Misidentification of as a true fungus (i

Misidentification of as a true fungus (i.e., initiates an infection (5). tissue resulting in keratitis and ulcer) (3, 4). Antifungal drugs are ineffective against antibodies, usually produce false-negative results from Rabbit polyclonal to ALKBH1 the serum of patients with ocular pythiosis. Molecular assays, Omeprazole based on PCR and sequence homology, require skilled personnel and sophisticated gear, which is not readily available in the regions where pythiosis is usually endemic. In addition, limited yield or degradation of the extracted DNA compromises the diagnostic performance of such assays. As alternatives, several investigators have developed immunohistochemical assays (IHCs) for the diagnosis of pythiosis. These assays are based on rabbit antiserum (as the primary antibody) and are raised against crude extracts (i.e., culture filtrate antigen [CFA] and soluble antigen from broken hyphae [SABH]) (28, 29). IHC showed good detection sensitivity but limited detection specificity due to cross-reactivity of the assay with some pathogenic fungi, i.e., and species (25, Omeprazole 29). Therefore, specificity of the IHCs needs to be improved. Elicitins form a group of proteins found only in a phylogenetically distinct group of microorganisms, the oomycetes, but are absent in all other microorganisms, including true fungi (30,C33). Recently, we reported a number of elicitins from the transcriptome, and one of them, ELI025, is highly expressed and appears at the pathogen cell surface (33,C35). Since the elicitins are unique to among the human pathogens, direct detection of ELI025 can aid in the development of a more specific IHC for pythiosis. In this study, we developed a new IHC using the rabbit anti-ELI025 antibody (anti-ELI) (33) for histodiagnosis of or other fungi (referred to as culture blocks) (Table 1) and from infected tissues (referred to as tissue blocks) (Table 2) for the evaluation of IHC. Nineteen strains of (reference codes CP01 to CP19 in Table 1; isolated from the environment [= 2] and patients with vascular pythiosis [= 9], ocular pythiosis [= 4], cutaneous pythiosis [= 2], and other forms of pythiosis [= 2]) and 31 isolates of other fungi (reference codes CC01 to CC31 in Table 1; served as controls and included spp. [= 8], spp. [= 4], spp. [= 3], spp. [= 2], spp. [= 2], spp. [= 2], spp. [= 2], and spp. [= 2] and one each of sp., sp., sp., sp., sp., and sp.) were obtained for culture block preparation. The identity of each organism was confirmed by culture. Each organism was grown in Sabouraud dextrose broth at 37C for up to 10 days. Merthiolate was added to the culture at the final concentration of 0.02% (wt/vol). The organism was harvested, fixed with 10% buffered formalin, and embedded in paraffin blocks at the Department of Pathology, Ramathibodi Hospital. TABLE 1 Results of the anti-CFA-based and anti-ELI-based immunohistochemical assays using culture blocksfor:sp??CC02sp??CC03sp??CC04sp??CC05sp??CC06sp??CC07sp??CC08sp??CC09sp??CC10sp??CC11sp??CC12sp??CC13sp??CC14sp??CC15sp??CC16sp??CC17sp??CC18sp??CC19sp??CC20sp??CC21sp??CC22sp??CC23sp??CC24sp??CC25sp??CC26sp??CC27sp??CC28sp??CC29sp??CC30sp??CC31sp?? Open in a separate window aParaffin-embedded blocks prepared from pure cultures of = 19) and true fungi (= 31). b+, Positive stain; ?, unfavorable stain. cRabbit anti-CFA (culture filtrate antigen) serum. dRabbit anti-ELI (ELI025) serum. TABLE 2 Results of the anti-CFA-based and anti-ELI-based immunohistochemical assays using tissue blocksfor:spSinus+??TC07spSinus+??TC08spNasal cavity+??TC09spNA+??TC14spLip+??TC15spCornea+??TC18spSkin++? Open in a separate window aParaffin-embedded blocks prepared from Omeprazole infected tissues of patients with pythiosis (= 19) and other mycoses (= 18). b+, Positive stain; ?, unfavorable stain. cRabbit anti-CFA (culture filtrate antigen) serum. dRabbit anti-ELI (ELI025) serum. eNA, data not available. fA pigmented fungus that causes a phaeomycotic cyst. A total of Omeprazole 37 paraffin-embedded tissue blocks, prepared from the infected tissues of 19 patients with pythiosis (reference codes TP01 to TP19 in Table 2) and 18 patients with other fungal infections (reference codes TC01 to TC18 in Table 2; served as negative controls and included [= 4], spp. [= 3], [= 3], [= 2], spp. [= 2], spp. [= 2], [= 1], and a phaeomycotic fungus [= 1]) were obtained from Ramathibodi Hospital, Siriraj Hospital, and Chulalongkorn Hospital. The identity of each organism in the infected tissues was confirmed by histological examination and culture identification. Each tissue or culture block was cut into 4-m slices using a microtome (Finesse 325; Thermo Scientific, USA). Paraffin-embedded sections were placed on glass slides for downstream IHC analyses. Grocott’s methenamine silver and immunohistochemical stains. Each paraffin-embedded section was analyzed with the Grocott’s methenamine silver (GMS) stain, as previously described (36), and was examined under a light microscope (Eclipse Ci; Nikon, Japan). Two different IHCs for detecting were.

Related Posts

Begin typing your search term above and press enter to search. Press ESC to cancel.

Back To Top