Diagnosis of Opisthorchiasis by Enzyme Linked Immunosorbent

Asian Pacific Journal of Allergy and Immunology (1990) 8 : 27-31

Diagnosis of Opisthorchiasis by Enzyme Linked Immunosorbent Assay Using Partially Purified Antigens.

Nltaya Poopyruchpong 1, Vlthoon Vlyanantl, E.S. Upatham 1 and Petcharln Srlvatanakul2

Opisthorchiasis is one of the SUMMARY Opisthorchis viverrini antigens were partially purified from adult diseases of public health and econo worms collected from liver and extrahepatic biliary system of Infected hamsters. I mic importance in Thailand. The Tegument fraction was obtained by chemical extraction, whereas other fractions prevalence of Opisthorchis viverrini were purified by Sephadex 0·200 gel filtration chromatography. Five fractions of infection among the population in O. viverrini antigens were obtained, namely tegument extract, somatic extract, the northeastern part of the country fraction 1 (Pi)' fraction 2 (P2) and fraction 3 (P3>, respectively. The enzym.llnked Immunosorbent assay technique was used to compare the reactivity of the five If has risen from 3.5 million cases in partially purified antigens. The sensitivity and specificity of all five antigens were 1965 to 5.4 million cases in 1981. I f compared by testing against the sera of 78 O. viverrlnl ·In'ected Individuals from o. Recently, it has been estimated that viverrini endemic areas and 70 Individuals from non·endemlc areas Infected with at least 7 million people are infected hookworm, Trichuris and Ascaris Including 49 Individuals with negative stool examl· with this parasite. 2 Current methods nation. The assays performed with tegument extract, somatic extract and P1 frac· for the diagnosis of O. viverrini infec tlon were found to have 100% sensitivity, whereas the sensitivities of those with II tion are based .~:.. the demonstration P2 and P3 were 98.1% and 83.3%, respectively. The tegument extract had the of Opisthorchis eggs in either stool, highest specificity as demonstrated by the lowest croaa.reactlvlty with other para· duodenal fluid or bile. However, altes. Our resulta Indicated that surface tegument la the most aultable antigen for the techniques are tedious and un use In Immunological dlagnoala of opisthorchiasis. reliable in some cases, especially in cases of light infection and in cases I! of bile duct obstruction. Several and to assess the immunodiagnostic one hour. After surface tegument ! attempts have been made to develop potential of these partially purified extraction, all adult worms were immunological methods for the antigens. ground and homogenized in a glass diagnosis of the liver fluke infec tissue grinder and sonicated five I MATERIALS AND METHODS times for 30 seconds each at 60 watts. I tion. 3-7 The results are still not satis f factory due to the low sensitivity and Opisthorchis viverrini antigens cross-reactivity of the techniques. f Adult worms were collected , It has been knowp. that a more from the liver of infected hamsters From the 1Center for Applied Malacology refined antigen would provide a after four weeks of infection. The and Entomology, Department of Biology, Faculty of Science, Mahidol University, surface tegument of adult worms more specific serological test for the Rama 6 Road. Bangkok 10400, Thailand <;\iagnosis of parasitic infection, in was prepared by extraction with non and 2Research Division, National Cancer cluding the caused by liver flukes. ionic detergent (Triton X-tOO solution Institute, Ministry of Public Health. Bangkok Therefore, the following study is an containing 50 mM Tris HCI pH 7.4, 10400, Thailand. attempt to isolate several purified 150 mM NaCi, 10 mM EDTApH 7.0) antigenic materials from O. viverrini at 5° C with continuous shaking for Correspondence: Dr. Vithoon Viyanant f • I. I ! i 28 POOPYRUCHPONG. ET AL.

The homogenate was centrifuged rin; infection by repeated stool exami and the tegument fractions gave at 5,000 rpm for 30 min at 4" C. An nation. Pool sera from the groups 1000;0 positive for all of the individuals aliquot of the supernatant designated of individuals with proved positive with O. viverrini infection regardless as somatic O. viverrini antigens was for O. viverrini infection by stool of the number of parasite eggs in further purified by Sephadex G-200 examination were used as the positive the faeces (Fig. 2,5,6). A rather poor gel filtration chromatography, using control. sensitivity was obtained with P2 and phosphate buffered saline solution P3 fractions (Figs. 3,4). When the (PBS,pH 7.2,0.15 M) as eluent. The RESULTS relationship between the number of fractions belonging to the same peaks The somatic antigen of O. viver parasite eggs per gram of faeces and were pooled, lyophilized, reconstituted rini was separated by gel fiItration the reciprocal of the antibody titers with distilled water and stored at chromatography into three fractions, were compared, the best correlation -70" C until use. The protein con namely PI, P2 and P3, respectively was observed with the tegument tents of each fraction of O. viverrini (Fig. I). Therefore, O. viverrini anti fraction (Fig. 6). antigens were determined by Folin gens used in this study were composed Ciocalteau method. 8 Table 1 shows the results of of tegument extract, somatic extract, ELISA assay among the group of pe0 and PI, P2 and P3 fractions of the Human sera ple with confirmed O. viverriniinfec somatic extract. tion, a group of people with other intes Serum samples were obtained The relationship between the tinal parasitic infections (hookworm, from three groups of inhabitants. egg count and the antibody titer to Trichuris and Ascaris) and a group Group 1 consists of the sera from 78 each of five antigens of O. viverrini of people with negative stool exami individuals from the endemic area in 197 individuals with and without nation. Although PI fraction was and has been found positive for O. O. viverrini eggs in faeces are shown found to have 100% sensitivity, it viverrini infection by stool examina in Figs. 2 to 6. When the cut off for also reacted with 52.8% of the sera tion. Group 2 consists of the sera ELISA reading was made at the serum from patients with other intestinal from 70 individuals reside in Pang-Nga titer of 1:40, PI, somatic extract parasites and 28.5% with sera from Province which is a non-endemic area for O. viverrini. Stool exami· nation for this group of people revealed infection with other parasites, such as hookworm, Trichuris trichiura or Ascaris lumbricoides. Group 3 consists of the sera from 49 indivi duals reside in both non-endemic and endemic areas of O. viverrini i' which has been found negative for ~ parasitic infection by stool exami ~,.. nation. Stool examinations were I" O.lS performed by the method of For ...'"-z Q malin-ether concentration technique 9 ;;J. and Stoll's egg counting technique. 10 U I" Q,. -0 Enzyme-Linked Immunosorbent Assay (ELISA) The indirect technique of en zyme-linked immunosorbent assay 20 80 100 for antibody to O. viverrini followed the method of Srivatanakul et al. 5 FRACTION NUMBER All fractions of the antigen were tested against the sera of groups 1, 2 Fig. 1 Gel filtration chromatography of O. viverrini somatic antigens and 3 individuals. A reference of on Sephadex G-200 column (5.3x75.0 em). Fractions of 3.0 ml the negative control was obtained by with a continuous flow rate of 21 ml/h were collected. using a pool of sera from a group of Fraction 1 (P1) fraction number 45-55 individuals who reside in non-endemic Fraction 2 (P2) fraction number 64-100 areas, who never consumed raw fish Fraction 3 (P3) fraction number 102-124 and were found negative for O. viver J I

t ELISA FOR DIAGNOSIS OF OPISTHORCHIASIS 29 ! I ! ! ,~ I I I, i 100.000 f I 100.000 · · 50.000 ! 50.000 • i I ! • 1

200 200

100 r • 0.385 100 • r = 0.329

....L ....L -' Negative 40 BO 160 320 640 12BO Negative 40 80 160 320 640 1,280 I , 1 I II 1 RECIPROCAl OF THE ANTIBODY TITER RECIPROCAL OF THE ANTIBODY TITER

Fig. 2 Correlation between the titer of antibodies Fig. 3 Correlation between the titer of antibodies to Pl fraction of O. viverrini antigens and the to P2 fraction of O. viverrini antigens and number of egg/G of faeces. the number of eggs/G of faeces.

Table 1. Results of ELISA assay among the group of individuals with confirmed opisthorchiasis, the group with other parasitic infections and the group with negative stool exam.

Group of individual Total number No. Pos. ELISA with various tested antigens tested (%)

Pl P2 P3 Somatic Tegument

Confirmed 78 78 75 65 78 78 opisthorchiasis (100) (96.1 ) (83.3) (100) (100) II Other parasitic 70 37 21 34 17 2 infections (52.8) (30.0) (48.5) (24.2) (2.8) III Negative stool 49 14 7 11 2 4 exam (28.5) (14.2) (22.4) (4.0) (8.1 ) 30 POOPYRUCHPONG, ET AL.

100,000 , IIlO,ooo 50,000 50.000 •

1 10,000 '"u ·• r1:'" 5,000 VI...., 10,000 ... u...., ...... ~ 5.000 loA. <:> ~ (J) , . ...co 1,000 ... ~ • ...'" m . • I.. 500 loA. • <:> 1.000 ....co:: • 200 • I r ~ 0.247 z: 500 100 200 !Ieg.the., 40 80 160 320 640 1280 2560 5120 10,240 r = 0.089 100 to RECIPROCAl OF THE ANTlBOOY TITER Fig. 5 Correlation between the titer of antibodies Nega t1 ve 40 80 160 320 640 1280 of somatic extract of O. viverrini antigens II 1 1 I and the number of eggs/G of faeces.

RECIPROCAL OF THE ANTIBODY TITER 100,000

50,000 Fig. 4 Correlation between the titer of antibodies to P3 fraction of O. viverrini antigens and the number of eggs/G of faeces.

.....VI 10,000 negative individuals. Somatic extract :rl also exhibited high sensitivity, but if 5.000 its specificity was relatively low since ~... it reacted with relatively large number , p of the sera from patients with other ~ parasites. The integument fraction ~ 1.000 was the only fraction found to have '"..... 500 the highest sensitivity and specificity. ~z: Among the five antigens shown to have positive correlation between 200 the titer of antibodies and the intensity of O. viverrini infection based on 100 r : 0.399 Stoll's egg counting technique, the tegument extract was found to have the highest correlation (correlation Negative 40 80 160 320 640 1280 2560 5120 coefficient, r =0.399) (Figs. 2 to 6). ••RECIPROCAL OF THE ANTIBODY TITER DISCUSSION Fig. 6 Correlation between the titer of antibodies to the tegument fraction of O. viverrini Antibodies to O. viverrini infec antigens and the number of eggs/G faeces. tion have been detected by various ELISA FOR DIAGNOSIS OF OPISTHORCHIASIS 31 I! t

immunologiCal techniques, such as a very low parasite infection which mediate hosts, transmission to man and I immunoelectrophoresis,2,3,7 and cannot be detected by stool examina geographical distribution in Thailand. ELISA.4-6 With regard to ELISA tion. Drug Res 1984; 34: 1164-7. II, assay, Feldheim and Knobloch 4 used It is apparent that while the 3. Janechaiwat J, Tharavanij S, Vajrasthira t a crude extract of O. viverrini and S, Chaicumpa W. The immunological tegument extract, P I fraction and reported that high sensitivity was diagnosis of human opisthorchiasis and somatic extract have similar sensitivity, obtained but low levels of cross the humoral immune response to opisthor the PI fraction and somatic extract reactivity with various species of chiasis infection in the hamster. J Med are less specific. The low specificity Assoc Thai 1980; 63: 439-47. I trematodes and nematodes still of P I fraction and somatic extract occurred. Similar results were also 4. Feldheim W, Knobloch J. Serodiagnosis as compared to the tegument extract obtained by Srivatanakul et al. 5, of Opisthorchis viverrini infestation by is probably due to the presence of I an enzyme immunoassay. Tropenmed i they reported that ELISA assay non-specific epitopes in the first two Parasitol 1982; 33 : 8-10. I detected 92.8070 of confirmed opis antigens. Since hookworm, Trichuris 5. Srivatanakul P, Viyanant V, Kurathong I thorchiasis individuals 46.5% but of and Ascaris are helminth parasites S, Tiwawech D. Enzyme-linked immu [ non-opisthorchiasis individuals also f that have been found throughobt nosorbent assay for detection of Opis ! were found to give positive results. Thailand, the test that shows less thorchis viverrini infection. Southeast ,I \ In our study, we showed that cross-reaction with these parasites Asian J Trop Med Public Health 1985; \ 16: 234-9. , by using partially purified antigens would be the most preferable. Based of O. viverrini, sensitivity and speci on our results, it can be concluded 6. Wongratanacheewin S, Bunnag D, ficity of the ELISA assay can be that the tegument extract is the most Sirisinha S. Enzyme-linked immuno ! sorbent assay for detection of antibodies improved. Among the five fractions suitable antigen to be used for immu to Opisthorchis viverrini. Asian Pac t of partially purified antigens used in nological assay for the diagnosis of J Allergy lmmunol1985; 3: 114. this study, the tegument extract, PI opisthorchiasis. 7. Viyanant V, Vivatanasesth P,Upatham antigen and somatic extract rendered E.S, Sornmani S, Siriteramongko1 S, 100% sensitivity. However, when ACKNOWLEDGEMENT I lmlarp S. Antibodies to opisthorchiasis ,i specificity of the five antigens were We are grateful to Mrs. Pissanee after treatment with praziquantel. J tested, the tegument extract was Ardsungnoen for her technical assis Parasitol Trop Med Ass Thailand 1985; found to have the highest specificity tance in examining the stool specimens 8: 20-4. I as indicated by the lowest cross for parasitic infections. 8. Lowry OH, Rosenbrough NJ, Farr SL, reactivity with antibody to other intes Randal RJ. Protein measurement with I tinal parasites (Table I). the occur the Folin phenol reagent. J Bioi Chern REFERENCES !, rence of false positive results of I. Preuksaraj S, Jeradit C, Sathitayatai A, 1951; 193: 265-75. , ELISA assay in the group of indi Kijvanee S, Sridonsrasmi T. Studies on 9. Ritchie, LS. An ether sedimentation t viduals with negative stool examina prevalence and intensity of intestinal technique for routine stool examination. tion was probably due to the fact infection in rural population of Thai Bull. United States Army Med Dept that the sera were obtained from land 1980-1981. Comm Dis J 1982; 8 : 1948; 8 : 326. heterogeneous groups of people from 245-69. 10. Stoll NR. An effective method of counting both non-endemic and endemic areas. 2. Harinasuta, C, Harinasuta, T. Opis hookworm eggs in faeces. Amer J Hyg Some of these individuals may have thorchis viverrini: Life cycle, inter 1923; 3 : 59.