title: Specific and quantitative detection and identification of Cryptosporidium hominis and C. parvum in clinical and environmental samples.
abstract: Cryptosporidium is an enteric protozoan parasite that is resistant to inactivation by commonly used drinking water disinfectants. Between 2004 and 2010, it was responsible for 60% of all waterborne protozoan parasitic outbreaks reported worldwide. Most sporadic infections in humans and almost all outbreaks are caused by Cryptosporidium parvum and Cryptosporidium hominis. We report the development and validation of a quantitative qPCR assay using minor groove binder (MGB)-probes targeting a unique Cryptosporidium specific protein-coding gene, that directly detects, quantitates and identifies C. hominis and C. parvum in environmental and faecal samples. An internal amplification control (IAC) was also developed and included in this assay. The qPCR assay was compared with an 18S nested PCR assay for sensitivity and specificity. The analytical sensitivity for the qPCR assay was 1 oocyst and 1-10 oocysts for the 18S assay. Evaluation of analytical specificity of the qPCR assay revealed no cross-reactions with other genera and detected all C. parvum and C. hominis isolates correctly. The diagnostic sensitivity and specificity of the qPCR was 100% compared to 96.9% and 98.4%, respectively for the 18S assay. The qPCR assay was also highly reproducible with RSD (relative standard deviation) values of 1.4-9.4%, when the assay was performed by four different technicians. When tested on water samples, the qPCR assay was more sensitive than the 18S assay, detecting positives in 37 of 138 water samples compared to 35 for the 18S locus. This qPCR assay should be a valuable tool for the detection and differentiation of C. hominis and C. parvum in both clinical and environmental samples.
authors.affiliation: School of Veterinary and Life Sciences, Murdoch University, Murdoch, WA 6150, Australia.
authors.forename: Rongchang
authors.initials: R
authors.lastname: Yang
authors.forename: Cain
authors.initials: C
authors.lastname: Murphy
authors.forename: Yong
authors.initials: Y
authors.lastname: Song
authors.forename: Josephine
authors.initials: J
authors.lastname: Ng-Hublin
authors.forename: Annika
authors.initials: A
authors.lastname: Estcourt
authors.forename: Nawal
authors.initials: N
authors.lastname: Hijjawi
authors.forename: Rachel
authors.initials: R
authors.lastname: Chalmers
authors.forename: Stephen
authors.initials: S
authors.lastname: Hadfield
authors.forename: Andrew
authors.initials: A
authors.lastname: Bath
authors.forename: Cameron
authors.initials: C
authors.lastname: Gordon
authors.forename: Una
authors.initials: U
authors.lastname: Ryan
mesh_terms: D000818:Animals; D002417:Cattle; D003457:Cryptosporidiosis / Q000175:diagnosis / Q000469:parasitology*; D003458:Cryptosporidium / Q000235:genetics / Q000302:isolation & purification*; D016785:Cryptosporidium parvum / Q000235:genetics / Q000302:isolation & purification*; D018153:Czech Republic; D016054:DNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D016053:RNA, Protozoan / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014867:Water / Q000469:parasitology*; D014904:Western Australia
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S; D014867:Water
keywords: 18S nested PCR; Clinical; Cryptosporidium; Environmental; qPCR