Open AccessMultiplex Real-Time PCR Assay Targeting Eight Parasites Customized to the Korean Population: Potential Use for Detection in Diarrheal Stool Samples from Gastroenteritis Patients
Author(s) -
Eun Jeong Won,
Soo Hyun Kim,
SeungJung Kee,
Jong Hee Shin,
Soon Pal Suh,
Jong Yil Chai,
DongWook Ryang,
Myung Geun Shin
Publication year - 2016
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0166957
Subject(s) - norovirus , multiplex , cryptosporidium , diarrhea , biology , blastocystis , dna extraction , polymerase chain reaction , entamoeba histolytica , microbiology and biotechnology , population , real time polymerase chain reaction , giardia lamblia , parasitology , virology , entamoeba , giardia , multiplex polymerase chain reaction , feces , medicine , gastroenterology , virus , bioinformatics , zoology , environmental health , gene , biochemistry
Intestinal parasitic diseases occur worldwide and can cause diarrhea or gastroenteritis; however, their diagnosis is quite difficult, especially in low-endemism countries. We developed a multiplex real-time PCR assay for detection of eight intestinal parasites and prospectively evaluated it for patients with gastroenteritis. The assay targeted Cryptosporidium parvum , Giardia lamblia , Entamoeba histolytica , Blastocystis hominis , Dientamoeba fragilis , Clonorchis sinensis , Metagonimus yokogawai , and Gymnophalloides seoi . Performance characteristics were evaluated based on recovery after DNA extraction, analytical sensitivity, specificity, reproducibility, cross-reactivity, and interference characteristics. Clinical performance was validated against microscopy on 123 diarrheal samples. The assay demonstrated strong correlations between DNA concentrations and C t values (R 2 , 0.9924–0.9998), and had a high PCR efficiency (83.3%–109.5%). Polymerase chain reactions detected as few as 10–30 copies of genomic DNA, and coefficient of variance was 0–7%. There was no cross-reactivity to the other 54 microorganisms tested. Interference occurred only in presence of high concentrations of erythrocytes or leukocytes. This assay had a higher correct identification rate (100.0% vs. 90.2%) and lower incorrect ID rate (0.0% vs. 9.8%) when compared to microscopy. Overall, this assay showed a higher sensitivity (100.0%; 95% confidence interval [CI] of 80.5–100.0) than microscopy (29.4%; 95% CI 10.31–55.96), and the specificity levels were comparable for both methods (100.0%; 95% CI 96.58–100.0). This newly developed multiplex real-time PCR assay offers a potential use for detecting intestinal parasitic pathogens customized to the Korean population.