Accurate Differentiation of Trichosporon Asahii and Trichosporon Asteroides from other Human Pathogenic Yeasts and Moulds by using Highly Specific Monoclonal Antibodies

Members of the genus Trichosporon are emerging opportunistic pathogens of humans, causing the invasive fungal disease trichosporonosis in immunocompromised patients, and summer-type hypersensitivity pneumonitis (SHP) in immunocompetent individuals through inhalation of arthroconidia. Trichosporonosis is frequently misdiagnosed as candidiasis or cryptococcosis due to a lack of awareness and the inaccuracy of immunodiagnostic tests for these yeast pathogens. Delays in identification and differentiation of Trichosporon spp. from other yeasts and timely administration of appropriate antifungal drug treatments add to the poor prognosis and high mortality rate associated with this trichosporonosis. This thesis describes the use of hybridoma technology to produce two highly specific murine monoclonal antibodies (MAbs), CA7 and TH1, for detection and differentiation of Trichosporon from other yeast pathogens. The MAbs react with extracellular antigens from T. asahii and T. asteroides, the two most common pathogenic agents of trichosporonosis. CA7 and TH1 do not recognise related Trichosporon spp., or unrelated pathogenic yeasts and moulds including Candida spp., Cryptococcus spp., species of Aspergillus, Fusarium, Scedosporium, and etiologic agents of mucormycosis. Immunofluorescence and western blotting studies show that MAb CA7, an immunoglobulin G1 (IgG1), binds to a major ~60kDa glycoprotein antigen produced on the surface of hyphae, while TH1, an immunoglobulin M (IgM), binds to an antigen produced on the surface of conidia. I show how the MAbs can be used with standard mycological growth medium (Sabouraud Dextrose Agar) and an enzyme-linked immunosorbent assay (ELISA) to accurately differentiate T. asahii from Candida albicans and Cryptococcus neoformans in single and mixed species cultures

Pre-clinical imaging of invasive candidiasis using ImmunoPET/MR

This is the final version of the article. Available from Frontiers Media via the DOI in this record.The human commensal yeast Candida is the 4th most common cause of hospital-acquired bloodstream infections, with C. albicans accounting for the majority of the >400,000 life-threatening infections annually. Diagnosis of invasive candidiasis (IC), a disease encompassing candidemia (blood-borne yeast infection) and deep-seated organ infections, is a major challenge since clinical manifestations of the disease are indistinguishable from viral, bacterial and other fungal diseases, and diagnostic tests for biomarkers in the bloodstream such as PCR, ELISA and pan-fungal β-D-glucan lack either standardisation, sensitivity or specificity. Blood culture remains the gold standard for diagnosis, but test sensitivity is poor and turn-around time slow. Furthermore, cultures can only be obtained when the yeast resides in the bloodstream, with samples recovered from hematogenous infections often yielding negative results. Consequently, there is a pressing need for a diagnostic test that allows the identification of metastatic foci in deep-seated Candida infections, without the need for invasive biopsy. Here, we report the development of a highly specific mouse IgG3 monoclonal antibody (MC3) that binds to a putative β-1,2-mannan epitope present in high molecular weight mannoproteins and phospholipomannans on the surface of yeast and hyphal morphotypes of C. albicans, and its use as a [64Cu]NODAGA-labeled tracer for whole-body pre-clinical imaging of deep-seated C. albicans infections using antibody-guided positron emission tomography and magnetic resonance imaging (immunoPET/MRI). When used in a mouse intravenous (i.v.) challenge model that faithfully mimics disseminated C. albicans infections in humans, the [64Cu]NODAGA-MC3 tracer accurately detects infections of the kidney, the principal site of blood-borne candidiasis. Using a strain of the emerging human pathogen Candida auris that reacts with MC3 in vitro, but which is non-infective in i.v. challenged mice, we demonstrate the accuracy of the tracer in diagnosing invasive infections in vivo. This pre-clinical study demonstrates the principle of antibody-guided molecular imaging for detection of deep organ infections in IC, without the need for invasive tissue biopsy.This work was supported, in part, by the European Union Seventh Framework Program FP7/2007-2013 under grant 602820

Aspergillus fumigatus and Its Allergenic Ribotoxin Asp f I: Old Enemies but New Opportunities for Urine-Based Detection of Invasive Pulmonary Aspergillosis Using Lateral-Flow Technology

Invasive pulmonary aspergillosis (IPA) caused by Aspergillus fumigatus is a life-threatening lung disease of immunocompromised patients. Diagnosis currently relies on non-specific chest CT, culture of the fungus from invasive lung biopsy, and detection of the cell wall carbohydrate galactomannan (GM) in serum or in BAL fluids recovered during invasive bronchoscopy. Urine provides an ideal bodily fluid for the non-invasive detection of pathogen biomarkers, with current urine-based immunodiagnostics for IPA focused on GM. Surrogate protein biomarkers might serve to improve disease detection. Here, we report the development of a monoclonal antibody (mAb), PD7, which is specific to A. fumigatus and related species in the section Fumigati, and which binds to its 18 kDa ribotoxin Asp f I. Using PD7, we show that the protein is secreted during hyphal development, and so represents an ideal candidate for detecting invasive growth. We have developed a lateral-flow device (Afu-LFD®) incorporating the mAb which has a limit of detection of ~15 ng Asp f I/mL urine. Preliminary evidence of the test’s diagnostic potential is demonstrated with urine from a patient with acute lymphoid leukaemia with probable IPA. The Afu-LFD® therefore provides a potential novel opportunity for non-invasive urine-based detection of IPA caused by A. fumigatus

Aspergillus fumigatus and Its Allergenic Ribotoxin Asp f I: Old Enemies but New Opportunities for Urine-Based Detection of Invasive Pulmonary Aspergillosis Using Lateral-Flow Technology.

Invasive pulmonary aspergillosis (IPA) caused by Aspergillus fumigatus is a life-threatening lung disease of immunocompromised patients. Diagnosis currently relies on non-specific chest CT, culture of the fungus from invasive lung biopsy, and detection of the cell wall carbohydrate galactomannan (GM) in serum or in BAL fluids recovered during invasive bronchoscopy. Urine provides an ideal bodily fluid for the non-invasive detection of pathogen biomarkers, with current urine-based immunodiagnostics for IPA focused on GM. Surrogate protein biomarkers might serve to improve disease detection. Here, we report the development of a monoclonal antibody (mAb), PD7, which is specific to A. fumigatus and related species in the section Fumigati, and which binds to its 18 kDa ribotoxin Asp f I. Using PD7, we show that the protein is secreted during hyphal development, and so represents an ideal candidate for detecting invasive growth. We have developed a lateral-flow device (Afu-LFD®) incorporating the mAb which has a limit of detection of ~15 ng Asp f I/mL urine. Preliminary evidence of the test’s diagnostic potential is demonstrated with urine from a patient with acute lymphoid leukaemia with probable IPA. The Afu-LFD® therefore provides a potential novel opportunity for non-invasive urine-based detection of IPA caused by A. fumigatus

Differentiation of the emerging human pathogens Trichosporon asahii and Trichosporon asteroides from other pathogenic yeasts and moulds by using species-specific monoclonal antibodies.

The fungal genus Trichosporon contains emerging opportunistic pathogens of humans, and is the third most commonly isolated non-candidal yeast from humans. Trichosporon asahii and T. asteroides are the most important species causing disseminated disease in immunocompromised patients, while inhalation of T. asahii spores is the most important cause of summer-type hypersensitivity pneumonitis in healthy individuals. Trichosporonosis is misdiagnosed as candidiasis or cryptococcosis due to a lack of awareness and the ambiguity of diagnostic tests for these pathogens. In this study, hybridoma technology was used to produce two murine monoclonal antibodies (MAbs), CA7 and TH1, for detection and differentiation of Trichosporon from other human pathogenic yeasts and moulds. The MAbs react with extracellular antigens from T. asahii and T. asteroides, but do not recognise other related Trichosporon spp., or unrelated pathogenic yeasts and moulds including Candida, Cryptococcus, Aspergillus, Fusarium, and Scedosporium spp., or the etiologic agents of mucormycosis. Immunofluorescence and Western blotting studies show that MAb CA7, an immunoglobulin G1 (IgG1), binds to a major 60 kDa glycoprotein antigen produced on the surface of hyphae, while TH1, an immunoglobulin M (IgM), binds to an antigen produced on the surface of conidia. The MAbs were used in combination with a standard mycological growth medium (Sabouraud Dextrose Agar) to develop an enzyme-linked immunosorbent assay (ELISA) for differentiation of T. asahii from Candida albicans and Cryptococcus neoformans in single and mixed species cultures. The MAbs represent a major advance in the identification of T. asahii and T. asteroides using standard mycological identification methods

Growth of yeasts for 24 h as single species or mixed species cultures and ELISA tests of soluble antigens using MAbs CA7 and TH1.

<p>(A) Sabouraud Dextrose Agar plates inoculated with different combinations of species: <i>T. asahii</i> var. <i>asahii</i> CBS8972 (<i>Ta</i>), <i>C. albicans</i> SC5314 (<i>Ca</i>), and <i>C. neoformans</i> var. <i>neoformans</i> CBS7779 (<i>Cn</i>). ELISA absorbance values of antigen solutions tested using MAb CA7 (B) and MAb TH1 (C). Each bar is the mean of three biological replicates ± standard error. Bars with the same letter are not significantly different at p<0.001 (ANOVA and Tukey-Kramer test).</p

Absorbance values from ELISA tests with MAbs CA7 and TH1 using periodate-treated antigens.

<p>Absorbance value significantly different (p<0.001) to control using ANOVA. Each value is the mean of eight biological replicates ± standard error.</p

Absorbance values from ELISA tests with MAbs CA7 and TH1 using protease-treated antigens.

<p>Absorbance value significantly different (p<0.001) to controls (buffer only) using ANOVA. Each value is the mean of eight biological replicates ± standard error.</p

Characterisation of antigens bound by MAbs CA7 and TH1.

<p>(A) ELISA of heat-treated antigens using MAb CA7 (broken line) and MAb TH1 (continuous line). Each point is the mean of three biological replicates ± standard errors. Asterisks show significant (p<0.001) decrease in absorbance value compared to respective controls (time point zero). (B) Western immunoblot with MAb CA7 using antigens from 3-day-old cultures of <i>T. asteroides</i> (lane 1), <i>T. asahii</i> (lane 4) and <i>T. inkin</i> (lane 5). Wells were loaded with 1.6 µg of protein. Lanes 2 and 3 contain broad range molecular mass markers (kDa); (C) DOT-BLOTS of <i>T. asahii</i> antigen treated with pronase or trypsin and then processed with MAbs TH1 or CA7. Each DOT-BLOT consists of antigen from two biological replicates. (D–I) Photomicrographs of <i>T. asahii</i> var. <i>asahii</i> CBS8972 cells, immunostained with MAb CA7 (D, E), TH1 (F, G) or TCM only (H, I) and anti-mouse polyvalent Ig fluorescein isothiocyanate. (D) Bright field image of germinated conidium with hypha; (E) Same field of view as panel D but examined under epifluorescence. Note the intense staining of the hyphal cell wall but not the conidiium. (F) Bright field image of germinated conidium with hypha and ungerminated conidia. (G) Same field of view as panel F but examined under epifluorescence. Note intense staining of ungerminated conidial cell wall but not hypha, and reduced fluorescence of germinated conidium. (H) Bright field image of germinated conidium with hypha. (I) Same field of view as panel H but examined under epifluorescence. Bar, 8 µm.</p

Aspergillus fumigatus and Its Allergenic Ribotoxin Asp f I: Old Enemies but New Opportunities for Urine-Based Detection of Invasive Pulmonary Aspergillosis Using Lateral-Flow Technology.

Invasive pulmonary aspergillosis (IPA) caused by Aspergillus fumigatus is a life-threatening lung disease of immunocompromised patients. Diagnosis currently relies on non-specific chest CT, culture of the fungus from invasive lung biopsy, and detection of the cell wall carbohydrate galactomannan (GM) in serum or in BAL fluids recovered during invasive bronchoscopy. Urine provides an ideal bodily fluid for the non-invasive detection of pathogen biomarkers, with current urine-based immunodiagnostics for IPA focused on GM. Surrogate protein biomarkers might serve to improve disease detection. Here, we report the development of a monoclonal antibody (mAb), PD7, which is specific to A. fumigatus and related species in the section Fumigati, and which binds to its 18 kDa ribotoxin Asp f I. Using PD7, we show that the protein is secreted during hyphal development, and so represents an ideal candidate for detecting invasive growth. We have developed a lateral-flow device (Afu-LFD®) incorporating the mAb which has a limit of detection of ~15 ng Asp f I/mL urine. Preliminary evidence of the test's diagnostic potential is demonstrated with urine from a patient with acute lymphoid leukaemia with probable IPA. The Afu-LFD® therefore provides a potential novel opportunity for non-invasive urine-based detection of IPA caused by A. fumigatus