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

Abstract

gCryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidiumpositive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.

High-Throughput Multiplex Quantitative Polymerase Chain Reaction Method for Giardia lamblia and Cryptosporidium Species Detection in Stool Samples

“…In fact, similar results have been obtained by others as well. 15,20 The high sensitivity of PCR is a clear advantage in the detection of parasites from stool samples. However, this makes PCR also prone to contamination that can happen if a positive sample contaminates negative samples that are analyzed in the same test run, or if the laboratory is contaminated by PCR products from previous amplifications.…”

Multicenter Evaluation of BD Max Enteric Parasite Real-Time PCR Assay for Detection of Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica

“…For APCR, 178-bp, 175-bp, and 338-bp regions of the small-subunit rRNA genes of Cryptosporidium spp., Giardia spp., and Entamoeba spp., respectively, were amplified based on previously described methods (10–12). All nucleic acid extraction and amplification reactions were performed by BD Diagnostics (Baltimore, MD).…”

Clues to uncommon and easily overlooked infectious diagnoses affecting the GI tract and distinction from their clinicopathologic mimics

“…168 Enzyme-linked immunosorbent assay tests have high sensitivity and can serve as an adjunct to microscopic evaluation of stool. 175 PCR assays also demonstrate high sensitivity and specificity 176,177 but are not as widely available.…”