Fluorescence detection of Cryptosporidium oocysts in human fecal specimens by using monoclonal antibodies

Abstract

With the discovery that the coccidian parasite Cryptosporidium sp. can cause severe symptoms in humans, implementation of many diagnostic techniques rapidly followed. The infection is self-limiting in patients with normal immune systems but chronic in the immunosuppressed patient. With the eventual development and use of therapeutic agents, it will become very important to find Cryptosporidium sp., even in low numbers, in fecal specimens. Production of a highly specific and sensitive antibody by use of cloning techniques has provided another diagnostic tool. Formalinized positive human fecal specimens (n = 99) and negative specimens (n = 198), of which 115 contained yeastlike fungi and other organisms, were tested in blind trials by use of a monoclonal antibody. Sensitivity was 100% with 3- to 4+ fluorescence on all cryptosporidial oocysts, both in light and heavy infections. The organisms were round and easily visible (4 to 6 micron), showing apple-green to yellow fluorescence against a dark background free of nonspecific fluorescence. Specificity was also 100% with all 99 positive Cryptosporidium sp. specimens exhibiting fluorescence and all 198 negative specimens showing no fluorescence. All positive and negative specimens were previously confirmed by the hot modified acid-fast technique. However, seven specimens previously considered negative by this acid-fast method were positive by the monoclonal antibody technique. These specimens were confirmed as positive, after extensive examination of additional smears prepared by the modified hot acid-fast method revealed rare organisms, emphasizing the increased sensitivity of the monoclonal antibody technique. Since acid-fast stains do not always consistently stain all oocysts, the increased sensitivity of the monoclonal reagent provides an excellent screening method.