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Home , Opisthorchiasis

I ASIAN PACIFIC JOURNAL OF ALLERGY AND IMMUNOLOGY (1997): 15: 115-122

Parasites Elicited Cross-Reacting Antibodies to vlverrlnl• ••

Yuwapom Sakolvaree. Launa Ybanez and Wanpen Chaicumpa

Opisthorchiasis viverrini, the SUMMARY Two batches of crude antigens extracted from adult Opis­ infection caused by Opis­ thorchis viverrini worms were compared. One was derived from adult thorchis viverrini is a disease of worms harvested from the livers of laboratory infected hamsters and public health concern of Thailand. another was obtained from worms sedimented from the faeces of opis­ thorchiasis patients following treatment with . SDS-PAGE The infection rarely produced acute and Coomassie brilliant blue staining revealed that the two prepara­ clinical disease which is different tions had similar protein components of which the predominant ones from the infection caused by 0. were the 17-18 kDa doublet. The antigens were used in an indirect felineus, but rather be chronic in ELISA for the detection of antibodies against O.viverrinl in the sera of nature and may persist for many four groups of patients, ie. patients with opisthorchiasis (group 1), pa­ 1 tients with mixed infections of O.viverrinl and other parasites (group years. The majority of infected in­ 2). patients with other parasitic infections (group 3), and normal­ dividuals are symptomless? Clinical heathy, parasite-free individuals (group 4). The sensitivity of the test manifestations of symptomatic was high (91-92%), regardless of the batch of the antigen used. How­ cases vary from mild to severe form ever, its specificity was relatively low (70-80%). Cross-reaction was ob­ depending upon the number of served with patients infected with heterotremus, Schisto­ soma spp.; Taenia spp.; ; Strongyloides stercoralis; worms present and the duration of ; Plasmodium spp.; hookworms and Plasmodium spp.; S. the infection. Severe cases are more stercoralis, Blastocystis hominis and yeast; and hookworms, Ascaris common in patients over 40 years lumbricoldes, and P.falciparum. Western blot analysis old and males are more affected revealed that sera of patients infected with these heterologous organ­ than females.3.4 Some patients may isms contained antibodies reactive to O.viverrlni antigenic components ranging from Ifr15.5 to 144. harbour thousands of flukes at any one time, suggesting that the mecha­ nism for parasite elimination is not Since Opisthorchis viverri­ light, examination of stool for eggs effective and/or that re-infeciton is ni infections are not pathognomonic, is uncertain. 0. viverrini eggs are common in this disease. Chronic diagnosis is based mainly on de­ difficult to distinguish from eggs of persistent infection with large num­ monstration of the fluke eggs in small intestinal flukes, ego Pros to- ber of worms can lead to chronic stool, duodenal fluid or bile. Al­ relapsing cholangitis and obstruc­ though examination of eggs in stool tive , S and may in some is reliable, it is useful only when From the Department of Microbiology and im­ cases eventually lead to cholangio­ intensity of infection is high. On the munology, Faculty of Tropical Medicine, Ma­ 6 hidol University, Bangkok. Thailand carcinoma. ,7 other hand, when the infection is Correspondence: Wanpen Chaicumpa 116 SAKOLVAREE. ET AL.

dendrium moienkampi, Phanaerop­ MATERIALS AND METHODS and once with distilled water. They solus bonnet, Haplorchis taichui were lyophilized. After the lyophili­ and H. pumillo. 8 In severe Preparation of O. viverrini antigen zation they were ground homoge­ infections, where there is a biliary neously in sterile distilled water obstruction, the eggs remain in the Collection of o. viverrini adult containing protease inhibitors. The gall bladder and/or biliary tract and worms preparation was subjected to an can not be found in the faeces. In Adult 0. viverrini worms MSE ultrasonication at maximum these instances, canulatlon of the were from two different sources. input at 4°C for 10 minutes. The su­ biliary canal is necessary for re­ Worms of the first batch were col­ pernatant was collected and the covering the eggs. However, such lected from infected individuals pellet was discarded. Protein con­ procedure is not simple and may tent of the supernatant was deter­ whereas the second batch was from . ed 15 pose some danger to the patient. ~. The preparation was kept infected hamsters. Alternative methods for diagnosis of m small aliquots at -20°C until opisthorchiasis included the use of used. DNA probe for detecting compli­ First batch. Patients with opisthor­ chiasis viverrini admitted at the mentary DNA of the fluke in stool 9 Serum samples the monoclonal antibody-based Bangkok Hospital for Tropical Dis­ eases were treated with Praziquantel ELISA for antigen (E-S/somatic) Serum samples were collected 9 10 at 40 mg/kg body weight and given detection • and various antibody from four groups of individuals, ie. · 1112 45 ml of saturated magnesium sul­ detectIon assays.' However group 1 from 22 patients with O. vi­ none of them meets the ideal criteri~ phate per orem seven hours later. Stools were collected within twelve verrini infection only; group 2 from on specificity and sensitivity as yet. 31 patients infected with O. viver­ The antibody detection assay, ego an hours after the treatment with Prazi­ quantel. Sedimentation was done on rint and other intestinal parasitic indirect ELISA may be as sensitive infections; group 3 were 141 pa­ 13 the stool specimens and adult 0. vi­ as the detection of eggs in stool tients with other parasitic infections' but cross-reaction with other para­ verrini were collected in normal saline solution (NSS). and group 4 from 24 normal' sitic infections are commonly and healthy, parasite-free individuals: frequently found when crude Second batch. Young adult ham­ The lists of serum specimens and parasite extract (which is the most the parasites found in patients of convenient and available source of sters were individually infected with 100 0. viverrini metacercariae. The groups 2 and 3 are tabulated m antigen) was used. The use of the Tables 1 and 2, respectively. parasite specific antigen, ego the 89 metacercariae were from muscles of kDa metabolic protein14 might be an laboratory-bred fresh water fishes b~longing to the genus Pontius pre­ Indirect ELISA a~~ative in order to increase spe­ VIOusly caught from the northeast of CifiCity of the antibody detection Crude O. vtverrini antigen at assay. However, sufficient amount Thailand. Two months after the in­ fection, the hamsters were sacrificed 2.5 J..Lg protein per ml of carbonate of such antigen for a routine work is bicarbonate buffer, pH 9.6 was not available by the conventional after ether euthanaesia and the liv­ used to coat all wells (100 J..Ll per method ofpreparation. ers were dissected out. The tissue was pressed in between two glass well) of microtitre plates (Greiner, Germany). The plates were incu­ As mentioned above, the val­ pads and examined for adult worms. bated at 37°C for one hour and at ue .of the antibody detection assay in The worms were carefully collected 4°C overnight. After the unbound opisthorchiasis is limited by the from the bile ducts using two surface of each well was blocked cross-reactive nature of the crude needles. They were collected in antigen used. In this communica­ sterile NSS. with 200 J!l of 1% bovine serum tion, the parasitic infections which albumin (BSA) in PBS, 100 J..LI of gives cross-reaction to the 0. viver­ Preparation ofantigen serum samples at 1:400 dilution rini crude antigen are reported. was added to appropriate wells ex­ The adult 0. viverrini worms cept the blank wells, incubated for were washed five times with NSS one hour, washed and 1: 1,000 ANTIBODY RESPONSES TO 117 •

adult 0. viverrini of the first batch. Similar pattern was also obtained Table 1. Patients of group 2 whose faecal samples revealed O. viverrini and other parasites when the second batch of antigen was testlXi. The components varied in molecular mass from Mr 17,000­ Patient no. Parasite(s) beside O. viverrini found in stools 71,000. A 17,000-18,000 kDa doublet were the most prominent 8 hookworms, Giardia lamblia components among all proteins. 12 , Entamoeba coli 24 81astocystis hpminis When the first batch of anti­ 32 Echinostoma spp. 34 Echinostoma spp., hookworms, 8. hominis gen was used in the indirect ELISA 45 Taenia spp. to detect antibodies in serum sam­ 47 Hookworms, E. nana, 8. hominfs 66 Trichomonas hominis ples of 24 normal controls, the mean 75 Hookworms, Strongyloides stercoralis, (X) of the optical densities was Entamoeba coli, 8. hominis 109 Hookworms, S. stercoralis, 8. hominis 0.030 and standard deviation (SO) 110 Hookworms, Echinostoma spp. was 0.012. If the X + 12 SO which 118 Hookworms, Echinostoma spp., G. lamblfa 124 Hookworms, 8. homfnis was 0.174 was used as the cutoff 130 Hookworms, S. stercoralis, 8. hominis limit between positive and negative 116,119 Hookworms, S. stercoralis 6,16 Hookworms, E. nana opisthorchiasis, it was found that all 2, 100 S. stercoralis of the 22 patients ofgroup 1 and 27 7,9,14, 15,20,27, Hookworms 28,30, 46, 49, 111 from 31 patients of group 2 were positive (Figure 2). The number of ELISA positive among group 3 was 33 from 141 patients. Thus the di­ agnostic sensitivity, diagnostic spe­ dilution of rabbit anti-human im­ stacking gel were used in the pro­ cificity and positive and negative munoglobulin-peroxidase conjugate cess. The separated components of predictive values were 92%, 80%, was added (100 J.LI per well). After the crude 0. viverrini antigen (40 60% and 97%, respectively. another incubation at 37°C for 30 f..lS per lane) was electroblotted onto minutes, the plates were washed and a sheet of nitrocellulose (NC) paper. When the second batch of the the substrate solution was added. The blot was put in a blocking solu­ antigen was used to capture the Enzyme-substrate reaction was al­ tion (3% BSA, 0.5% gelatin in antibodies in the patients' sera, lowed to proceed for 20 minutes. PBS, pH 7.4) for one hour at room similar results of the ELISA were The reaction was stopped by adding temperature with gentle rocking. obtained. If the 00 0.174 was used 50 III of 1 N NaOH to each well. The NC strip was washed three as the cutoff optical density between Optical densities of the contents of times with washing buffer (0.1 M positive and negative opisthorchia­ all wells were read against the PBS, pH 7.4 containing 0.05% sis, 19 patients of group 1, 27 pa­ blanks at 492 nm using an ELISA Tween-20), then it was put into the tients of group 2 and 50 patients of reader. serum specimen diluted at 1:400 for group 3 were positive (Figure 3). one hour at room temperature. The The diagnostic sensitivity, diagnos­ SDS-PAGE and Western blot strip was then washed and the im­ tic specificity and predictive and analysis mune complexes were visualized negative values were 91 %, 70%, using peroxidase conjugated rabbit 49% and 96%, respectively. The Sodium dodecyl sulphate anti -human immunoglobulins and parasites found in patients whose polyacrylamide gel electrophoresis its substrate. serum samples were positive by was carried out in vertical slab gel ELISA using the second batch of apparatus (Bio-Rad Laboratories, RESULTS antigen are given in Table 3. USA) according to the system of Laemnli. A 10% acrylamide sepa­ Figure 1 illustrates protein Figure 4 illustrates represen­ rating gel and 4% acrylamide components of crude extract of tative Western blot patterns of 118 SAKOLVAREE, ET AL.

SDS-PAGE separated 0. vivemm antigen reacted with serum samples Table 2. List of organisms found in 118 patients of group 3 of patients which cross-reacted by ELISA to the antigen. Table 4 sum­ marizes the serum reactivities of pa­ Patient no. Parasite(s) beside O. viverrini found in stools tients with opisthorchiasis, patients with other parasitic infections and 36 T. trichiura 37 A. lumbricoides, hookworm, T. trichiura, P. fa/ciparum healthy, parasite-free controls 38 S. stercoraHs, P. falciparum against the 0. viverrini extract as 39 T. hominis, A. lumbricoides, T. trichiura, P. fa/ciparum 40 Hookworms, 8, hominis, P. fa/ciparum revealed by SDS-PAGE and West­ 41 T. trichiura, hookworms, S. stercoralis ern blot analysis. 52 S. stercoralis 53 Hookworms, P. fa/ciparum 55 S. stercora lis, T. hominis, yeasts, P. fa/ciparum 56 Hookworms, G. lamblia, P. fa/ciparum DISCUSSION 60 Hookworms, S. stercoralis, A. lumbricoides, P. falciparum 64 S. stercoralis, P. fa/ciparum The most predominant com­ 65 T. saginata 69 S. stercoralis, hookworms, P. fa/ciparum ponents of the crude extract of 0. 70 Hookworms, P. fa/ciparum viverrini adult worms were the 17­ 73 Hookworms, P. fa/ciparum 77 A. lumbricoides, T. trichiura, hookworms, 18 kDa doublet. This finding is con­ P. fa/ciparum sistent with that reported by Ruppel 78 A. lumbricoides, T. trichiura 16 79 S. stercoralis, hookworms et al. and Wongratanacheewin et 80 S. stercoralis. hookworms, P. fa/ciparum al!4 who observed further that this 2.1-2.21 Paragonimus heterotremus doublet were glycoproteins and 3.1-3.10 spp. 4.1-4.21 Trichinella spiralis were most likely localized in the 5.1-5.13 Taenia spp. surface tegument of the parasite. 6.1-6.20 Strongyloides spp. 7.1-7.12 Hookworms 8.1-8.3 Entamoeba histolytica Sensitivities of the indirect 9.1-9.17 Plasmodium spp. 10.1-10.3 spinigerum ELISA were 92% and 91 % using 11.1 Filaria the first and second batches of crude extracts prepared from adult 0. vi­ verrini derived from two distinct sources, respectively. SDS-PAGE of the two antigens also revealed the same patterns of fractionated com­ Table 3. Parasites found in patients whose serum samples gave false positive ELISA for the detection of antibodies to O. viverrini ponents. Thus one source of the 0. crude antigen (second batch) viverrini adult worms can be an alternative of another. Four out of 53 patients of groups 1 and 2 gave Parasites Number of cases which gave false positive reactionltotal false negative ELISA when the first batch of antigen was used. The Paragonimus heterotremus 14/21 same four cases and additional 1 Schistosoma spp. 2110 case gave false negative result when Taenia spp. 4/13 Trichinella spiralis 16/21 the second batch of antigen was Strongyloides stercoralis 8121 used. These cases had stool egg Hookworms 1/12 Plasmodium spp. 1/17 counts of less than 800 EPGF at the Hookworms and Plasmodium spp. 2/3 time the serum samples were col­ S. stercoralis , B. homlnis and yeast 1/1 Hookworms, Ascaris lumbricoldes. 1/1 lected thus classified as cases with T. trlchiura and P. falciparum light infection. 16 Egg output is known to correlate with the worm burden. 3 In the present study, the J';o"'...."~"""~__""~~_",,>.t"""'''''''."''''....-...,,',:,'''''''',,',...,..''''''''"~"h<"••"..",'-''~~.,.-''''....:.''',' ..."'""";'.'-'":""'_"'''''."'''"''''''_'"''''-~''''A"'.'''''•. ,''''''''''''_~' .,-.~"., •• ,

~ -I iiio o -< :;u Table 4. Serum samples of patients with parasitic infections and normal individuals of opisthorchiasis endemic (Thai) and non-endemic men (Swedes)! areas which contained antibodies reactive to antigenic components of 0, viveninisomatic antigens (3 z en Serum sample which Antigenic component (mol.wt x 1()"3 ) enm gave reaction 144 112 94 90 84 76 72 ~ ro ~ ~ ~ ~ 47 45 43 42 41 40 38 -I Opisthorchiasis ...... o ...... o ...... ~, ...... Trichinellosis ...... ~ Hook worm infection ...... N ...... :::r ...... ea' ... ~. Filanasls (W.b) ...... CD ...... Malana (P.I.) ...... ~: Malana (P.v.) ...... Amoebiasis ...... Normal Thai Normal SWedes

Serum sample which Antigenic component (mol. wt x 10'3) gave reaction 37 36 35 34 32 30 29 28 27 25.5 24.5 22 21.5 20.4 19.7 18.5 18 17 16.5 15.5

Opisthorchiasis ...... ± ± ...... Paragonimiasis ...... Schistosomiasis ...... Gnathostomiasis ...... Trichlnellosis ...... Hook worm imection ...... Strongyloidiasis ...... Trichuriasis ...... capillariasis ... (W b.) ...... Taeniasis ...... Malana ...... Malaria (P v) ...... Amoebiasis ...... Normal Thai ...... Normal SWedes

.... CO

122 SAKOLVAREE, ET AL

after the wonns had been removed 5 Harinasuta C. Opisthorchiasis in Thai­ lysis of Opisthorchis viverrlnl by Praziquantel treatment. 18,23 land. Southeast Asian J Trop Med antigens by immunoprecipitation and Public Health 1978; 9: 281. polyacrylamide gel electrophoresis. 6. Haswell-Elkins MR, Sithithawom P, Parasitology 1988; 96: 119-28. The significant difference p < Elkins D. Opisthorchis viverrini and IS. Lowry OH, Rosebrough NJ, Farr AL, 0.05) in ELISA positive of those in northeast Randall R1. Protein measurement with with 0. viverrini infection (groups 1 Thailand. Parasitol Today 1992; 8: 86­ the Folin-phenol reagent. J Bioi Chern and 2) and those without the infec­ 9. 1951; 193: 265-75. 7. Sithithawom P, Tesana S, Pipitgool 16. Sadun EH. Study on Opisthorchis tion (groups 3 and 4) suggests that V, Kaewkes S, Pairojkul C, Sripa B, viverrini in Thailand. Am J Hyg the test could be used as a screening Puapairoj A, Thaiklar K. Relation­ 1955; 62: 81-115. method for opisthorchiasis. How­ ship between faecal egg count and worm burden of Opisthorchis viverri­ 17. Phillipson RF, McFadzean JA. Clo­ ever, the test could not be used norchis, Opisthorchis and Paragoni­ alone for diagnosis as it could not ni in human autopsy cases. Parasitol 1991; 277-81. mus gel diffusion study. Tran R Soc differentiate between the present 8. Radomyos P, Bunnag D, Harinasuta Trop Med Hyg 1962; 56: 13. and past cure infections and could T. Worms recovered in stool fol­ 18. Feldheim W, Kobloch 1. Serodiagno­ give also some false positive results. lowing praziquantel treatment. sis of Opisthorchis viverrini infes­ For definite diagnosis of the present Anzneirnittel Forch 1984; 34: 1215-7. tation by an enzyme immune assay. 9. Sirisinha S, Chawengkirttikul R, Tropenmed Parasit 1982; 33: 8-10. infection it is required that either the Sermswan R, Amornpant S, 19. Wongratanacheewin S, Bunnag D, parasite specific antigens and/or Mongkolsuk S, Panyim S. Detection Vaeusom N. et al. Characterization of eggs are present in the patient's of Opisthorchis viverrini by mono­ humoral immune response in the specimen. clonal antibody-based ELISA and serum and bile of patients with opis­ DNA hybridization. Am J Trop Med thorchiasis and its application in diag­ Hyg 1991; 44: 140-5. nosis. Am J Trop Moo Hyg 1988: 38: REFERENCES 10. Chaicumpa W, Ybanez L, Kittikoon 356-62. V, Pungpak S, Ruangkunapom Y, 20. Sirisinha S, Tuti S, Vichasri S et al. Kurathong S, Lerdvirasirikul P, Chongsa-nguan M. Detection of Humoral immune response in ham­ Wongpaitoon V et al. Opisthorchis Opis-thorchis viverrini antigens in sters infected with OpisthorchiS viverrini infection in rural and urban stools using specific monoclonal vivemm. Southeast Asian J Trop communities in northeast Thailand. antibody. Int J Parasitol 1992; 22: Med Public Health 1983; 14: 243-51. Trans Roy Soc Trop Med Hyg 1987; 527-31. 21. Chawengkirttikul R, Sirisinha S. An­ 81 :411-4.. II. Sirisinha S. Immunodiagnosis of tibodies in serum and bile of hamsters 2 Bunnag D, Harinsuta T. Opisthor­ human liver fluke infections. Asian experimentally infected with Opis­ chiasis, and paragoni­ Pac J Allergy Immuno11986; 4: 81-8. thorchis viverrini. J Parasitol 1988; miasis. In: Warren K, Mahmouds A 12. Sirisinha S, Chawengkirttikul R, Has­ 18: 721-7. (eds.). Tropical and Geographical well-Elkins MR, Elkins DB, Kaewkes 22. Janechaiwat J, Tharavanij S, Vajra­ Medicine. New York. McGraw-Hill S, Sithithawom P. Evaluation of a sthira S, Chaicumpa W. The immno­ Book Company 1984; 461-70. monoclonal antibody based ELISA for logical diagnosis of human opisthor­ 3 Pungpak S, Harinasuta T, Bunnag D, the diagnosis ofOpisthorchis viverrini chiasis and the humoral immune res­ Chindanond D, Radomyos P. Faecal infection in an endemic area. Am J ponse to Opisthorchis infection in the egg output in relation to worm burden Trop Med Hyg 1995; 52: 521-4. hamster. J Med Assoc Thailand and clinical features in human opis­ 13. Sriwatanakul P, Viyanant V, Kura­ 1980; 63: 439-46. thorchiasis. Southeast Asian J Trop thong S, Tiwawesh D. Enzyme-linked 23. Ruangkunapom Y, Akai P, Chongsa­ Med Public Health 1990; 21: 275-80. immunosorbent assay for detection of nguan M, Sri Yeythong S, Kitikoon V, 4 Pungpak S, Riganti M, Bunnag D, Opisthorchis viverrini infection. Chaicumpa W. Changes in serum anti­ Harinasuta T. Clinical features in Southeast Asian J Trop Moo Public bodies to O. vivem·ni in humans and severe opisthorchiasis viverrini. Health 16: 234-9. hamsters following treatment of opis­ Southeast Asian J Trop Med Public 14. Wongratanacheewin S, Chawengkirt­ thorchiasis. Asian Pac J Allergy Health 1985; 16: 405-9. tikul R, Bunnag D, Sirisinha S. Ana­ Immuno11994; 11: 83-4.