title: Multiplex Real-Time PCR Assay Targeting Eight Parasites Customized to the Korean Population: Potential Use for Detection in Diarrheal Stool Samples from Gastroenteritis Patients.
abstract: 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 Ct values (R2, 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.
authors.affiliation: Departments of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University Hospital, Gwangju, Republic of Korea.
authors.forename: Eun Jeong
authors.initials: EJ
authors.lastname: Won
authors.affiliation: Departments of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University Hospital, Gwangju, Republic of Korea.
authors.forename: Soo Hyun
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Departments of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University Hospital, Gwangju, Republic of Korea.
authors.forename: Seung Jung
authors.initials: SJ
authors.lastname: Kee
authors.affiliation: Departments of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University Hospital, Gwangju, Republic of Korea.
authors.forename: Jong Hee
authors.initials: JH
authors.lastname: Shin
authors.affiliation: Departments of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University Hospital, Gwangju, Republic of Korea.
authors.forename: Soon Pal
authors.initials: SP
authors.lastname: Suh
authors.affiliation: Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Jong Yil
authors.initials: JY
authors.lastname: Chai
authors.affiliation: Departments of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University Hospital, Gwangju, Republic of Korea.
authors.forename: Dong Wook
authors.initials: DW
authors.lastname: Ryang
authors.affiliation: Departments of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University Hospital, Gwangju, Republic of Korea.|Brain Korea 21 Plus Project, Chonnam National University Medical School, Gwangju, Republic of Korea.|Environmental Health Center for Childhood Leukemia and Cancer, Chonnam National University Medical School and Chonnam National University Hwasun Hospital, Hwasun, Jeollanam-do, Republic of Korea.
authors.forename: Myung Geun
authors.initials: MG
authors.lastname: Shin
mesh_terms: D016054:DNA, Protozoan; D003967:Diarrhea / Q000175:diagnosis* / Q000469:parasitology*; D005243:Feces / Q000469:parasitology*; D006801:Humans; D007411:Intestinal Diseases, Parasitic / Q000175:diagnosis*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D056910:Republic of Korea; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan