Multicenter Validation of Commercial Antigenuria Reagents To Diagnose Progressive Disseminated Histoplasmosis in People Living with HIV/AIDS in Two Latin American Countries.

Histoplasmosis is an important cause of mortality in patients with AIDS, especially in countries with limited access to antiretroviral therapies and diagnostic tests. However, many disseminated infections in Latin America go undiagnosed. A simple, rapid method to detect Histoplasma capsulatum infection in regions where histoplasmosis is endemic would dramatically decrease the time to diagnosis and treatment, reducing morbidity and mortality. The aim of this study was to validate a commercial monoclonal Histoplasma galactomannan (HGM) enzyme-linked immunosorbent assay (Immuno-Mycologics [IMMY], Norman, OK, USA) in two cohorts of people living with HIV/AIDS (PLHIV). We analyzed urine samples from 589 people (466 from Guatemala and 123 from Colombia), including 546 from PLHIV and 43 from non-PLHIV controls. Sixty-three of these people (35 from Guatemala and 28 from Colombia) had confirmed histoplasmosis by isolation of H. capsulatum Using the standard curve provided by the quantitative commercial test, the sensitivity was 98% (95% confidence interval [CI], 95 to 100%) and the specificity was 97% (95% CI, 96 to 99%) (cutoff = 0.5 ng/ml). Semiquantitative results, using a calibrator of 12.5 ng/ml of Histoplasma galactomannan to calculate an enzyme immunoassay index value (EIV) for the samples, showed a sensitivity of 95% (95% CI, 89 to 100%) and a specificity of 98% (95% CI, 96 to 99%) (cutoff ≥ 2.6 EIV). This relatively simple-to-perform commercial antigenuria test showed a high performance with reproducible results in both countries, suggesting that it can be used to detect progressive disseminated histoplasmosis in PLHIV in a wide range of clinical laboratories in countries where histoplasmosis is endemic.

Urine Galactomannan-to-Creatinine Ratio for Detection of Invasive Aspergillosis in Patients with Hematological Malignancies.

Galactomannan (GM) testing of urine specimens may provide important advantages, compared to serum testing, such as easy noninvasive sample collection. We evaluated a total of 632 serial urine samples from 71 patients with underlying hematological malignancies and found that the urine GM/creatinine ratio, i.e., (urine GM level × 100)/urine creatinine level, which takes urine dilution into account, reliably detected invasive aspergillosis and may be a promising diagnostic tool for patients with hematological malignancies. (This study has been registered at ClinicalTrials.gov under registration no. NCT01576653.).

Serum and urine galactomannan testing for screening in patients with hematological malignancies.

Testing for serum galactomannan (GM) has been established as an important method for diagnosing invasive aspergillosis (IA); however, limited data exist regarding the application of urine GM testing. The objective of this study was to evaluate the performance of GM screening of urine specimens and to compare results with serum GM. The study was performed between July 2012 and March 2013 in adult patients with underlying hematological malignancies who were hospitalized at the Medical University of Graz, Austria. Serum and urine screening samples were collected and tested twice weekly (always on the same day). In total, 242 serum samples and a similar number of urine samples were collected from 75 patients. A total of 21/242 (8.7%) serum samples from 13 patients were GM positive. Sensitivity, specificity, positive predictive value, and negative predictive value using a 0.1 optical density index cutoff for urine samples (compared with same-day serum results) were as follows: 47.6%, 86%, 24.4%, and 94.5%, respectively. In 8/10 patients with probable IA, at least one positive GM result was found with this cutoff. After calculating clinical performance of the urine GM test, we found that sensitivity increased to 71.4% and specificity to 88.2%. Spearman-Rho correlation analysis revealed a significant positive correlation between serum and urine samples (P < 0.001; ρ = 0.252). In conclusion, GM detection in urine might be a promising method for IA screening. However, further studies are needed.

[Sero-diagnosis for pulmonary aspergillosis--its utility in early diagnosis].

The clinical utility of Pastorex Aspergillus, a commercially available circulating aspergillus galactomannan antigen detection kit was evaluated. In animal models of pulmonary aspergillosis, it was extremely sensitive for detection of not only A. fumigatus antigen but also antigens of A. flavus and A. niger both in serum and urine. In a preliminary study in autopsied or clinically proven cases with aspergillus infections a high positive rate of antigen detection was obtained when it was applied prospectively. A total of 1,373 clinical samples obtained from patients with suspected aspergillus infection were examined. Among 67 patients giving antigen-positive results in their clinical samples including serum, urine, sputum and others, 17 patients proved to have aspergillus infection by histological or cultural studies. Sixteen patients were judged to be suspicious of aspergillus infection. Measurement of serum (1 --> 3) beta-D glucan by G-test in these cases was well correlated to the results of the antigen detection test. However, the serum showed often antigen-negative even when it was examined at the peak of illness. The rapid clearance of the antigen from circulation was considered to be one of the reasons for this phenomenon. In these cases, antigen detection in samples other than serum, such as urine, sputum, and pleural fluid was also useful. In conclusion, when using Pastorex Aspergillus to obtain the early diagnosis of aspergillus infection frequent examination in different types of samples is recommended.

Vegetable cells in urinary samples of patients with bricker ileal conduit.

During routine cytopathological evaluation of urines for malignant cells we have occasionally noticed vegetable cells that were only present in patients with Bricker ileal conduit. We wanted to identify the means and sources of contamination of urinary samples from these patients. During the period between May and November 2010, 637 urinary samples were routinely evaluated for malignant cells. Among them were 13 urinary samples from Bricker ileal conduit which we rescreened. We prepared all urinary samples by membrane filtration and stained them according to Papanicolaou. Subsequently, we prepared samples from ostomy adhesives made by Coloplast and by ConvaTec which are used to secure the ostomy bag onto urostomy. We also took samples from different constituents (hydrocolloids) of ostomy adhesives. On the cytopathological review, we found vegetable cells along with intestinal mucosa cells in urinary samples of seven patients with Bricker ileal conduit. With the light microscopic examination of the samples prepared from different ostomy adhesives, we found vegetable cells only in Coloplast adhesives. In preparations of hydrocolloids, we found vegetable cells only in guar gum. They were morphologically identical to those found in urine samples of patients with Bricker ileal conduit and in Sensura and Sensura Xpro (Coloplast) ostomy adhesives. We determined that the origin of vegetable cells in urines from Bricker ileal conduit is the ostomy adhesive. The vegetable cells differ from human intestinal epithelial cells regarding size, shape, and color so it is difficult to misinterpret them as dysplastic cells.

Detection of urinary excreted fungal galactomannan-like antigens for diagnosis of invasive aspergillosis.

Mortality associated with invasive aspergillosis (IA) remains high, partly because of delayed diagnosis. Detection of microbial exoantigens, released in serum and other body fluids during infection, may help timely diagnosis. In course of IA, Aspergillus galactomannan (GM), a well established polysaccharide biomarker, is released in body fluids including urine. Urine is an abundant, safely collected specimen, well-suited for point-of-care (POC) testing, which could play an increasing role in screening for early disease. Our main objective was to demonstrate GM antigenuria as a clinically relevant biological phenomenon in IA and establish proof-of-concept that it could be translated to POC diagnosis. Utilizing a novel IgM monoclonal antibody (MAb476) that recognizes GM-like antigens from Aspergillus and other molds, we demonstrated antigenuria in an experimental animal IA model (guinea pig), as well as in human patients. In addition, we investigated the chemical nature of the urinary excreted antigen in human samples, characterized antigen detection in urine by immunoassays, described a putative assay inhibitor in urine, and indicated means of alleviation of the inhibition. We also designed and used a lateral flow immunochromatographic assay to detect urinary excreted antigen in a limited number of IA patient urine samples. In this study, we establish that POC diagnosis of IA based on urinary GM detection is feasible. Prospective studies will be necessary to establish the performance characteristics of an optimized device and define its optimal clinical use.

Sensitivity and specificity of a blood and urine galactomannan antigen assay for diagnosis of systemic aspergillosis in dogs.

BACKGROUND: Diagnosis of canine systemic aspergillosis requires fungal culture from a sterile site, or confirmatory histopathology from a nonsterile site. Invasive specimen collection techniques may be necessary. OBJECTIVE: To evaluate the sensitivity and specificity of a serum and urine Aspergillus galactomannan antigen (GMA) ELISA assay for diagnosis of systemic aspergillosis in dogs. DESIGN: Multicenter study. ANIMALS: Thirteen dogs with systemic aspergillosis and 89 dogs with other diseases. Thirty-seven of the 89 dogs had signs that resembled those of systemic aspergillosis and 52 dogs were not suspected to have aspergillosis. PROCEDURE: The GMA ELISA was performed on serum specimens from all dogs and urine specimens from 67 dogs. Galactomannan indices (GMI) ≥ 0.5 were considered positive. Results for dogs in each group were compared. RESULTS AND CONCLUSIONS: The sensitivity and specificity of the assay for serum were 92 and 86%, respectively, and for urine were 88 and 92%, respectively. False negatives were seen only in dogs with localized pulmonary aspergillosis. Use of a cutoff GMI of 1.5 increased specificity to 93% for both serum and urine without loss of sensitivity for diagnosis of disseminated infection. High-level false positives (> 1.5) occurred in dogs with other systemic mycoses and those treated with Plasmalyte. CLINICAL RELEVANCE: Serum and urine Aspergillus GMA ELISA is a noninvasive, sensitive, and specific test for the diagnosis of disseminated aspergillosis in dogs when a cutoff GMI of ≥ 1.5 is used.

Aspergillus antigenuria compared to antigenemia in bone marrow transplant recipients.

The detection of galactomannan antigen in urine was investigated in 26 bone marrow transplant recipients using an Aspergillus latex agglutination test (Pastorex). After modification of the method, which was originally devised for serum testing, the detection limit in native urine was approximately 20 ng/ml. Antigen was found in 79 (36.4%) of 217 serial urine samples, compared to 40 (11.8%) of 340 serum samples. As a rule, antigenuria preceded antigenemia and was more persistent. The sensitivity, specificity, positive predictive value and negative predictive value of antigenuria for autopsy-proven aspergillosis and clinically suspected Aspergillus infection were 57%, 53%, 31% and 77%, respectively, while those of antigenemia were 43%, 53%, 25% and 71%. It is concluded that urine testing is more reliable than serum testing for the detection of Aspergillus galactomannan. The detection of antigen, however, whether in serum or in urine, allows no clear distinction between Aspergillus infection and exposure to non-infectious Aspergillus antigens.

Galactomannan antigenemia and antigenuria in aspergillosis: studies in patients and experimentally infected rabbits.

Purified galactomannan (GM) from Aspergillus fumigatus was used in both a radioimmunoassay and an enzyme-linked immunoassay for antigen detection. Results of the two tests seemed interchangeable. By one or both assays, GM was detected in serum from four of 12 rabbits lethally infected with A. fumigatus in concentrations ranging from 108 to 356 ng/ml. Serum antigen was detected in only two of 12 patients with invasive aspergillosis. Results of assay for GM in urine were far more encouraging. Urinary GM was detectable throughout the course of lethal aspergillosis in all 16 rabbits, in concentrations of 24-1,900 ng/ml. Urine from seven of 13 patients with invasive aspergillosis had GM concentrations of 1-83 ng/ml. Antigen excretion roughly paralleled extent of disease.

A new sensitive sandwich enzyme-linked immunosorbent assay to detect galactofuran in patients with invasive aspergillosis.

A double-direct sandwich enzyme-linked immunosorbent assay that uses a rat anti-galactomannan monoclonal antibody as the acceptor and detector antibody was designed. This immunoassay, which detects less than 1 ng of galactomannan per ml, was assessed in a retrospective study with samples from patients with invasive aspergillosis. Serum is more appropriate than urine for use in the search for circulating galactomannan. Antigenemia does not have a transient character. Galactomannan can be detected at least 39 days before the death of the patients.

Receptor-mediated clearance of Aspergillus galactomannan.

The fate of radiolabeled Aspergillus fumigatus galactomannan was studied after intravenous injection into rabbits and rats. At 1 hr, the liver contained 35% of the injected dose in rabbits and 30% in rats. Excretion of galactomannan into the urine, measured in rabbits, was another major catabolic route and accounted for 35% of the dose by 24 hr. Immunization of rabbits increased hepatic uptake and decreased urinary excretion. Hepatic uptake in unimmunized rats could be decreased by Saccharomyces cerevisiae mannan, alpha-methyl mannoside, and N-acetylglucosamine, known inhibitors of the macrophage mannose receptor. Autoradiography showed hepatic radiolabeled galactomannan to be concentrated in Kupffer cells, which express the mannosyl receptor for glycoproteins. Macrophage mannosyl receptors may constitute a general mechanism for clearing fungal mannans from the bloodstream.

Biodisposition of FITC-labeled aloemannan in mice.

Biodisposition of FITC-labeled aloemannan (F-AM) with the homogenate from some organs in mice was demonstrated. F-AM was metabolized only by the mucosa from the large intestine into smaller molecules that were effectively absorbed in mice. The homogenate from the other tissues did not affect the metabolism of F-AM. The degraded product (1) of F-AM after incubation with 10% feces homogenate for 24 h was chromatographed on a highly porous polymer and a Sephadex LH-20 column to provide an FITC-degraded fraction (2), which was shown to have a molecular weight of 800 D on Sephadex G-25 gel permeation. Metabolite 2 was examined by physicochemical methods and shown to be a mixture of FITC-hexose and -2 hexose on FAB-MS. An FITC-degraded fraction (3) with a molecular weight of 3 KD was obtained by 6-h incubation with 10% feces homogenate on Sephadex G-25 column chromatography and was shown to be a mixture of FITC-9 and 12 x hexose on TOF-MS.