Allergenic cross-reactivity ofCurvularia lunatawith other airborne fungal species

A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the Candida Panel for the identification of Candida species, and the Filamentous Fungi Panel for the identification of Aspergillus species and Rhizopusarrhizus. The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes.

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Development of Murine Monoclonal Antibodies for the Immunohistochemical Diagnosis of Systemic Bovine Aspergillosis

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879600800111 ◽

1996 ◽

Vol 8 (1) ◽

pp. 68-75 ◽

Cited By ~ 31

Author(s):

H. E. Jensen ◽

B. Aalbaek ◽

P. Lind ◽

H. V. Krogh ◽

P. L. Frandsen

Keyword(s):

Monoclonal Antibodies ◽

Polyclonal Antibodies ◽

Fungal Species ◽

Cross Reactivity ◽

Water Soluble ◽

Enzyme Linked Immunosorbent Assay ◽

Immunohistochemical Techniques ◽

Immunohistochemical Diagnosis ◽

Murine Monoclonal Antibodies

Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and-4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and-2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions ( n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.

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Cross-reactivity of non-Aspergillus fungal species in the Aspergillus galactomannan enzyme immunoassay

Diagnostic Microbiology and Infectious Disease ◽

10.1016/j.diagmicrobio.2007.04.022 ◽

2007 ◽

Vol 59 (1) ◽

pp. 113-115 ◽

Cited By ~ 81

Author(s):

Jessica R. Cummings ◽

Ginger R. Jamison ◽

Jan W. Boudreaux ◽

Merry J. Howles ◽

Thomas J. Walsh ◽

...

Keyword(s):

Enzyme Immunoassay ◽

Fungal Species ◽

Cross Reactivity

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Multicenter Clinical Validation of a Cartridge-Based Real-Time PCR System for Detection of Coccidioides spp. in Lower Respiratory Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.01277-17 ◽

2017 ◽

Vol 56 (2) ◽

Cited By ~ 9

Author(s):

Michael A. Saubolle ◽

Bette R. Wojack ◽

Anne M. Wertheimer ◽

Atehkeng Z. Fuayagem ◽

Stephen Young ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Fungal Species ◽

Cross Reactivity ◽

Clinical Test ◽

Target Sequence ◽

Fungal Culture ◽

Content Type ◽

Bronchial Wash

ABSTRACT Available methods for the diagnosis of coccidioidomycosis have significant shortcomings relative to accuracy and timeliness. We retrospectively and prospectively evaluated the diagnostic performance and reproducibility of a new cartridge-based real-time PCR assay for Coccidioides spp. directly in lower respiratory secretions and compared them to today's “gold standard,” fungal culture. The GeneSTAT Coccidioides assay uses a 106-bp target sequence repeated multiple times (∼60×) per genome, thus lowering the limit of detection (LOD) for extracted DNA to 10 genome equivalents/ml. A total of 332 prospective and retrospective individual patient specimens were tested. The retrospective samples consisted of 100 bronchoalveolar lavage or bronchial wash (BAL/BW) (51 positive and 49 negative by culture) specimens that had been collected previously and stored at −70°C. These samples were tested by the GeneSTAT Coccidioides assay across three clinical test sites. The sensitivity was 100%, and the specificity ranged between 93.8% and 100%. There was minimal variance in the percent agreement across the three sites, 95.6% to 100%. Additionally, a total of 232 fresh (prospective) deidentified BAL/BW specimens were tested across the three clinical sites, which included a number of specimens from Southern California to provide a diversity of isolates. Specimens were tested by fungal culture, with any isolates of Coccidioides, except for one, being confirmed by molecular means (AccuProbe). The sensitivity of the GeneSTAT Coccidioides assay across the three sites was 100% (4/4) for positive fresh specimens, and the overall specificity of the assay was 99.6% (227/228), ranging from 98.1% to 100%. In testing for cross-reactivity, the assay was 100% specific when screened against 47 different bacterial, viral, and fungal species.

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Immunohistochemical Cross-Reactivity Between Arthrographis kalrae and Highly Pathogenic Coccidioides posadasii, Histoplasma capsulatum, and Paracoccidioides Fungal Species

Mycopathologia ◽

10.1007/s11046-019-00348-5 ◽

2019 ◽

Vol 184 (3) ◽

pp. 393-402

Author(s):

Godai Shumoto ◽

Luciene Airy Nagashima ◽

Eiko Nakagawa Itano ◽

Tomoko Minakawa ◽

Keiichi Ueda ◽

...

Keyword(s):

Histoplasma Capsulatum ◽

Fungal Species ◽

Cross Reactivity ◽

Coccidioides Posadasii ◽

Highly Pathogenic

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Genetic Susceptibility to Fungal Infections and Links to Human Ancestry

Frontiers in Genetics ◽

10.3389/fgene.2021.709315 ◽

2021 ◽

Vol 12 ◽

Author(s):

Bharati Naik ◽

Sumayyah M. Q. Ahmed ◽

Suparna Laha ◽

Shankar Prasad Das

Keyword(s):

Fungal Infections ◽

Fungal Species ◽

Cross Reactivity ◽

Nucleotide Polymorphisms ◽

Host Genetics ◽

Symbiotic Associations ◽

Immunological Status ◽

Feasible Option ◽

Human Proteins ◽

The Individual

Over the ages, fungi have associated with different parts of the human body and established symbiotic associations with their host. They are mostly commensal unless there are certain not so well-defined factors that trigger the conversion to a pathogenic state. Some of the factors that induce such transition can be dependent on the fungal species, environment, immunological status of the individual, and most importantly host genetics. In this review, we discuss the different aspects of how host genetics play a role in fungal infection since mutations in several genes make hosts susceptible to such infections. We evaluate how mutations modulate the key recognition between the pathogen associated molecular patterns (PAMP) and the host pattern recognition receptor (PRR) molecules. We discuss the polymorphisms in the genes of the immune system, the way it contributes toward some common fungal infections, and highlight how the immunological status of the host determines fungal recognition and cross-reactivity of some fungal antigens against human proteins that mimic them. We highlight the importance of single nucleotide polymorphisms (SNPs) that are associated with several of the receptor coding genes and discuss how it affects the signaling cascade post-infection, immune evasion, and autoimmune disorders. As part of personalized medicine, we need the application of next-generation techniques as a feasible option to incorporate an individual’s susceptibility toward invasive fungal infections based on predisposing factors. Finally, we discuss the importance of studying genomic ancestry and reveal how genetic differences between the human race are linked to variation in fungal disease susceptibility.

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Detection of Aspergillus Flavus in Wheat Grains Using Anti-Mannoprotein (MP1) and Spore Proteins Polyclonal Antibodies

10.21203/rs.3.rs-530983/v1 ◽

2021 ◽

Author(s):

Ranjana Kumari ◽

Ananta Ghosh

Keyword(s):

Aspergillus Flavus ◽

Polyclonal Antibodies ◽

Protein A ◽

Fungal Species ◽

Cross Reactivity ◽

Soluble Form ◽

Minimum Concentration ◽

Wheat Grains ◽

Stored Wheat ◽

Spore Proteins

Abstract Cell wall mannoprotein (MP1) gene of an aflatoxigenic strain of Aspergillus flavus, isolated from stored wheat grains, was cloned and sequenced. MP1 protein was expressed in E. coli in soluble form and purified. Polyclonal antibodies were raised against recombinant MP1 protein and inactivated spores of this fungus in rabbit, and purified by ammonium sulphate precipitation, Protein A sepharose and antigen affinity chromatography. The minimum concentration of purified mycelial or spore proteins that could be detected by ELISA was determined as 100 ng using 2 µg of these antibodies. The anti-MP1 antibody was found more sensitive than anti-spore protein antibody. Western blot and immunofluorescence analysis showed reactivity of these antibodies to various proteins (30 kDa to 200 kDa) distributed throughout the surface of mycelia and spore of A. flavus. Cross reactivity of these antibodies was detected with fungi belonging to different Aspergillus, Rhizopus and Alternaria species out of fourteen different fungal species tested. In fungal contaminated wheat grains these antibodies could detect presence of as low as 1 µg mycelia or 103 spores per gram of wheat grains using ELISA. The results suggest that the developed antibodies could be successfully applied for the detection of predominant fungal infestation in stored wheat grains.

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Rapid electrochemical identification of pathogenic Candida species

Journal of Medical Microbiology ◽

10.1099/jmm.0.009183-0 ◽

2009 ◽

Vol 58 (9) ◽

pp. 1182-1189 ◽

Cited By ~ 10

Author(s):

Alastair Muir ◽

A. Toby A. Jenkins ◽

Gordon Forrest ◽

John Clarkson ◽

Alan Wheals

Keyword(s):

Fungal Pathogens ◽

Limit Of Detection ◽

Pathogenic Fungi ◽

18S Rrna Gene ◽

Fungal Species ◽

Cross Reactivity ◽

Its2 Region ◽

Rrna Gene ◽

Human Dna ◽

Species Specific

This study describes the development of a novel assay to detect fungal DNA and identify the most clinically relevant invasive human pathogenic fungi to the species level using oligonucleotide probes, labelled with electrochemically active groups, and solid-state electrodes. A panfungal probe designed against the 18S rRNA gene region, capable of detecting all fungal pathogens tested, and species-specific probes, designed against the ITS2 region for detection of the five Candida species most commonly encountered in the clinical setting (Candida albicans, Candida glabrata, Candida parapsilosis species complex, Candida krusei and Candida tropicalis), are described. When tested with PCR-amplified DNA from both type and clinical strains of the relevant species, the probes were able to positively identify the relevant fungi, indicated by production of a current significantly elevated above the background reading. No cross-reactivity was observed with any of the species-specific probes when compared with nine non-target Candida species or in the presence of human DNA equivalent to an equal number of ITS2 targets. The panfungal probe gave results that were similarly positive against 15 other fungal species and also did not cross-react with human DNA. The limit of detection of the assay was shown to be approximately 1 genome equivalent for all probes using extracted genomic DNA.

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