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

Abstract

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.

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.…”

“…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). Real-time PCR presently is the most extensively used method, allowing for both accurate quantification of the molecular target and typing under technical conditions that avoid cross contamination and DNA carryover (25,26). However, DNA extraction remains a limiting condition for PCR technologies, since DNA polymerase inhibitors (which often are found in environmental and clinical samples) are copurified with nucleic acids and the yield of extraction depends on the technical conditions (27,28).…”

Molecular Testing for Clinical Diagnosis and Epidemiological Investigations of Intestinal Parasitic Infections

“…Hence, the assay allowed direct detection of C. hominis and C. parvum, and in the event of a positive genus-specific (SSU rDNA) result in the absence of a positive result from either of the two LIB loci, the 300-bp-long SSU rRNA gene product could be sequenced for identification of the species present. It has been argued that SSU rDNA PCR is compromised in its ability to detect mixed species due to preferential amplification of the predominant species in a sample (153,163,164). Therefore, the setup developed by Hadfield et al does not circumvent this potential problem, since this genus-specific PCR is based on SSU rRNA gene amplification.…”

“…One reaction targeted the entire genus by amplification of the SSU rRNA gene coupled with a reaction targeting the C. parvum-specific LIB13 locus (163). The second reaction targeted the C. hominis-specific LIB13 locus and also included an internal process control.…”

Application of a qPCR Assay with Melting Curve Analysis for Detection and Differentiation of Protozoan Oocysts in Human Fecal Samples from Dominican Republic

“…Primers to amplify this region, originally designed by Morgan and others, 15 have also been modified by others and used in real-time PCR to detect and differentiate Cryptosporidium spp. 16 The melt temperatures determined for the Cryptosporidium species detected in this study differ by a small margin of 0.27 to 0.42 and therefore, would require high-resolution melt curve analysis for reliable species identification. An additional benefit to using a broad-specificity universal primer set is that unknown, emerging, or opportunistic coccidian pathogens may be discovered if this assay was used for difficult-to-diagnose cases or with immunocompromised patients.…”