Archive | 2021

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



Background: Intestinal capillariasis is a disease caused by Capillaria philippinensis. Human infections became more prevalent in many countries including Egypt. This nematode is related to Trichinella and Trichuris species, all of them belong to Trichinelloidea superfamily. Diagnosis of intestinal capillariasis may be missed by stool examination. Objective: This study aimed to use enzyme-linked immunosorbent assay (ELISA) and immunoblotting in screening for intestinal capillariasis as a practical and rapid diagnostic test. Subjects and Methods: Trichuris muris and Trichinella spiralis adult worms were isolated from infected 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 infected patients. The study evaluated cross-reactivity between T. spiralis and T. muris antigens and sera from cases infected with C. philippinensis using ELISA and immunoblotting. Results: In ELISA, T. muris crude antigen cross reacted with 100% of capillariasis sera with 100% sensitivity and specificity and cross reacted with 10% of sera from bancroftian filariasis. T. spiralis crude antigen cross reacted with 50% of capillariasis sera, and 9% of sera from bancroftian filariasis. Neither T. muris nor T. spiralis crude antigens reacted with sera from control group. Immunoblotting results showed that IgG antibodies from control group didn t recognize specific proteins in T. muris antigen, while in W. bancrofti group, only one band in one sample appeared (100-135 kDa). IgG antibodies from capillariasis cases recognized multiple common protein bands (35-180 kDa). IgG antibodies from capillariasis, control and W. bancrofti sera did not recognize specific proteins in T. spiralis crude antigen. Conclusion: Antigens from T. muris and T. spiralis can be used successfully to detect infection with C. philippinensis using serum samples of cases by ELISA. Crude antigen of 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. Immunodiagnosis of capillariasis Ali et al., 179 through many sophisticated, expensive, and sometimes invasive techniques to be diagnosed[10]. This study is based on the concept of similarity between members of the Trichinelloidea superfamily, and the possible cross reactivity between patient sera and antigens from members of the superfamily, which 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 C. philippinensis, as the direct methods are not always positive[18]. SUBJECTS AND METHODS This descriptive analytical study was conducted at Medical Parasitology Department, Faculty of Medicine, and Department of Zoology, Immunity Division, Faculty of Science, Beni-Suef University from 2018 to 2020. Preparation of adult worm somatic extracts: T. muris adult worms were isolated from laboratory infected mice and the homogenate preparation was carried out as previously described[19,20]. Briefly, worms were washed thoroughly in PBS, homogenized in 50 mM Tris HCl (pH 8.8) containing 0.15 M NaCl, 1 mM EDTA, 1 mM 1,4-Dithiothreitol and 50 mM Phenylmethylsulfonyl fluoride using motor driven homogenizer (REMI, Maharashtra, India) at 4°C. The extract was centrifuged at 5000 × g followed by 15,000 x g for 30 min and the clear supernatant was separated. For T. spiralis antigen, adult worms were collected from the small intestine of experimentally infected Wistar rats at 4 days after experimental infection. The collected adult worms were washed several times in PBS and then homogenized in lysis buffer containing 7 M urea, 2 M thiourea, 4% 3-(3-Cholamidopropyl) dimethylammonio-1-propanesulfonate hydrate 65 mM Tris, 2% DTT, and 1% Bio-Lyte (pH 3-10). The adult worms lysates were centrifuged at 20,000 × g at 4°C for 60 min and the supernatant was collected and used as adult extract[21]. The protein content for both adult worm extracts was determined by Bicinchoninic acid assay kit (Sigma)[22]. Human sera: Human blood samples were collected from 20 patients who had been diagnosed by stool samples analysis as having C. philippinensis infection by detecting eggs, adults and/or larvae in stool; from 20 control individuals who had not experienced any parasitic diseases; and 10 samples were from patients with W. bancrofti infections. Stool examination of the last two groups showed no other nematodes eggs such as A. lumbricoides, A. duodenale, and T. trichuria. ELISA: The optional dilutions of various reagents were determined using checkerboard titration. The assay was performed as previously described[23]. Briefly, 96-well ELISA plates (Corning, USA) were coated with adult worm antigens (2.0 μg/ml) in 100 μl of bicarbonate buffer (pH 9.6) overnight at 4°C. After blocking with PBS 0.1% Tween 20 (PBST) containing 5% fetal calf serum at room temperature for 2 h, the following reagents were sequentially added and incubated at 37°C for 1 h: (1) human sera diluted 1:100 in PBST, and (2) horseradish peroxidase-labeled goat anti-human (HRP) IgG antibody (Sigma, USA) diluted 1:5000. The reactions were detected by the addition of the substrate o-phenylenediamine dihydrochloride (OPD; Sigma, USA) plus H2O2 and stopped with 50 μl/ well of 2 M H2SO4. Optical density (OD) values at 490 nm were measured with a microplate reader (TECAN, Austria). All samples were run in duplicate. The cut-off value of the ELISA was evaluated for the two antigen preparations based on a calculation of mean control OD + (2 x SD). The recorded ODs above the cut-off value were regarded as positive. Western blotting: Immunoblotting was carried out as previously described[24]. Adult worm extracts were separated in SDS-10% polyacrylamide gels and transferred to nitrocellulose membranes (0.45 μm; Heidelberg; Serva Electrophoresis GmbH, Germany) by electroblotting. Membranes were washed in PBS/ Tween buffer (PBS containing 0.3% Tween-20) and incubated for 1 h at room temperature in blocking buffer containing 5% non-fat milk in PBS/Tween-20, followed by washing and incubation with the human serum (1:100) in washing buffer overnight at 4°C. Formed immunocomplexes were detected by HRP anti-human IgG antibody (1:5000; KPL, Maryland, USA). After 2 h of incubation at room temperature, bands were developed by adding substrate (50 mg 3,3′-Diaminobenzidine tetrahydrochloride and 100 μl H2O2 in 100 ml PBS). Statistical analysis: SPSS (version 20) statistical program (SPSS Inc., Chicago, IL) was used to carry out one-way analysis of variance (ANOVA) on our data. When significant differences by ANOVA were detected, analysis of differences between the means of the OD values was performed by Dunnett’s t-test. The sensitivity and specificity of ELISA were assessed according to the following formulas: Sensitivity = no. of true positives/(no. of true positives + no. of false negatives) × 100; and specificity = no. of true negatives/ (no. of false positives + no. of true negatives) × 100[25]. Positive and negative predictive values and diagnostic accuracy were also calculated. P values < 0.5 were considered significant. Ethical approval: The study was approved by the Research Ethical Committee of the Beni-Suef University, Faculty of Medicine, Egypt. Signed informed consent was obtained from all individuals included in the study after explaining the purpose of the study. PARASITOLOGISTS UNITED JOURNAL

Volume None
Pages None
DOI 10.21608/puj.2021.76982.1121
Language English
Journal None

Full Text