Multiplex Real-Time PCR Assay Targeting Eight Parasites Customized to the Korean Population: Potential Use for Detection in Diarrheal Stool Samples from Gastroenteritis Patients

<div><p>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 <i>Cryptosporidium parvum</i>, <i>Giardia lamblia</i>, <i>Entamoeba histolytica</i>, <i>Blastocystis hominis</i>, <i>Dientamoeba fragilis</i>, <i>Clonorchis sinensis</i>, <i>Metagonimus yokogawai</i>, and <i>Gymnophalloides seoi</i>. 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 (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.</p></div

PCR efficiency (%) obtained from slope of dependence of DNA concentrations on threshold cycle (Ct) values.

<p>PCR efficiency (%) obtained from slope of dependence of DNA concentrations on threshold cycle (Ct) values.</p

Analytical sensitivity (limit of detection, LoD) and analytical precision of the multiplex qPCR assay.

<p>Analytical sensitivity (limit of detection, LoD) and analytical precision of the multiplex qPCR assay.</p

Diagnostic performance from microscopic examination and multiplex qPCR in terms of detection of the major parasites causing gastroenteritis in stool samples from patients with diarrhea.

<p>Diagnostic performance from microscopic examination and multiplex qPCR in terms of detection of the major parasites causing gastroenteritis in stool samples from patients with diarrhea.</p

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

<p>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 <i>Cryptosporidium parvum</i> (tube 1) and <i>Metagonimus yokogawai</i> (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 <i>Blastocystis hominis</i> (tube 1), <i>Entamoeba histolytica</i> (tube 2), and <i>Dientamoeba fragilis</i> (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 <i>Gymnophalloides seoi</i> (tube 1), <i>Giardia lamblia</i> (tube 2), and <i>Clonorchis sinensis</i> (tube 3) were observed in the FAM channel. Patient No. 35’s sample showed a positive curve within tube 3 according to FAM signals.</p

Established combination of primers and probes using the multiplex real-time PCR assay for the major parasites causing gastroenteritis.

<p>Established combination of primers and probes using the multiplex real-time PCR assay for the major parasites causing gastroenteritis.</p

Relationship between the fluconazole usage and the percentage of isolates with non-susceptible to fluconazole, or isolates with decreased susceptibility to fluconazole (MIC ≥4 μg/ml) at nine university hospitals in Korea.

<p>The usage of fluconazole, defined as the daily dose/1,000 patient days (DDD/1,000 PD) at the individual hospital was represented by the grey columns. The percentage of isolates with non-susceptible to fluconazole (closed circle with solid line) and the percentage of isolates with decreased susceptibility to fluconazole (MIC ≥4 μg/ml) (open rectangle with dotted line) showed positive correlations with the usage of fluconazole at the individual hospitals.</p