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BACKGROUND: Culture can be used for diagnosis and antifungal susceptibility testing in animals with fungal infections. Limited information is available regarding the diagnostic performance of culture and the susceptibility patterns of Histoplasma spp. isolates. HYPOTHESIS/OBJECTIVES: Describe the clinical utility of culture and the susceptibility patterns of Histoplasma spp. isolates causing histoplasmosis in cats and dogs. ANIMALS: Seventy-one client-owned animals, including 33 cats and 19 dogs with proven or probable histoplasmosis. METHODS: Culture was attempted from tissue or fluid samples. Diagnostic performance of culture, cytopathology, and antigen detection were compared with final diagnosis. Susceptibility to antifungal agents was determined for a subset (11 from dogs, 9 from cats) of culture isolates. RESULTS: Culture had a diagnostic sensitivity of 17/33 (52%; 95% confidence interval [CI], 34%-69%) and 15/19 (79%; 95% CI, 61%-97%) and specificity of 6/6 (100%; 95% CI, 54%-100%) and 10/10 (100%; 95% CI, 69%-100%) in cats and dogs, respectively. Culture was not positive in any animal in which cytopathology and antigen testing were negative. Target drug exposure (area under the concentration curve [AUC]/minimum inhibitory concentration [MIC] >25) should be easily achieved for all isolates for itraconazole, voriconazole, or posaconazole. Five of 20 (25%) isolates had fluconazole MIC ≥32 µg/mL and achieving target drug exposure is unlikely. CONCLUSIONS AND CLINICAL IMPORTANCE: Fungal culture did not improve diagnostic sensitivity when used with cytopathology and antigen detection. Susceptibility testing might help identify isolates for which fluconazole is less likely to be effective.

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BACKGROUND: Histoplasma antigen and anti-Histoplasma antibody detection are used to support the diagnosis of histoplasmosis. There is a paucity of published data on antibody assays. OBJECTIVES: Our primary hypothesis was that anti-Histoplasma immunoglobulin G (IgG) antibody detection using enzyme immunoassay (EIA) will be more sensitive as compared to immunodiffusion (ID). ANIMALS: Thirty-seven cats and 22 dogs with proven or probable histoplasmosis; 157 negative control animals. METHODS: Residual stored sera were tested for anti-Histoplasma antibodies using EIA and ID. Results of urine antigen EIA were reviewed retrospectively. Diagnostic sensitivity was calculated for all three assays and compared between immunoglobulin G (IgG) EIA and ID. The diagnostic sensitivity of urine antigen EIA and IgG EIA, interpreted in parallel, was reported. RESULTS: Sensitivity of IgG EIA was 30/37 (81.1%; 95% confidence interval [CI], 68.5%-93.4%) in cats and 17/22 (77.3%; 95% CI, 59.8%-94.8%) in dogs. Diagnostic sensitivity of ID was 0/37 (0%; 95% CI, 0%-9.5%) in cats and 3/22 (13.6%; 95% CI, 0%-28.0%) in dogs. Immunoglobulin G EIA was positive in all animals (2 cats and 2 dogs) with histoplasmosis but without detectable antigen in urine. Diagnostic specificity of IgG EIA was 18/19 (94.7%; 95% CI, 74.0%-99.9%) in cats and 128/138 (92.8%; 95% CI, 87.1%-96.5%) in dogs. CONCLUSION AND CLINICAL IMPORTANCE: Antibody detection by EIA can be used to support the diagnosis of histoplasmosis in cats and dogs. Immunodiffusion has an unacceptably low diagnostic sensitivity and is not recommended.

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BACKGROUND: Antibody detection is the main method for diagnosis of coccidioidomycosis, but it has limitations. The Coccidioides antigen enzyme immunoassay is recommended for testing cerebrospinal fluid in suspected meningitis. Reports on urine and serum antigen detection evaluated small numbers of patients who were mostly immunocompromised. The purpose of this study was to assess the accuracy of combined antibody and antigen detection for diagnosis. METHODS: A retrospective study, including all patients in whom Coccidioides antigen detection in serum was performed between January 2013 and May 2017, was conducted at Valleywise Health Medical Center (formerly Maricopa Integrated Health System). Sensitivity and specificity of antigen and antibody were evaluated in 158 cases and 487 controls. RESULTS: The sensitivity of antibody detection by immunodiffusion (ID) was 84.2%. The sensitivity of antigen detection was 57.0% if both urine and serum were tested and 36.7% if urine alone was tested. The sensitivity of combining antigen and ID antibody detection was 93.0%. The sensitivity of urine and serum antigen detection was 55.4% in proven and 58.7% in probable cases, 79.1% in disseminated and 41.6% in pulmonary cases, and 74.7% in immunocompromised and 40.0% in immunocompetent patients. Specificity was 99.4% for antigen detection and 96.5% for ID antibody detection. Diagnostic accuracy was 95.4% for ID antibody and antigen detection, 93.6% for ID antibody alone, and 89.1% for pathology or culture. CONCLUSIONS: These findings support combined antibody and antigen detection for diagnosis of progressive coccidioidomycosis. The diagnosis may have been missed if antigen detection was not performed.

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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.

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Rabbit antithymocyte globulin (rATG) is known to yield false-positive Histoplasma antigenemia. The fourth generation MiraVista Histoplasma antigen assay was modified to block this effect (MiraVista Diagnostics, Indianapolis, Indiana). We report a case of rATG-induced false-positive Blastomyces and Histoplasma antigenemia in a lung transplant recipient despite modifications of these antigen assays.

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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.

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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.

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Histoplasmosis is known to be endemic to the Midwestern United States, but cases have been reported throughout much of the world. Somali, Hmong, and Burmese (ethnically Karen) persons make up some of the largest refugee populations coming the United States in recent years. Yet, information about risk of Histoplasma capsulatum infection amongst these populations is limited. This study used the CDC Migrant Serum Bank to test ~100 samples from each of Somali, Burmese, and Hmong U.S.-bound refugees. Samples were tested by enzyme immunoassay for Histoplasma capsulatum IgG. Overall 1% (2/299) of refugee serum samples were positive for H. capsulatum IgG. One of 99 samples obtained from Hmong refugees was positive, and the other positive sample came from among 100 Burmese refugee samples. H capsulatum IgG positivity was detected at low levels in Hmong and Burmese refugees. No IgG positivity was detected among 100 Somali refugees.

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Coccidioides is a soil-dwelling fungus that causes coccidioidomycosis, a disease also known as Valley fever, which affects humans and a variety of animal species. Recent findings of Coccidioides in new, unexpected areas of the United States have demonstrated the need for a better understanding of its geographic distribution. Large serological studies on animals could provide important information on the geographic distribution of this pathogen. To facilitate such studies, we used protein A/G, a recombinant protein that binds IgG antibodies from a variety of mammalian species, to develop an enzyme immunoassay (EIA) that detects IgG antibodies against Coccidioides in a highly sensitive and high-throughput manner. We showed the potential of this assay to be adapted to multiple animal species by testing a collection of serum and/or plasma samples from dogs, mice, and humans with or without confirmed coccidioidomycosis. We then evaluated the performance of the assay in dogs, using sera from dogs residing in a highly endemic area, and found seropositivity rates significantly higher than those in dogs of non-endemic areas. We further evaluated the specificity of the assay in dogs infected with other fungal pathogens known to cross-react with Coccidioides. Finally, we used the assay to perform a cross-sectional serosurvey investigating dogs from Washington, a state in which infection with Coccidioides has recently been documented. In summary, we have developed a Coccidioides EIA for the detection of antibodies in canines that is more sensitive and has higher throughput than currently available methods, and by testing this assay in mice and humans, we have shown a proof of principle of its adaptability for other animal species.

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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.

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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.

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OBJECTIVE: To evaluate the sensitivity and specificity of an enzyme immunoassay (EIA) for antibodies to a recombinant Blastomyces adhesin-1 repeat antigen (rBAD-1) to aid in the diagnosis of blastomycosis in dogs and compare the findings with results from other tests used for this purpose. DESIGN: Prospective analytic study. SAMPLE: Serum and urine from 70 dogs with and without blastomycosis. PROCEDURES: Serum and urine samples were collected from dogs with blastomycosis (n = 21), histoplasmosis (8), or nonfungal pulmonary disease (21) and from healthy control dogs living in a blastomycosis-endemic area (20). Serum was tested for antibodies against Blastomyces dermatitidis with the rBAD-1 antibody EIA and an A-antigen antibody agar gel immunodiffusion (AGID) assay. Serum and urine were tested for B dermatitidis antigen with a quantitative EIA. RESULTS: Sensitivity of the quantitative antigen EIA was 100% in serum and urine samples from dogs with blastomycosis, with specificity of 95% in urine samples from dogs with nonfungal pulmonary disease and 100% in urine samples from healthy dogs. Sensitivity of the rBAD-1 antibody EIA (95%) was significantly greater than that of the A-antigen antibody AGID assay (65%). Specificity of the antibody EIA was 88% in dogs with histoplasmosis, 95% in healthy dogs, and 100% in dogs with nonfungal pulmonary disease. CONCLUSIONS AND CLINICAL RELEVANCE: The rBAD-1 antibody EIA had greater sensitivity than the A-antigen antibody AGID assay in dogs with blastomycosis. This antibody EIA may assist in distinguishing histoplasmosis from blastomycosis. Further evaluation in a larger prospective study is needed to verify these results.

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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.

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Serologic tests for antibodies to Blastomyces dermatitidis are not thought to be useful for the diagnosis of blastomycosis, in part due to the low sensitivity of immunodiffusion and complement fixation. Earlier studies have shown that the enzyme immunoassay improves the sensitivity of antibody detection for the diagnosis of blastomycosis. Microplates coated with the B. dermatitidis surface protein BAD-1 were used for testing sera from patients with proven blastomycosis or histoplasmosis and controls. Semiquantification was accomplished by using standards containing human anti-B. dermatitidis antibodies. The antibodies were detected in 87.8% of the patients with blastomycosis by the enzyme immunoassay compared to 15.0% by immunodiffusion. The specificities were 99.2% for patients with nonfungal infections and healthy subjects and 94.0% for patients with histoplasmosis. The results were highly reproducible on repeat testing. When combined with antigen testing, antibody testing improved the sensitivity from 87.8% to 97.6%. Enzyme immunoassay detection of antibodies against BAD-1 is highly specific, has greatly improved sensitivity over immunodiffusion, and may identify cases with negative results by antigen testing. This assay has the potential to aid in the diagnosis of blastomycosis.

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Indoleamine 2,3 dioxygenase (IDO) plays an important role in immunoregulation as it is involved in downregulating immune responses to infections. We sought to characterize IDO activity in histoplasmosis and to do so, C57Bl6 mice were infected intranasally with Histoplasma capsulatum. After infection, lung and spleen IDO activity was assessed by HPLC and IDO expression by qRT-PCR. The distribution of IDO was determined by immunohistochemical staining. Cytokine levels were measured in lung and spleen homogenates using cytokine bead array. Fungal burden was quantified by culture. Subcutaneous pellets containing methyltryptophane (1-MT) were employed to inhibit IDO in vivo. Histoplasma infection strongly induced functional lung IDO, with activity at its highest at weeks 1 and 2 and then decreased thereafter as the mice cleared the infection. Lung IDO activity positively correlated with the fungal burden (Rho = 0.845), interferon-γ (Rho = 0.839) and tumor necrosis factor-α (Rho = 0.791) levels, P < 0.001. In contrast, spleen IDO activity was not induced despite high infection burden and cytokine levels. IDO expressing cells were predominately located at the ring edge of Histoplasma-induced granulomas. IDO inhibition prior to infection reduced fungal burdens and inflammation in lungs and spleen. Histoplasma preferentially induces lung IDO, as early as one week after infection. IDO appears to modulate the immune response to Histoplasma infection.

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Antigen detection has been reported to be a promising method for rapid diagnosis of coccidioidomycosis in humans. Coccidioides antigen detection has not been previously reported in dogs with coccidioidomycosis and was evaluated in 60 cases diagnosed based on detection of anti-Coccidioides antibodies at titers of 1:16 or more in serum. Controls included dogs with presumed histoplasmosis or blastomycosis, other fungal infections, or nonfungal diseases and healthy dogs. Urine and serum specimens were tested using an enzyme immunoassay for Coccidioides galactomannan antigen. Antibody testing was performed at commercial veterinary reference laboratories. Antigen was detected in urine or serum of 12 of 60 (20.0%), urine only in 2 of 57 (3.5%), and serum only in 11 of 58 (19.0%) dogs with coccidioidomycosis. Antigen was detected in the urine of 3 of 43 (7.0%) and serum of 1 of 37 (2.7%) dogs with histoplasmosis or blastomycosis but not in 13 dogs with other fungal infections (serum, 9; urine, 13), 41 dogs with nonfungal diseases (urine, 41; serum, 18), or healthy dogs (serum, 21; urine, 21). Detection of antigen was an insensitive method for diagnosis of coccidioidomycosis in dogs in which the diagnosis was based primarily upon detection of antibodies at titers of 1:16 or higher, and the highest sensitivity was in serum.

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We demonstrated that sodium gluconate was the factor causing false-positive galactomannan (GM) antigenemia of Plasma-Lyte hydration solution. Infusion of sodium gluconate-containing solution but not gluconate-free Plasma-Lyte resulted in positive serum GM antigenemia. Serum GM concentrations also correlated with the volume and in vitro concentrations of GM within gluconate-containing solutions of infused Plasma-Lyte.

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Micafungin alone and combined with liposomal amphotericin B was evaluated against two strains of Histoplasma capsulatum. Micafungin was active in vitro against the mold but not the yeast form but was ineffective in vivo. Micafungin appears to be ineffective in treatment of histoplasmosis.

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Blastomycosis is a serious and potentially fatal infection, and diagnosis can be difficult at times. We evaluated the diagnostic utility of a commercially available assay for detection of Blastomyces dermatitidis antigen, recently modified to permit quantitation, in subjects with newly diagnosed blastomycosis. Twenty-three of 27 (85.1%) subjects had detectable B. dermatitidis antigenuria. In 2 of these 23, positive results were obtained after concentration of the urine specimen. Nine of 11 (81.8%) subjects had detectable B. dermatitidis antigen in serum, including 3 subjects with negative results before treatment of serum with ethylenediaminetetraacetic acid (EDTA) and positive results after EDTA treatment. B. dermatitidis antigen was not detected in specimens from 50 control subjects but was detected in 15 patients with histoplasmosis. B. dermatitidis antigen was detected in most of the patients with blastomycosis and can be a useful tool for timely diagnosis.

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During a Histoplasma outbreak in a colony of fruit bats at a southern United States zoo, it was observed that although Histoplasma was recovered in culture from multiple sites at necropsy, none of the samples collected from those bats tested positive for Histoplasma antigen (HAg). Five of the Histoplasma isolates from the bats were subsequently identified as Latin American (LA) clade A, restriction fragment length polymorphism (RFLP) class 6. These observations raised concern as to whether the commercially available HAg test could detect Histoplasma antigen not of the North American clade upon which the HAg test had been developed. To evaluate this concern, a murine model of disseminated histoplasmosis was established, and mice were infected with multiple LA Histoplasma isolates, including clinical isolates recovered from Brazilian AIDS patients (RFLP class 5 and class 6) and isolates recovered from the bats during the outbreak (RFLP class 6). Histoplasma antigen was detected in all infected mice in our experiments, even when Histoplasma was not recovered in culture. Because the currently available HAg test is able to detect Histoplasma antigen in mice infected with Latin American isolates, this suggests that bat host factors rather than differences among Histoplasma RFLP classes were responsible for the inability to detect HAg in infected bats.

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