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Abstract

Schistosoma mekongi infection is endemic in countries along the Mekong River and certain of its tributaries in the lower Mekong basin, especially in Lao People's Democratic Republic and Cambodia. Diagnosis of schistosomiasis is crucial before treatment and epidemiological surveys before and/or after an intervention, such as a mass drug administration. A newly developed immunochromatographic test (ICT) for the diagnosis of schistosomiasis mekongi, based on antiparasite antibody detection in human sera, was evaluated. The schistosomiasis mekongi-ICT (Smk -ICT) strip was developed using somatic antigen from adult S. mekongi. In total, 209 serum samples were examined, including 14 from parasitologically proven schistosomiasis mekongi patients, 30 from schistosomiasis japonica patients, other parasitosis (n = 135), and healthy volunteers (n = 30) from areas not endemic for S. mekongi. Eleven schistosomiasis mekongi samples were positive according to the Smk -ICT, whereas all healthy control samples were negative. Cross-reactions with paragonimiasis heterotremus, sparganosis, trichinellosis, and taeniasis saginata samples were observed at 2.4% (4/165). The diagnostic sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 78.6% (95% confidence interval [CI] 49.2–95.3), 97.6% (95% CI 93.9–99.3), 73.3% (95% CI 44.9–92.2), 98.2% (95% CI 94.7–99.6), and 96.1% (95% CI 92.1–98.4), respectively. The Smk -ICT kit might be useful to assess the prevalence of disease before establishing transmission control and mass deworming campaigns in countries in the Mekong River subregion.

Introduction

Schistosoma mekongi causes schistosomiasis mekongi in humans and is endemic in the southern parts of the Lao People's Democratic Republic (Lao PDR) and in northeastern Cambodia along the Mekong River (Muth et al. 2010, Gordon et al. 2019, Sayasone et al. 2020). The parasite is also zoonotic, infecting dogs and pigs (Ohmae et al. 2004, Muth et al. 2010). More than 140,000 people are estimated to be at risk of infection in these two countries, where morbidity levels are high (Urbani et al. 2002, Chai and Jung 2019, Sayasone et al. 2020). Although geographically restricted in parts of Lao PDR and Cambodia, S. mekongi affects not only people living in endemic areas but also immigrants and travelers to the endemic areas (Leshem et al. 2009).

Humans are infected by the percutaneous penetration of the infective cercariae that are released from the intermediate host snail (Neotricula aperta) into freshwater (Attwood and Upatham 2012, Limpanont et al. 2015).

The mature worms are paired, and the females lay eggs in the superior mesenteric veins of the host. The eggs are carried by blood flow from the mesenteric veins into the liver, where they are trapped and can cause local granuloma formation, and pathology can progress to hepatosplenomegaly, enlarged periportal vein, and portal hypertension (Biays et al. 1999, Ohmae et al. 2004, Sinuon et al. 2007, Colley et al. 2014, Sayasone et al. 2020). The pathology of S. mekongi infection is similar to that of Schistosoma japonicum, and infection with either species can have serious outcomes if left untreated (Zhou et al. 2010, McManus et al. 2018). These two Schistosoma species are very closely related (Attwood et al. 2015).

The regional action plan for neglected tropical diseases in the Western Pacific Region for years 2012–2016 recommended targeting schistosomiasis mekongi for elimination (WHO Regional Office for the Western Pacific 2013). The first step in attacking any parasitic diseases is to establish methods for accurate diagnosis. This is crucial before treatment and also for epidemiological surveys of schistosomiasis in endemic areas to determine the prevalence of infection before and/or after an intervention, such as mass drug administration (Ross et al. 2001, McManus et al. 2018).

Various techniques have been established to diagnose schistosomiasis mekongi in humans and animals. These include parasitological (Sayasone et al. 2015), immunological (Ittiprasert et al. 2000, Ohmae et al. 2004, Zhu et al. 2005, Nickel et al. 2015, Sangfuang et al. 2016, Vonghachack et al. 2017), and molecular methods (Sanpool et al. 2012, Kongklieng et al. 2013, Thanchomnang et al. 2013). The parasitological approach to detect Schistosoma eggs in human stool samples, particularly the Kato–Katz thick smear technique, is time consuming and might overlook light infections.

However, this method has been utilized for control programs in Lao PDR and Cambodia (Vonghachack et al. 2017, Sayasone et al. 2020). Therefore, highly sensitive, user-friendly, and rapid test platforms to provide point of care (POC) testing are required, especially for detection of light infections, which are common during late stages of transmission control programs. A POC lateral flow immunochromatographic test (ICT), based on detection of Schistosoma mansoni and Schistosoma haematobium circulating antigens in urine, has been utilized for the diagnosis of S. mekongi infections and showed considerably higher sensitivity than the Kato–Katz thick smear (van Dam et al. 2004, Vonghachack et al. 2017, Homsana et al. 2020).

However, false-positive results were seen among patients with opisthorchiasis and urinary tract infections and hematuria (Homsana et al. 2020). Until now, there has been no report of a lateral flow device “ICT” for detecting anti- S. mekongi-specific IgG antibodies in the serum for diagnosis of human schistosomiasis mekongi. In this study, we describe the development of an ICT for the POC diagnosis of schistosomiasis mekongi (Smk -ICT) using adult S. mekongi somatic antigen and evaluate its accuracy.

Materials and Methods

Ethical approval

All procedures performed in studies involving human participants were in accordance with ethical standards of the Khon Kaen University Ethics Committee for Human Research (HE621265) and with the 2013 Declaration of Helsinki and its later amendments or comparable ethical standards. Experimental mice and N. aperta snails were handled according to the Guidelines for Animal Experimentation of the National Research Council of Thailand and approved by Faculty of Tropical Medicine Animal Care and Use Committee at Mahidol University in Bangkok, Thailand (FTM-ACUC 009/2020E).

Preparation of S. mekongi antigen

Frozen S. mekongi adult worms were obtained from the Applied Malacology Center, Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Thailand. The preparation of the antigen was performed as follows: briefly, the worms (∼200 mg wet weight) were homogenized with a tissue grinder in a small volume of distilled water (500 μL) containing proteinase inhibitors (Mini EASYpack Protease Inhibitor Cocktail Tablets; Roche, Basel, Switzerland), and the homogenate was centrifuged at 10,000 × g at 4°C. The protein concentration of the resultant supernatant was determined using a standard method (Lowry et al. 1951).

Human sera

In total, 209 serum samples were supplied from the Frozen Serum Bank at the Faculty of Medicine, Khon Kaen University and Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan. Fourteen serum samples were collected from schistosomiasis mekongi patients located in the Champasak Province, southwestern Lao PDR (their demographic data are presented in Table 1 and Supplementary Table S1), and 30 samples were from schistosomiasis japonica patients located in Leyte Island, the Philippines (Table 1 and Supplementary Table S2).

Table 1.

Types of Human Sera Examined and Results of Screening these Using the Smk-Immunochromatographic Test Kit

Type of serum samples	No. positive cases using Smk-ICT kit/total no. examined	Positive case band intensity^a
Schistosomiasis mekongi	11/14	1–4
Male = 10 (71.4%), female = 4 (28.6%)
Age range (mean ± SD) = 4–54 (16 ± 13) years
Schistosomiasis japonica	22/30	1–4
Male = 24 (80.0%), female = 6 (20.0%)
Age range (mean ± SD) = 10–49 (24 ± 11) years
Healthy controls	0/30	None
Opisthorchiasis viverrini	0/10	None
Clonorchiasis sinensis	0/10	None
Paragonimiasis	1/10	1
Sparganosis	1/5	3
Taeniasis saginata	1/10	2
Cysticercosis	0/10	None
Strongyloidiasis	0/10	None
Ascariasis	0/10	None
Hookworm infections	0/10	None
Trichuriasis	0/10	None
Capillariasis philippinensis	0/10	None
Angiostrongyliasis	0/10	None
Gnathostomiasis	0/10	None
Trichinellosis	1/10	1

Band intensity at the T-line was evaluated according to the reference color card.

Smk-ICT, schistosomiasis mekongi-immunochromatographic test.

Schistosomiasis mekongi patients were schistosome egg positive in stool samples according to the Kato–Katz thick smear method (Katz et al. 1972), whereas schistosomiasis japonica patients were schistosome egg positive in stool samples according to the direct smear technique using a total of 1 g of feces (Beaver et al. 1984). The remaining 135 serum samples were from patients with other parasitic diseases (Table 1), with infection status confirmed by parasitological and/or serological methods.

Cases of opisthorchiasis, paragonimiasis, taeniasis saginata, ascariasis, hookworm infections, trichuriasis, capillariasis philippinensis, and strongyloidiasis were confirmed by detection of parasite eggs or larvae in stool samples using the modified formalin ethyl acetate concentration method (Elkins et al. 1986). Cases of clonorchiasis were confirmed by the modified Kato–Katz technique (Katz et al. 1972). Cysticercosis was diagnosed using computed tomography scanning and immunodiagnostic tests (Intapan et al. 2008). Gnathostomiasis cases were confirmed using information from a combination of serological methods, clinical manifestations, and a history of risky dietary preferences (Intapan et al. 2010).

Angiostrongyliasis cases were confirmed by serology and clinical manifestations (Somboonpatarakun et al. 2020). Trichinellosis cases were confirmed by detection of larvae in muscles and serology (Morakote et al. 1991). Sparganosis was diagnosed by histopathological investigation (Boonyasiri et al. 2014). Thirty samples were included from healthy volunteers who resided outside endemic areas for S. mekongi and who proved to be negative for intestinal parasitic infections based on the formalin ethyl acetate concentration technique (Elkins et al. 1986) at the time of blood collection.

Preparation of the lateral flow immunochromatography strip

The Smk -ICT strip was developed using adult S. mekongi somatic antigen. The membrane strip was prepared as reported previously (Sadaow et al. 2020) with some modifications. Briefly, 2 mg/mL of antigen was absorbed at the test line (“T”), and goat anti-mouse IgG antibody 1 mg/mL (Lampire Biological Laboratories, Pipersville, PA) was absorbed at the control line (“C”). Both were dispensed onto the nitrocellulose membrane (Sartorius Stedim Biotech, Goettingen, Germany) using a XYZ3210 Dispenser (Bio-Dot, Irvine, CA) at a spray rate of 0.1 μL/mm.

Colloidal gold (40 nm) conjugated with mouse anti-human IgG (Kestrel BioSciences Co., Pathum Thani, Thailand) was sprayed on a piece of glass fiber (GF33; Whatman Schleicher & Schuell, Dassel, Germany) to form the conjugate pad at a rate of 6 μg/mL. The coated membrane, conjugate pad, sample pad, and absorbent pad were laminated and pasted overlapping onto a backing card and then cut lengthwise and divided into strips measuring 4 × 60 mm using a guillotine cutter (CM5000 Guillotine; Bio-Dot). Each strip was placed in a plastic cassette (Fig. 1a) and stored at room temperature. The test kit was composed of the immunochromatographic cassette, sample buffer for diluting the serum sample and running buffer for chromatography.

FIG. 1.

The lateral flow immunochromatographic test for schistosomiasis mekongi. (a) The assembled device. (b) The interpretation card. The cutoff level is ≥1. (c) Interpretation of results, from left to right, two bands appear at both the C and T lines in a positive case, whereas only the control band appears in negative cases. The test is invalid when the control band does not appear. Color images are available online.

Testing of the Smk-ICT

The optimized Smk -ICT kit is shown (Fig. 1a). The serum sample was diluted (1:5) in sample buffer (Kestrel BioSciences Co.), and 5 μL of the diluted serum was added into a sample well (“S”), followed by 90 μL of running buffer in the buffer well (“B”). The result was visually interpreted 15 min after the addition of the running buffer (Kestrel BioSciences Co.). The band intensity was evaluated visually according to the interpretive color card (Fig. 1b, with 1 as the cutoff level). Appearance of red bands at both the T and C lines indicates a positive result (Fig. 1c). The diagnostic values including sensitivity, specificity, and positive and negative predictive values were calculated as previously described (Galen 1980). The STATA software package version 10.1 (StataCorp LP, College Station, TX) was used to perform the analysis.

Results

After optimization, Smk -ICT strips were tested using individual sera. The results are summarized in Table 1. No Smk -ICT yielded positive results in 30 negative controls. The diagnostic values are shown in Table 2.

Table 2.

Diagnostic Values of the Smk -Immunochromatographic Test for Schistosomiasis Mekongi, Schistosomiasis Japonica, and a Combination of the Two Forms of Schistosomiasis

Diagnostic value	Schistosomiasis mekongi^a	Schistosomiasis japonica^b	Combined schistosomiasis^c
Sensitivity (%)	78.6 (49.2–95.3)	73.3 (54.1–87.7)	75.0 (59.7–86.8)
Specificity (%)	97.6 (93.9–99.3)	97.6 (93.9–99.3)	97.6 (93.9–99.3)
Positive predictive value (%)	73.3 (44.9–92.2)	84.6 (65.1–95.6)	89.2 (74.6–97.0)
Negative predictive value (%)	98.2 (94.7–99.6)	95.3 (90.9–97.9)	93.6 (88.8–96.8)
Positive likelihood ratio	32.4 (11.8–88.6)	30.2 (11.2–81.6)	30.9 (11.6–82.7)
Negative likelihood ratio	0.22 (0.08–0.60)	0.27 (0.15–0.49)	0.26 (0.15–0.43)
Accuracy (%)	96.1 (92.1–98.4)	93.8 (89.5–96.8)	92.8 (88.4–95.9)

Numbers in parenthesis indicate ranges of 95% confidence intervals.

Diagnostic value calculation for schistosomiasis mekongi only ( n = 179).

Diagnostic value calculation for schistosomiasis japonica only ( n = 195).

Diagnostic value calculation when combining schistosomiasis mekongi and schistosomiasis japonica as a composite disease entity ( n = 209).

The diagnostic sensitivity and specificity values for schistosomiasis mekongi were calculated as 78.6% (95% confidence interval [CI] 49.2–95.3) and 97.6% (95% CI 93.9–99.3), respectively. Most of the serum samples from schistosomiasis japonica cases were positive (73.3%, 22/30), and relevant diagnostic values are shown in Table 2. Combining S. mekongi and S. japonicum infections as a composite disease entity yielded values for diagnostic sensitivity and specificity of 75.0% (95% CI 59.7–86.8) and 97.6% (95% CI 93.9–99.3), respectively. Cross-reactions were observed in 2.4% (4/165) of samples from individuals who did not have schistosomiasis (Table 1): these samples came from cases of paragonimiasis heterotremus, sparganosis, trichinellosis, and taeniasis saginata.

Discussion

Human schistosomiasis is a tropical helminthic disease, which remains prevalent in several nations (McManus et al. 2018). In Asia, S. japonicum, S. mekongi, and very rarely Schistosoma malayensis are zoonotic species causing schistosomiasis (Gordon et al. 2019). The present study focused on the development of a diagnostic system for the detection of schistosomiasis mekongi patients in the Mekong River basin, especially in Lao PDR and Cambodia.

Previously, serological assays were developed for diagnosis of S. mekongi infection (Ittiprasert et al. 2000, Ohmae et al. 2004, Zhu et al. 2005, Nickel et al. 2015, Sangfuang et al. 2016, Vonghachack et al. 2017). Ittiprasert et al. (2000) reported 100%, 35.2%, and 50%, respectively, of sensitivity, specificity, and accuracy for antibody detection by enzyme-linked immunosorbent assay (ELISA) using a crude adult S. mekongi antigen. Furthermore, an ELISA to detect antibodies against soluble S. japonicum egg antigens, used in a survey of communities along the Mekong River in Cambodia, gave positive results varying from 40% to 90% (Ohmae et al. 2004).

A dipstick dye immunoassay kit, using S. japonicum soluble egg antigen for the diagnosis of schistosomiasis mekongi, had sensitivity of 97.1%, but cross-reaction occurred in 18.3% of patients infected with Opisthorchis viverrini (Zhu et al. 2005). We observed no cross-reaction with opisthorchiasis samples. This could be due to the use of a different antigen type from that used by Zhu et al. (2005) and the fact that we collected opisthorchiasis sera from areas nonendemic for schistosomiasis mekongi. However, cautious interpretation should be made in areas where both S. mekongi and O. viverrini co-exist. The calculated sensitivity of a S. mansoni -based ELISA and indirect immunofluorescence assay for detection of S. mekongi antibodies was 94.5% (Nickel et al. 2015).

Recently, a survey in villages in Lao PDR and Cambodia, using ELISA to detect antibody against S. mansoni antigens, gave positive results for 43.1% and 26.5% of those tested, respectively (Vonghachack et al. 2017). In the present study, the diagnostic values of our kit were calculated as 78.6%, 97.6%, and 96.1% for sensitivity, specificity, and accuracy, respectively. This kit can also detect schistosomiasis japonica cases, a result expected given their shared antigenic epitopes (Zhu et al. 2005). Combining results from schistosomiasis mekongi and schistosomiasis japonica samples, the sensitivity was 75.0% and the accuracy was 92.8%.

Despite their being S. mekongi egg positive, our Smk-ICT gave negative results in three individuals. One possible reason is that these serum samples were collected from cases soon after S. mekongi infection, resulting in low antibody responses. Serial blood collections following infection are required to determine when seroconversion occurs. In S. mansoni and S. haematobium infections, seroconversion took at least 6 months, while eggs were detected much earlier (Bottieau et al. 2006). Another possibility is variation in host genetic factors affecting immune responses, that is, relating to major histocompatibility complex class II phenotypes in infected populations as revealed in another schistosomiasis endemic area (Waine et al. 1998).

Cross-reactions were observed in paragonimiasis heterotremus, sparganosis, trichinellosis, and taeniasis saginata serum samples collected from areas not endemic for schistosomiasis mekongi. Previous workers on diagnosis of human schistosomiasis have made similar observations (Ittiprasert et al. 2000, Yu et al. 2011, Jiang et al. 2014). Despite the relatively low cross-reaction rate we found (2.4%), which is much lower than that in those studies, this is still a concern with the POC tool. A questionnaire about risk behaviors for parasite infections can support the interpretation of results in a population-level study. Moreover, clinical symptoms of other parasitic infections usually differ from those of schistosomiasis at the symptomatic stage (Biays et al. 1999, Gottstein et al. 2009, Blair 2014, Liu et al. 2015, Sanpool et al. 2017).

In addition to the limitations detailed above, there are investigations and enhancements that could be pursued in the future. The Smk-ICT has been developed as a POC tool for the rapid diagnosis of human schistosomiasis mekongi (and schistosomiasis japonica). The test is only semi-quantitative: intensity of color at the T-line can be read directly by the naked eye. However, quantification of color intensity should be performed using a machine such as a chromogenic test reader (Mewamba et al. 2021), and possible correlation of T-line intensity with Schistosoma egg counts in fecal samples should be investigated.

Prospective users of these kits should also be aware that only a small number (n = 14) of human schistosomiasis mekongi sera were used for sensitivity evaluation: additional schistosomiasis mekongi sera are required to confirm the reliability of the assays. The performance of the Smk-ICT kits may be affected by batch-to-batch variation of S. mekongi somatic antigens (Viana et al. 2019, Graeff-Teixeira et al. 2021). Sensitive and specific recombinant antigens that can be mass produced in the future will likely replace native antigens, such as we used here, and will lead to uniform performance.

Our Smk-ICT could be used for screening travelers who visit endemic areas (Lao PDR and Cambodia) or local residents crossing borders from these countries. The test is easy to use in the field or rural areas without any sophisticated equipment. If the test is positive in individuals who are schistosome egg negative, then re-evaluation by parasitological or nucleic acid-based techniques could be used to identify false-positive results (Chen et al. 2021). This is increasingly important as prevalence becomes low during eradication programs (Chen et al. 2021).

Conclusions

This rapid testing tool can be used to determine the seroprevalence of S. mekongi infection in endemic areas in countries along the Mekong River and certain of its tributaries in the lower Mekong basin. Thus, data can be provided on the real boundaries of schistosomiasis mekongi in these areas and act as ground truthing for geographical information systems. Although our Smk-ICT showed no cross-reactions with opisthorchiasis and clonorchiasis in the present study, further evaluation is necessary to verify this.

Footnotes

Acknowledgments

We would like to thank David Blair for valuable suggestions and assistance with the presentation of this article via Publication Clinic Khon Kaen University, Thailand.

Authors' Contributions

All authors contributed to the study conception and design. R.R., L.S., S.L., Y.L., P.C., H.O., Z.L., and H.Y. performed the material preparation and the sample collection. R.R., L.S., O.S., P.B., T.T., P.J., H.O., H.Y., P.M.I., and W.M. performed the data collection, methodology, investigation, and analysis. R.R., Z.L., H.Y., P.M.I., and W.M. contributed to the first draft of the article. All authors contributed to the editing, and reviewed and approved the final article.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This study was supported by a Distinguished Research Professor Grant from the Thailand Research Fund (O.S., P.M.I., and W.M.; grant number DPG6280002), a grant from Khon Kaen University Research and Graduate studies (O.S., P.M.I., and W.M.; Research Program Grant), a Scholarship under the Post-Doctoral Training Program from Khon Kaen University (R.R., grant number PD2564-09), and grants from the Khon Kaen University Faculty of Medicine (W.M. and O.S., grant numbers DR63101and RG63301). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Supplementary Material

Supplementary Table S1

Supplementary Table S2

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Supplementary Material

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Development and Accuracy Evaluation of Lateral Flow Immunoassay for Rapid Diagnosis of Schistosomiasis Mekongi in Humans

Abstract

Introduction

Materials and Methods

Ethical approval

Preparation of S. mekongi antigen

Human sera

Preparation of the lateral flow immunochromatography strip

Testing of the Smk-ICT

Results

Discussion

Conclusions

Footnotes

Acknowledgments

Authors' Contributions

Author Disclosure Statement

Funding Information

Supplementary Material

References

Supplementary Material