Background: Central nervous system (CNS) histoplasmosis is a life-threatening condition and represents a diagnostic and therapeutic challenge. Isolation of Histoplasma capsulatum from cerebrospinal fluid (CSF) or brain tissue is diagnostic; however, culture is insensitive and slow growth may result in significant treatment delay. We performed a retrospective multicenter study to evaluate the sensitivity and specificity of a new anti-Histoplasma antibody enzyme immunoassay (EIA) for the detection of IgG and IgM antibody in the CSF for diagnosis of CNS histoplasmosis, the primary objective of the study. The secondary objective was to determine the effect of improvements in the Histoplasma galactomannan antigen detection EIA on the diagnosis of Histoplasma meningitis. Methods: Residual CSF specimens from patients with Histoplasma meningitis and controls were tested for Histoplasma antigen and anti-Histoplasma immunoglobulin G (IgG) and immunoglobulin M (IgM) antibody using assays developed at MiraVista Diagnostics. Results: A total of 50 cases and 157 controls were evaluated. Fifty percent of patients with CNS histoplasmosis were immunocompromised, 14% had other medical conditions, and 36% were healthy. Histoplasma antigen was detected in CSF in 78% of cases and the specificity was 97%. Anti-Histoplasma IgG or IgM antibody was detected in 82% of cases and the specificity was 93%. The sensitivity of detection of antibody by currently available serologic testing including immunodiffusion and complement fixation was 51% and the specificity was 96%. Testing for both CSF antigen and antibody by EIA was the most sensitive approach, detecting 98% of cases. Conclusions: Testing CSF for anti-Histoplasma IgG and IgM antibody complements antigen detection and improves the sensitivity for diagnosis of Histoplasma meningitis.

Detection of (1,3)-ß-d-Glucan in Cerebrospinal Fluid in Histoplasma Meningitis.

The diagnosis of central nervous system (CNS) histoplasmosis is often difficult. Although cerebrospinal fluid (CSF) (1,3)-ß-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 Histoplasma 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 (P < 0.05) and 82 pg/ml for controls with other causes of fungal meningitis (P = 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 Histoplasma meningitis.

In vitro activity of isavuconazole against fluconazole-resistant isolates of Histoplasma capsulatum.

No clinical trials for histoplasmosis have been performed with the newer azoles, leaving itraconazole as the azole of choice. In vitro studies suggest that Histoplasma capsulatum from patients that relapse on fluconazole has higher minimum inhibitory concentrations (MICs) to fluconazole and voriconazole but not itraconazole and posaconazole. The newest azole, isavuconazole, shares structural similarity to voriconazole, but to date nobody has explored emergence of resistance. In vitro susceptibilities to isavucoanzole and fluconazole were performed on previously obtained isolates from the patients who relapsed on fluconazole therapy. Susceptibilities were determined by NCCLS method M27A developed for yeasts, as modified for H. capsulatum. The change in the MIC from the primary to the relapse isolate was tested using Wilcoxon Rank-Sum for paired data. Among the primary isolates, the median MICs were 1.0 (range 0.25 to 4.0) µg/ml for fluconazole and ≤0.007 (range ≤0.007 to 0.015) µg/ml for isavuconazole. In the group of relapsed isolates, the median MICs rose to 8.0 (range 0.25 to 64.0) µg/ml for fluconazole and remained unchanged at ≤0.007 (range ≤0.007 to 0.015) µg/ml for isavuconazole (P < .001). Only one isolate exhibited a nonsignificant increase in MIC to isavuconazole. Histoplasma isolates from patients who relapsed on fluconazole did not have an elevation in MICs to isavuconazole. This differs from the results previously seen with voriconazole and suggests that despite a closer structural similarity to voriconazole than itraconazole and posaconazole, isavuconazole has a higher barrier to resistance and may be effective as therapy for histoplasmosis.

Clinical and epidemiological characterization of histoplasmosis cases in a nonendemic area, Connecticut, United States.

We performed a retrospective analysis of histoplasmosis cases diagnosed at our institution in New Haven, Connecticut, from 2005 to 2015. Among 12 cases of active histoplasmosis, seven were immunosuppressed and five had human immunodeficiency virus (HIV). Eleven patients reported travel to potentially endemic areas at a median of 105 days prior to presentation; travel to the Caribbean was most common (n = 6). Median time to diagnosis from symptom onset and first Histoplasma antigen testing were 41 and 28 days, respectively. Consistent with reports from other non-endemic areas, our findings suggest that the epidemiology of histoplasmosis may differ in Connecticut, potentially contributing to delayed diagnoses.

Enhanced Antibody Detection and Diagnosis of Coccidioidomycosis with the MiraVista IgG and IgM Detection Enzyme Immunoassay.

Coccidioidomycosis is a common cause of community-acquired pneumonia in areas of the southwestern United States in which the disease is endemic. Clinical presentations range from self-limited disease to severe disseminated disease. Therefore, early and accurate diagnosis is essential to ensure appropriate treatment and monitoring. Currently available diagnostic tests have variable accuracy, particularly in certain patient populations, and new tests may offer improved accuracy for the diagnosis of coccidioidomycosis. Serum samples from 103 cases of coccidioidomycosis and 373 controls were tested for IgG and IgM antibodies using the MVista anti-Coccidioides antibody enzyme immunoassay. Serum specimens from 170 controls from areas in which the disease is endemic and 44 cases were tested by immunodiffusion at MiraVista Diagnostics. The sensitivity of the MVista antibody assay was 88.3%, and the specificity was 90%. The sensitivity was maintained in the presence of immunocompromising conditions or immunosuppressive therapies. The sensitivity of immunodiffusion was 60.2%, and the specificity was 98.8%. The sensitivity of complement fixation (62 cases) was 66.1%, but the specificity could not be determined. The MVista anti-Coccidioides antibody enzyme immunoassay offers improved sensitivity, compared with immunodiffusion and complement fixation, is not impaired in immunocompromised patients, and permits highly reproducible semiquantification.

Improved Diagnosis of Acute Pulmonary Histoplasmosis by Combining Antigen and Antibody Detection.

BACKGROUND: Acute pulmonary histoplasmosis can be severe, especially following heavy inoculum exposure. Rapid diagnosis is critical and often possible by detection of antigen, but this test may be falsely negative in 17% of such cases. Antibody detection by enzyme immunoassay (EIA) may increase sensitivity and permit the measurement of immunoglobulin M (IgM) and immunoglobulin G (IgG) classes of antibodies separately. METHODS: Microplates coated with Histoplasma antigen were used for testing of serum from patients with acute pulmonary histoplasmosis and controls in the MVista Histoplasma antibody EIA. Results for IgG and IgM were reported independently. RESULTS: IgG antibodies were detected in 87.5%, IgM antibodies in 67.5%, and IgG and/or IgM antibodies in 88.8% of patients with acute pulmonary histoplasmosis in this assay, while immunodiffusion, complement fixation, and antigen testing showed sensitivities of 55.0%, 73.1%, and 67.5%, respectively (n = 80). Combining antigen and antibody detection increased the sensitivity to 96.3%. CONCLUSIONS: The MVista Histoplasma antibody EIA offers increased sensitivity over current antibody tests while also allowing separate detection of IgG and IgM antibodies and complementing antigen detection. Combining antigen and EIA antibody testing provides an optimal method for diagnosis of acute pulmonary histoplasmosis.

Seroprevalence of histoplasmosis in Kampala, Uganda.

Histoplasmosis is endemic to the Midwestern United States, but cases have been reported nearly worldwide. A 1970 study found 3.8% skin test sensitivity to Histoplasma capsulatum in Uganda but no systemic study of histoplasmosis exposure has occurred since the onset of the human immunodeficiency virus (HIV) pandemic. This study investigated the seroprevalence of H. capsulatum and sought previously undetected cases of histoplasmosis in Kampala, Uganda. Serum, cerebrospinal fluid (CSF) and/or urine specimens were obtained from HIV-infected persons with suspected meningitis. Specimens were tested for H. capsulatum IgG and IgM by enzyme immune assay and Histoplasma antigen. 147 of the 257 subjects who were enrolled had cryptococcal meningitis. Overall, 1.3% (2/151) of subjects were serum Histoplasma IgG positive, and zero of 151 were IgM positive. Antigen was not detected in any serum (n = 57), urine (n = 37, or CSF (n = 63) samples. Both subjects with serum Histoplasma IgG positivity had cryptococcal meningitis. Histoplasma capsulatum IgG was detected at low levels in persons with HIV/AIDS in Kampala, Uganda. Histoplasmosis is not widespread in Uganda but microfoci do exist. There appears to be no cross-reactivity between Cryptococcus neoformans and Histoplasma antigen testing, and cryptococcosis appears to be at most, a rare cause of positive Histoplasma IgG.

Histoplasmosis complicating tumor necrosis factor-α blocker therapy: a retrospective analysis of 98 cases.

BACKGROUND: Histoplasmosis may complicate tumor necrosis factor (TNF)-α blocker therapy. Published case series provide limited guidance on disease management. We sought to determine the need for long-term antifungal therapy and the safety of resuming TNF-α blocker therapy after successful treatment of histoplasmosis. METHODS: We conducted a multicenter retrospective review of 98 patients diagnosed with histoplasmosis between January 2000 and June 2011. Multivariate logistic regression was used to evaluate risk factors for severe disease. RESULTS: The most commonly used biologic agent was infliximab (67.3%). Concomitant corticosteroid use (odds ratio [OR], 3.94 [95% confidence interval {CI}, 1.06-14.60]) and higher urine Histoplasma antigen levels (OR, 1.14 [95% CI, 1.03-1.25]) were found to be independent predictors of severe disease. Forty-six (47.4%) patients were initially treated with an amphotericin B formulation for a median duration of 2 weeks. Azole treatment was given for a median of 12 months. TNF-α blocker therapy was initially discontinued in 95 of 98 (96.9%) patients and later resumed in 25 of 74 (33.8%) patients at a median of 12 months (range, 1-69 months). The recurrence rate was 3.2% at a median follow-up period of 32 months. Of the 3 patients with recurrence, 2 had restarted TNF-α blocker therapy, 1 of whom died. Mortality rate was 3.2%. CONCLUSIONS: In this study, disease outcomes were generally favorable. Discontinuation of antifungal treatment after clinical response and an appropriate duration of therapy, probably at least 12 months, appears safe if pharmacologic immunosuppression has been held. Resumption of TNF-α blocker therapy also appears safe, assuming that the initial antifungal therapy was administered for 12 months.

(1,3)-ß-d-glucan in cerebrospinal fluid for diagnosis of fungal meningitis associated with contaminated methylprednisolone injections.

Prompt diagnosis and treatment of fungal meningitis are critical, but culture is insensitive. (1,3)-ß-d-Glucan (BDG) testing is FDA approved for serological diagnosis of invasive fungal disease; however, BDG testing is not approved for cerebrospinal fluid (CSF), and the appropriate cutoff value is unknown. We aimed to validate the diagnostic accuracy of CSF BDG measurements for fungal meningitis among patients exposed to contaminated methylprednisolone acetate (MPA). A retrospective observational study was conducted at St. Joseph Mercy Hospital and Vanderbilt University from November 2013 to February 2014. Patients were included if they had received a contaminated MPA injection. Cases were classified as probable or proven meningitis according to Centers for Disease Control and Prevention guidelines. CSF BDG testing was performed according to the package insert instructions for serum samples, and results were validated using Clinical and Laboratory Standards Institute procedures (MiraVista Diagnostics). Of 233 patients, 45 had meningitis (28 proven cases), 53 had spinal/paraspinal infections (19 proven cases), and 135 did not develop disease. Using the manufacturer's cutoff value (≥80 pg/ml), the sensitivity and specificity were 96% and 95%, respectively, for proven meningitis and 84% and 95% for probable or proven meningitis. Receiver operating characteristic analysis identified the optimal cutoff value for proven meningitis to be 66 pg/ml (sensitivity, 100%; specificity, 94%) and that for probable or proven meningitis to be 66 pg/ml (sensitivity, 91%; specificity, 92%). Our results suggest that CSF BDG measurements are highly sensitive and specific for the diagnosis of fungal meningitis associated with contaminated MPA injections. Further study on the utility of CSF BDG testing for other types of fungal meningitis is needed.

Positive (1-3) B-d-glucan and cross reactivity of fungal assays in coccidioidomycosis.

Fungal antigen testing in immunosuppressed patients has emerged as a powerful diagnostic tool. Some assays are relatively nonspecific, and misinterpretation can have severe clinical consequences. Additionally, when new assays become commercially available it is important to evaluate the potential for cross reactivity. We recently observed several immunosuppressed patients with positive (1→3)-ß-D-glucan (BG) who were eventually diagnosed with coccidioidomycosis in the endemic area of Tucson, Arizona. Although the BG assay is known to detect glucans of many fungal pathogens, reports of cross-reactivity with Coccidioides remain sparsely reported. To test the cross-reactivity of fungal antigens in detection assays, serum samples from patients with coccidioidomycosis testing positive for Coccidioides antigen were evaluated for BG. Of 12 samples positive for Coccidioides antigen (≥0.07 ng/ml), 11 (92%) were positive by BG (>80 pg/ml), and of 11 positive for Aspergillus galactomannan, 10 (91%) were positive by BG (>80 pg/ml). We conclude that the BG assay is nonspecific, detecting glucans from many fungal pathogens, including Coccidioides. In the endemic area, a positive BG warrants further specific testing.

Histoplasmosis: Up-to-Date Evidence-Based Approach to Diagnosis and Management.

Histoplasmosis is the most common endemic mycosis in the North America, Central America, and many countries of South America and also occurs in China, India, Southeast Asia, Africa, Australia, and Europe. Clinical syndromes are not specific and histoplasmosis often is overlooked in the evaluation of patients with community-acquired pneumonia, chronic cavitary pneumonia resembling tuberculosis or anaerobic infection, granulomatous inflammatory diseases such as sarcoidosis or Crohn disease, and malignancy. The diagnosis depends on understanding the geographic distribution, common clinical presentations, and tests used for diagnosis of histoplasmosis. While histoplasmosis resolves without treatment in most patients, treatment is indicated in all immunocompromised patients and those with progressive disseminated disease or chronic pulmonary disease. Treatment is appropriate in most patients with acute pulmonary disease but rarely in those with other pulmonary or mediastinal manifestations. The preferred agents include liposomal amphotericin B for more severe cases and itraconazole for milder cases and "step-down" therapy following response to amphotericin B.

Cerebrospinal fluid Coccidioides antigen testing in the diagnosis and management of central nervous system coccidioidomycosis.

The goal of this study was to report on the potential utility of cerebrospinal fluid (CSF) Coccidioides antigen testing in the diagnosis and management of Coccidioides meningitis. We retrospectively reviewed medical records of seven patients with Coccidioides meningitis who had Coccidioides antigen tests performed on CSF. In two severely immunocompromised patients, CSF Coccidioides antigen testing was helpful in the diagnosis when other testing modalities were negative. Coccidioides antigen testing was also useful in the management of patients who had progression of disease due to non-adherence, development of resistance, failure of therapy and the presence of vasculitis. Changing antigen levels helped identify disease complications in three patients that led to alterations in therapy or management. On the basis of our review of these seven patients with Coccidioides meningitis, we concluded that the Coccidioides antigen test contributed to the diagnosis and management of patients with Coccidioides meningitis.

Glycosylation and immunoreactivity of the Histoplasma capsulatum Cfp4 yeast-phase exoantigen.

The yeast phase of Histoplasma capsulatum is the virulent form of this thermally dimorphic fungal pathogen. Among the secreted proteome of Histoplasma, culture filtrate protein 4 (Cfp4) is a heavily glycosylated factor produced abundantly and specifically by Histoplasma yeast cells, suggesting its role in pathogenesis. We have generated three monoclonal antibodies as tools for characterization and detection of Cfp4 and determined the epitope each recognizes. Through site-directed mutagenesis of Cfp4, we identified three asparagines that function as the principal sites of N-linked glycan modification. To test the function of Cfp4 in Histoplasma pathogenesis, we generated Cfp4-deficient strains by insertional mutagenesis and by RNA interference. Cfp4-deficient strains are not attenuated in virulence in human macrophages or during lung infection in a murine model of histoplasmosis. Coinfection of differentially marked Cfp4-producing and Cfp4-deficient strains demonstrates that production of Cfp4 does not confer a fitness advantage to Histoplasma yeasts during murine lung infection. Despite no apparent role in acute virulence in mice, secretion of the Cfp4 glycoprotein by yeast cells is consistent across clinical and laboratory isolates of the North American type 1 and type 2 phylogenetic groups as well as a strain from Panama. In addition, human immune sera recognize the Histoplasma Cfp4 protein, confirming Cfp4 production during infection of human hosts. These results suggest the potential utility of Cfp4 as a diagnostic exoantigen for histoplasmosis.