Enzyme-linked immunoassay for detection of Cryptosporidium antigens in fecal specimens

Abstract

Cryptosporidium sp. is a ubiquitous 4- to 6-micron protozoan parasite infecting the intestinal tract of humans. It causes mild to fulminant diarrhea in patients, especially immunocompromised persons, and it may be hard to detect by microscopic fecal examination. An indirect, double-antibody enzyme-linked immunosorbent assay (ELISA) was developed using specifically produced goat and rabbit antisera to detect Cryptosporidium antigens in human feces. Of 62 frozen stools from patients with cryptosporidiosis, as detected by at least two microscopic diagnostic techniques, 51 were positive by ELISA; all ELISA-negative specimens came from patients with fewer than five oocysts per 0.01 ml of concentrated fecal sample examined after modified acid-fast or fluorescent monoclonal antibody staining. A total of 182 specimens from persons without Cryptosporidium infection were negative by ELISA in 176 instances; 3 ELISA-positive specimens came from patients with cryptosporidiosis diagnosed earlier. The sensitivity of the assay was 82.3%, and specificity was 96.7%. The predictive value of a positive ELISA was 89.5%, and the predictive value of a negative ELISA was 94.2%. The ELISA was not affected by the presence of eight other intestinal parasites but was sometimes affected by repeated freezing and thawing of fecal specimens. All fecal specimens were heated to 100 degrees C for 2 min to reduce proteolytic enzyme activity, although the necessity of this step needs further evaluation. This first-generation ELISA is a simple, rapid, easily standardized test for Cryptosporidium antigens in stool samples which will be useful for diagnosis and for large-scale epidemiologic studies.

Evaluation of Different Diagnostic Approaches for Detection of Cryptosporidium in Stools of Diarrheic Children

“…They attributed decreased sensitivity to repeated freezing and thawing of specimens. (34) The diagnostic performance of the ELISA in the present study revealed moderate sensitivity and specificity. The PPV, which indicates the probability of having cryptosporidiosis among diarrheic children was relatively high (68.7%).…”

Identification of a 15-kilodalton surface glycoprotein on sporozoites of Cryptosporidium parvum

“…Spleens were removed, B-lymphocytes were fused with FOX.NY myeloma cells, and the cells were grown in Dulbecco's modified Eagle's medium (DMEM), as described before (6). Supernatants were tested for anti-Cryptosporidium antibodies by enzyme-linked immunosorbent assay (ELISA) (34,37). Hybridomas producing anti-C. parvum antibody were cloned by limiting dilution and further assessed for reactivity by Western immunoblot analysis (see below).…”

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.…”