Comparison of DNA Extraction Methods and Real-Time PCR Assays for the Detection of Blastocystis sp. in Stool Specimens

Abstract

Diagnosis of Blastocystis in stool may be challenging, as microscopic examination and culture-based methods have demonstrated low sensitivity. Molecular detection assays are now available for this enteric parasite, based on “in-house” or commercial-developed techniques. The aim of this study was to assess and compare the performance of (i) two DNA extraction methods (manual versus automated), and (ii) four qPCR assays (three “in-house” and one commercialized), for detection of Blastocystis sp. in human stools. One hundred and forty stools were included, among which 76 were confirmed to be positive for Blastocystis. The manual DNA extraction method allowed for the identification of significantly more positive specimens than the automated method (p < 0.05). In particular, specimens with a low parasite load were negative when DNA was extracted with the automated process. The four qPCR assays also had variable performances, with the commercialized assay being the most sensitive (84%) but the least specific (82%). Overall, for all qPCR assays, the specificity decreased when the sensitivity increased. Blastocystis’ subtype, notably the subtype 4, influenced these performances. Our results indicate that the positivity rate for the detection of Blastocystis in stools could be variable according to the DNA extraction method and the qPCR assay used. These pitfalls need to be considered for the selection of method and interpretation of results, particularly considering the search of this intestinal parasite in a donor before fecal microbiota transplantation.

Comparison of molecular diagnostic approaches for the detection and differentiation of the intestinal protist Blastocystis sp. in humans

“…Surprisingly, it appears that this is the very first study comparing the sensitivity between the commonly used cPCR protocol [ 20 ] and qPCR [ 25 ] for the detection of Blastocystis sp. Previously, some studies showed higher sensitivity of qPCR in comparison with classical methods such as direct-light microscopy or xenic in vitro culture [ 14 , 15 , 25 ]. The study by Nourrisson et al [ 14 ] compared four qPCR protocols for detection of Blastocystis sp.…”

“…Previously, some studies showed higher sensitivity of qPCR in comparison with classical methods such as direct-light microscopy or xenic in vitro culture [ 14 , 15 , 25 ]. The study by Nourrisson et al [ 14 ] compared four qPCR protocols for detection of Blastocystis sp. and found that they differed in specificity and sensitivity.…”

“…Nevertheless, accurate detection and distinction of Blastocystis subtypes is essential to understand the transmission and the role of this protist in human health. Due to their high sensitivity and specificity, molecular methods such as conventional PCR (cPCR) or real-time PCR (qPCR) are often used [ 14 , 22 , 25 ]. In addition, next-generation sequencing (NGS) is gaining prominence in detection of Blastocystis and its subtypes [ 4 , 17 , 27 ].…”

Evaluation of the Use of Singleplex and Duplex CerTest VIASURE Real-Time PCR Assays to Detect Common Intestinal Protist Parasites

“…The diagnostic sensitivity and specificity of The VIASURE Giardia assay were 0.94 and 0.99, respectively. Overall, these figures were superior to those obtained with other commercial qPCR assays, including the Allplex Gastrointestinal Panel-Parasite assay (Seegene, Seoul, Korea; sensitivity: 0.84-1; specificity: 0.81-082) [49,50] or the LIGHTMIX Gastro Parasite assay (Tib MolBiol, Berlin, Germany; sensitivity: 0.99; specificity: 0.89) [49]. Here it should be noted that the diagnostic performance of the latter assay did not consider the subtype of the isolates investigated.…”