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

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

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

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

Genetic relatedness by microsatellite analysis and virulence in the <i>G</i>. <i>mellonella</i> model of 15 respiratory and 16 ear isolates of <i>A</i>. <i>terreus</i> from six hospitals.

<p>Genetic relatedness by microsatellite analysis and virulence in the <i>G</i>. <i>mellonella</i> model of 15 respiratory and 16 ear isolates of <i>A</i>. <i>terreus</i> from six hospitals.</p

Antifungal susceptibilities, microsatellite genotypes and virulence of 31 <i>A</i>. <i>terreus</i> isolates from six hospitals in South Korea.

<p>Antifungal susceptibilities, microsatellite genotypes and virulence of 31 <i>A</i>. <i>terreus</i> isolates from six hospitals in South Korea.</p

Genetic relationships of 31 <i>A</i>. <i>terreus</i> isolates according to source.

<p>The dendrogram is based on a categorical analysis of seven microsatellite markers in combination with unweighted pairgroup method using the arithmetic average (UPGMA) clustering. The number on the tree indicates the branch length, showing the difference along a branch. All 31 isolates (R1 to R15 and E1 to E16) comprised 27 distinct genotypes (GT 1 to GT 27) by 7 microsatellite markers. However, when a cluster is defined as the isolation of two or more strains with a branch length distance of < 0.63, ear isolates comprise clusters II and V, and the respiratory isolates comprise clusters I, III, and IV, suggesting a closer genetic relatedness among isolates from the same body site (ear or respiratory tract). Five isolates (R7, R15, E1, E4, and R12) were unique to a single isolate, which did not cluster with other isolate as a branch length distance of < 0.63. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186086#pone.0186086.t002" target="_blank">Table 2</a> for detailed information on each isolate.</p

Diversity of clinical isolates of <i>Aspergillus terreus</i> in antifungal susceptibilities, genotypes and virulence in <i>Galleria mellonella</i> model: Comparison between respiratory and ear isolates

<div><p>We analyzed the antifungal susceptibility profiles, genotypes, and virulence of clinical <i>Aspergillus terreus</i> isolates from six university hospitals in South Korea. Thirty one isolates of <i>A</i>. <i>terreus</i>, comprising 15 respiratory and 16 ear isolates were assessed. Microsatellite genotyping was performed, and genetic similarity was assessed by calculating the Jaccard index. Virulence was evaluated by <i>Galleria mellonella</i> survival assay. All 31 isolates were susceptible to itraconazole, posaconazole, and voriconazole, while 23 (74.2%) and 6 (19.4%) showed amphotericin B (AMB) minimum inhibitory concentrations (MICs) of ≤ 1 mg/L and > 4 mg/L, respectively. Notably, respiratory isolates showed significantly higher geometric mean MICs than ear isolates to AMB (2.41 <i>vs</i>. 0.48 mg/L), itraconazole (0.40 <i>vs</i>. 0.19 mg/L), posaconazole (0.16 <i>vs</i>. 0.08 mg/L), and voriconazole (0.76 <i>vs</i>. 0.31 mg/L) (all, <i>P</i> <0.05). Microsatellite genotyping separated the 31 isolates into 27 types, but the dendrogram demonstrated a closer genotypic relatedness among isolates from the same body site (ear or respiratory tract); in particular, the majority of ear isolates clustered together. Individual isolates varied markedly in their ability to kill infected <i>G</i>. <i>mellonella</i> after 72 h, but virulence did not show significant differences according to source (ear or respiratory tract), genotype, or antifungal susceptibility. The current study shows the marked diversity of clinical isolates of <i>A</i>. <i>terreus</i> in terms of antifungal susceptibilities, genotypes and virulence in the <i>G</i>. <i>mellonella</i> model, and ear isolates from Korean hospitals may have lower AMB or triazole MICs than respiratory isolates.</p></div