Intensities of <i>Clonorchis sinensis</i> infection by EPG ranges.

<p>Intensities of <i>Clonorchis sinensis</i> infection by EPG ranges.</p

Correlation analysis between numbers of recovered worms and EPG counts.

<p>Data of egg-positive subjects (n = 50) were used for analysis. The correlation coefficient (Spearman's rho, <i>r</i>) usually presents a weak (<i>r</i><0.5), moderate (0.5<<i>r</i><0.8) and strong (0.8<<i>r</i>) relationship. A Student's t test was used to examine significance of the correlation coefficient and <i>P</i> value less than 0.05 was considered statistically significant.</p

Adult worms of <i>C. sinensis</i> recovered from patients.

<p>(A) Three kinds of worms with different colors were confirmed. (B, C and D) A few worms were collected with partial maceration (arrowheads) from different subjects.</p

<i>Clonorchis sinensis</i> infection by age and sex.

<p><i>Clonorchis sinensis</i> infection by age and sex.</p

Application of a loop-mediated isothermal amplification (LAMP) assay targeting <i>cox1</i> gene for the detection of <i>Clonorchis sinensis</i> in human fecal samples

<div><p>Background</p><p>Clonorchiasis is prevalent in the Far East, and a major health problem in endemic areas. Infected persons may experience, if not treated, serious complications such as bile stone formation, pyogenic cholangitis, and even cholangiocarcinoma. Early diagnosis and treatment are important to prevent serious complications and, therefore, the simple and reliable diagnostic method is necessary to control clonorchiasis in endemic areas, where resources for the diagnosis are limited.</p><p>Methodology/Principle findings</p><p>The loop-mediated isothermal amplification (LAMP) assay has been applied for the detection of <i>Clonorchis sinensis</i> DNA. Six primers targeting eight locations on the cytochrome c oxidase subunit 1 gene of <i>C</i>. <i>sinensis</i> were designed for species-specific amplification using the LAMP assay. The LAMP assay was sensitive enough to detect as little as 100 fg of <i>C</i>. <i>sinensis</i> genomic DNA and the detection limit in 100 mg of stool was as low as one egg. The assay was highly specific because no cross-reactivity was observed with the DNA of other helminths, protozoa or <i>Escherichia coli</i>. Then, LAMP assay was applied to human fecal samples collected from an endemic area of clonorchiasis in Korea. Using samples showing consistent results by both Kato-Katz method and real-time PCR as reference standards, the LAMP assay showed 97.1% (95% CI, 90.1–99.2) of sensitivity and 100% (95% CI, 92.9–100) of specificity. In stool samples with more than 100 eggs per gram of feces, the sensitivity achieved 100%.</p><p>Conclusions</p><p>To detect <i>C</i>. <i>sinensis</i> in human fecal samples, the LAMP assay was applied and achieved high sensitivity and specificity. The LAMP assay can be utilized in field laboratories as a powerful tool for diagnosis and epidemiological survey of clonorchiasis.</p></div

Sensitivity of the loop-mediated isothermal amplification (LAMP) assay and PCR for the detection of <i>Clonorchis sinensis</i> genomic DNA.

<p>Ten-fold serial dilutions starting from 1 ng of genomic DNA (lane 1) down to 1 fg (lane 7) were tested. (A) Naked eye detection of LAMP products using SYBR Green I. A green color indicates a positive reaction, and an orange color indicates a negative reaction. (B) Fluorescence of LAMP products after using SYBR Green I followed by detection under UV light. (C) Agarose gel electrophoresis of LAMP products followed by ethidium bromide staining and detection under UV light. (D) PCR with outer primers F3 and B3. Values in the left are in base pairs. Lane 8, non-template control (NTC); lane M, molecular marker.</p

Amplification of <i>Clonorchis sinensis</i> DNA in 120 stool samples by loop-mediated isothermal amplification (LAMP) according to the infection intensity presented as eggs per gram of feces (EPG).

<p>Amplification of <i>Clonorchis sinensis</i> DNA in 120 stool samples by loop-mediated isothermal amplification (LAMP) according to the infection intensity presented as eggs per gram of feces (EPG).</p

Sensitivity of the loop-mediated isothermal amplification (LAMP) assay for the detection of <i>Clonorchis sinensis</i> eggs in feces experimentally spiked with a known number of eggs in ten-fold serial dilutions from 10,000 eggs (lane 1) to 1 egg (lane 5).

<p>(A) Naked eye detection of LAMP products using SYBR Green I. A green color indicates a positive reaction, and an orange color indicates a negative reaction. (B) Fluorescence of LAMP products after using SYBR Green I followed by detection under UV light. (C) Agarose gel electrophoresis of LAMP products followed by ethidium bromide staining and detection under UV light. Values in the left are in base pairs. Lane 6, negative stool DNA; lane M, molecular marker.</p

Primer sets used for amplification of the <i>Clonorchis sinensis</i> cytochrome c oxidase subunit 1 (<i>cox1</i>) gene by the loop-mediated isothermal amplification (LAMP) technique.

<p>(A) Locations of the primer sequences. (B) Names, length and sequences of six primers. F3 and B3 represent forward and backward external primers, respectively; FIP and BIP represent forward and backward internal primers, respectively; and LF and LB represent forward and backward loop primers, respectively. Primer FIP consists of F1 complementary sequence and F2 direct sequence. Primer BIP consists of B1 direct sequence and B2 complementary sequence.</p

The AUCs based on methylation (<i>AHRR</i> and <i>F2RL3</i>) and urine cotinine in predicting current smoker from never-smoker status.

<p>The AUCs based on methylation (<i>AHRR</i> and <i>F2RL3</i>) and urine cotinine in predicting current smoker from never-smoker status.</p