Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

Abstract

This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR). A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

Prevalence and genetic characterization of Cryptosporidium spp. and Cystoisospora belli in HIV-infected patients

“…Most of these published papers have shown that C. parvum and C. hominis are the most prevalent species 1,15,25 . With respect to C. belli, only one study demonstrated genetic polymorphisms among clinical isolates obtained from HIV(+) patients 23 .…”

Method verification study for detection of Cryptosporidium spp. by realtime PCR method

“…PCR tabanlı yöntemler, az sayıda Cryptosporidium'un saptanması için oldukça duyarlı ve özgüldür, zaman ve maliyet açısından da etkilidir (15) . Ayrıca geleneksel yöntemler tür düzeyinde tanımlama yapamadığından, bu amaçla PCR, real-time PCR, Restriction Fragment Lenght Polymorphism (RFLP), mikroarray, melting analizi, sekans analizi gibi moleküler yöntemler geliştirilmiştir (2) . Günümüzde bu nedenlerle moleküler yöntemlerle tanı koyan laboratuvarların sayısı artmaktadır.…”

“…Gastrointestinal sistem epitel hücrelerini enfekte eder, akut ve bol sulu ishale neden olur (1) . İshal, immun sistemi sağlam bireylerde kendi kendini sınırlarken, immun sistemi baskılanmış konaklarda kronik ve yaşamı tehdit edici olabilir (1,2) . Parazit özellikle gıda ve su kaynaklı ishal nedenleri arasında önemli bir yer tutmaktadır (1,(3)(4)(5) .…”

“…İnsanlarda olguların çoğuna Cryptosporidium hominis (C. hominis) ve Cryptosporidium parvum (C. parvum) neden olmaktadır. Bunun dışında, Cryptosporidium meleagridis, Cryptosporidium felis, Cryptosporidium canis de bildirilmiştir (2,3,6,10,11) . C. hominis esas olarak insanları enfekte ederken, en yaygın zoonotik tür olan C. parvum çok sayıda hayvanı ve insanı enfekte ederek zoonotik bulaşta rol oynamaktadır (1,6) .…”

Specific and quantitative detection and identification of Cryptosporidium hominis andC. parvum in clinical and environmental samples

“…Jothikumar et al, (2008) designed a dual TaqMan assay to detect C. hominis and C. parvum in stool specimens based on an unknown locus (AF190627), which had a specificity of 94 %. More recently, TaqMan assays based on the LIB13 (function unknown) and 18S loci were developed to detect and differentiate C. parvum and C. hominis in human faecal samples Rolando et al, 2012;Mary et al, 2013). …”