Detection ofClonorchis sinensisin stool samples using real-time PCR

Abstract

Human clonorchiasis, caused by infection with the trematode Clonorchis sinensis, is a common health problem in East Asia. In an attempt to develop a new, sensitive method for the diagnosis of the disease, the use of a real-time PCR (targeting the internal-transcribed-spacer-2 sequence of the parasite) to detect C. sinensis-specific DNA in faecal samples has recently been evaluated. The PCR-based assay, which included an internal control to detect any inhibition of the amplification by faecal constituents in the sample, was performed on stool samples and on DNA controls representing a wide range of intestinal microorganisms. The assay appeared very specific, only showing positivity with C. sinensis and Opisthorchis felineus. The sensitivity of the assay was explored by testing 170 preselected samples of human faeces, from an endemic area of South Korea, which had known (microscopically-determined) densities of C. sinensis eggs. The sensitivity of the assay was 100% for the 74 samples that each had > 100 eggs/g and 91.4% for the other 70 samples found egg-positive by microcopy (i.e. those that had <or= 100 eggs/g). Three of the 26 samples that appeared egg-negative by microscopy were found PCR-positive. Encouragingly, the PCR cycle-threshold values, which reflect parasite-specific DNA loads, showed significant correlation with the egg counts. The real-time PCR used in this study therefore appears to be a powerful tool for both the detection and quantification of C. sinensis infections.

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

“…Three additional positive samples were detected out of 26 samples in which no eggs were found by microscopy. The C T values were strongly correlated with the intensity of infections, as determined by egg counts in Kato-Katz smears (369). A similar approach with primers and a hydrolysis probe designed from the C. sinensis ITS1 sequence showed specific (with the exception of O. viverrini) and sensitive detection of C. sinensis in a small number of fecal samples and fish tissue samples (370) Primer sets for the detection of O. viverrini DNA in stool samples using LAMP were designed from the mitochondrial nad1 sequence and from the ribosomal ITS1 sequence of O. viverrini (373,374).…”

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis

“…Some of the examples are multiplex PCR approaches (Le et al 2006), PCR and sequencing of nuclear ribosomal and mitochondrial DNA (Park 2007) and nuclear marker sequences (PM-int9) (Shekhovtsov et al 2009), PCR-RFLP analysis of the 18S-ITS1-5.8S nuclear ribosomal DNA region (Kang et al 2008), and PCR targeting ribosomal DNA ITS regions (Sato et al 2009). A rapid, high-throughput, specific, and sensitive realtime PCR has been applied for the detection of C. sinensis (Kim et al 2009;Cai et al 2012;Rahman et al 2011) and O. viverrini (Intapan et al 2008a, 2008b, 2009aSuksumek et al 2008;Sri-Aroon et al 2011;Janwan et al 2011) DNA. However, those methods have been applied for detecting both parasites in separate assays and are useful in endemic areas of either C. sinensis or O. viverrini alone but may be problematic where C. sinensis and O. viverrini are co-endemic.…”

A Pentaplex Real-Time Polymerase Chain Reaction Assay for Detection of Four Species of Soil-Transmitted Helminths

“…26,[29][30][31] These diagnostic limitations can be avoided with real-time PCR because it allows simultaneous detection and quantification of different targets and has been shown to be highly sensitive and specific for detection of many intestinal parasites. 9,[12][13][14][15][32][33][34][35][36][37] In this study, a pentaplex real-time PCR was evaluated to detect infections with the human roundworm, two species of hookworm and threadworm. An additional fifth primer and probe set was used for the amplification and detection of an internal control for the detection of possible inhibition of the amplification reaction by fecal contaminants.…”