Screening of Capillaria philippinensis infection using Trichuris muris and Trichinella spiralis antigens

Original Mona I Ali1, Nadia A El-Dib2, Mahmoud Abdel-Latif3, Waleed M Arafa4, Samah S Article Abdel Gawad1 1 and Cairo2 Universities, Immunity Division, Department of Zoology, Faculty of Science3 and Medical Departments of Medical Parasitology Faculty4 of Medicine, Beni-Suef

Parasitology, Faculty of VeterinaryABSTRACT Medicine , Beni-Suef University, Egypt

Background: Intestinal capillariasis is a disease caused by Capillaria philippinensis. Human infections Trichinella and Trichuris species, all of them belong to Trichinelloidea superfamily. Diagnosis of intestinal capillariasis becamemay be missed more prevalent by stool examination. in many countries including Egypt. This nematode is related to Objective: screening for intestinal capillariasis as a practical and rapid diagnostic test. Subjects and This Methods: study aimed Trichuris to use muris enzyme-linked and Trichinella immunosorbent spiralis adult assay worms (ELISA) were and isolated immunoblotting from infected in mice and crude antigens were prepared. The protein content for both adult worm extracts was determined. Human blood samples were collected from 20 capillariasis patients, 20 control individuals and 10 W. bancrofti T. spiralis and T. muris antigens and sera from cases infected with C. philippinensis Results: infected patients.T. muris The crude study antigen evaluated cross cross-reactivity reacted with between100% of capillariasis sera with 100% using ELISA and immunoblotting. T. spiralis crude In ELISA, T. sensitivitymuris nor T. and spiralis specificity crude antigensand cross reacted reacted with with sera 10% from of sera control from group. bancroftian Immunoblotting filariasis. results showed antigen cross reacted with 50% of capillariasis sera, and 9% of sera from bancroftianT. muris antigen,filariasis. while Neither in W. bancrofti that IgG antibodies from control group didn't recognize specific proteins in and W. bancrofti group, only one band in one sample appeared (100-135T. spiralis kDa). crude IgG antigen. antibodies from capillariasis Conclusion:cases recognized Antigens multiple from common T. muris protein and T. bands spiralis (35-180 can be kDa). used IgG successfully antibodies to from detect capillariasis, infection controlwith C. philippinensis sera did not recognize specific proteins in T. muris gave better results than that of T. spiralis. Immunoblotting can be used for diagnosis of capillariasis by using crude antigen of T. muris. using serum samples of cases by ELISA. Crude antigen of Keywords: 21 May, 2021, 10 August, 2021. Received: ELISA, immunodiagnosis,Accepted: intestinal capillariasis, Trichinelloidea, western blotting. Corresponding Author: Mona I. Ali, Tel.: +20 1221969196, E-mail: [email protected]

Print ISSN: Online ISSN: Vol. 14, No. 2, August, 2021.

1687-7942, 2090-2646, INTRODUCTION [8-13] The genus Capillaria is a member of the superfamily andEgypt Thailand. Egypt[10]. showed the highest number of cases Trichinelloidea. From this group only three major detected outside the endemic areas in the Philippines genera: Trichuris, Trichinella and Capillaria cause Infection with C. philippinensis is mainly acquired human diseases. All Trichinnelloidae members have a through ingestion of the parasite larva in raw, unique structure of the esophagus with the presence of stichocytes that differentiates them from other [10]. nematodes[1]. C. philippinensis undercooked, small fresh water or brackish water fish eating birds that causes intestinal capillariasis in intestinalor by contaminated villi with fingers subsequent during fishmalabsorption evisceration of is a nematode of fish- Infection leads to inflammation and atrophy of the the disease seems to be endemic[2]. In Thailand, a enteropathy[14,15] humans. It first appeared in the Philippines[3]. Subsequent where usuallynutrients suffer resulting from chronic in extreme diarrhea protein-deficient[16]. The disease cases were diagnosed in many parts of the world can be fatal if diagnosis. Patients and with treatment intestinal arecapillariasis delayed. includinglarge number Korea of [4]cases, Taiwan, were identifiedand China[5,6]. Youssef et al.[7] to lack of knowledge of the nature of this disease[17]. sporadic cases were diagnosed particularly in Upper InSome non-endemic cases are notareas, easily diagnosis diagnosed is often and skipped had to pass due reported the first case in Egypt after which Personal non-commercial use only. PUJ copyright © 2021. All rights reserved DOI: 10.21608/puj.2021.76982.1121

178 Immunodiagnosis of capillariasis Ali et al., through many sophisticated, expensive, and sometimes was performed as previously described[23] invasive techniques to be diagnosed[10]. . Briefly, This study is based on the concept of similarity 96-well ELISA plates (Corning, USA) were coated between members of the Trichinelloidea superfamily, with adult worm antigens (2.0 μg/ml) in 100 μl of and the possible cross reactivity between patient sera 5%bicarbonate fetal calf bufferserum (pHat room 9.6) temperature overnight at for 4°C. 2 h, After the and antigens from members of the superfamily, which followingblocking with reagents PBS 0.1% were Tween sequentially 20 (PBST) added containing and can add a new diagnostic method for the detection of the disease. The aim of the study is to establish a rapid, easy, and accurate diagnostic tool for infection with incubated at 37°C for 1 h: (1) human sera diluted 1:100 C. philippinensis, as the direct methods are not always 1:5000.in PBST, The and reactions(2) horseradish were detected peroxidase-labeled by the addition goat positive[18]. anti-human (HRP) IgG antibody (Sigma, USA) diluted

of the substrate o-phenylenediamine dihydrochloride SUBJECTS AND METHODS (OPD; Sigma, USA) plus H2O2 and stopped with 50 μl/ well of 2 M H2SO4. Optical density (OD) values at 490 This descriptive analytical study was conducted at nm were measured with a microplate reader (TECAN, Austria). All samples were run in duplicate. The cut-off and Department of Zoology, Immunity Division, Faculty value of the ELISA was evaluated for the two antigen Medical Parasitology Department, Faculty of Medicine, werepreparations regarded based as positive. on a calculation of mean control OD + (2 x SD). The recorded ODs above the cut-off value Preparationof Science, Beni-Suef of adult University worm somatic from extracts:2018 to 2020. T. muris Western blotting: Immunoblotting was carried adult worms were isolated from laboratory infected out as previously described[24]. Adult worm extracts mice and the homogenate preparation was carried out as previously described[19,20] were separated in SDS-10% polyacrylamide gels and . Briefly, worms were transferred to nitrocellulose membranes (0.45 µm; washed thoroughly in PBS, homogenized in 50 mM Tris Heidelberg; Serva Electrophoresis GmbH, Germany) HCl (pH 8.8) containing 0.15 M NaCl, 1 mM EDTA, 1 mM incubatedby electroblotting. for 1 h Membranesat room temperature were washed in blocking in PBS/ 1,4-Dithiothreitol and 50 mM Phenylmethylsulfonyl Tween buffer (PBS containing 0.3% Tween-20) and atfluoride 5000 × usingg followed motor by 15,000 driven x homogenizer g for 30 min and (REMI, the followed by washing and incubation with the human clearMaharashtra, supernatant India) was at separated.4°C. The extract was centrifuged buffer containing 5% non-fat milk in PBS/Tween-20,

For T. spiralis antigen, adult worms were collected serum (1:100) in washing buffer overnight at 4°C. from the small intestine of experimentally infected Formed immunocomplexes were detected by HRP Wistar rats at 4 days after experimental infection. The anti-human IgG antibody (1:5000; KPL, Maryland, collected adult worms were washed several times in USA). After 2 h of incubation at room temperature, bands were developed by adding substrate (50 mg 3,3′-Diaminobenzidine tetrahydrochloride and 100 μl PBS and then homogenized in lysis buffer containing StatisticalH2O2 in 100 analysis: ml PBS). 7 M urea, 2 M thiourea, 4% 3-(3-Cholamidopropyl) dimethylammonio-1-propanesulfonate hydrate 65 mM SPSS (version 20) statistical forTris, 60 2% min DTT, and andthe supernatant 1% Bio-Lyte was (pH collected 3-10). The and adultused program (SPSS Inc., Chicago, IL) was used to carry asworms adult lysates extract were[21]. The centrifuged protein content at 20,000 for both × g at adult 4°C detected,out one-way analysis analysis of differences of variance between (ANOVA) the onmeans our data. When significant differences by ANOVA were [22]. worm extracts was determined by Bicinchoninic acid accordingof the OD to values the following was performed formulas: by Sensitivity Dunnett’s t-test.= no. Humanassay kit sera:(Sigma) Human blood samples were collected The sensitivity and specificity of ELISA were assessed from 20 patients who had been diagnosed by stool samples analysis as having C. philippinensis infection of true positives/(no. of true positives + no. of false[25]. by detecting eggs, adults and/or larvae in stool; from negatives) × 100; and specificity = no. of true negatives/ 20 control individuals who had not experienced any accuracy(no. of false were positives also calculated. + no. of true P negatives)values < 0.5× 100 were parasitic diseases; and 10 samples were from patients Positive and negative predictive values and diagnostic with W. bancrofti infections. Stool examination of the last two groups showed no other nematodes eggs such Ethicalconsidered approval: significant. The study was approved by the as A. lumbricoides, A. duodenale, and T. trichuria.

ELISA: The optional dilutions of various reagents were wasResearch obtained Ethical from Committee all individuals of the includedBeni-Suef in University, the study determined using checkerboard titration. The assay afterFaculty explaining of Medicine, the purpose Egypt. of Signed the study. informed consent

179 PARASITOLOGISTS UNITED JOURNAL RESULTS

Detection of cross-reactive human IgG using Table 1. ELISA: When T. muris crude antigen was tested for T. muris Ag and T. spiralis Ag among capillariais patients. Diagnostic yield and accuracy of ELISA results using T. muris Ag T. spiralis Ag reactions were found in 100% from capillariasis and 100% 50% serodiagnostic cross-reacting human IgG, sero-positive Sensitivity Specificity 100% 100% 10% from bancroftian filariasis cases. Calculated ELISA PPV 100% 100% sensitivity and specificityCapillaria for capillariasis or Capillaria were and both W. NPV 100% 66.7% bancrofti100% (Table 1). The difference betweenP the means Accuracy 100% 75% of OD for control and Trichinella were statistically significant ( ˂0.01 and human˂0.001, IgG respectively) recorded positive (Fig. 1). reactions In addition, in 50% from T. muris crude antigen. crude antigen tested for serodiagnostic cross-reacting In contrast, IgG antibodies from capillariasis cases Capillaria recognize specific proteins in capillariasis and 9% from bancroftian filariasis (Fig. W.2). Thebancrofti mean of OD for P the reaction with (n=20) could recognize multiple commonW. bancrofti protein was significantly higher than reaction with control and bands ranging between 35-180 kDa (35, 48, 63, 75, respectively for infections T. spiralis ( ˂0.05) (Fig. 2). Calculated T.100, muris 135,180 crude antigen kDa). IgG except antibodies in only fromone serum sample sensitivity and specificity were 50% and 100% antisera (n=10) did not recognize specific proteins in Detection of specifically(Table recognized 1). proteins in adult worm antigens using immunoblotting: IgG other(No. 8), W. that bancrofti recognized a protein band (100-135 kDa) proteins(Fig. 3). IgGin T. antibodies spiralis crude from antigen. capillariasis, control and infections did not recognize specific antibodies from selected controls (n=20) did not

T. muris crude antigen

0.6 100% 0.5

0.4

0.3 P < 0.01* P < 0.001*

0.2 10% Cut-off = 0.11 0%

Human IgG (OD 450 nm) 0.1

0.0 Control Capillaria Infections Fig. 1. T. muris *: P crude antigen cross reacted with 100% of capillariasis sera, and 10% of other parasitic infections (bancroftian filariasis); Sifnificant ( < 0.05). T. spiralis crude antigen

0.9 50% 0.8 0.7 0.6 P < 0.05* P < 0.05* 0.5 9% 0.4 0% Cut-off = 0.34 0.3 0.2 0.1 Human IgG (OD 450 nm) 0.0 Control Capillaria Infections

Fig. 2. T. spiralis *: P crude antigen cross reacted with 50% of capillariasis and 9% of other parasitic infections (bancroftian filariasis); Sifnificant ( < 0.05).. 180 Immunodiagnosis of capillariasis Ali et al.,

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

180 135 100 75

63 48

35

Control Capillaria Bancroftian filariasis Fig. 3. T. muris crude antigen. IgG antibodies from Capillaria

Western blot. Sera from control did not recognize specific proteins in positive cases could recognize multiple common protein bands ranging between 35-180 kDa. There is an apparent band (100-135 DISCUSSIONkDa) in serum no. (8) in bancroftian filariasis group.

The real prevalence of C. philippinensis may be higher than predicted, but the lack of awareness of the capillariasis cases could recognize multiple common parasite and the outcome of infection by physicians proteinBy bands Immunoblotting, in T. muris IgGcrude antibodies antigen ranging from and laboratory technicians is delaying the diagnosis[3]. Immunodiagnosis may allow early detection of the parasite. Using it as an extra diagnostic tool helps between 35-180 kDa (35,et al 48,.[29] 63, used 75, C. 100,philippinensis 135,180 to detect C. philippinensis infection[18]. In our study, coproantigenkDa). No bands and were egg detected antigen in thefor controldiagnosis group. of the reactivity of serum samples from 20 cases intestinalSimilarly, Abdel-Rahmancapillariasis utilizing the immunoblotting diagnosed as human C. philippinensis infection was T. group, while multiple protein bands were recognized muris and T. spiralis formethod. both antigens Results showedin capillariasis no bands cases. in These the control bands C.examined philippinensis by ELISA cases and were immunoblotting reactive with against T. muris ranged from 10 to 148 KDa for C. philippinensis antigen and 50% of antigens. cases reacted By ELISA, with 100% T. spiralis of the the other hand, in our study IgG antibodies from W. T. muris bancrofticoproantigen and from 60 to 62 for egg antigen. On antigenantigens while with 9% statistical reacted significance. with T. spiralis Only antigens, 10% of in T. muris crude antigen except in only serum sample indicatingbancroftian the filariasis superiority antisera of the reactedformer as with a diagnostic antisera did not recognize specific proteins antigen. The similarity of stichocytes between the members of the family Trichinelloidae, allows for the infectionsthat recognized and other a singlenematodes protein including band Capillaria (100-135 use of their antigens in immunological reactions[26]. askDa). tested There by Joekelmay be et alcross.[30] who reactivity proved between immunological filarial Dirofilaria and Capillaria These cells are rich in antigens, that can be used aerophila in expermintally infected dogs. for the serodiagnosis of capillariasis[27]. Similar cross-reactions between studies consistent with our results were conducted In conclusion, this study showed that antigen worldwide. Intapan et al.,[27] diagnosed intestinal from T. muris and T. spiralis can be used successfully capillariasis using T. spiralis to detect infection with C. philippinensis using serum reacted positively with 75% of trichinellosis. Another et antigen al.[28] proved by ELISA that that C. of T. muris gave better results than crude antigens of philippinensis sera were also reactive with T. spiralis T.samples spiralis of. Western cases by blot ELISA technique technique. also canCrude be usedantigen for andstudy T. conductedvulpis antigens. by Banzon diagnosis of capillariasis by utilizing crude antigen of T. muris. To avoid the false negative diagnosis of infection with C. philippinensis in the absence of eggs in stool, an Author contributions: All authors contributed to et al.[18] in the study conception and design, the practical work, which coproantigen was extracted from the stools of results interpretation, statistical analysis and writing infectedimmunological patients. study Hyperimmune was carried serum by El-Dib was prepared the manuscript. against this antigen and used successfully to detect Conflict of interest: All authors declare that they the copro antigen of C. philippinensis in stool samples Funding: have no conflict of interest. of suspected cases by sandwich ELISA. No funding. 181 PARASITOLOGISTS UNITED JOURNAL REFERENCES 17.

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