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Having reported that pretreatment of serum samples with EDTA at 100°C improved the sensitivity for the detection of Histoplasma antigenemia, we have evaluated this method for the detection of Coccidioides antigenemia. Urine and serum samples from patients with coccidioidomycosis were tested using the MVista Coccidioides enzyme immunoassay, and serum samples with and without EDTA-heat treatment were tested. Antigenemia was detected in 28.6% of patients whose samples were not EDTA-heat treated and in 73.1% of those whose samples were treated. Antigenuria was detected in 50% of patients. Specificity of 100% was obtained in healthy subjects, but cross-reactions were seen in 22.2% of patients with histoplasmosis or blastomycosis. EDTA-heat treatment improves the sensitivity for the detection of Coccidioides antigenemia.

Detection of galactomannan antigen in serum or bronchoalveolar lavage fluid is useful for diagnosis of invasive aspergillosis, but the current serologic tests are not useful. (1-->3)-beta-D-glucan can be detected in the serum of patients who have aspergillosis, candidiasis, and a few other mold infections, but false-positive results limit its reliability for diagnosis. None of these methods is useful for diagnosis of zygomycosis and certain other less common mold infections. This article focuses on the detection of antigen and beta-D-glucan for diagnosis of invasive aspergillosis and candidiasis.

The endemic mycoses are often overlooked in the evaluation of community-acquired pneumonia, and appropriate testing is not performed until the patient has failed to improve on antibacterial therapy. Antigen detection and fungal serology may be helpful in diagnosis of endemic mycoses as causes for pneumonia. Guidelines for recognition and evaluation of endemic mycoses as causes for community-acquired pneumonia are proposed. Performance of antigen testing on bronchial washings or lavage fluid may improve the sensitivity for diagnosis over microscopic examination and the speed of diagnosis over culture. This article focuses on antigen detection and serology for diagnosis of endemic mycoses. Of note is that isolation of the fungus by culture remains the only method for definitive diagnosis, and the only method for diagnosis in some patients.

Introduction Endemic mycoses are major causes of morbidity and mortality in the Americas, Africa, and Asia. Coccidioidomycosis and blastomycosis are often overlooked in patients diagnosed with community-acquired pneumonia [1,2]. Patients with acute pulmonary histoplasmosis caused by a high inoculum exposure often present with diffuse pulmonary involvement that may progress to respiratory insuffi ciency [3,4]. Histoplasmosis usually causes progressive disseminated disease in immunosuppressed patients. In each of these mycoses, rapid diagnosis may allow the physician to initiate therapy early, potentially reducing morbidity and even preventing death. Antigen detection can be a sensitive and specifi c method for rapid diagnosis of the endemic mycoses, with a sensitivity of approximately 75% for extensive pulmonary infections and more than 90% for disseminated disease, and a specifi city of approximately 98%. The use of the Histoplasma antigen enzyme immunoassay (EIA) for diagnosis and management of acute pulmonary and progressive disseminated histoplasmosis is well documented [5]. Experience with the Blastomyces antigen EIA is growing and supports a similar role in the management of blastomycosis. A Coccidioides antigen EIA was developed in 2008 (Durkin, unpublished data), offering an additional approach to the rapid diagnosis of coccidioidomycosis. Whereas molecular methods have been described for detection of fungal nucleic acid in specimens from patients with endemic mycoses, superiority over other rapid methods has not been established, and reagents are not commercially available for routine use. Their role in the diagnosis of histoplasmosis is not yet established. This review updates information previously reported concerning the diagnosis of histoplasmosis and blastomycosis and presents new information on coccidioidomycosis. Earlier reviews provide more in-depth discussion of other aspects of fungal antigen detection [6], and other reviews provide information on the use of antigen detection for diagnosis of cryptococcosis, aspergillosis, and candidiasis [6–10].

The second-generation Histoplasma antigen immunoassay is semiquantitative, expressing results as a comparison to a negative control, which requires repeat testing of the prior specimen with the current specimen to accurately determine a change in antigen. Reporting results in this manner often is confusing to the ordering physician and laboratory. Development of a quantitative assay could improve accuracy, reduce interassay variability, and eliminate the need to test the prior sample with the current sample in the same assay. Calibrators with known concentrations of Histoplasma antigen were used to quantitate antigen in specimens from patients with histoplasmosis and from controls. Samples from cases of disseminated histoplasmosis or other mycoses and controls were tested to evaluate the performance characteristics of the quantitative assay. Paired specimens were evaluated to determine if quantitation eliminated the need to test the current and prior specimens in the same assay to assess a change in antigen. The sensitivity in samples from patients with AIDS and disseminated histoplasmosis was 100% in urine and 92.3% in serum. Cross-reactions occurred in 70% of other endemic mycoses, but not in aspergillosis. Specificity was 99% in controls with community-acquired pneumonia, medical conditions in which histoplasmosis was excluded, or healthy subjects. A change in antigen level categorized as an increase, no change, or decrease based on antigen units determined in the same assay agreed closely with the category of change in nanograms/milliliter determined from testing current and prior specimens in different assays. Sensitivity, specificity, and interassay precision are excellent in the new third-generation quantitative Histoplasma antigen immunoassay.

We review the experience at our institution with galactomannan (GM) testing of bronchoalveolar lavage (BAL) fluid in the diagnosis of invasive pulmonary aspergillosis (IPA) among solid-organ transplant recipients. Among 81 patients for whom BAL GM testing was ordered (heart, 24; kidney, 22; liver, 19; lung, 16), there were five cases of proven or probable IPA. All five patients had BAL GM of ≥2.1 and survived following antifungal therapy. The sensitivity, specificity, and positive and negative predictive values for BAL GM testing at a cutoff of ≥1.0 were 100%, 90.8%, 41.7%, and 100%, respectively. The sensitivity of BAL GM testing was better than that of conventional tests such as serum GM or BAL cytology and culture. Moreover, a positive BAL GM test diagnosed IPA several days to 4 weeks before other methods for three patients. Twelve patients had BAL GM of ≥0.5 but no evidence of IPA. Among these, lung transplant recipients accounted for 41.7% (5/12) of the false-positive results, reflecting frequent colonization of airways in this population. Excluding lung transplants, the specificity and positive predictive value for other solid-organ transplants increased to 92.9% and 62.5%, respectively (cutoff, ≥1.0). In conclusion, BAL GM testing facilitated more-rapid diagnoses of IPA and the institution of antifungal therapy among non-lung solid-organ transplant recipients and helped to rule out IPA.

Galactomannans (GalMs) are complex polysaccharides composed of D-galactose and D-mannose. Alkaline-extracted GalMs from Aspergillus fumigatus contain a core of (136)--D-mannopyranosyl residues, with (135)-linked -galactofuranosyl units. The pathogenic fungus Cryptococcus neoformans also contains complex capsular polysaccharides composed of glucuronoxylomannan (GXM) and galactoxylomannans (GalXM). GXM makes up about 90% of the capsular mass and is a polymer of up to six different repeating units that consist mostly of a linear (133)-mannan trisaccharide with side groups consisting of (132)-glucopyranosyluronic acid and (132)- and (134)xylopyranosyl (2, 6). Additionally, the mannan backbone of GXM is modified by acetyl groups. GalXM constitutes about 7% of the capsular mass (2) and has an (136) galactan backbone containing four potential short oligosaccharide branch structures. The branches are 3-O

SummaryAntigen detection is a useful adjunct for the diagnosis of histoplasmosis. The purpose of this study was to evaluate antigen detection in bronchoalveolar lavage (BAL) fluid using an improved second-generation Histoplasma antigen assay. Antigen was detected in 16 of 19 (84%) cases of histoplasmosis and 5 of 6 (83.3%) blastomycosis cases using the second-generation assay vs. 13 of 19 (68%) and 4 of 6 (66.7%), respectively, in the original assay. Ten-fold concentration permitted detection of antigen in an additional case of histoplasmosis and another with blastomycosis, for an overall sensitivity of 23 of 25 (92.0%). Specificity was 98.2% in both assays in controls with other pulmonary infections. These findings support the diagnostic utility of the second-generation assay in patients with pulmonary histoplasmosis and blastomycosis.

Invasive fungal infections are often difficult to diagnose using radiology, culture methods, histopathology, or serology, and turnaround time is often lengthy. Antigen testing has been developed for several important invasive mycoses, offering rapid turnaround time and sensitivity comparable or superior to those of other methods. This review provides an update on rapid antigen testing for several invasive fungal infections, including histoplasmosis, blastomycosis, coccidioidomycosis, and aspergillosis and is intended as an addendum to previous reviews on the subject.

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.

Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Background. The utility of Aspergillus galactomannan (GM) and β-D-glucan (BG) in liver transplant recipients remains uncertain. Methods. As part of a randomized, double-blind trial of antifungal prophylaxis in liver transplant recipients at risk for invasive fungal infections (IFIs), GM and BG were assessed in 199 patients at baseline (enrollment) and weekly thereafter for the duration of study drug. Receiver operating characteristic (ROC) analysis was used to evaluate the accuracy of these for the diagnosis of IFIs. Results. Overall, 46.4% of the patients at baseline had positiveGM(index ≥ 0.5) and 89.6%had BG of 80 pg/mL or greater with BG level of 500 pg/mL or greater in 31.8%. Patients with invasive aspergillosis (IA) (3/3) had positive GM at baseline as did 45.5% of those without IA (P = 0.098); the area under the ROC curve for the diagnosis of IA was 0.77 (fair test, ie, good sensitivity but poor specificity). Using BG cutoff of 80 pg/mL or higher, 100%(12/12) of the patients with IFI had positive baseline BGand as did 88.9% (160/180) of those without IFI (P = 0.618); the area under the ROC curve for predicting IFIs was 0.56 (poor test). In multivariate analyses, GM positivity was associated with study site (P = 0.041), and BG positivity with renal replacement therapy (P = 0.05) and study site (P = 0.01). The GM and BG levels declined over time; positivity at subsequent time points was lower in comparison with baseline (P < 0.001). Conclusions. The GM and BG tests had significant center variability and limited accuracy for the diagnosis of IFIs in high-risk liver transplant recipients.

To better understand clinical and epidemiologic patterns of blastomycosis, we report on a large series of blastomycosis in Indiana.All microbiologically and histopathologically confirmed cases of blastomycosis from four hospitals serving Central Indiana from 1985 to 2014 were identified. Available data were collected. Data on population estimates, annual precipitation, and construction in Indiana were evaluated for correlations with incidence rates of blastomycosis.A total of 114 patients were identified. The mean age was 44.4 years; 27% had diabetes mellitus, and 16% were immunosuppressed. Most presented with pneumonia (90%); 48% had extrapulmonary disease (CNS involvement in 9%), and 15% developed ARDS. Cultures, cytopathology, and histopathology were positive in 86%, 27%, and 85% of the sample, respectively, and fungal antigen was positive in 76%. Amphotericin B was administered in 49%, and 87% received an azole. Total mortality was 12%. Immunosuppression (OR = 3.0), diabetes mellitus (OR = 2.9), and multilobar pneumonia (OR = 2.9) were associated with increased likelihood of ICU admission. There was a significant increase in incidence over time in Marion County. There was no correlation with amount of precipitation, but the rise in incidence coincided with a 2005 state initiative to expand Indiana's highway infrastructure.The incidence of blastomycosis in Central Indiana may be on the rise. Physicians in endemic areas should be aware of the potentially fulminant consequences of the disease.

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 micro-foci 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.

Objectives: To determine the activity of newer triazoles against strains of Histoplasma capsulatum resistant to fluconazole. Methods: Susceptibility testing was performed on 17 paired pre- and post-treatment H. capsulatum isolates from patients with AIDS who failed fluconazole. Results: The median MICs of fluconazole, voriconazole, and posaconazole and ravuconazole for the pre-treatment isolates were 1 mg/L, 0.015 mg/L and

We developed a colorimetric microtiter plate polymerase chain reaction enzyme immunoassay (PCR-EIA) for the detection of Histoplasma capsulatum in urine. The specificity of the PCR assay was confirmed using H. capsulatum (positive control) and Blastomyces dermatitidis (negative control) isolates. The analytical sensitivity of the PCR assay was determined by testing urine samples spiked with freshly grown H. capsulatum organisms and was 2 yeasts per reaction in urine and 0.2 yeasts per reaction in urine sediment after centrifugation. Fifty-one urine specimens positive for H. capsulatum antigen and 25 urine specimens from healthy volunteers were tested blindly. Patient specimens also were cultured for H. capsulatum. The PCR assay was positive in 4 (7.8%) of 51 urine specimens containing antigen and negative in urine specimens from healthy volunteers. The positive PCR results occurred in 4 of 5 urine specimens that were positive by culture, and each exhibited high level of antigenuria (>20 U). Urine cultures were not positive in 24 urine specimens with an antigenuria of 1-19.9 U, but were positive in 5 of 27 urine specimens with antigenuria >20 U. Thus, positive PCR results in urine specimens correlate with positive culture results, but not with antigenuria. The low sensitivity of this PCR assay in urine limits its use in the diagnosis of disseminated histoplasmosis.

Until recently, the diagnosis of invasive aspergillosis was most often made based on clinical and radiologic findings, which areinsensitive and non-specific.The Platelia Aspergillus galactomannan enzyme immunoassay, which has been available in Europe for nearly a decade and in the United States since 2003, provides an aid to the diagnosis of invasive aspergillosis. Part I of this article described the test methodology and those factors that may affect test performance. Part II of this article discusses guide lines and indications for performing the test and comparison of the galactomannan assay with other rapid methods.

Pulmonary manifestations are the hallmark of histoplasmosis. Clinical syndromes range from asymptomatic infection to diffuse alveolar disease causing respiratory difficulty and even death. Serologic tests for antibodies and antigen detection are especially helpful in the diagnosis of histoplasmosis but are frequently overlooked. Detection of Histoplasma capsulatum antigen in bronchoalveolar lavage fluid may be particularly helpful in patients with acute pulmonary histoplasmosis or disseminated disease with pulmonary involvement. Topics of special importance for pulmonary disease specialists include the approach to the exclusion of histoplasmosis in the evaluation of patients with suspected sarcoidosis, differentiation of pulmonary histoplasmosis and malignancy in those with lung masses or mediastinal lymphadenopathy, and recognition and management of chronic pulmonary and mediastinal manifestations of histoplasmosis. Although histoplasmosis is mild and self-limited in most healthy individuals, antifungal therapy is indicated in those with acute diffuse pulmonary infection, chronic pulmonary histoplasmosis, progressive disseminated disease, and perhaps mediastinal adenitis accompanied by obstructive symptoms. Antifungal therapy to prevent reactivation of histoplasmosis during immunosuppressive therapy, or transition of mediastinal adenitis to fibrosing mediastinitis, although controversial, is not recommended. Several new drugs active against H. capsulatum offer alternatives in patients failing or intolerant of current therapies.

Anti-tumor necrosis factor-alpha (TNF-alpha) antibodies are frequently used to treat inflammatory diseases. However, these drugs also have immunosuppressive effects. We report on three patients who developed disseminated histoplasmosis on therapy with TNF-alpha inhibitors. In vitro assays were used to characterize the role of these agents in host defense against Histoplasma capsulatum. Intracellular proliferation of H. capsulatum was measured in alveolar macrophages and peripheral monocytes of normal volunteers in the presence and absence of the TNF-alpha antibody, infliximab. Both infliximab and control antibody enhanced fungal growth in monocytes and alveolar macrophages, suggesting this was a nonspecific antibody response. Despite similar intracellular fungal loads in the presence of both antibodies, lymphocyte proliferation in response to blood monocytes and alveolar macrophages infected with H. capsulatum was inhibited by the addition of physiologic doses of infliximab, whereas control antibody had no effect. The production of H. capsulatum-induced interferon-gamma and TNF-alpha was assessed in 5-day cultures containing lymphocytes and alveolar macrophages or monocytes. Interferon-gamma secretion was significantly reduced in the presence of infliximab. In summary, patients receiving anti-TNF-alpha therapy are at risk for developing disseminated histoplasmosis. This may be due to a defect in the TH1 arm of cellular immunity.

Demographic and clinical parameters among patients with acquired immunodeficiency syndrome and histoplasmosis in Brazil and United States were compared. The Brazilian isolates were typed by restriction-fragment length polymorphism analysis and were DNA fingerprinted by random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR). Skin lesions occurred in 66% of Brazilian case patients, compared with 1%-7% of US case patients. Of 21 treated case patients, 4 (19%) died, a rate similar to that of the US case patients (5%-13%). By nuclear gene typing, the Brazilian isolates were equally divided between South American classes 5 and 6, and RAPD-PCR showed 18 distinct genetic fingerprints in 20 isolates. Skin lesions are more common in infection with class 5 or 6 organisms than with class 2 Histoplasma capsulatum. The role of genetic differences in the organism as a cause for the clinical differences requires investigation.

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.

Pneumonia with pleuritis is a rare presentation of histoplasmosis infection. We present a 12-year old previously healthy boy in whom histoplasmosis presented with pleuritis, confirmed by detection of antigen in empyema fluid.

Prompt diagnosis and treatment of fungal meningitis are critical, but culture is insensitive. (1,3)-β- 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.

Infective endocarditis is an uncommon manifestation of infection with Histoplasma capsulatum. The diagnosis is frequently missed, and outcomes historically have been poor. We present 14 cases of Histoplasma endocarditis seen in the last decade at medical centers throughout the United States. All patients were men, and 10 of the 14 had an infected prosthetic aortic valve. One patient had an infected left atrial myxoma. Symptoms were present a median of 7 weeks before the diagnosis was established. Blood cultures yielded H. capsulatum in only 6 (43%) patients. Histoplasma antigen was present in urine and/or serum in all but 3 of the patients and provided the first clue to the diagnosis of histoplasmosis for several patients. Antibody testing was positive for H. capsulatum in 6 of 8 patients in whom the test was performed. Eleven patients underwent surgery for valve replacement or myxoma removal. Large, friable vegetations were noted at surgery in most patients, confirming the preoperative transesophageal echocardiography findings. Histopathologic examination of valve tissue and the myxoma revealed granulomatous inflammation and large numbers of organisms in most specimens. Four of the excised valves and the atrial myxoma showed a mixture of both yeast and hyphal forms on histopathology. A lipid formulation of amphotericin B, administered for a median of 29 days, was the initial therapy in 11 of the 14 patients. This was followed by oral itraconazole therapy, in all but 2 patients. The length of itraconazole suppressive therapy ranged from 11 months to lifelong administration. Three patients (21%) died within 3 months of the date of diagnosis. All 3 deaths were in patients who had received either no or minimal (1 day and 1 week) amphotericin B.

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.

In sequential clinical trials of treatment for histoplasmosis in patients with acquired immunodeficiency syndrome, therapy with fluconazole failed in a higher proportion of patients than did therapy with itraconazole. To determine the cause for failure with fluconazole, antifungal susceptibility testing that used modified National Committee on Clinical Laboratory Standards procedures was performed on all baseline and failure isolates. Failure occurred more frequently in patients with baseline isolates with fluconazole minimum inhibitory concentrations (MICs) > or =5 microg/mL versus lower MICs; 29% versus 3%, respectively. There was at least a 4-fold increase in fluconazole MIC in the isolates from 10 (59%) of 17 patients for whom paired pretreatment and failure or relapse isolates were available. Cross-resistance to itraconazole was not seen. In conclusion, fluconazole is less active than itraconazole for Histoplasma capsulatum and induces resistance during therapy, which accounted for treatment failure in some patients.

Objective The objective of this study was to determine whether a triple therapy regimen incorporating twice-daily saquinavir is as effective as a three-times daily regimen. Methods This was an open-label, Phase III, multicentre, 48-week study involving 837 HIV-1-infected patients randomised to one of the following: saquinavir soft gel capsule (SGC) 1200 mg three-times daily, plus two nucleoside reverse transcriptase inhibitors (NRTIs) (arm A); saquinavir SGC 1600 mg twice-daily, plus two NRTIs (arm B); saquinavir SGC 1200 mg twice-daily and nelfinavir 1250 mg twice-daily, plus a single NRTI (arm C). The primary outcome measure was the virological response in arm A versus B and in arm A versus C with respect to the percentage of patients whose plasma HIV-1 RNA levels fell below the level of quantification for the Amplicor assay (Results At 48 weeks, the percentage of patients with plasma HIV-1 RNA levels Conclusions A twice-daily triple therapy regimen incorporating saquinavir SGC plus two NRTIs was of equivalent efficacy to the three-times daily regimen studied. All regimens were generally well tolerated.

The nucleotide sequences of internal transcribed spacer (ITS) regions of rRNA genes of 24 isolates of Histoplasma capsulatum were examined. The results indicate that the sequences of ITS regions in different isolates are not identical. Sequence variations were found at 20 positions in the 496 bp that were sequenced. Ten different sequence patterns, designated types A through H, were observed when the sequences from the 24 isolates were aligned. Twelve isolates from Indianapolis were classified into four different types. Two isolates from New York belonged to type G. Three isolates from different cities were type F. The remaining six isolates were of different types.

We describe a 14-year-old girl with hyperimmunoglobulin E (Job) syndrome who presented with fatigue, abdominal pain, fever, and weight loss. Endoscopic examination of the terminal ileum revealed ulceration, edema, and erythema. Histopathologic findings of the terminal ileum demonstrated intracellular yeast forms compatible with Histoplasma capsulatum. The patient was treated with oral itraconazole and had a rapid and complete response.

Histoplasmosis is a common endemic mycosis in the Americas, often causing severe disease in patients with AIDS. Antigen detection has become an important method for rapid diagnosis of histoplasmosis in the United States but not in Central or South America. Isolates from patients in the United States are predominantly found to be class 2 isolates when typed using the nuclear gene YPS3, while isolates from Latin America are predominantly typed as class 5 or class 6. Whether infection with these Latin American genotypes produces positive results in the Histoplasma antigen assay has not been reported. In this study, we have compared the sensitivity of antigen detection for AIDS patients from Panama who had progressive disseminated histoplasmosis to that for those in the United States. Antigenuria was detected in the MVista Histoplasma antigen enzyme immunoassay (EIA) in 95.2% of Panamanian cases versus 100% of U.S. cases. Antigenemia was detected in 94.7% of the Panamanian cases versus 92% of the U.S. cases. Two clinical isolates from Panama were typed using YPS3 and were found to be restriction fragment length polymorphism class 6. We conclude that the MVista Histoplasma antigen EIA is a sensitive method for diagnosis of histoplasmosis in Panama. Progressive disseminated histoplasmosis (PDH) is a common and serious opportunistic disease among patients with AIDS in the United States and Latin America. In Panama, PDH occurred in 8% of hospitalized patients from 1997 to 2003 and was fatal in 12% of cases (6). The clinical picture was similar to that reported elsewhere and usually included fever, weight loss, and respiratory symptoms, often in association with diarrhea, as well as hepatosplenomegaly, hepatic enzyme elevation, and pancytopenia. Skin lesions were noted in 17% of the Panamanian cases (6), lower than the observed rate for cases from Brazil (66%) but higher than that for cases from the United States (3%) (7). Disease in patients in Mexico and Latin America has been described as being caused by isolates of Histoplasma capsulatum var. capsulatum that exhibit distinct genetic profiles in comparison to those from the United States (3, 8, 9). Keath et al. (9) first described typing based upon restriction fragment length polymorphism (RFLP) in the YPS3 gene. They showed that Panamanian strains were typed as YPS3 class 3, 5, or 6, while the North American strains were predominantly class 2. Later, Kasuga et al. used the DNA sequence substitution rates in four independent protein-coding genes to identify at least eight different genotypes, or clades (8). North American class 2 was the predominant genotype in the United States and Latin American group A in Latin America. Latin American group B was found only in Columbia and Argentina. Based on only four isolates from Panama included in the study by Kasuga et al., Panamanian strains included Latin American group A and a lone lineage, designated H81. The diagnosis of PDH relies on demonstration of the organism in clinical specimens or detection of antigen in body fluids (2). Histopathology may be falsely negative in up to 50% of patients, caused by sampling error, paucity of organisms, or inexperience of the pathologist. Culture may require several weeks to isolate and identify the organism and may be falsely negative for 15% of patients (13). Antigenuria can be detected in 95% of cases in patients with AIDS in the United States (4, 13) but has not been evaluated in Latin America. Whether genetic differences in Latin American isolates would affect the sensitivity for diagnosis by antigen detection is unknown. The prevalence of PDH in AIDS patients from Panama offered the opportunity to address this question.

We evaluated the Fungitell beta-glucan (BG) test with specimens from patients with presumed histoplasmosis. The sensitivity of the test was 87% in histoplasmosis cases and had a specificity of 68% with controls. Histoplasmosis should be considered as a possible cause for a positive BG test, but such results may be found with many other conditions that are clinically similar to this fungal disease. Therefore, there is a need to conduct confirmatory tests for histoplasmosis in the appropriate clinical setting.

We observed false-positive results in the Platelia Aspergillus enzyme-linked immunoassay (EIA) for specimens from patients with histoplasmosis and mice with experimental infection. Platelia Aspergillus EIA-positive specimens were negative in the second-generation Histoplasma antigen EIA. Care must be taken to exclude histoplasmosis for patients with positive Platelia Aspergillus EIA results.

Although disseminated blastomycosis is a rare complication in pregnancy, delay in diagnosis and treatment can be fatal. We investigate the use of the Blastomyces urine antigen in diagnosis following disease progression in the intrapartum, postpartum, and neonatal periods. We describe a case of disseminated blastomycosis in a pregnant adolescent and review the pertinent literature regarding treatment and monitoring blastomycosis in pregnancy and the neonatal periods. This is the first reported case in which the Blastomyces urine antigen is utilized as a method of following disease activity during pregnancy confirming absence of clinically evident disease in a neonate. Urine antigen detection for blastomycosis can be useful for following progression of disease in patients with disseminated blastomycosis in both the intrapartum and postpartum periods.

Purpose: Oropharyngeal candidasis (thrush) is the most common opportunistic infection in individuals who are positive for the human immunodeficiency virus (HIV) and those who have progressed to AIDS. Itraconazole has a broad in vitro spectrum of activity, including a wide variety of Candida species. Our study determined the relative efficacy of a new oral solution formulation of itraconazole and fluconazole tablets in the treatment of oropharyngeal candidiasis. Patients and Methods: This was a prospective randomized, third-party–blind, multicenter trial conducted at 12 centers in the United States. One hundred seventy-nine HIV-positive patients with mycologically documented oropharyngeal candidiasis were treated with itraconazole oral solution 200 mg/day for 7 or 14 days, or fluconazole tablets 100 mg/day for 14 days. Severity of disease was scored clinically before treatment and at clinical evaluations on days 3, 7, 14, 21, 35, and 42. Semiquantitative cultures of mouth washings were also obtained on these days. Results: Both 14-day and 7-day regimens of itraconazole oral solution were equivalent to fluconazole for most efficacy parameters. The clinical response rate was 97% after 14 days of itraconazole and 87% after 14 days of fluconazole. Itraconazole oral solution given for 7 days was also equivalent to fluconazole treatment for 14 days. Approximately one half of patients in all three groups relapsed by 1 month after completion of treatment. There were few adverse reactions to either drug. Conclusion: Itraconazole oral solution is well tolerated and offers an alternative at least as effective as fluconazole in the treatment of oropharyngeal candidiasis.

Blastomycosis is an uncommonly recognized disease in pediatric patients. We describe 4 cases of pediatric blastomycosis that presented to our children's hospital, 2 with isolated pulmonary blastomycosis and 2 with disseminated blastomycosis. Because of variable clinical presentations and morbidity if treatment is delayed, physicians must maintain a high index of suspicion and obtain appropriate diagnostic tests promptly. For the first time, we report the effect of therapy on Blastomyces antigen clearance. In our experience, the urine antigen detection for B. dermatitidis is useful for diagnosis and follow up during therapy.

To improve our understanding of risk factors, management, diagnosis, and outcomes associated with histoplasmosis after solid organ transplant (SOT), we report a large series of histoplasmosis occurring after SOT.All cases of histoplasmosis in SOT recipients diagnosed between 1 January 2003 and 31 December 2010 at 24 institutions were identified. Demographic, clinical, and laboratory data were collected.One hundred fifty-two cases were identified: kidney (51%), liver (16%), kidney/pancreas (14%), heart (9%), lung (5%), pancreas (2%), and other (2%). The median time from transplant to diagnosis was 27 months, but 34% were diagnosed in the first year after transplant. Twenty-eight percent of patients had severe disease (requiring intensive care unit admission); 81% had disseminated disease. Urine Histoplasma antigen detection was the most sensitive diagnostic method, positive in 132 of 142 patients (93%). An amphotericin formulation was administered initially to 73% of patients for a median duration of 2 weeks; step-down therapy with an azole was continued for a median duration of 12 months. Ten percent of patients died due to histoplasmosis with 72% of deaths occurring in the first month after diagnosis; older age and severe disease were risk factors for death from histoplasmosis. Relapse occurred in 6% of patients.Although late cases occur, the first year after SOT is the period of highest risk for histoplasmosis. In patients who survive the first month after diagnosis, treatment with an amphotericin formulation followed by an azole for 12 months is usually successful, with only rare relapse.

Background Central nervous system (CNS) histoplasmosis is a life-threatening condition, and represents a diagnostic and therapeutic challenge. Although CSF (1,3)-β-d-glucan (CSF BDG) is available as a biologic marker for diagnosis of fungal meningitis, there are limited data on its use for diagnosis of Histoplasma meningitis. We evaluated CSF BDG detection using the Fungitell assay in patients with CNS histoplasmosis and controls. Methods Patients were classified as cases if there was CNS inflammation (CSF WBC ≥ 5 mm3/ml) plus laboratory confirmation of H. capsulatumin CNS samples or from extra-CNS sites with no alternative etiology for CSF pleocytosis. Controls were patients with histoplasmosis but no evidence of CNS involvement, an alternative diagnosis, or other fungal meningitis. Results In total, 47 cases and 153 controls were evaluated (Table 1). Forty-nine percent of patients with CNS histoplasmosis and 43.8% of controls were immunocompromised. CSF BDG was positive in 25 (53.2%) cases using a level of ≥80 pg/ml, the median CSF BDG level was 140.5 pg/ml (range from Conclusion A positive CSF BDG supports the diagnosis of fungal meningitis but cannot distinguish among the different etiologies. The sensitivity and specificity of detection of CSF BDG was lower than that of antigen detection. Disclosures L. J. Wheat, MiraVista: President and owner, equity and Salary

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, P0.001. 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.

Donor-derived fungal infections can be associated with serious complications in transplant recipients. Most cases of donor-derived candidiasis have occurred in kidney transplant recipients in whom contaminated preservation fluid is a commonly proposed source. Donors with cryptococcal disease, including those with unrecognized cryptococcal meningoencephalitis may transmit the infection with the allograft. Active histoplasmosis or undiagnosed and presumably asymptomatic infection in the donor that had not resolved by the time of death can result in donor-derived histoplasmosis in the recipient. Potential donors from an endemic area with either active or occult infection can also transmit coccidioidomycosis. Rare instances of aspergillosis and other mycoses, including agents of mucormycosis may also be transmitted from infected donors. Appropriate diagnostic evaluation and prompt initiation of appropriate antifungal therapy are warranted if donor-derived fungal infections are a consideration. This document discusses the characteristics, evaluation and approach to the management of donor-derived fungal infections in organ transplant recipients.

Diagnosis of Histoplasma capsulatum infection in cats traditionally relies upon identification of organisms in circulating monocytes or in tissue specimens from affected organs. In this retrospective study, results of a urine antigen assay were compared with standard diagnostic methods in cats with clinical signs suggestive of histoplasmosis. Antigenuria was detected in 17/18 cats with a histopathologic or cytopathologic diagnosis of histoplasmosis. This preliminary evaluation of the Histoplasma urine antigen test suggests it may be a useful aid in diagnosing this disease in cats.

The second-generation MVista Blastomyces antigen enzyme immunoassay was not quantitative; therefore, specimens obtained previously were tested in the same assay as new specimens to assess the change in antigen levels. Furthermore, the sensitivity in serum had not been fully evaluated. The purpose of this study was to evaluate a quantitative Blastomyces antigen assay and detection of antigen in serum. Calibrators containing known concentrations of Blastomyces galactomannan were used to quantify antigen in urine and serum from patients with proven blastomycosis and from controls. Paired current and previously obtained urine specimens were tested to determine if quantification eliminated the need for concurrent testing to assess change in antigen. Pretreatment of serum with EDTA at 104°C was evaluated to determine if dissociation of immune complexes improved detection of antigenemia. Antigenuria was detected in 89.9% of patients with culture- or histopathology-proven blastomycosis. Specificity was 99.0% in patients with nonfungal infections and healthy subjects, but cross-reactions occurred in 95.6% of patients with histoplasmosis. Change in antigen level categorized as increase, no change, or decrease based on antigen units determined in the same assay agreed closely with the category of change in ng/ml determined from different assays. Pretreatment increased the sensitivity of detection of antigenemia from 35.7% to 57.1%. Quantification eliminated the need for concurrent testing of current and previously obtained specimens for assessment of changes in antigen concentration. Pretreatment increased the sensitivity for detection of antigenemia. Differentiation of histoplasmosis and blastomycosis is not possible by antigen detection.

It is well known that cross reactions with other fungal pathogens including Histoplasma capsulatum can occur with the use of the Platelia™ Aspergillus galactomannan assay. We report two patients with confirmed blastomycosis whose bronchoalveolar lavage (BAL) fluid tested positive for Aspergillus galactomannan despite no evidence of aspergillosis.

Sporotrichosis is considered a “global health problem” in immunocompromised patients (7). Although Sporothrix schenckii is the main human pathogen, genetic sequencing indicates that the S. schenckii complex is comprised of several species: S. albicans, S. brasiliensis, S. globosa, S. luriei, S. mexicana, and S. schencki (7, 8). The diagnosis of sporotrichosis can be suspected by demonstration of small yeast cells (3 to 5 m) with multiple buds, some of which are cigar-shaped or show large spherical bodies (Fig. 1A). While serological diagnosis by detection of antibodies to mycelial antigens has been described, cross-reactions occurred in about one-quarter of patients with histoplasmosis (1). Antigen detection has proven useful for diagnosis of other fungal infections but has not been reported in sporotrichosis. Cross-reactivity in the MVista Histoplasma antigen enzyme immunoassay (EIA) occurs in 70 to 90% of patients with blastomycosis, paracoccidioidomycosis, and penicilliosis marneffei (2) and 59% of patients with coccidioidomycosis (5). Herein, cross-reactivity is reported in a patient with sporotrichosis. A 34-year-old African-American male from Amarillo, Texas, with untreated AIDS was admitted with a fluctuating of level of consciousness. He had complained of dysphagia, but additional information, including epidemiological history, was not obtainable because of impaired mental status. Examination showed extensive ulcers and nodules on the oral mucosa, face, hands, and chest and impaired mental status. The CD4 count was 11 cells/ml, and the HIV viral load was 110,911 copies/ml. Tests for anti-Blastomyces and anti-Coccidioides antibodies and cryptococcal antigenemia were negative, but Histoplasma antigenuria was detected at 1.7 U (reference range, below 1.0 U). Skin biopsy showed yeast cells, some with multiple buds and spherical bodies (Fig. 1B), and Sporothrix was isolated by culture from the skin and blood. Identification was based upon using lactophenol cotton blue staining of colonies showing narrow hyphae with slender tapering conidiophores at right angles to the hyphae and tear-shaped conidia arranged in rosette-like clusters at the apex of the conidiophores, as well as conversion of the mold to yeast with typical cigar bodies at 37°C. The patient was treated with amphotericin B based upon the skin biopsy findings, but developed multiorgan failure and died after 6 days of treatment. Repeat blood cultures were negative after 24 h of antifungal therapy. The antigen detected in specimens from patients with histo