Tracking the Emerging Human PathogenPseudallescheria boydiiby Using Highly Specific Monoclonal Antibodies

Abstract

Pseudallescheria boydii has long been known to cause white grain mycetoma in immunocompetent humans, but it has recently emerged as an opportunistic pathogen of humans, causing potentially fatal invasive infections in immunocompromised individuals and evacuees of natural disasters, such as tsunamis and hurricanes. The diagnosis of P. boydii is problematic since it exhibits morphological characteristics similar to those of other hyaline fungi that cause infectious diseases, such as Aspergillus fumigatus and Scedosporium prolificans. This paper describes the development of immunoglobulin M (IgM) and IgG1 -light chain monoclonal antibodies (MAbs) specific to P. boydii and certain closely related fungi. The MAbs bind to an immunodominant carbohydrate epitope on an extracellular 120-kDa antigen present in the spore and hyphal cell walls of P. boydii and Scedosporium apiospermum. The MAbs do not react with S. prolificans, Scedosporium dehoogii, or a large number of clinically relevant fungi, including A. fumigatus, Candida albicans, Cryptococcus neoformans, Fusarium solani, and Rhizopus oryzae. The MAbs were used in immunofluorescence and doubleantibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to accurately differentiate P. boydii from other infectious fungi and to track the pathogen in environmental samples. Specificity of the DAS-ELISA was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of environmental isolates.Pseudallescheria boydii is an infectious fungal pathogen of humans (7,16,40,58,59). It is the etiologic agent of white grain mycetoma in immunocompetent humans (7) and has emerged over recent years as the cause of fatal disseminated infections in individuals with neutropenia, AIDS, diabetes, renal failure, bone marrow or solid organ transplants, systemic lupus erythematous, and Crohn's disease; in those undergoing corticosteroid treatment; and in leukemia and lymphoma patients (1,2,3,18,27,31,32,34,36,37,38,47,49,52). The fungus is the most prevalent species after Aspergillus fumigatus in the lungs of cystic fibrosis patients (8), where it causes allergic bronchopulmonary disease (5) and chronic lung lesions simulating aspergillosis (24). Near-drowning incidents and recent natural disasters, such as the Indonesian tsunami in 2004, have shown P. boydii and the related species Scedosporium apiospermum and Scedosporium aurantiacum to be the causes of fatal central nervous system infections and pneumonia in immunocompetent victims who have aspirated polluted water (4,11,12,21,22,25,30,33,57). Its significance as a potential pathogen of disaster evacuees has led to its recent inclusion in the Centers for Disease Control and Prevention list of infectious etiologies in persons with altered mental statuses, central nervous system syndromes, or respiratory illness.P. boydii is thought to be an underdiagnosed fungus (60), and misidentification is one of the reasons that the mortality rate due to invasive pseudallescheriasis is high. Detection of inv...

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“…Bound antibody was visualized by incubating wells with tetramethyl benzidine (T-2885; Sigma) substrate solution [24], [25] for 30 min. The reactions were stopped by the addition of 3 M H 2 SO 4 .…”

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