Elguezabal, J. for and have garnered experimental support (7, 13). Among these, the ability GSK621 of the organism to convert morphogenically from blastoconidia to filamentous forms has been the subject of intensive study (7). and are unique within the genus in that, in addition to the pseudohyphal form, they also form germ tubes and true hyphae (7). Like blastoconidia, pseudohyphae form by budding; however, the new cell remains attached to the parent and elongates with constrictions where cells meet. In contrast, germ tubes are outgrowths of the blastoconidia that grow by apical extension and form septae behind the growing tip. The germ tube is the precursor to the true hypha, with parallel wall morphology. The conversion to hyphae is accompanied by expression of novel antigens on the filamentous form, identified by using both polyclonal sera and monoclonal antibodies (5, 8-12, 14, 18, 25, 28, 31, 37, 39-41, 43, 49-53). The expansion of medical technology and the increased survival of immunocompromised patients has led to a general increase in mycoses over the past several decades (15, 21). Although remains the most virulent species, infections caused by other species, including in both phenotype and genotype, was first described in 1995, when it was associated with oral infection in patients with human immunodeficiency virus (48). This organism has since been described across diverse geographic regions and has been associated with both superficial and systemic infections (33). Because of its similarities to from has been challenging. A variety of phenotypic assays have been employed, but none have proven completely reliable (47). Growth of is inhibited at 45C, but this method requires several days’ growth on synthetic media (38). The most accurate methods currently available Mouse monoclonal to DDR2 for differentiating these species are molecular techniques such as PCR, DNA fingerprinting, and pulsed-field gel electrophoresis, but these are time-consuming and difficult to apply to large numbers of isolates (47). A novel approach using fluorescent in situ hybridization with peptide nucleic acid probes (PNA-FISH) to detect differences in the rRNA sequences between these species has been reported and holds promise (36). Other recent efforts have been made to exploit serologic differences between these two species, with some success (3, 28, 32), offering the potential for immunologic reagents to provide more efficient and rapid identification. We have been using phage display technology to study surface antigen expression on (17). In the present study, we have applied this technology to identify single-chain antibody fragments (scFv) that specifically recognize the filamentous form of antigens during morphogenesis, and it has yielded reagents that can distinguish from Ca613p,MRO2-O,MRO4-O, MRO9-R, MRO17-O, SC5314, CAH7-1ACBS7988, CBS8500+++?colonization in mothers and neonates at Strong Memorial Hospital, Rochester, N.Y., and were kindly provided by W. Watson, formerly of the Department of Pediatrics, University of Rochester School of Medicine and Dentistry. dstrains CAH7-1A (strains were purchased from the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, and were GSK621 kindly provided by S. Spinelli, Department of Chemistry, University of Rochester, Rochester, N.Y. fT481 was kindly provided by J. S. Butler, Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry. gstrains grown in serum had a more pleiomorphic appearance than when grown in Medium 199, but they retained their activity with the scFv. hND, not determined. Identification and enrichment of strain 3153A was used as the starting strain for panning the phage library (17). To isolate scFv against determinants expressed on the filament of strain TG1. Ampicillin-resistant transductants were regrown and infected with helper phage for a subsequent round of enrichment. Populations of phage that exhibited increased binding to the filaments after two rounds of enrichment were analyzed for sequence diversity by PCR and restriction enzyme digestion. The scFv coding sequence was amplified GSK621 by using primers that flank the cloning sites followed by restriction digestion with strains. Blastoconidia of strains were induced to form filaments via inoculation at 5 105 cells/ml either in Medium 199 in six-well culture dishes on the surfaces of glass coverslips or in human serum in 24-well culture dishes on the surfaces.