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

Journal of Clinical Microbiology (Impact Factor: 3.99). 11/1985; 22(4):587-9.
Source: PubMed


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 less than 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 (chi 2 = 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.

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    • "To detect C. parvum infection, several methods were developed such as sedimentation-flotation technique (McNabb et al., 1985), oocysts staining (O'Donoghue, 1995), PCR amplification (Laxer et al., 1991). However, these approaches could not be applied widely because of its high cost (Wang et al., 2009). "
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    ABSTRACT: We cloned the cp23 gene coding P23 (glyco)protein from Cryptosporidium parvum isolated from Thua Thien Hue province, Vietnam. The coding region of cp23 gene from C. parvum is 99% similar with cp23 gene deposited in NCBI (accession number: U34390). SDS-PAGE and Western blot analysis showed that the cp23 gene in E. coli BL21 StarTM (DE3) produced polypeptides with molecular weights of approximately 37, 40 and 49 kDa. These molecules may be non- glycosylated or glycosylated P23 fusion polypeptides. Recombinant P23 protein purified by GST (glutathione S-transferase) affinity chromatography can be used as an antigen for C. parvum antibody production as well as to develop diagnostic kit for C. parvum.
    Full-text · Article · Sep 2014
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    • "A portion of each specimen was examined by microscopy to detect Cryptosporidium oocysts and Giardia cysts using the Sheather's sugar flotation technique (McNabb et al., 1985) and Lugol's iodine stain method (Hendrix, 2002), respectively. Wet smears were examined using a bright-field microscope with 100Â and 400Â magnification. "
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    ABSTRACT: Non-human primates (NHPs) are commonly infected with Cryptosporidium spp. and Giardia duodenalis. However, molecular characterisation of these pathogens from NHPs remains scarce. In this study, 2,660 specimens from 26 NHP species in China were examined and characterised by PCR amplification of 18S rRNA, 70 kDa heat shock protein (hsp70) and 60 kDa glycoprotein (gp60) gene loci for Cryptosporidium; and 1,386 of the specimens by ssrRNA, triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) gene loci for Giardia. Cryptosporidium was detected in 0.7% (19/2660) specimens of four NHP species including rhesus macaques (0.7%), cynomolgus monkeys (1.0%), slow lorises (10.0%) and Francois’ leaf monkeys (6.7%), belonging to Cryptosporidium hominis (14/19) and Cryptosporidium muris (5/19). Two C. hominis gp60 subtypes, IbA12G3 and IiA17 were observed. Based on the tpi locus, G. duodenalis was iden- tified in 2.2% (30/1,386) of specimens including 2.1% in rhesus macaques, 33.3% in Japanese macaques, 16.7% in Assam macaques, 0.7% in white-headed langurs, 1.6% in cynomolgus monkeys and 16.7% in olive baboons. Sequence analysis of the three targets indicated that all of the Giardia-positive specimens belonged to the zoonotic assemblage B. Highest sequence polymorphism was observed at the tpi locus, including 11 subtypes: three known and eight new ones. Phylogenetic analysis of the subtypes showed that most of them were close to the so-called subtype BIV. Intragenotypic variations at the gdh locus revealed six types of sequences (three known and three new), all of which belonged to so-called subtype BIV. Three specimens had co-infection with C. hominis (IbA12G3) and G. duodenalis (BIV). The presence of zoonotic genotypes and subtypes of Cryptosporidium spp. and G. duodenalis in NHPs suggests that these animals can potentially contribute to the transmission of human cryptosporidiosis and giardiasis.
    Full-text · Article · Aug 2014
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    • "A total of 770 samples were used in the study, including those from animals aged < 90, 90–180, 181–360, and > 360 days (Table 2). Fecal samples of at least 25 g were examined for the presence of Cryptosporidium oocysts using bright-field microscopy of the fecal material that had been concentrated with the formalin–ethyl acetate sedimentation method and Sheather’s sugar flotation technique [24]. Cryptosporidium-positive samples were stored in 2.5% potassium dichromate at 4°C. "
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    ABSTRACT: Cryptosporidiosis in dogs has been reported worldwide, involving both asymptomatic and diarrheic dogs. Large-scale surveys of Cryptosporidium infection in dogs have been performed in some countries using differents diagnostic methods. But, few data are available on the infection rate and molecular characteristics of Cryptosporidium spp. in dogs in China.Result: In this study, 770 fecal samples from 66 locations in Henan Province were examined. The average Cryptosporidium infection rate was 3.8%, with dogs in kennels having the highest rate of 7.0% (chi2 = 14.82, P < 0.01). The infection rate was 8.0% in dogs younger than 90 days, which was significantly higher than that in the other age groups (1.1-3.8%;chi2 = 18.82, P < 0.01). No association was noted between the infection rate and the sex of the dogs. Twenty-nine Cryptosporidium-positive samples were amplified at the small subunit rRNA (SSU rRNA), 70-kDa heat shock protein (HSP70), and actin loci using PCR. Sequence analysis of these amplicons identified only Cryptosporidium canis, which showed 100% identity with the published sequences of the SSU rRNA, HSP70, and actin genes. Our results confirm that C. canis is popular in the dog population in China, considering the large number of dogs in China and the close contact between dogs and humans, the role of C. canis in the transmission of human cryptosporidiosis warrants attention.
    Full-text · Article · Jan 2014 · BMC Veterinary Research
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