Two Monoclonal Antibodies That Specifically Recognize Aspergillus Cell Wall Antigens and Can Detect Circulating Antigens in Infected Mice.

Invasive aspergillosis (IA) is a life-threatening disease mainly caused by Aspergillus fumigatus and Aspergillus flavus. Early diagnosis of this condition is crucial for patient treatment and survival. As current diagnostic techniques for IA lack sufficient accuracy, we have raised two monoclonal antibodies (1D2 and 4E4) against A. fumigatus cell wall fragments that may provide a platform for a new diagnostic approach. The immunoreactivity of these antibodies was tested by immunofluorescence and ELISA against various Aspergillus and Candida species in vitro and by immunohistochemistry in A. fumigatus infected mouse tissues. Both monoclonal antibodies (mAbs) showed intensive fluorescence with the hyphae wall of A. fumigatus and A. flavus, but there was no staining with other Aspergillus species or Candida species. Both mAbs also showed strong immunoreactivity to the cell wall of A. fumigatus hyphae in the infected liver, spleen and kidney of mice with IA. The antigens identified by 1D2 and 4E4 might be glycoproteins and the epitopes are most likely a protein or peptide rather than a carbohydrate. An antibody-based antigen capture ELISA detected the extracellular antigens released by A. fumigatus, A. flavus, A. niger and A. terreus, but not in Candida species. The antigen could be detected in the plasma of mice after 48 h of infection by double-sandwich ELISA. In conclusion, both 1D2 and 4E4 mAbs are potentially promising diagnostic tools to investigate invasive aspergillosis.

Rapid detection of the aspergillosis biomarker triacetylfusarinine C using interference-enhanced Raman spectroscopy.

Triacetylfusarinine C (TAFC) is a siderophore produced by certain fungal species and might serve as a highly useful biomarker for the fast diagnosis of invasive aspergillosis. Due to its renal elimination, the biomarker is found in urine samples of patients suffering from Aspergillus infections. Accordingly, non-invasive diagnosis from this easily obtainable body fluid is possible. Within our contribution, we demonstrate how Raman microspectroscopy enables a sensitive and specific detection of TAFC. We characterized the TAFC iron complex and its iron-free form using conventional and interference-enhanced Raman spectroscopy (IERS) and compared the spectra with the related compound ferrioxamine B, which is produced by bacterial species. Even though IERS only offers a moderate enhancement of the Raman signal, the employment of respective substrates allowed lowering the detection limit to reach the clinically relevant range. The achieved limit of detection using IERS was 0.5 ng of TAFC, which is already well within the clinically relevant range. By using an extraction protocol, we were able to detect 1.4 µg/mL TAFC via IERS from urine within less than 3 h including sample preparation and data analysis. We could further show that TAFC and ferrioxamine B can be clearly distinguished by means of their Raman spectra even in very low concentrations.

Triacetylfusarinine C: A urine biomarker for diagnosis of invasive aspergillosis.

OBJECTIVES: Early diagnosis of invasive aspergillosis (IA) remains challenging, with available diagnostics being limited by inadequate sensitivities and specificities. Triacetylfusarinine C, a fungal siderophore that has been shown to accumulate in urine in animal models, is a potential new biomarker for diagnosis of IA. METHODS: We developed a method allowing absolute and matrix-independent mass spectrometric quantification of TAFC. Urine TAFC, normalized to creatinine, was determined in 44 samples from 24 patients with underlying hematologic malignancies and probable, possible or no IA according to current EORTC/MSG criteria and compared to other established biomarkers measured in urine and same-day blood samples. RESULTS: TAFC/creatinine sensitivity, specificity, positive and negative likelihood ratio for probable versus no IA (cut-off ≥ 3) were 0.86, 0.88, 6.86, 0.16 per patient. CONCLUSION: For the first time, we provide proof for the occurrence of TAFC in human urine. TAFC/creatinine index determination in urine showed promising results for diagnosis of IA offering the advantages of non-invasive sampling. Sensitivity and specificity were similar as reported for GM determination in serum and bronchoalveolar lavage, the gold standard mycological criterion for IA diagnosis.

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.).

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.

[Mycotoxin research in humans]. / Investigación del efecto de las micotoxinas en el ser humano.

This study investigates the occurrence of aflatoxins in Ecuador. Early investigators proved the presence of aflatoxins in human and animal food, but the disturbing data lead to the formation of two research teams at Guayaquil University and the Agrarian University of Ecuador to investigate aflaxotins and other mycotoxins in food and their relationship to human health. Because the concept of mycotoxicosis as a result of the secondary metabolites produced by different species of moulds could cause different clinical patterns, the research team includes Aspergillus metabolites found in the urine of a patient with pulmonary aspergilloma. We considered that the body itself could create secondary metabolites. An ELISA method was used to detect mycotoxins with the specific reactive compounds using a company base assay. This allows the detection quantitative of such metabolites in 24 h collected urine. The patient was treated with itraconazole for nine months, after clinical, radiological and aflatoxins testing. We also investigated three other cases in children with a second level of malnutrition and only with vomitoxins results and in three investigated cases of otomycosis caused by Aspergillus niger only in one case traces of aflatoxins were found.

Investigación del efecto de las micotoxinas en el ser humano / Micotoxins research in humans

El propósito de esta investigación es estudiar la presencia de las aflatoxinasen Ecuador. El escaso conocimiento en nuestro país a este respecto,nos llevó a un equipo de investigadores de las universidades de Guayaquil yAgraria del Ecuador, a realizar un proyecto de investigación, titulado“Las aflatoxinas y otras micotoxinas en los alimentos y su relación con lasalud humana en nuestro medio”. Siendo la micotoxicosis aquella intoxicaciónresultante de la ingesta de los metabolitos secundarios producidos pordiferentes especies de mohos que generan diferentes cuadros clínicos,¿por qué no investigar metabolitos de Aspergillus en la orina de una pacientecon aspergiloma pulmonar? Consideramos que el propio organismo podríagenerar metabolitos secundarios, por lo que incluimos en este estudio nuestrocaso de aspergiloma pulmonar comprobado. La determinación de lasmicotoxinas se realizó por ELISA, utilizando reactivos específicos de NeogenCorporation, que permiten determinaciones cuantitativas en orina. Se instauróa la paciente tratamiento con itraconazol durante nueve meses, realizandocontroles clínicos, radiológicos y determinación de aflatoxinas. Se estudiaron,también, tres casos de niños con desnutrición en grado III, detectándoseúnicamente la presencia de vomitoxina. Además, se encontraron trazas deaflatoxinas en uno de tres casos de otomicosis por Aspergillus niger(AU)

Micotoxins research in humansThis study investigates the occurrence of aflatoxines in Ecuador. Earlyinvestigators proved the presence of aflatoxins on human and animal food, butthe disturbing data lead to the formation of two research teams at GuayaquilUniversity and the Agrarian University of Ecuador to investigate aflaxotins andother mycotoxins on food and its relationship to human health. Because theconcept of mycotoxicosis as a result of the secondary metabolites producedby different species of moulds could cause different clinical patterns, theresearch team includes Aspergillus metabolites found in the urine for a patientwith pulmonary aspergilloma. We considered that the body itself could createsecondary metabolites. An ELISA method was used to detect mycotoxins withthe specific reactive compounds using a company base assay. This allows thedetection quantitative of such metabolites in 24 h collected urine. The patientwas treated with itraconazol for nine months, after clinical, radiological andaflatoxins testing. We also investigated three other cases in children with asecond level of malnutrition and only with vomitoxins results and on threeinvestigated cases of otomycosis caused by Aspergillus niger only in one casetraces of aflatoxins was found(AU)

Isolated renoureteric aspergilloma due to Aspergillus flavus: case report and review of the literature.

In this paper we describe a case in which acute renal colic was associated with elimination of multiple hyphal masses of Aspergillus flavus. Also, we reviewed the literature on similar cases and we found a similar pattern characterized by a marked male predominance, association with at least one underlying medical condition that predisposes to fungal infection, the presence of local symptoms resembling acute ureteral colic, and the absence of systemic manifestations. Moreover, our data suggest that Aspergillus balls must be suspected when a diabetic and intravenous drug user presents with acute renal colic and that non-obstructive renal aspergillosis may be initially treated with itraconazole.

Chronic necrotizing pulmonary aspergillosis with concomitant aspergilluria.

This paper reports a case of chronic necrotizing pulmonary aspergillosis in a patient without underlying disease. Aspergilluria was the starting point in the search for the origin of the pulmonary disease, later confirmed by an open lung biopsy.

Secretion of a potential virulence factor, a fungal ribonucleotoxin, during human aspergillosis infections.

We show by cloning and nucleotide sequence analysis that the 18kDa antigen found in the urine of patients suffering from aspergillosis is related to the fungal protein toxins restrictocin and mitogillin. These are inhibitors of translation which act by catalytic inactivation of eukaryotic ribosomes; they may be implicated in the pathogenesis of the disease.

Acute ureteral colic from an obstructing renal aspergilloma.

Fungus balls of the renal collecting system are rarely of organisms other than Candida. A case of obstructing aspergilloma associated with acute ureteral colic is presented. The clinical features of this case are characteristic of renal aspergillomas in 10 additional cases described in the literature. Ten of the 11 patients were male. Each of the patients had an underlying disease that predisposed to fungal infection. Although all 11 patients were cured, diagnostic evaluation was often protracted as commoner causes of ureteral obstruction were excluded. Successful therapy required evacuation of the obstructing hyphal mass; open surgical procedures were necessary in five instances. Systemic amphotericin B should be reserved for patients with residual infection following removal of the fungus ball. This report emphasizes the need to consider aspergilloma in the differential diagnosis of acute ureteropelvic obstruction in the appropriate patient population.

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.

[Changes of the clinico-biochemical indices in the rumirid juice and urine in experimental aflatoxicosis of dairy cows]. / Zmeny klinicko-biochemických ukazatelu v brachorové tekutine a moci pri experimentální aflatoxikóze dojnic.

The health condition and course of fermentation processes in the rumen were studied in four cows of the Red Spotted breed at the age of four to nine years. The clinico-biochemical indices in the rumen liquor and urine were used. The experimental animals were exposed to a mixture of aflatoxins applied in the dose of 200 mg B1 and 80 mg B2. The toxic action of aflatoxins manifested itself as inappetence, increased temperature, changes in the pulse and respiration rate and reduced activity of the proventriculi. Diarrhoea was observed in two animals. The pH value, total acidity and ammonia level in rumen liquor ranged within the limits of reference values. The significant drop of the production of volatile fatty acids with changes in their proportions and a reduction of the acetic acid level with a simultaneous increase of the percentage of butyric acid testity to a disorder in the activity of rumen microflora. The reduction of the number of infusorians as a biological indicator of fermentation processes proves the correctness of this assumption. During the elimination of aflatoxins through the kidneys the function of the kidneys is impaired, showed proteinuria, ketonuria, glycosuria and haematuria.