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.

Fig 1. Representative reaction curves of the multiplex real-time PCR assay.

A) Multiple reaction curves indicate amplification of the positive controls and clinical samples obtained from patients with gastroenteritis. B) Positive curves of two positive controls for Cryptosporidium parvum (tube 1) and Metagonimus yokogawai (tube 2) were observed in the Quasar 670 channel. Patient No. 106’s sample showed a positive curve within tube 1 according to Quasar 670 signals. C) Positive curves of two positive controls for Blastocystis hominis (tube 1), Entamoeba histolytica (tube 2), and Dientamoeba fragilis (tube 3) were observed in the VIC/HEX channel. Patient No. 112’s sample showed a positive curve within tube 1 according to the VIC/HEX signals. D) Positive curves of two positive controls for Gymnophalloides seoi (tube 1), Giardia lamblia (tube 2), and Clonorchis sinensis (tube 3) were observed in the FAM channel. Patient No. 35’s sample showed a positive curve within tube 3 according to FAM signals.