Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of Schistosoma japonicum.

BACKGROUND: With the continuous decline in prevalence and intensity of Schistosoma japonicum infection in China, more accurate and sensitive methods suitable for field detection become much needed for schistosomiasis control. Here, a novel rapid and visual detection method based on the combination of recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) was developed to detect S. japonicum DNA in fecal samples. RESULTS: The LFD-RPA assay targeting SjR2 could detect 5 fg S. japonicum DNA, which was identical to qPCR and real-time RPA assay, and showed no cross-reaction with other parasites. The detection could be finished within 15-20 min at a wide temperature range (25-45 °C), and the results could be visualized by naked eye. The diagnostic validity of LFD-RPA assay was further assessed with 14 fecal samples of infected patients diagnosed by Kato-Katz method and 31 fecal samples of healthy persons, and compared with that of Enzyme-linked immunosorbent assay (ELSIA) and Indirect Hemagglutination Assay (IHA). The LFD-RPA assay showed 92.68 % sensitivity, 100 % specificity and excellent diagnostic agreement with the gold standard Kato-Katz test (k = 0.947, Z = 6.36, P < 0.001), whereas ELISA showed 85.71 % sensitivity, 93.55 % specificity, and substantial diagnostic agreement (k = 0.793, Z = 5.31, P < 0.001), and IHA showed 78.57 % sensitivity, 83.87 % specificity, and moderate diagnostic agreement (k = 0.600, Z = 4.05, P < 0.001), indicating that the LFD-RPA was much better than the traditional methods. CONCLUSIONS: The LFD-RPA assay established by us is a sensitive, specific, rapid and convenient method for the diagnosis of schistosomiasis, and shows a great potency in field application.

Evaluation of banked urine samples for the detection of circulating anodic and cathodic antigens in Schistosoma mekongi and S. japonicum infections: a proof-of-concept study.

In Asia, Schistosoma japonicum is the predominant schistosome species, while Schistosoma mekongi is confined to limited foci in Cambodia and Lao People's Democratic Republic. While the People's Republic of China has been successful in controlling schistosomiasis, the disease remains a major public health issue in other areas. In order to prioritise intervention areas, not only accurate diagnosis is important but also other factors, such as practicality, time-efficiency and cost-effectiveness, since they strongly influence the success of control programmes. To evaluate the highly specific urine-based assays for the schistosome circulating cathodic antigen (CCA) and the circulating anodic antigen (CAA), banked urine samples from Cambodia (n=106) and the Philippines (n=43) were examined by the upconverted phosphor lateral flow (UCP-LF) CAA assay and the point-of-care (POC)-CCA urine assay. Based on 250 µl urine samples, UCP-LF CAA sensitivity outcomes surpassed a single stool examination by the Kato-Katz technique. The banked urine samples in the current study did not allow the evaluation of larger volumes, which conceivably should deliver considerably higher readings. The sensitivity of a single urine POC-CCA was in the same order as that of a single Kato-Katz thick smear examination, while the sensitivity approached that of triplicate Kato-Katz when a combination of both CAA and CCA assays was used. The promising results from the current proof-of-concept study call for larger investigations that will determine the accuracy of the urine-based CCA and CAA assays for S. mekongi and S. japonicum diagnosis.

Application of an immunomagnetic bead ELISA based on IgY for detection of circulating antigen in urine of mice infected with Schistosoma japonicum.

Schistosomiasis is an important zoonosis and some livestock especially bovine and swine play a crucial role on the disease transmission in endemic areas. The gold standard for animal Schistosoma japonicum infection is fecal examination although indirect agglutination assay (IHA) is so far mostly used in field survey and laboratory examination. Lack of sensitivity, poor practicality and high false positivity limit the use of those methods for routine veterinary detection as well as human diagnosis. A novel immunomagnetic bead ELISA based on IgY (egg yolk immunoglobulin) was developed for detection of circulating schistosomal antigen (CSA) in sera of hosts infected with S. japonicum. To assess the application of this method for diagnosis of domestic animal schistosomiasis with urine sample, the immunomagnetic bead ELISA based on IgY (IgY-IMB-ELISA) was employed in the present study to detect CSA in urine of murine schistosomiasis with either light (10 S. japonicum cercariae infection per mouse) or heavy infection (30 S. japonicum cercariae infection per mouse). The results showed that the CSA levels in urine of heavily and lightly infected mice reached a peak in 8 and 10 weeks after infection, respectively, remaining at a constant plateau in both groups by the end of the experiment (14 weeks after infection). The CSA level in urine of heavily infected mice was much higher than that of lightly infected mice from 8 to 14 weeks after infection. The effect of praziquantel treatment on the CSA level in urine of heavily infected mice was also investigated. It was found that the CSA level in urine of heavily infected mice with treatment was much lower than that of untreated mice at 4 weeks post-treatment, although still higher than that of control mice, and then gradually descended to the background level by 8 weeks after treatment. Our findings suggested that the IgY-IMB-ELISA may be an efficient and practical tool in non-invasive diagnosis of schistosome infection based on antigen detection, and evaluation of the efficacy of chemotherapy as well.

[Detection of urine antibodies to Schistosoma japonicum by magnetic particle affinity immunoassay].

OBJECTIVE: To explore a non-invasive method for detection of urine antibodies to Schistosoma japonicum. METHODS: The urine antibodies to S. japonicum were detected by magnetic particle affinity immunoassay (MPAIA) in 158 cases of schistosomiasis japonica and 100 health persons, and their serum antibodies to S. japonicum were also detected at the same time. RESULTS: The sample of urine by MPAIA was 10 microl original urine without any special treatment. The positive rate of urine and serum were 48.10% (76/158)and 88.61% (140/158), respectively. There was difference between the performance of two methods (chi2 = 60.24, P < 0.05). However, both of their specificity were 100% (100/100). CONCLUSION: MPAIA is viable for detection of urine antibodies to S. japonicum, but its sensitivity should be improved.

System level metabolic effects of a Schistosoma japonicum infection in the Syrian hamster.

A metabolic profiling strategy was used to investigate the metabolic responses of Syrian hamsters (SLAC) to a Schistosoma japonicum infection using high resolution 1H nuclear magnetic resonance (NMR) spectroscopy and pattern recognition. In two independent experiments, male hamsters were each infected with 100 S. japonicum cercariae. At days 34-36 post-infection, urine was obtained from hamsters housed individually in metabolism cages. At the same time, urine was collected from age- and sex-matched infection-free control hamsters. The main biochemical effects of a S. japonicum infection in the hamster consisted of reduced levels of urinary tricarboxylic acid cycle intermediates, including citrate and succinate and increased levels of pyruvate. In addition, a range of microbial-related metabolites, such as hippurate, p-cresol glucuronide, phenylacetylglycine and trimethylamine were also associated with a S. japonicum infection. Most of the observed biochemical effects were in common with those previously characterized for a S. mansoni infection in a mouse host. The major distinguishing consequence of a S. japonicum infection in the hamster was the inhibition of manufacture or utilization of short-chain fatty acids, when compared to a S. mansoni infection in the mouse.

Production of monoclonal antibodies using an electrophoretically separated diagnostic marker from the urine of individuals infected with Schistosoma japonicum.

In this paper, electrophoresis was successfully used to screen a diagnostic marker (a glycoprotein of 30 kDa) from the urine of individuals infected with Schistosoma japonicum, and then with the marker as antigen, two lines of monoclonal antibodies (mAbs) were obtained, NP56 and NP54. NP56 was of better immunoreactivity, its immunoglobulin isotype being IgG2b. NP56 reacted with soluble egg antigen and adult worm antigen and miracidia in eggs of S. japonicum. Using NP56 as a probe in indirect ELISA, the sensitivity and specificity (median egg excretion per gram of feces 69) was 90% and 100% in concentrated urine samples and 50% and 100% in original urine samples, respectively. The results indicate that the method is feasible for producing mAbs with diagnostic markers separated electrophoretically from the urine of individuals infected with pathogen.

Comparative studies on detecting CAg in urine of acute schistosomiasis patients by mAb-RIHA and mAb-DotELISA.

Urine was concentrated 20-fold for assay for CAg of Schistosoma japonicum. mAb-RIHA and mAb-DotELISA were positive in 78, 31% and 65.06% of cases respectively, of 83 patients with acute schistosomiasis. The false positive rates in 101 healthy controls were 14.85% and 0%, respectively. Cross-reactions (using mAb-RIHA) were seen in 16.36% and 14.28% of patients with clonorchiasis, 49 patients with ankylostomiasis, respectively. Corresponding figures for mAb-DotELISA were 0% and 0%.