Development of an immunochromatographic point-of-care test for detection of IgG antibody in serodiagnosis of human trichinellosis

  • Tongjit Thanchomnang
    Affiliations
    Faculty of Medicine, Mahasarakham University, Maha Sarakham 44000, Thailand

    Mekong Health Science Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand
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  • Lakkhana Sadaow
    Affiliations
    Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand

    Mekong Health Science Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand
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  • Oranuch Sanpool
    Affiliations
    Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand

    Mekong Health Science Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand
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  • Pewpan M. Intapan
    Affiliations
    Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand

    Mekong Health Science Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand
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  • Rutchanee Rodpai
    Affiliations
    Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand

    Mekong Health Science Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand
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  • Patcharaporn Boonroumkaew
    Affiliations
    Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand

    Mekong Health Science Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand
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  • Penchom Janwan
    Affiliations
    Department of Medical Technology, School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat 80161, Thailand
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  • Somjintana Tourtip
    Affiliations
    Faculty of Medicine, Mahasarakham University, Maha Sarakham 44000, Thailand
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  • Wanchai Maleewong
    Correspondence
    Corresponding author: Wanchai Maleewong, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand ORCID iD: 0000-0002-6726-3759
    Affiliations
    Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand

    Mekong Health Science Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand
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Open AccessPublished:August 27, 2021DOI:https://doi.org/10.1016/j.ijid.2021.08.056

      Highlights

      • ∙ Development of an immunochromatographic test for serodiagnosis of human trichinellosis.
      • ∙ Using Trichinella spiralis larval somatic antigen.
      • ∙ Valuable for a rapid, uncomplicated point-of-care testing tool.
      • ∙ A sensitive and specific tool for screening and detection of infection.
      • ∙ Useful in laboratories where sophisticated equipment is lacking.

      Abstract

      Objective: Trichinellosis is a globally distributed food-borne parasitic disease. Initially, diagnosis is usually made based on clinical signs, symptoms, and history of eating raw or undercooked meat. In the present study, an immunochromatographic test (ICT) kit was developed to diagnose trichinellosis by detecting IgG antibodies in sera of infected humans.
      Methods: Somatic extract from Trichinella spiralis larvae was used as the antigen for the ICT kit development. Diagnostic efficacy was evaluated using human serum samples from proven trichinellosis patients, healthy persons, and those with other parasitic infections.
      Results: The diagnostic sensitivity, specificity, positive and negative predictive values and accuracy of the ICT kit were 100.0% (95%; CI 88.8 to 100.0%), 92.5% (95% CI; 86.9 to 96.2%), 73.8% (95% CI; 58.0 to 86.1%), 100.0% (95% CI; 97.3 to 100.0%) and 93.8% (95% CI; 89.2 to 96.9%), respectively.
      Conclusions: This ICT diagnostic kit can be used as a testing tool for human trichinellosis. This test should permit rapid detection of infection to enable prompt anthelminthic treatment. It can also be used for retrospective diagnoses and field surveys based at laboratories where sophisticated equipment is lacking.

      Keywords

      Introduction

      Trichinellosis is one of the most critical food-borne parasitic zoonoses worldwide (
      • Murrell KD
      • Pozio E.
      Trichinellosis: the zoonosis that won't go quietly.
      ). Humans acquire trichinellosis by ingesting raw or undercooked meats containing the infective Trichinella larvae (
      • Pozio E.
      World distribution of Trichinella spp. infections in animals and humans.
      ;
      • Zarlenga D
      • Thompson P
      • Pozio E.
      Trichinella species and genotypes.
      ). There are ten named species of Trichinella, and three additional genotypes (T6, T8, and T9) have been reported to date (
      • Zarlenga D
      • Thompson P
      • Pozio E.
      Trichinella species and genotypes.
      ). The named species are Trichinella spiralis, T. nativa, T. britovi, T. pseudospiralis, T. murrelli, T. nelsoni, T. papuae, T. zimbabwensis, T. patagoniensis, and T. chanchalensis (
      • Pozio E
      • Zarlenga D.
      International Commission on Trichinellosis: Recommendations for genotyping Trichinella muscle stage larvae.
      ;
      • Zarlenga D
      • Thompson P
      • Pozio E.
      Trichinella species and genotypes.
      ). The most common species which can cause human disease is Trichinella spiralis (
      • Murrell KD
      • Pozio E.
      Trichinellosis: the zoonosis that won't go quietly.
      ;
      • Gottstein B
      • Pozio E
      • Nöckler K.
      Epidemiology, diagnosis, treatment, and control of trichinellosis.
      ), which occurs worldwide and is commonly associated with domestic pigs (
      • Murrell KD
      • Pozio E.
      Trichinellosis: the zoonosis that won't go quietly.
      ). In Asia, there have been reports of outbreaks from China, Japan, Lao PDR, Vietnam, Korea, and other countries, including Thailand (
      • Stehr-Green JK
      • Schantz PM.
      Trichinosis in Southeast Asian refugees in the United States.
      ;
      • Takahashi Y
      • Mingyuan L
      • Waikagul J.
      Epidemiology of trichinellosis in Asia and the Pacific Rim.
      ). In human infections, the severity of clinical symptoms is proportional to the number of larvae ingested and the Trichinella species involved (
      • Clausen MR
      • Meyer CN
      • Krantz T
      • Moser C
      • Gomme G
      • Kayser L
      • et al.
      Trichinella infection and clinical disease.
      ;
      • Dupouy-Camet J
      • Bruschi F.
      • Dupouy-Camet J
      • Murrell KD
      FAO/WHO/OIE guidelines for the surveillance, management, Prevention, and Control of Trichinellosis.
      ;
      • Teunis PF
      • Koningstein M
      • Takumi K
      • van der Giessen JW.
      Human beings are highly susceptible to low doses of Trichinella spp.
      ). Symptoms are diverse and may include generalized fever, abdominal pain, diarrhea, vomiting, myalgia, periorbital edema, and prostration. Severe trichinellosis cases have been associated with prolonged symptoms and cardiovascular, pulmonary, and neurological complications, which sometimes prove fatal (
      • Dupouy-Camet J
      • Kociecka W
      • Bruschi F
      • Bolas-Fernandez F
      • Pozio E.
      Opinion on the diagnosis and treatment of human trichinellosis.
      ;
      • Dupouy-Camet J
      • Bruschi F.
      • Dupouy-Camet J
      • Murrell KD
      FAO/WHO/OIE guidelines for the surveillance, management, Prevention, and Control of Trichinellosis.
      ).
      There are two classes of methods for the detection of Trichinella infection in humans or animals. Methods such as trichinoscopy (compression method) and the artificial digestion method directly test for the presence of parasites in tissues or digests. The other class of methods indirectly demonstrates infection by detecting specific antibodies using serological methods such as enzyme-linked immunosorbent assay (ELISA) (
      • Morakote N
      • Sukhavat K
      • Khamboonruang C
      • Siriprasert V
      • Suphawitayanukul S
      • Thamasonthi W.
      Persistence of IgG, IgM, and IgE antibodies in human trichinosis.
      ,
      • Morakote N
      • Khamboonruang C
      • Siriprasert V
      • Suphawitayanukul S
      • Marcanantachoti S
      • Thamasonthi W.
      The value of enzyme-linked immunosorbent assay (ELISA) for diagnosis of human trichinosis.
      ;
      • Gómez-Morales MA
      • Ludovisi A
      • Amati M
      • Cherchi S
      • Pezzotti P
      • Pozio E.
      Validation of an enzyme-linked immunosorbent assay for diagnosis of human trichinellosis.
      ;
      • Hu CX
      • Jiang P
      • Yue X
      • Zeng J
      • Zhang XZ
      • Song YY
      • et al.
      Molecular characterization of a Trichinella spiralis elastase-1 and its potential as a diagnostic antigen for trichinellosis.
      ), indirect immunofluorescence assay (
      • Bruschi F
      • Gómez-Morales MA
      • Hill DE.
      International Commission on Trichinellosis: Recommendations on the use of serological tests to detect Trichinella infection in animals and humans.
      ), enzyme immunohistochemical technique (
      • Bruschi F
      • Gómez-Morales MA
      • Hill DE.
      International Commission on Trichinellosis: Recommendations on the use of serological tests to detect Trichinella infection in animals and humans.
      ), and immunoblotting (
      • Gómez-Morales MA
      • Mazzarello G
      • Bondi E
      • Arenare L
      • Bisso MC
      • Ludovisi A
      • et al.
      Second outbreak of Trichinella pseudospiralis in Europe: clinical patterns, epidemiological investigation, and identification of the etiological agent based on the western blot patterns of the patients' serum.
      ,
      • Gómez-Morales MA
      • Ludovisi A
      • Amati M
      • Cherchi S
      • Tonanzi D
      • Pozio E.
      Differentiation of Trichinella species (Trichinella spiralis/Trichinella britovi versus Trichinella pseudospiralis) using western blot.
      ,
      • Gómez-Morales MA
      • Ludovisi A
      • Amati M
      • Blaga R
      • Zivojinovic M
      • Ribicich M
      • et al.
      A distinctive Western blot pattern to recognize Trichinella infections in humans and pigs.
      ). However, these serological methods are laborious, requiring sophisticated equipment that is not always available in local laboratories. Such limitations often delay appropriate diagnosis, especially for people in outlying or rural areas. To overcome these drawbacks, there is a need for rapid processing of analytes using a point-of-care test (POCT) tool that is easy to use, portable, and suitable for field situations. An immunochromatographic strip test was developed by
      • Fu BQ
      • Li WH
      • Gai WY
      • Yao JX
      • Qu ZG
      • Xie ZZ
      • et al.
      Detection of anti-Trichinella antibodies in serum of experimentally-infected swine by immunochromatographic strip.
      to detect anti-Trichinella antibodies in serum of experimentally infected swine. This test could detect anti-Trichinella antibodies for different infection doses, and the result was also in agreement with detection by ELISA (
      • Fu BQ
      • Li WH
      • Gai WY
      • Yao JX
      • Qu ZG
      • Xie ZZ
      • et al.
      Detection of anti-Trichinella antibodies in serum of experimentally-infected swine by immunochromatographic strip.
      ). Here, we report the development of an immunochromatographic test (ICT) kit based on antibody detection to diagnose human trichinellosis. The use of this kit is simple, requiring no expensive equipment.

      Materials and Methods

       Parasites and antigens

      Antigen was prepared from the T. spiralis strain ISS62. This strain, originally from a domestic pig and now maintained in mice, caused an outbreak of human trichinellosis in Thailand's Mae Hong Son Province in 1986 (
      • Pozio E
      • Khamboonruang C.
      Trichinellosis in Thailand: epidemiology and biochemical identification of the aetiological agent.
      ). The muscles of mice, one month after oral inoculation with Trichinella larvae, were digested with pepsin-HCl. Trichinella spiralis larvae were harvested using a modified Baermann technique (
      • Justus DE
      • Morakote N.
      Mast cell degranulation associated with sequestration and removal of Trichinella spiralis antigens.
      ) and were washed several times in distilled water. The extraction of somatic antigen from these larvae was previously described (
      • Intapan PM
      • Maleewong W
      • Sukeepaisarnjaroen W
      • Morakote N.
      Potential use of Trichinella spiralis antigen for serodiagnosis of human capillariasis philippinensis by immunoblot analysis.
      ). Briefly, the T. spiralis larvae (500 mg wet weight) were homogenized with a tissue grinder in a small volume (1000 µL) of distilled water containing proteinase inhibitors (Complete ULTRA Tablets, Mini EASYpack Protease Inhibitor Cocktail Tablets, Roche, Basel, Switzerland). The extract was then sonicated with an ultrasonic disintegrator and centrifuged at 10,000 × g for 30 min at 4°C. The protein concentration in the supernatant was assayed using the QuickStart Bradford Protein Assay (Bio-Rad Laboratories Inc., CA). The supernatant was kept as the source of antigen and stored at -70°C until used.

       Human sera

      Human serum samples were used for the evaluation of the diagnostic properties of the test. These sera were supplied by the frozen sample bank (-70°C) at the Faculty of Medicine, Khon Kaen University. The samples were divided into three groups: 1) the negative control group (n = 30) comprised samples from healthy adult volunteers who were free (based on stool examination using the formalin ethyl-acetate concentration technique (FECT) (
      • Elkins DB
      • Haswell-Elkins M
      • Anderson RM.
      The epidemiology and control of intestinal helminths in the Pulicat Lake region of Southern India. I. Study design and pre-and post-treatment observations on Ascaris lumbricoides infection.
      )) from any parasitic intestinal infection at the time of blood collection; 2) the trichinellosis group (n = 31), which comprised samples from outbreak cases in northern Thailand. These cases had been confirmed by detection of intramuscular T. spiralis larvae and were also antibody-positive according to an ELISA test (
      • Morakote N
      • Khamboonruang C
      • Siriprasert V
      • Suphawitayanukul S
      • Marcanantachoti S
      • Thamasonthi W.
      The value of enzyme-linked immunosorbent assay (ELISA) for diagnosis of human trichinosis.
      ), and 3) serum samples (n = 116) from patients with parasitic infections other than trichinellosis (Table 1). In this last group, diagnoses of opisthorchiasis viverrini, paragonimiasis, taeniasis saginata, ascariasis, hookworm infections, strongyloidiasis, and trichuriasis were confirmed by parasitological detection using the FECT method. Fascioliasis was confirmed by recovery of worms and serological methods (
      • Wongkham C
      • Tantrawatpan C
      • Intapan PM
      • Maleewong W
      • Wongkham S
      • Nakashima K.
      Evaluation of immunoglobulin G subclass antibodies against recombinant Fasciola gigantica cathepsin L1 in an enzyme-linked immunosorbent assay for serodiagnosis of human fasciolosis.
      ), cysticercosis was confirmed by serology and computed tomography (
      • Intapan PM
      • Khotsri P
      • Kanpittaya J
      • Chotmongkol V
      • Maleewong W
      • Morakote N.
      Evaluation of IgG4 and total IgG antibodies against cysticerci and peptide antigens for the diagnosis of human neurocysticercosis by ELISA.
      ), gnathostomiasis was confirmed by serological methods together with clinical manifestations and relevant dietary histories (
      • Intapan PM
      • Khotsri P
      • Kanpittaya J
      • Chotmongkol V
      • Sawanyawisuth K
      • Maleewong W.
      Immunoblot diagnostic test for neurognathostomiasis.
      ), angiostrongyliasis cantonensis was diagnosed based on serological methods and clinical manifestations (
      • Somboonpatarakun C
      • Intapan PM
      • Sadaow L
      • Rodpai R
      • Sanpool O
      • Maleewong W.
      Development of an immunochromatographic device to detect antibodies for rapid diagnosis of human angiostrongyliasis.
      ), and sparganosis was confirmed by detection of plerocercoids (Table 1). Pooled serum samples from healthy individuals and trichinellosis patients were also used as negative and positive controls.
      Table 1Human sera studied and results of the immunochromatographic test kit using somatic antigen extracted from Trichinella spiralis larvae.
      Type of serum sampleNumber positive/Total number
      Proven trichinellosis31/31
      Healthy controls0/30
      Trichuriasis0/10
      Ascariasis0/10
      Hookworm infections0/10
      Strongyloidiasis0/10
      Angiostrongyliasis0/10
      Gnathostomiasis1/10
      Taeniasis saginata0/10
      Cysticercosis1/10
      Sparganosis0/6
      Opisthorchiasis viverrini0/10
      Paragonimiasis3/10
      Fascioliasis6/10

       Immunochromatographic test kit

      The ICT device for diagnosing human trichinellosis was designed based on the specificity of antibodies against T. spiralis larval somatic antigens. In the device, 1 µg/µL of the antigen and goat anti-mouse IgG (Lampire Biological Laboratories, Pipersville, PA) were sprayed at a flow rate of 0.1 µL/mm onto a nitrocellulose membrane (Sartorius Stedim Biotech, Goettingen, Germany) to form the test and control lines, respectively. Colloidal gold-labeled mouse anti-human IgG (Kestrel BioSciences Co., Pathumthani, Thailand) was sprayed at a flow rate of 1 µL/mm onto the conjugate pad made from a piece of glass microfiber filter GF33 (Whatman Schleicher & Schuell, Dassel, Germany). The strip was covered with a plastic case and stored in an aluminum foil pouch. Each kit consisted of an immunochromatographic device, buffer (25mM Tris-HCl, pH 8.0, including 0.1% sodium azide) for diluting the serum sample, facilitating chromatography, and a card to assist interpretation of the color intensity at the test line. For testing, each serum sample was diluted with the buffer in the optimum ratio of 1:30 by using checkerboard titration. Five microliters of the diluted serum were applied to the sample well, followed by an additional 90 µL of the buffer. The result is judged as positive if red bands appear on both the test (T) and control (C) lines after 15 min, while the appearance of a red band at only the C line indicates a negative reaction (Figure 1). The level of response was estimated by comparing the intensity of positive lines with those shown on the reference card (Figure 2), with level 1 taken as the cutoff level. The precision of the method was investigated by performing the test on the same positive and negative control samples on different days: no day-to-day variation was seen. The diagnostic values, including sensitivity, specificity, and positive and negative predictive values, were calculated as previously described (
      • Galen RS.
      Predictive value and efficiency of laboratory testing.
      ). Stata software (version 10) (StataCrop LP, Lakeway Drive College Station, TX) was used to perform the analysis.
      Figure 1
      Figure 1Schematic diagram of the immunochromatographic test strip. An absorbent pad, immobilized nitrocellulose membrane, conjugate pad, and sample pad were glued together on a plastic adhesive backing card. At the test line, the Trichinella spiralis antigen and control line, the anti-mouse IgG antibody were fixed on the nitrocellulose membrane.
      Figure 2
      Figure 2Representative results of the Ts-ICT kit (left). Sensitivity and specificity were evaluated using P, pooled positive trichinellosis human sera; N, pooled healthy control sera; Ts01-05, sera from trichinellosis cases; Hc01-05, sera from healthy individuals; Ac, angiostrongyliasis; Al, ascariasis; Hw, hookworm infections; Ss, strongyloidiasis; Tt, trichuriasis; Ov, opisthorchiasis viverrini; Se, sparganosis; Tn, taeniasis saginata; Gn, gnathostomiasis; Cc, cysticercosis; Fg, fascioliasis; and Ph, paragonimiasis. B, buffer area; S, sample area; T, test line; C, control line. The interpretation card for determining the cutoff level (right).

      Results

      The trichinellosis ICT (Ts-ICT) kit was evaluated using sera from trichinellosis patients, healthy controls, and patients with other parasitic diseases (Table 1, Figure 2). All 31 trichinellosis serum samples yielded positive results using a cutoff value of 1. In contrast, none of the 30 healthy control sera showed positive results. Cross-reactivity was observed in serum samples from cases of gnathostomiasis (1 of 10), cysticercosis (1 of 10), paragonimiasis (3 of 10), and fascioliasis (6 of 10). The diagnostic values of the Ts-ICT were calculated with the results from all serum samples. Sensitivity, specificity, and positive, negative predictive values and accuracy were 100.0% (95% CI 88.8 to 100.0%), 92.5% (95% CI 86.9 to 96.2%), 73.8% (95% CI 58.0 to 86.1%), 100% (95% CI 97.3 to 100.0%) and 93.8% (95% CI 89.2 to 96.9%), respectively. The positive and negative likelihood ratios were 13.3 (95% CI 7.5 to 23.4) and 0, respectively. The detection limit of the Ts-ICT kit was found to be stable when stored in an aluminum foil bag containing silica gel desiccant for 12 months at ambient temperature (25°C) and for 18 months at 4°C.

      Discussion

      Diagnosis of human trichinellosis is based on clinical signs, symptoms, and history of raw or undercooked meat consumption, muscle biopsy, and serological methods (
      • Diaz JH
      • Warren RJ
      • Oster MJ.
      The Disease Ecology, Epidemiology, Clinical Manifestations, and Management of Trichinellosis Linked to Consumption of Wild Animal Meat.
      ). The definitive diagnostic test for trichinellosis in humans is demonstrating the presence of the muscle larvae in a tissue biopsy (
      • Intapan PM
      • Chotmongkol V
      • Tantrawatpan C
      • Sanpool O
      • Morakote N
      • Maleewong W.
      Molecular identification of Trichinella papuae from a Thai patient with imported trichinellosis.
      ). However, this direct method has low sensitivity in light and moderate infections (
      • Dupouy-Camet J
      • Bruschi F.
      • Dupouy-Camet J
      • Murrell KD
      FAO/WHO/OIE guidelines for the surveillance, management, Prevention, and Control of Trichinellosis.
      ) and is harmful to patients; for these reasons, serology is essential. Many serological diagnostic assays have been developed for trichinellosis. The International Commission on Trichinellosis accepted ELISA methods using Trichinella excretory/secretory (E/S) antigens for serological tests for Trichinella infection in animals and humans (
      • Bruschi F
      • Gómez-Morales MA
      • Hill DE.
      International Commission on Trichinellosis: Recommendations on the use of serological tests to detect Trichinella infection in animals and humans.
      ) but suggested that immunoblotting should be used to confirm ELISA-positive sera (
      • Bruschi F
      • Gómez-Morales MA
      • Hill DE.
      International Commission on Trichinellosis: Recommendations on the use of serological tests to detect Trichinella infection in animals and humans.
      ). The E/S-based ELISA is an excellent tool for epidemiological studies and monitoring Trichinella exposure (
      • Bruschi F
      • Gómez-Morales MA
      • Hill DE.
      International Commission on Trichinellosis: Recommendations on the use of serological tests to detect Trichinella infection in animals and humans.
      ). In addition, detection of antibodies against Trichinella E/S antigen in serum of clinically positive pig blood samples (
      • Zhang GP
      • Guo JQ
      • Wang XN
      • Yang JX
      • Yang YY
      • Li QM
      • et al.
      Development and evaluation of an immunochromatographic strip for trichinellosis detection.
      ) and experimentally infected swine using an immunochromatographic strip has been reported: the strip detected anti-Trichinella antibodies for different infection doses (
      • Fu BQ
      • Li WH
      • Gai WY
      • Yao JX
      • Qu ZG
      • Xie ZZ
      • et al.
      Detection of anti-Trichinella antibodies in serum of experimentally-infected swine by immunochromatographic strip.
      ). The result was also in agreement with ELISA (
      • Zhang GP
      • Guo JQ
      • Wang XN
      • Yang JX
      • Yang YY
      • Li QM
      • et al.
      Development and evaluation of an immunochromatographic strip for trichinellosis detection.
      ;
      • Fu BQ
      • Li WH
      • Gai WY
      • Yao JX
      • Qu ZG
      • Xie ZZ
      • et al.
      Detection of anti-Trichinella antibodies in serum of experimentally-infected swine by immunochromatographic strip.
      ).
      In this study, we developed a new Ts-ICT using crude somatic antigen from T. spiralis larvae to detect anti-T. spiralis immunoglobulin G (IgG) antibody in human serum samples. The Ts-ICT exhibited high sensitivity (100%) and specificity (92.5%). Preparation of crude somatic antigens, such as we used, is easier than for E/S antigens. Preparation of the latter is expensive, labor-intensive, and time-consuming, ultimately yielding only a small amount of ES product and requiring in vitro culture of live parasites. For these reasons, and given the high sensitivity and specificity of the Ts-ICT described here, we are satisfied that crude somatic antigens are perfectly adequate.
      A further development that would improve this diagnostic tool is to ensure a stable supply of antigens and to simplify quality control. Recently, a recombinant T. spiralis elastase-1 was developed to detect anti-Trichinella IgG in human trichinellosis: the sensitivity was 97.4%, and the specificity was 99.1% using ELISA (
      • Hu CX
      • Jiang P
      • Yue X
      • Zeng J
      • Zhang XZ
      • Song YY
      • et al.
      Molecular characterization of a Trichinella spiralis elastase-1 and its potential as a diagnostic antigen for trichinellosis.
      ). This or other promising recombinant Trichinella antigens should be developed. Consideration should be given to developing the diagnostic kit for the detection of trichinellosis using whole-blood samples. These improvements would likely lead to increased diagnostic sensitivity and specificity and a more user-friendly and rapid platform in resource-limited settings.
      Cross-reactions of the Ts-ICT were different from those reported for the ELISA using Trichinella E/S antigen (
      • Gómez-Morales MA
      • Ludovisi A
      • Amati M
      • Cherchi S
      • Pezzotti P
      • Pozio E.
      Validation of an enzyme-linked immunosorbent assay for diagnosis of human trichinellosis.
      ;
      • Taher EE
      • Méabed EMH
      • El Akkad DMH
      • Kamel NO
      • Sabry MA.
      Modified dot-ELISA for diagnosis of human trichinellosis.
      ), possibly due to differences in the types and numbers of infected serum samples for specificity evaluation. Moreover, the present Ts-ICT was utilized to test with a trichinellosis serum sample caused by Trichinella papuae (
      • Intapan PM
      • Chotmongkol V
      • Tantrawatpan C
      • Sanpool O
      • Morakote N
      • Maleewong W.
      Molecular identification of Trichinella papuae from a Thai patient with imported trichinellosis.
      ), and the result was also positive (Supplementary Figure 1). Cross-reactions between T. spiralis and T. papuae are not unexpected; the Ts-ICT kit is likely to give a positive result for any species of Trichinella. Some cross-reactions did occur when the Ts-ICT was used with sera from gnathostomiasis, cysticercosis, paragonimiasis, and fascioliasis patients. The positive result obtained for one gnathostomiasis and one cysticercosis case could be due to subclinical trichinellosis. False positives in paragonimiasis and fascioliasis cases could be due to these trematodes sharing antigenic epitopes with Trichinella. Whatever the explanation, this should not be a serious problem in clinical settings due to differences in clinical presentation among these parasitic diseases.(
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      • Intapan PM
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      ;
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      Food Microbiology Series: Biology of Food-borne Parasites.
      ). Trichinella spiralis shares some larval somatic antigens with Capillaria philippinensis, making it possible to use T. spiralis antigens to diagnose intestinal capillariasis cases (
      • Intapan PM
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      • Thanchomnang T
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      • Phosuk I
      • et al.
      Development and evaluation of a rapid diagnostic immunochromatographic device to detect antibodies in sera from intestinal capillariasis cases.
      ,
      • Intapan PM
      • Maleewong W
      • Sukeepaisarnjaroen W
      • Morakote N.
      An enzyme-linked immunosorbent assay as screening tool for human intestinal capillariasis.
      ,
      • Intapan PM
      • Maleewong W
      • Sukeepaisarnjaroen W
      • Morakote N.
      Potential use of Trichinella spiralis antigen for serodiagnosis of human capillariasis philippinensis by immunoblot analysis.
      ). When the present Ts-ICT was evaluated with sera from proven intestinal capillariasis cases, seven out of ten samples were positive (data not shown). This was not unexpected. Both parasites are in the superfamily Trichuroidea (
      • Roach TI
      • Wakelin D
      • Else KJ
      • Bundy DA.
      Antigenic cross-reactivity between the human whipworm, Trichuris trichiura, and the mouse trichuroids Trichuris muris and Trichinella spiralis.
      ), and both contain stichocytes, which are an important source of diagnostic antigens of Trichinella (
      • Despommier DD
      • Müller M.
      The stichosome and its secretion granules in the mature muscle larva of Trichinella spiralis.
      ;
      • Takahashi Y
      • Mizuno N
      • Shimazu K
      • Araki T.
      Ultrastructure, antigenicity, and histochemistry of stichocyte granules of adult Trichinella spiralis.
      ;
      • Boireau P
      • Vayssier M
      • Fabien JF
      • Perret C
      • Calamel M
      • Soulé C.
      Characterization of eleven antigenic groups in Trichinella genus and identification of stage and species markers.
      ). Such cross-reactions should not pose a problem to physicians as intestinal capillariasis patients lack signs and symptoms of trichinellosis (
      • Limsuwan S
      • Siriprasert V.
      A clinical study on trichinosis in Changwat Phayao.
      ).
      The Ts-ICT takes less time to perform than any other serological diagnostic format, is straightforward, and does not require special equipment or elaborate training from the healthcare provider/test operator. It is suitable as a POCT and is valuable for clinical and epidemiological studies, mainly when the source of infection is unknown.
      In conclusion, the rapid Ts-ICT kit for the serodiagnosis of human trichinellosis will be helpful in early diagnosis to permit prompt anthelminthic treatment, perform retrospective diagnoses, and conduct epidemiological surveys (
      • Ljungström I.
      Immunodiagnosis in man.
      ). However, prospective users of the kit should be aware of the following attributes and constraints. First, the high sensitivity in our hands was based on the use of sera from patients with infection confirmed by the detection of intramuscular larvae. In acute trichinellosis cases, there may be a low antibody response, requiring caution in interpretation. Serial blood collections following infection are required to determine when seroconversion occurs. Second, the performance of the Ts-ICT kit may vary between batches of T. spiralis somatic antigen. Identification and mass production of sensitive and specific recombinant antigens will solve this problem in the future. Third, given the occasional false-positive result, diagnosis using parasitological approaches should be considered in some cases.

      Funding

      This study was supported by a Distinguished Research Professor Grant from the Thailand Research Fund [Grant no. DPG6280002] (OS, PMI, and WM), a grant from Khon Kaen University [Research and Graduate Studies Affairs Grant no. RP64010] (OS and WM), a grant from Khon Kaen University (KKU) under scholarships under the Doctoral Training Program of the Graduate School and Research and Graduate Studies Affairs, [Grant no. 60164] (LS), a grant from the Khon Kaen University Faculty of Medicine [grant numbers DR63101 and RG63301] (WM and OS), and the Faculty of Medicine, Mahasarakham University (TT). The funders had no role in study design, data collection, interpretation, or the decision to submit the work for publication.

      Conflict of interest

      The authors declare no conflicts of interest.

      Contributions

      Conceptualization and study design: TT, LS, OS, RR, PMI, ST, and WM.
      Data collection and Methodology: TT, LS, OS, RR, PB, PJ, and WM.
      Resource: TT, LS, RR, OS, PB, and PMI.
      Formal analysis and investigation: TT, LS, RR, OS, PB, PJ, ST, PMI, and WM.
      Writing - original draft preparation: TT, LS, RR, PJ, and WM.
      Writing - review and editing: TT, LS, RR, PJ, and WM.
      Supervision: PMI and WM.
      All authors read and approved the final manuscript.

      Ethical approval

      All procedures performed involving human participants were in accordance with the Khon Kaen University Ethics Committee for Human Research (HE621265). Experiments involving the use of animals were approved by the 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).

      Acknowledgments

      We thank Prof. David Blair for editing English via Publication Clinic Khon Kaen University, Thailand.

      Appendix. Supplementary materials

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