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. Author manuscript; available in PMC: 2022 Aug 1.
Published in final edited form as: Parasitol Res. 2021 Jul 14;120(8):2839–2846. doi: 10.1007/s00436-021-07224-6

Comparative assessment of immunochromatographic test kits using somatic antigens from adult Opisthorchis viverrini and IgG and IgG4 conjugates for serodiagnosis of human opisthorchiasis

Weeraya Phupiewkham 1,2, Lakkhana Sadaow 1, Oranuch Sanpool 1, Rutchanee Rodpai 1, Hiroshi Yamasaki 3, Wannaporn Ittiprasert 4, Victoria H Mann 4, Paul J Brindley 4,*, Wanchai Maleewong 1, Pewpan M Intapan 1,*
PMCID: PMC8554465  NIHMSID: NIHMS1741573  PMID: 34259939

Abstract

Chronic infections of humans with Opisthorchis viverrini and Clonorchis sinensis spanning decades may lead to life-threatening pathology prior to cholangiocarcinoma (CCA), which usually has a poor prognosis. Serological tools can support the parasitological examination in clinical diagnosis and support screening for risk of CCA. We developed novel immunochromatographic test kits using a soluble, somatic tissue extract of adult O. viverrini worms as an antigen and colloidal-gold labeled conjugates of IgG and IgG4 antibodies and evaluated the diagnostic values of both the OvSO-IgG and OvSO-IgG4 kits. For diagnosis of human opisthorchiasis individually, the diagnostic sensitivity, specificity, positive and negative predictive values with 95% confidence intervals in the OvSO-IgG kit were 86.6% (78.9-92.3), 89.5% (84.2-93.5), 82.9% (74.8-89.2), and 91.9% (87.0-95.4), respectively, while the 75% (65.9-82.7), 98.4% (95.5-99.7), 96.6% (90.3-99.3), and 87% (81.7-91.2), respectively, for the OvSO-IgG4 kit at the prevalence of infection of 37.1%. Twenty-three (76.7%) and 14 (46.7%) of 30 clonorchiasis sera showed positive reactivity with the OvSO-IgG and OvSO-IgG4 kits, respectively. There was 84.1% (κ-value = 0.649) concordance between the two kits, which was statistically significant (p < 0.001). Both ICT kits can be employed as quick and easy point-of-care diagnostic tools, and hence the OvSO-IgG and OvSO-IgG4 kits can support expanded capacity for clinical diagnosis of human opisthorchiasis and clonorchiasis. These kits may find utility in large-scale surveys in endemic areas where there are limited sophisticated medical facilities or capacity.

Keywords: Opisthorchiasis, Serodiagnosis, Somatic antigen, IgG, IgG4, Immunochromatographic test kits

Introduction

Chronic infections in humans with the fish-borne liver flukes, Opisthorchis viverrini and Clonorchis sinensis, may cause fatal bile duct cancer (cholangiocarcinoma; CCA) and liver cancer (hepatocarcinoma) (Honjo et al. 2005; Sripa et al. 2010; IARC 2012; Zheng et al. 2017). Approximately 600 million people globally are at risk of infection with O. viverrini or C. sinensis (Sripa et al. 2010). More than 10 million people are estimated to be infected with O. viverrini in the lower Mekong River sub-region, i.e., Cambodia, Lao People's Democratic Republic, southern Vietnam, Myanmar, and Thailand (Sripa et al. 2010; Aung et al. 2017). In clonorchiasis, over 200 million people are at risk of clonorchiasis, 15-20 million are infected and 1.5-2 million people (approximately 10% of infected people) present symptoms and complications in Northeast and East Asia (northern Vietnam, China, and Korea) (Sripa et al. 2010). Diagnosis of human infections is accomplished by the demonstration of eggs in feces, bile or duodenal contents and it is the gold standard. However, this tool is laborious and requires proficient, practiced technicians and microscopists (Sripa et al. 2010). Serologic and molecular tools have been used to support diagnosis and screening (McCarthy et al. 2012; Saijuntha et al. 2018) of opisthorchiasis (Wongratanacheewin et al. 1988a, 2003; Akai et al. 1995; Laha et al. 2008; Sripa et al. 2012; Teimoori et al. 2015) and clonorchiasis (Sirisinha et al. 1990; Hong et al. 1999; Zhao et al. 2004; Li et al. 2011, 2012; Kim et al. 2019).

Recently, a rapid diagnostic immunochromatographic test (ICT) kit using an excretory-secretory (ES) antigen from adult O. viverrini was developed for specific IgG detection in human opisthorchiasis and clonorchiasis (Sadaow et al. 2019). The preparation of ES antigen is comparatively tedious and expensive due to the necessity of in vitro maintenance of the adult parasites, which in turn are sourced from experimental infection of laboratory hamsters. The yield is also limited. By contrast, the preparation of somatic antigen is less expensive than ES antigen, yield greater quantities of usable antigen, and worm culture is not needed. In this study, the ICT kit (OvSO-kit) using somatic extract from adult O. viverrini worms was developed for the detection of specific IgG antibody, with a key we have termed the OvSO-IgG kit, in sera from human cases confirmed with opisthorchiasis. IgG antibody responses predominate in opisthorchiasis sera (Wongratanacheewin et al. 1988b) while IgG4 antibody provides high diagnostic specificity for human opisthorchiasis (Tesana et al. 2007). Accordingly, an OvSO-kit termed the OvSO-IgG4 kit also was developed for the detection of specific IgG4 antibody. The diagnostic performance of these two kits was investigated and assessed in this study.

Materials and methods

Parasite and antigen

Opisthorchis viverrini metacercariae were collected from naturally infected cyprinid fish from a water reservoir in Amphur Muang, Khon Kaen Province, Thailand. The fish were purchased from a local fish market in Khon Kaen, Thailand. Following collection, O. viverrini metacercariae were examined under a dissecting microscope and identified as described (Sripa et al. 2010) and used for infection of laboratory hamsters. The experimental infection of hamsters were performed as described (Intapan and Maleewong 2006; Sadaow et al. 2019). Adult O. viverrini worms were recovered from the livers and bile ducts of the hamsters three months later. Adult somatic antigen was obtained as described (Intapan and Maleewong 2006). Briefly, the adult worms were homogenized with tissue grinder in a small volume of 0.1 M phosphate-buffered saline, pH 7.4, containing proteinase inhibitors (cOmpleteTM ULTRA Tablets, Mini EASYpack Protease Inhibitor Cocktail Tablets, Roche, Basel, Switzerland). The suspension was sonicated with an ultrasonic disintegrator and centrifuged at 10,000 x g for 30 min at 4°C. The protein concentration of the antigen was determined (Bradford 1976). The supernatant as the antigen was aliquoted and stored at −80°C until used.

Human sera

Human opisthorchiasis sera used in this study were obtained from the serum biobank of the Department of Parasitology, Faculty of Medicine, Khon Kaen University. Serum samples from cases of clonorchiasis had been stored in the biobank at the Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan. There were four categories of sera included in our study: 1) 30 samples of negative control from healthy volunteers from northeastern Thailand who were proven to be free of any intestinal parasitic infection at the time of blood collection; 2) 112 samples opisthorchiasis sera from persons infected O. viverrini in northeastern provinces of Thailand, a region endemic area for O. viverrini; 3) 30 samples of clonorchiasis from persons infected with C. sinensis in Guangi Zhuang Autonomous Region, China, a locality endemic from infection with C. sinensis (Yu et al. 2003); and 4) 160 samples from cases diagnosed with other parasites besides O. viverrini and C. sinensis. The healthy volunteers, opisthorchiasis, paragonimiasis, taeniasis saginata, ascariasis, hookworm infections, trichuriasis, capillariasis phillipinensis, strongyloidiasis, giardiasis, blastocystosis and minute intestinal flukes infections were confirmed by parasitological detection in stool samples using the modified formalin ethyl acetate concentration method (Elkins et al. 1986). Clonorchiasis was confirmed by the modified Kato-Katz technique (Katz et al. 1972) and ELISA (Yu et al., 2003). Fascioliasis was confirmed by recovery of worms and serological methods (Wongkham et al. 2005), cysticercosis was confirmed by serology and computed tomography (Intapan et al. 2008), gnathostomiasis was confirmed by serological methods with clinical manifestation and histories of dietary preferences (Intapan et al. 2010), angiostrongyliaisis cantonensis was diagnosed by serological methods with clinical manifestation (Somboonpatarakun et al. 2020), trichinellosis cases were confirmed by detection of intramuscular larvae and serological methods (Morakote et al. 1991), and sparganosis was confirmed by detection of plerocercoids (Table 1). Sera pooled from the healthy individuals and opisthorchiasis cases served as the negative and positive controls, respectively, to ensure between day precision. Informed consent was obtained from adult participants and from parents or legal guardians of minors. The reporting of experimental data has been performed using the criteria of the STARD 2015 list for reporting diagnostic accuracy studies (Cohen et al. 2016).

Table 1.

Comparison of reactivities using OvSO-IgG and OvSO-IgG4 kits.

Type of samples Number of positive cases/total cases examined
(intensity level of bandsa)
OvSO-IgG kit OvSO-IgG4 kit
Thai healthy control 1/30 (0.5) 0/30
Opisthorchiasis 97/112 (0.5-4) 84/112 (0.5-8)
Clonorchiasis 23/30 (0.5-3) 14/30 (0.5-3)
Paragonimiasis 6/10 (0.5) 0/10
Fascioliasis 4/10 (0.5-2) 0/10
Cysticercosis 0/10 0/10
Sparganosis 0/10 1/10 (1)
Taeniasis saginata 0/10 0/10
Ascariasis 0/10 0/10
Hookworms 0/10 0/10
Angiostrongyliasis 2/10 (0.5) 1/10 (0.5)
Trichinellosis 0/10 1/10 (2)
Trichuriasis 0/10 0/10
Capillariasis philippinensis 0/10 0/10
Gnathostomiasis 2/10 (0.5-1) 0/10
Strongyloidiasis 1/10 (0.5) 0/10
Giardiasis 1/10 (1) 0/10
Blastocystosis 1/10 (2) 0/10
Infection with minute intestinal flukes 2/10 (0.5-1) 0/10
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a

The intensity level of band at the T line was evaluated according to the interpretation card reference.

Immunochromatographic tests

The OvSO-IgG kit was optimized according to the method previously reported (Sadaow et al. 2019) with some modification by using the antigen (1 mg/mL) absorbed in the T-line at a flow rate of 0.1 μL/mm. The antimouse IgG (Lampire Biological Laboratories, Pipersville, PA) was dispensed onto nitrocellulose membrane (Sartorius Stedim Biotech SA, Goettingen, Germany) to serve as the C-lines. The colloidal gold-conjugated mouse monoclonal anti-human IgG (Kestrel BioSciences Co., Pathumthani, Thailand) was sprayed onto a glass microfiber filter (GF33; Whatman Schleicher & Schuell, Dassel, Germany) to form the conjugate pad. Samples of sera were diluted with sample buffer at a ratio of 1:30, and 5 μL of diluted serum were loaded into the port (S) and 90 μL of chromatography buffer were added into the buffer well (B), as marked in supplementary Fig. 1. The OvSO-IgG4 kit was also optimized using a method like that described above, except the concentration of antigen (2 mg/mL), colloidal gold-conjugated monoclonal anti-human IgG4 (ZyMAX, Invitrogen, Camarillo, CA), and the sera were undiluted when tested in the kit. The intensity of positive bands with both kits was estimated visually (unaided) by comparison with the reference board with level ≥ 0.5 as the cut-off level (Supplementary Fig. 1).

Statistical analysis

The diagnostic values were calculated as described (Galen, 1980). These values were calculated and expressed as follows: sensitivity was equal to the number of true positives/(number of true positives + number of false negatives) x 100; specificity was equal to the number of true negatives/(number of false positives + number of true negatives) x 100; positive predictive value was equal to the number of true positives/(number of false positives + number of true positives) x 100; negative predictive value was equal to the number of true negatives/(number of false negatives + number of true negatives) x 100; accuracy was equal to the number of true positives + number of true negatives/(number of true positives + number of false negatives + number of false positives + number of true negatives) x 100; true negative was the number of control samples (other parasitoses and healthy controls) that were negative by the assay; true positive was the number of proven opisthorchiasis or clonorchiasis or boths samples that were positive by the assay; false positive was the number of control samples that were positive by the assay; and false negative was the number of proven opisthorchiasis or clonorchiasis or both samples that were negative by the assay. Positive likelihood ratio was calculated as sensitivity/(100 – specificity) and negative likelihood ratio as (100 – sensitivity)/specificity. Sensitivity, specificity and cross-reactivity of OvSO-IgG and OvSO- IgG4 kits were compared using McNemar’s test (The SPSS Statistics Data Editor, SPSS Inc., Chicago, IL). Total concordance was calculated using Cohen's kappa coefficient. Stata version 13.1 was used to perform the analysis.

Results

The diagnostic values and intensities of positive bands in the OvSO-IgG kit and the OvSO-IgG4 kits calculated for diagnosis of opisthorchiasis, clonorchiasis and both diseases were compared (Tables 1 and 2).

Table 2.

The diagnostic test statistical parameters of the OvSO-IgG and OvSO-IgG4 test kits.

Number OvSO-IgG kit
 OvSO-IgG4 kit
OVa CSb OV & CSc OVa CSb OV & CSc
True positive 97 23 120 84 14 98
False positive 20 20 20 3 3 3
True negative 170 170 170 187 187 187
False negative 15 7 22 28 16 44
Diagnostic values (95% Confidence Interval);
Sensitivity (%) 86.6 (78.9-92.3) 76.7 (57.7-90.1) 84.5 (77.5-90.0) 75.0 (65.9-82.7) 46.7 (28.3-65.7) 69.0 (60.7-76.5)
Specificity (%) 89.5 (84.2-93.5) 89.5 (84.2-93.5) 89.5 (84.2-93.5) 98.4 (95.5-99.7) 98.4 (95.5-99.7) 98.4 (95.5-99.7)
Positive predictive value (%) 82.9 (74.8-89.2) 53.5 (37.7-68.8) 85.7 (78.8-91.1) 96.6 (90.3-99.3) 82.4 (56.6-96.2) 97.0 (91.6-99.4)
Negative predictive value (%) 91.9 (87.0-95.4) 96.0 (92.0-98.4) 88.5 (83.2-92.7) 87.0 (81.7-91.2) 92.1 (87.5-95.4) 81.0 (75.3-85.8)
Positive likelihood ratio 8.2 (5.4-12.5) 7.3 (4.6-11.5) 8.0 (5.3-12.2) 47.5 (15.4-146.7) 29.6 (9.0-96.8) 43.7 (14.1-135.0)
Negative likelihood ratio 0.15 (0.09-0.24) 0.26 (0.14-0.50) 0.17 (0.12-0.26) 0.25 (0.18-0.35) 0.54 (0.39-0.76) 0.31 (0.25-0.40)
Accuracy (%) 88.4 (84.3-91.8) 87.7 (82.6-91.7) 87.3 (83.3-90.7) 89.7 (85.7-92.9) 91.4 (86.8-94.7) 85.8 (81.6-89.4)
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a

OV as an Opisthorchiasis individually

b

CS as Clonorchiasis individually

c

OV&CS as Opisthorchiasis/Clonorchiasis as a combined disease entity

Representative results obtained in OvSO-IgG kit and the OvSO-IgG4 kit were shown in Supplementary Fig. 2. Low level cross-reactive intensities were also detected and graded using the reference board (Supplementary Fig. 1). For diagnosis of opisthorchiasis individually, the sensitivity, specificity, positive and negative predictive values with 95% confidence intervals in the OvSO-IgG kit were 86.6% (78.9-92.3), 89.5% (84.2-93.5), 82.9% (74.8-89.2), and 91.9% (87.0-95.4), respectively, while for the OvSO-IgG4 kit were 75% (65.9-82.7), 98.4% (95.5-99.7), 96.6% (90.3-99.3), and 87% (81.7-91.2), respectively, at the prevalence of infection of 37.1%. Twenty-three (76.7%) and 14 (46.7%) of 30 clonorchiasis sera were positively reactive as assessed with the OvSO-IgG and OvSO-IgG4 kits, respectively .The differences in performance between the both tests were statistically significant (p < 0.001) with Cohen’s kappa agreement 84.1 % (κ-value = 0.649) for opisthorchiasis individually (Table 3), 84.6 % (κ-value = 0.363) for clonorchiasis individually (Table 4), and 81.6 % (κ-value = 0.608) for opisthorchiasis/clonorchiasis as a combined disease entity (Table 5).

Table 3.

Comparison of diagnostic parameters in both ICT kits: opisthorchiasis individually.

Test type and resultsa OvSO-IgG4 kit
OvSO-IgG4 kit Number of positives Number of negatives Total
Number of positives 78 39 117
Number of negatives 9 176 185
Total 87 215 302
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a

These results showed statistically significant differences between both kits (Exact McNemar’s test; p < 0.001), with Cohen’s kappa agreement 84.1 % (κ-value = 0.649, (p < 0.001).

Table 4.

Comparison of diagnostic parameters in both ICT kits: clonorchiasis individually.

Test type and resultsa OvSO-IgG4 kit
OvSO-IgG4 kit Number of positives Number of negatives Total
Number of positives 13 30 43
Number of negatives 4 173 177
Total 17 203 220
Open in a new tab
a

These results showed statistically significant differences between both kits (Exact McNemar’s test; p < 0.001), with Cohen’s kappa agreement 84.6 % (κ-value = 0.363, p < 0.001).

Table 5.

Comparison of diagnostic parameters in both ICT kits: opisthorchiasis/clonorchiasis as a combined disease entity.

Test type and resultsa OvSO-IgG4 kit
OvSO-IgG4 kit Number of positives Number of negatives Total
Number of positives 90 50 140
Number of negatives 11 181 192
Total 101 231 332
Open in a new tab
a

These results showed statistically significant differences between both kits; (Exact McNemar’s test; p < 0.001), with Cohen’s kappa agreement 81.6 % (κ-value = 0.608, p < 0.001).

Discussion

POC tests based on chromatographical lateral flow approaches have found utility in a broad range of applications including the diagnosis of infections including parasitic diseases (Drancourt et al. 2016). Recently, an ICT kit for serodiagnosis of human opisthorchiasis and clonorchiasis was developed based on IgG antibody detection in sera using soluble ES antigen from adults O. viverrini worms (Sadaow et al. 2019). The diagnostic sensitivity, specificity, positive, and negative predictive values of the ICT based on ES antigen were 94.6%, 91.2%, 89.7% and 95.4%, respectively (Sadaow et al. 2019). In the present study, we developed new ICT kits, termed OvSO-kits, using soluble lysates of adult O. viverrini somatic tissues as the antigen for detection of IgG and IgG4 antibodies. Both the OvSO-IgG and OvSO-IgG4 kits can be used for serodiagnosis of opisthorchiasis. These kits can also detect some clonorchiasis cases. This is expected given the close phylogenetic relationship of these two fish-borne trematodes and, consequently, their shared antigenic epitopes (Sirisinha et al. 1990). The sensitivity values of both OvSO-kits were lower than for ES antigen while the specificity of OvSO-IgG4 kit gave higher values. However, the limitations of preparing O. viverrini ES antigen include its expense, and its labor intensive and time-consuming nature, and low yield of ES product (~0.3 μg/adult worm). By contrast, desirable attributes of the new OvSO-kits that use O. viverrini somatic antigen include its less laborious aspect, higher yield of soluble antigen, and that it obviates the requirement for in vitro culture of the flukes. Notably, the yield for somatic antigen (~30 μg/adult worm) is ~100 times greater the yield of the ES products from similar numbers of adult O. viverrini.

Elevated IgG, IgA and IgE antibody titers in serum and bile have been reported during opisthorchiasis especially in serum IgG antibody which predominates in opisthorchiasis (Wongratanacheewin et al. 1988b) and clonorchiasis (Li et al. 2018). IgG4 antibody levels are also correlated with chronic helminth infections and intensity of infection (Grogan et al. 1996; Shin et al. 2016). Moreover, IgG and IgG4 antibodies have been used to diagnose of other helminth infections including gnathostomiasis (Anantaphruti et al. 2005), strongyloidiasis (Boonroumkaew et al. 2020), and fascioliasis (Wongkham et al. 2005). IgG4 antibody has a high diagnostic specificity for human opisthorchiasis (Tesana et al. 2007) and clonorchiasis (Hong et al. 1999). With respect to heavy opisthorchiasis or clonorchiasis, the disease manifestation may be subclinical but often induces jaundice, cholangitis, cholecystitis, enlarged liver and gallbladder, and in chronic stages, frequently leads to biliary adenocarcinoma (Honjo et al. 2005; Sripa et al. 2010; IARC 2012; Zheng et al. 2017). Accordingly, access to rapid detection of O. viverrini or C. sinensis using OvSO-IgG and OvSO-IgG4 kits can provide valuable tools to diagnose and monitor for treatment and reduce the risk of development of CCA.

As noted, the OvSO-IgG kit exhibited a higher sensitivity than the OvSO-IgG4 kit, which may be due to the IgG antibody produced in both stages of early/acute and chronic infection stages (Kanamura et al. 1979). Whereas IgG4 represents only one subclass of IgG in human sera (Hamano et al. 2001), it predominate in the chronic infection stage (Aalberse et al. 1983). However, the diagnostic usefulness/availability from both tests was markedly different (p < 0.001) (Table 2): the OvSO-IgG kit exhibits high sensitivity that should be suitable for use in the initial screening of early O. viverrini and possibly also of C. sinensis infections whereas the OvSO-IgG4 kit, with its high specificity, can support and ensure serodiagnosis. Therefore, both kits can be together used for serodiagnosis of opisthorchiasis and/or clonorchiasis at the bedside and for sero-epidemiological investigations. Moreover, negative results were seen with opisthorchiasis sera (Table 1), perhaps being the consequence of light infection.

These are some limitations to this study. The OvSO-IgG test kit showed cross-reactions with several other parasitic infections such as paragonimiasis (6/10), fascioliasis (4/10), angiostrongyliasis (2/10), gnathostomiasis (2/10), infection with minute intestinal flukes (2/10), strongyloidiasis (1/10), giardiasis (1/10), and blastocystosis (1/10), while the OvSO-IgG4 kit showed cross-reaction with sparganosis (1/10), angiostrongyliasis (1/10) and trichinellosis (1/10). The OvSO-IgG test kit gave higher sensitivity but lower specificity than the OvSO-IgG4 kit. This outcome may have arisen because O. viverrini somatic antigen preparation includes more than 30 antigenic polypeptides and hence may share epitopes with other parasites. Also, subclinical infection with liver flukes may have contributed to these findings because the sera for testing the control specificity were mostly from residents from northeast Thailand, which is a region endemic for opisthorchiasis (Sripa et al. 2010). These limitations should not obstruct the application of these tools given the clinically different setting of opisthorchiasis and clonorchiasis (Blair et al. 1999; Tsai et al. 2001; Gottstein et al. 2009; Boonyasiri et al. 2014; Nawa et al. 2015; Nutman 2017). Nevertheless, it is necessary to consider the possibility of Fasciola infection in the regions where fascioliasis occurs, and to comprehensively diagnose the disease including other examination data, such as clinical signs, stool examination, radio-imaging, etc. (Mas-Coma et al. 2015).

Prospective users of these kits should also be aware of the following attributes and constraints. First, since some clonorchiasis cases can be detected, the kits will be useful as rapid diagnostic tests in highly O. viverrini endemic regions such as Cambodia and north-east Thailand but that the results should be carefully interpreted in highly C. sinensis endemic regions, where opisthorchiasis is not found, such as north Vietnam, south-east and north-west China. Second, since no data were available on the duration of infections of the cases in the present study, additional, control parasite-infection sera, for which more specific information on time since parasite exposure and/or treatment is available, will be needed to more fully evaluate the reliability of the assays, for example during acute infection and following praziquantel therapy. Third, the performance of the OvSO-IgG and OvSO-IgG4 kits may be affected between batch to batch variation during production of O. viverrini somatic antigen. Fourth, it will be informative to evaluate the utility of the kits for diagnosis of infection with Opisthorchis felineus in Western Siberia, Russian Federation, and adjacent territories (Fedorova et al. 2020). Fifth, because both positive and false negative results can affect their diagnostic values, diagnosis by parasitological approaches should to be considered. And, lastly, the future identification and mass production of sensitive and specific recombinant antigens can be expected to supersede methods that use complex mixtures of native antigens as used here.

In conclusion, we have developed new ICT tools that are rapid, easy to use, and can support the stool examination in the alternative ways for clinical diagnosis of opisthorchiasis and/or clonorchiasis. The production of the OvSO-IgG and OvSO-IgG4 kits appear to be less costly, less time consuming, and are far easier to produce than the OvES-kit. The OvSO-IgG and OvSO-IgG4 kits likely also are promising tools for antibody screening for risk of CCA at the bedside, in the hospitals or health centers with sub-optimal medical capacity in the regions where both opisthorchiasis and clonorchiasis are endemic.

Supplementary Material

1741573_Sup_material

Acknowledgements

We express our gratitude to Jitaporn Harasan from the Mekong Health Science Research Institute for providing such an effective immunochromatographic technique. We thank Drs. Masanori Kawanaka and Yasuyuki Morishima for granting permission to use the clonorchiasis serum samples and for providing its information.

Financial support

This study was supported by Distinguished Research Professor Grant, Thailand Research Fund (TRF) (PMI and WM, grant no. DPG 6280002); Khon Kaen University, Research and Graduate Studies Affairs, (OS, PMI and WM, grant no. RP64010); Faculty of Medicine, Khon Kaen University (PMI and WM, grant no. DR63101 and RG63301); a scholarship under the Doctoral Training Program of the Graduate School and Research and Graduate Studies Affairs, Khon Kaen University (KKU) (LS, grant no 60164); and the National Cancer Institute, National Institutes of Health (NIH), USA (PJB and PMI, award no. RO1CA164719). The contents of this report are solely the responsibility of the authors and do not necessarily represent the official views of the TRF, KKU or the NIH.

Footnotes

Conflicts of interest The authors declare that they have no conflict of interest.

Ethical standards This study was approved by the research ethics committee of the Khon Kaen University Ethics Committee for Human Research (Ethics number: HE611507, approved 2 November 2018). Animal Ethics Committee of the Khon Kaen University, according to the Ethic of Animal Experimentation of the National Research Council of Thailand (AEMDKKU 002/2018, approved 4 January 2018).

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