Detection and differentiation of Cryptosporidium hominis and Cryptosporidium parvum by dual TaqMan assays

Abstract

Rapid identification of the two major species of Cryptosporidium associated with human infections, Cryptosporidium hominis and Cryptosporidium parvum, is important for investigating outbreaks of cryptosporidiosis. This study reports the development and validation of a real-time PCR TaqMan procedure for detection of Cryptosporidium species and identification of C. hominis and C. parvum in stool specimens. This procedure comprised a generic TaqMan assay targeting the 18S rRNA for sensitive detection of Cryptosporidium species, as well as two other TaqMan assays for identification of C. hominis and C. parvum. The generic Cryptosporidium species assay can be duplexed with the C. parvum-specific assay. The generic Cryptosporidium species assay was able to detect ten Cryptosporidium species and did not cross-react with a panel of ten other protozoan parasites. The generic Cryptosporidium species assay could detect 1-10 oocysts in a 300 ml stool specimen, whilst each of the species-specific TaqMan assays had detection sensitivities that were approximately tenfold higher. The 18S rRNA assay was found to detect Cryptosporidium species in 49/55 DNA extracts from stool specimens containing either C. hominis or C. parvum. The C. hominis TaqMan assay correctly identified C. hominis in 24/31 validation panel specimens containing this species. The C. parvum-specific assay correctly identified C. parvum in 21/24 validation panel specimens containing this species. This real-time PCR procedure was used to detect and identify C. hominis and C. parvum in stool specimens from outbreak investigations in the USA and Botswana, resulting in identification of C. hominis and/or C. parvum in 66/67 stool specimens shown to be positive for these species using other techniques. From the outbreak specimens tested, the TaqMan procedure was found to have a specificity of 94 %. This TaqMan PCR procedure should be a valuable tool for the laboratory diagnosis of cryptosporidiosis caused by C. hominis and C. parvum during outbreak investigations.

Multicentric Evaluation of a New Real-Time PCR Assay for Quantification of Cryptosporidium spp. and Identification of Cryptosporidium parvum and Cryptosporidium hominis

“…The actual sensitivity was estimated to be about 300 oocysts per g of stool but might be lower with an extraction protocol leading to a more-concentrated DNA solution. Other PCR methods based on the same target sequence displayed comparable sensitivity values (22,26). The sensitivity of the 18S PCR determined by Stroup et al (34) was 100 to 1,000 oocysts/200 mg of stool; that technique used the QIAamp DNA Stool kit and Scorpion probes.…”

“…The most commonly used targets are the 18S rRNA gene (18), the Cryptosporidium oocyst wall protein (19), the 60-kDa glycoprotein (20), heat shock protein 70, the Laxer locus, and microsatellite loci (reviewed in reference 21). Several studies agree on the higher sensitivity of PCR targeting the 18S rRNA gene, in relation to its copy number (22). Nested PCR has been put forward as a means of improving the sensitivity of detection (23) and reverse transcription-PCR as a method for studying viability in environmental samples (24).…”

Detection and Differentiation of Cryptosporidium spp. in Human Clinical Samples by Use of Real-Time PCR

“…The LIB13 locus has been used previously as an alternative to specifically detect C. hominis and C. parvum (20,36). Separate PCRs were performed for these species to reduce amplification competition in mixed infections of C. hominis and C. parvum.…”

“…Real-time PCRs for detection of Cryptosporidium spp. and genotypes in human clinical samples have been described (2,17,18,20,22,36,38). However, none identify C. hominis and C. parvum and detect other Cryptosporidium spp.…”

Frequency of Emerging Parasites in HIV/AIDS and Oncological Patients Stool by Coprological and Molecular Analysis

“…Cryptosporidium spp. [23] were amplified from 500 ng of template DNA in a reaction volume of 25 μL, consisting of 1 × PCR amplification buffer with dATP, dGTP, dCTP, and dTTP (1.2 µM each), 0.5 ng each forward primer JVAF (5'-CCAATTACAAAACCAAAA-AGTCC-3') and reverse primer JVAR (5'-ATGAC-GGGTAACGGGGAAT-3') and 1.5 U of Taq DNA polymerase (Roche, Mannheim, Germany). Thirty-five cycles of amplification were performed in a Maxi-gene thermal cycler (Axygen, Union City, CA, USA).…”