Comparison of sedimentation and flotation techniques for identification of Cryptosporidium sp. oocysts in a large outbreak of human diarrhea

Abstract

Cryptosporidiosis, previously seen mostly among immunocompromised patients, is now recognized among immunocompetent patients. During a large outbreak of cryptosporidiosis in two day-care centers, we compared two procedures for the demonstration of the organism in preserved stool specimens. Of 703 stool specimens tested by both techniques, Sheather sucrose flotation (SSF) identified 127 (18.1%) as positive for Cryptosporidium sp. oocysts. Ritchie Formalin-ethyl acetate sedimentation (F/EA) plus a modified cold Kinyoun acid-fast stain (MCK) of the sediment identified 129 (18.4%) as positive for Cryptosporidium sp. oocysts. The degree of agreement between the two tests was statistically highly significant (P < 0.0001). A total of 161 (22.9%) were positive by one technique or the other; 95 (13.5%) were positive by both techniques. A total of 32 specimens were positive by SSF but negative by F/EA plus MCK, and 34 specimens were positive by F/EA plus MCK but negative by SSF. The discrepancies between the two techniques occurred in stool specimens that contained rare to a few oocysts. Other parasitic forms were found by both techniques. F/EA plus trichrome staining recovered 126 (17.9%) specimens with Giardia lamblia, whereas SSF recovered only 42 (6.0%) specimens with G. lamblia. No association (X2 = 0.02, P = 0.89) was observed between the presence of G. lamblia and Cryptosporidium sp. in these stool specimens. We concluded that F/EA plus MCK of the sediment was as effective in the concentration and identification of Cryptosporidium sp. oocysts as SSF. F/EA plus MCK may be advantageous as a single concentration method for general parasitology when Cryptosporidium sp. is also being sought.

Cryptosporidium Spp. And Cryptosporidiosis

“…In one study in Bangkok, polymicrobial infections were not found more frequently with Cryptosporidium than with other gastrointestinal pathogens (255). Although some have suggested that a significant association exists between Giardia lamblia and Cryptosporidium (136,300), others have disagreed (121,174), and such a synergistic association is not supported by the composite survey studies (from references in Table 4).…”

“…Stool concentration techniques include flotation in Sheather sucrose solution, in zinc sulfate (33% to saturated), or in sodium chloride (36% to saturated) or sedimentation with Formalin ether (modified Ritchie) or Formalin ethyl acetate. One comparison of four techniques found no appreciable difference among methods (6), while another found Formalin ether to be the most sensitive for sedimentation and sodium chloride the most sensitive for flotation (56); others have found Sheather sucrose as or more effective than Formalin ether or Formalin ethyl acetate (161,174,308). Sheather solution allows immediate recognition of pink-tinged oocysts by using high-power magnification, but oocysts begin to disrupt and lose their characteristic spherical shape after as little as 15 min (161,174,308); furthermore, they may not adhere to glass slides for permanent record and staining may be altered.…”

Prevalence of Intestinal Cryptosporidiosis in Malaysian Children with Malignancies

“…Moreover, the sugar flotation technique is more cumbersome to perform, and the presence of the sugar solution of Sheather"s (Sheather"set al, 1923) could inhibit staining procedures (Casemoreet al, 1985). Similarly, epidemiological data did not support the superiority of Sheather's sugar flotation for the detection of Cryptosporidium oocysts in stool specimens (McNabb et al, 1985). Recently, the use of several copro-antigen tests or antigen detection assays have been employed as another diagnostic modality in detecting Cryptosporidium antigens in stool samples (Jexet al, 2008).…”

Cryptosporidiosis.

“…Some workers have found no differences among these methods, whereas others have found the formalin-ether and sodium chloride flotations to be the most sensitive (5, 53,112). Some researchers think that the Sheather's sugar solution gives results equal to or better than those obtained with formalin-ether or formalin-ethyl acetate (88,213,357). When Sheather's sugar solution is used, oocysts appear pink-tinged by bright-field microscopy (Fig.…”