Identification of genotypically mixed Cryptosporidium parvum populations in humans and calves

Tanriverdi, Sultan; Arslan, Mükremin Özkan; Akiyoshi, Donna E.; Tzipori, Saul; Widmer, Giovanni

doi:10.1016/s0166-6851(03)00138-5

Cited by 73 publications

(50 citation statements)

References 24 publications

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“…DNA was isolated from semi-purified oocyst suspensions prepared from stool samples by saturated-salt solution centrifugation and extracted using QIAamp DNA mini kit spin columns (Qiagen Ltd, Crawley, UK) as previously described by Elwin et al (2001). For each sample, the speciation was performed by PCR-RFLP of the Cryptosporidium oocyst wall protein (COWP) gene as previously described by Spano et al (1997) and by real-time PCR using simplex Lib 13 primers for C. parvum and C. hominis as described by Tanriverdi et al (2003). For this study, we tested the DNA of 5 C. hominis and 5 C. parvum isolates as these two species are the cause of the majority of human cases of cryptosporidiosis.…”

Section: A T E R I a L S A N D M E T H O D Smentioning

confidence: 99%

Whole genome amplification (WGA) for archiving and genotyping of clinical isolates ofCryptosporidiumspecies

et al. 2009

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S U M M A R YClinical and environmental isolates of pathogens are often unique and may be unculturable, yielding a very limited amount of DNA for genetic studies. Cryptosporidium in particular are difficult to propagate. Whole genome amplification (WGA) is a valuable technique for amplifying genomic material. In this study, we tested 5 WGA commercial kits using Cryptosporidium clinical isolates. DNA of 5 C. hominis and 5 C. parvum clinical isolates and C. parvum IOWA reference strain were used. The majority of the samples were amplified by all of the kits tested. The integrity and fidelity of the amplified genomic DNA were assessed by sequence analysis of several PCR products of varying length. We found evidence that one kit in particular may be more error prone while another seemed the more suitable kit for Cryptosporidium clinical samples, generating high molecular weight DNA from all the samples with high fidelity. Thus WGA was found to be a useful technique for producing amplified DNA suitable for downstream genotyping techniques and archiving of Cryptosporidium clinical isolates.

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Section: A T E R I a L S A N D M E T H O D Smentioning

confidence: 99%

Whole genome amplification (WGA) for archiving and genotyping of clinical isolates ofCryptosporidiumspecies

et al. 2009

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“…Cryptosporidium izolatı elde etmek amacıyla yapılan saha çalışmalarında 2011 yılında buzağılarda bu protozoonun %5'ler düzeyine kadar düştüğü görülmüştür 15,19 . Kars'ta ishalli buzağılarda C. parvum tip II saptanmıştır 20,21 . Yapılan bu çalışma ile bölgede ishalli ve sağlıklı buzağılarda ve ineklerde C. parvum'un subtipleri tespit edilmiştir.…”

Section: Tartişma Ve Sonuçunclassified

“…Türkiye'de ilk olarak bu araştır-ma ile C. parvum türünün subtipleri saptanmıştır. Kars yö-resinde daha önce yapılan araştırmalarda 20,21 bildirilen C. parvum tip II genotipi bu çalışma da da buzağılarda ve daha yaşlı sığırlarda bulunmuştur. Bu çalışma ile ishalli buzağılarda da C. parvum IIaA15G2R1 subtipinin sorumlu etiyolojik ajan olduğu saptanmıştır.…”

Section: Tartişma Ve Sonuçunclassified

“…Cryptosporidium etkenleri ishalli üç aylığa kadar olan buzağılarda daha sık görülmekte ve klinik olgulara da rastlanmaktadır 15,19 . Kars yöresinde buzağılarda C. parvum tip II genotipi belirlenmiş ve çiftliklerde multilokuslu genotipler yüksek bulunmuştur 20,21 . Ancak bugüne kadar C. parvum türünün subtipleri ile ilgili ne Kars yöresinde ne de Türkiye'de bir araştırma bulunmamaktadır.…”

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Determination of Cryptosporidium parvum Subtypes in Cattle in Kars Province of Turkey

Arslan¹,

Ekinci²

2009

Kafkas Univ Vet Fak Derg

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“…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.…”

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

et al. 2011

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Real-time PCR has the potential to streamline detection and identification of Cryptosporidium spp. in human clinical samples. In the present article, we report the first such assay to allow not only detection and differentiation of the most common human pathogens, Cryptosporidium hominis and Cryptosporidium parvum, but also simultaneous amplification of a region of the small subunit (SSU) rRNA gene, permitting direct sequence analysis to identify any Cryptosporidium species. An internal control is incorporated to identify the presence of PCR inhibitors. Analytical sensitivity was determined to be as low as 200 oocysts per gram of feces processed, equivalent to 2 oocysts per PCR. The C. hominis and C. parvum PCRs specifically detected only species/genotypes in their respective target clades. Diagnostic sensitivity and specificity, evaluated against a widely used conventional nested SSU rRNA gene PCR as a nominated gold standard using a panel of 258 (151 positive and 107 negative) samples, were 100% and 99.1%, respectively. The assay agreed with PCR-restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein gene for 134 of 136 (98.5%) samples tested prospectively and typed two additional isolates. The real-time PCR assay was sensitive, specific, and reproducible and significantly improved laboratory work flow and turnaround times.

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Identification of genotypically mixed Cryptosporidium parvum populations in humans and calves

Cited by 73 publications

References 24 publications

Whole genome amplification (WGA) for archiving and genotyping of clinical isolates ofCryptosporidiumspecies

Whole genome amplification (WGA) for archiving and genotyping of clinical isolates ofCryptosporidiumspecies

Determination of Cryptosporidium parvum Subtypes in Cattle in Kars Province of Turkey

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

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