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.

Molecular Epidemiology ofCryptosporidiumandGiardiain Humans on Prince Edward Island, Canada: Evidence of Zoonotic Transmission From Cattle

“…In previous reports that found a lower prevalence, testing for Cryptosporidium oocysts was carried out using acid-fast staining (Chai et al, 1996;Park et al, 2006;Mirzaei, 2007;Cheun et al, 2010), whereas in our study, a method was used based on monoclonal antibody capture (direct fluorescence antibody staining). This method is considered more sensitive and specific, and it therefore reduces the likelihood of false-negative and false-positive results (Garcia et al, 1987). Given that many of the human faecal samples were preserved, Cryptosporidium DNA may have been inaccessible, resulting in the low number of isolates amenable to genotyping (Cheun et al, 2010).…”

Detection of Cryptosporidium oocysts in fresh and frozen cattle faeces: comparison of three methods

“…The sensitivity and specificity of these methods vary, depending on the protocol used. Various screening methods for Cryptosporidium oocysts from, primarily, fresh human faecal samples have been studied (Garcia et al 1987;Ungar 1990;Siddons et al 1992;MacPherson and McQueen 1993;Tee et al 1993;Aarnaes et al 1994;Parisi and Tierno 1995;Garcia and Shimizu 1997;Johnston et al 2003;Magi et al 2006). These studies have produced conflicting results and it is reasonable to deduce that there is currently no available gold standard diagnostic method.…”

Severe cryptosporidiosis in a seven‐year‐old renal transplant recipient – Case report and review of the literature

“…Modified acid-fast staining will also screen for Isospora and Cyclospora which are important stool pathogens in immunocompromised hosts (19). Direct fluorescent antibodies that identify Cryptosporidium oocysts have a published sensitivity of 100% and specificity of 100% (20). Direct immunofluorescence using monoclonal antibodies is now the gold standard for stool examination for Cryptosporidium spp.…”