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Home , Gnathostoma spinigerum, Gnathostomiasis

ELISA FOR IMMUNODIAGNOSIS OF

PRAVAN SUNTHARASAMAI, VARUNEE DESAKORN, SRICHAROEN MIGASENA, DANAI BUNNAG and TRANAKCHIT HARINASUTA Department of Clinical Tropical Medicine and Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok, .

INTRODUCTION using larval antigens. The specificity and sensitivity of the test were evaluated. Human gnathostomiasis caused by Gna­ thostoma spinigerum is an endemic disease in MATERIALS AND METHODS Thailand (Daengsvang, 1980). The diagnosis is usually presumptive on the basis of clinical Antigens were prepared from G. spinigerum features, with laboratory findings of eosino­ third-stage larvae recovered from liver, sto­ philia in the peripheral blood and by exclusion mach, intestine and body muscles of mice of other diseases (Swanson, 1971). A con­ after one month of experimental firmed or parasitologic diagnosis is rare since with oral administration of second-stage the parasite is recovered from only a small larvae in infected . The larvae were percentage of the patients by surgical removal cleaned by several washes with normal saline of the worm or spontaneous emergence of the and finally suspended in distilled water. A worm through , gingiva or in the urine. crude water-extract of the third-stage larvae was prepared according to the method pre­ A number of immunological tests have viously described by Sawada and co-workers, been applied to the diagnosis of gnathosto­ (1965). Aliquots of 5 ml of the extract were miasis(Cross, 1975), but theresu1ts have been kept at -20oC after lyophilization. unsatisfactory due to insensitivity or non­ specificity i.e. cross reaction with other para­ Sera were collected from patients with sitic diseases (Tada et a/., 1966; Morisita cutaneous migratory swelling presumably due eta/., 1969; Punyagupta and Pacheco, 1961; to gnathostomiasis, patients with a clinical Kasemsuth et a/., 1981). Recently, the en­ diagnosis of eosinophilic meningo-myelo-en­ zyme-linked immunosorbent assay (ELISA) cephalitis due to G. spinigerum (Punyagupta (Engvall and Perlmann, 1972) have been eta/., 1968; Boongird et a/., 1977), patients used to detect antibodies associated with with eosinophilic meningo- due various helminthic infection such as , to Angiostrongy/us cantonensis based on clini­ , (Kagan, 1984), cal criteria (Punyagupta et a/., 1970) and a and (Jaroonvesama et a/., history of consuming poorly cooked Pi/a 1985). Since the results appeared to be en­ snails that resulted in an outbreak of the couraging, the ELISA might be useful in the illness, and patients with other parasitic immunodiagnosis of gnathostomiasis. In diagnosed by stool or sputum this study, an ELISA system for gnathosto­ examinations. The other negative control mal antibodies in human sera was developed sera were from healthy blood donors in Bangkok. Sera from parasitological con­ This study was partly supported by a research grant from the SEAMEO-TROPMED project. firmed cases of cutaneous gnathostomiasis

274 Vol. 16 No. 2 June 1985 eLiSA FOR lMMUNODIAGNOSIS OF ;fUMAN GNATHOSTOMIASIS without evidence of other parasitic infections RESULTS as revealed by stool examination served as positive controls. Sera from 160 patients with cutaneous ·migratory swelling (CMS) presumably due In preparing the enzyme-labelled antihuman to gnathostomiasis, I 0 patients with a clinical lgG, go;tt antihuman lgG (Kallestad Lot, diagnosis of gnathostomal eosinophilic­ 204 G20999) was labelled with alkaline phos­ meningo-myeloencephalitis (EME-Gs) and 4 phatase (Type VII, Sigma Chemical company, parasitological confirmed cases of gnathosto­ St. Louis, Mo: U.S.A.: specific activity 1090 miasis were tested for G. .Ypinigerum anti­ units/mg protein) by the method described bodies by ELISA. The results are shown in by Engvall and Perlmann, (1971). Table 1. Sera from all the parasitological The ELISA used was essentially that of confirmed patients and patients with clinical Engvall and Perlmann, (1972) employing EME-Gs showed an ELISA titre of 1 : 400 disposable polystyrene plates (Cooke Micro­ and above, whilst only 56~~ of the CMS cases titer M220-29A, Dynatech Laboratories). tested gave this result. Thus, at the cut-point The titre was determined visually and con­ titre of I : 400, the sensitivity of the test was firmed spectrophotometrically at 405 nm reduced from I 00% to 59/"~ when the CMS against the reading for control negative cases were included (Table 3). serum. Same positive serum from a parasito­ Sera from 67 healthy blood donors, 24 ligical confirmed case of cutaneous migratory patients with eosinophilic meningo-encepha­ swelling and negative serum from one single litis due to angiostrongyliasis (EME-Ac) and individual was used throughout the period 92 patients that were positive for other parasi­ of study for quality control purpose. toses were tested to determine the specificity By checkerboard titration with reference of the test. The results are shown in Table 2. positive (a parasitological confirmed case of At the titre of 1 : 400 and above, positive gnathostomiasis) and control negative serum, results were observed in 1.5/-'~ of blood do­ the optimum dilution of enzyme-labelled anti­ nors, 33% of EME-Ac and 23% of other human lgG used was 1 : 500, and the opti­ parasitic infections including , mum N-concentration of antigen used was Strongyloides sterco/aris, Enterobius 1'ermi­ 5 f.lg(ml. cularis, , Trichuris trichiu-

Table I Frequency distribution of ELISA titres in sera from cases of cutaneous migratory swelling (CMS), eosinophilic meningo-myeloencephalitis, typically due to gnathostomiasis (EME-Gs) and CMS with parasitological proof (PCMS).

Sources Reciprocal ELISA Titre ----- ~-~--·------·-- of sera neg 25 100 400 1600 6400 Total

PCMS 2 2 4 EME-Gs 4 6 10 CMS 31 12 28 55 33 160 ------Total 31 12 28 61 41 174

Yol. In No. 2 June 1985 n:; SoutmAsr ASIAN''· TRoP. Mu>. Pull. HLTIL

Table 2 Frequency distribution of ELISA titres in ser.1 from healthy blood donors, cases of eosinophilic meningo-encephalitis due to A. cantonensis (El\1E-Ac) and cases of other parasitic infections tested against G. sjl"nigerum antigen.

-~-~-===.:: -~==---======- -::-.::..=--======..=::.::::=:.:=.:.::~_:_:__ __ Sources Reciprocal ELISA titre ------·------. ------. of sera neg 25 I 00 400 1600 Total

EME-Ac 2 6 8 8 24 Infection with Hookworm 4 S. stercolaris £. l'ermicularis 1 1 0. viverrini 7 3 4 2 17 Lung-fluke 1 5 4 10 Taenia spp. W. bancrofi i 2 B. malayi & hookworm ' P. falciparum 3 2 7 Mixed* 8 11 18 8 2 47 Blood donors 49 8 9 1 67 ------·------·------Total 71 33 49 27 3 183 * including: hookworm, S. stercolaris, A. lumbricoides, T. trichiura, 0. 1•iverrini, lung-fluke, Taenia spp., F. huski, Echinostoma spp., P. (alciparum.

Table 3 specificity of the test was 84 ~~ at the titre of Sensitivity and specificity of ELISA in sera I : 400 and above (Table 3) when all samples from patients with and without other than CMS and EME-Gs were consi­ gnathostomiasis. dered negative. In contrast, the specificity of the test for EME-Ac against EME-Gs was ! infection ------67 %. Table 3 shows that when the propor­ ELISA Confirmed N . tion of gnathostomiasis (174) in the sample . egahve > 1 :400 or presumptive ( _ 183) (357) was 0.49, the predictive value at the (n=l74) n- titre of 1 : 400 or above was 77 %. However in EME samples where the proportion of Positive (n = 133) 103 30 EME-Gs was 0.29, the predictive value was Negative (n = 244) 71 153 56~{. at this titre. Sensitivity (103/174) = 59.0% Specificity (153/183) = 84.0~~) DISCUSSION Predictive value (103/133) = 77.0~~ The present study evaluated the sensitivity ra, , lungfluke, Taenia and specificity of the ELISA employing crude spp., , buski, extract of the third-stage larvae of G. spini­ Echinostoma spp., , Bru­ gerum in immunodiagnosis of the gnathosto­ gia malayi and Plasmodium falciparum. The miasis. In most cases of human gnathosto-

276 Vol. 16 No. 2 June 1985 ELISA FOR IMMUNODIAGNOSIS OF HUMAN GNATHOSTOMIASIS miasis the worms obtained surgically or from Apted, 1982). Hence some of the false- spontaneous emergence rarely reach maturity; positive may actually be true-positives. only third-stage larvae or immature adults The ELISA for gnathostomiasis as per­ are recovered from patients (Daengsvang, formed in this study is not yet applicable for 1980). It is generally believed that antigens general diagnostic purposes. In the majority used in a serodiagnostic test should be pre­ of patients, a reliable clinical diagnosis can pared from the same stage of parasite as that be made from history and clinico-pathological which is recovered from patients; that was features and the place of domicile of the the rationale in the selection of third-stage patient. The ELISA would be helpful when larvae for antigen preparation in this study. such clinical evidence is lacking. On rare Sensitivity of the ELISA was evaluated occasions, the serological test may be used in from parasitologically confirmed cases (PC the differential diagnosis of the intestinal mass MS) as well as presumptive cases i.e. CMS (Sirikulchayanonta and Chongchitnant, 1979; and EME-Gs. The overall sensitivity of the Laohapand eta!., 1981). A change in ELISA test was 59%. which might be an underesti­ titre might also be useful as an indicator of mation since the criteria for CMS cases diseases activity, especially when specific depended on exclusion of other helminth­ treatment for gnathostomiasis become avail­ associated or allergic swelling based only on able. clinical features and geographical distribution. In conclusion the ELISA technique is Some of the false negatives might actually be potentially useful for the immunodiagnosis of non-gnathostomal. On the other hand, it was gnathostomiasis. Nevertheless improvement also possible that antibody levels in CMS, if of sensitivity and specificity is needed. The they were exclusively due to the gnathostomes, specificity might be improved by purification may be lower than those in EME-Gs where and fractionation of antigens. Simultaneous the worm migrates from the interstitial tissue assay of patients' sera against a panel of anti­ to an enclosed CNS space or in PCMS where gens derived from G. spinigerum, A. canto­ the worm sought to leave the through nensis, Paragonimus heterotremus etc. might the skin. help in making an immunodiagnosis of helminthic infection in that homologous titre In determining the specificity, ELISA should be higher than heterologous titre. titre of sera from healthy blood donors, EME­ Other immunodiagnostic approach includes Ac patients and patients with other parasitosis the detection of circulating antigen or antigens were used to detect nonspecific reaction; at in blood or CSF using highly specific anti­ the titre of 1 : 400 and above the specificity bodies such as monoclonal antibodies. of the test was 84 %. When only sera from patients with EME-Ac were tested against SUMMARY EME-Gs, the specificity was reduced to 67 %­ As in the case of sensitivity, the specificity An ELISA for immunodiagnosis of human might be an underestimation; the non­ gnathostomiasis using a crude water extract gnathostomal controls may have subclinical of third-stage larvae of G. spinigerum as anti­ or latent infections with gnathostomes since gen, and alkaline phosphatase labelled goat patients with gnathostomiasis and those antihuman IgG in the indicator system was with angiostrongyliasis, and developed and evaluated. At the titre of share the habit of eating raw 1 : 400 and above positive results were ob­ or half-cooked meat (Manson-Bahr and served in 100% of 4 parasitological confirmed

Vol. 16 No. 2 June 1985 277 SOUTHEAST ASIAN J. TROP. MED. PuB. lfLTH. and 10 eosinophilic meningo-encephalitis DAENGSVANG, S., (1980). Monograph on the (EME) typical of gnathostomiasis cases, 56 % genus Gnathostoma and gnathostomiasis of 160 cutaneous migratory swelling cases, in Thailand. p. 21. Southeast Asian 33% of 24 cases with EME typical of A. Medical Information Center, Internatio­ cantonensis infections, 23% of 92 cases with nal Medical Foundation of , other parasitic infections and 1.5% of blood Tokyo, Japan. donors. The overall sensitivity was 59% and ENGVALL, E. and PERLMANN, P., (1971). specificity 84 %. The predictive value was Enzyme-linked immunosorbent assay, 77 %. The results indicated that ELISA is (ELISA). Quantitative assay of immuno­ potentially useful for immunodiagnosis of globulin G. Immunochem., 8 : 871. gnathostomiasis but improvement of sensi­ ENGVALL, E. and PERLMANN, P., (1972). tivity and specificity is needed. Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific ACKNOWLEDGEMENTS antibodies by enzyme-labeled anti­ immunoglobulin in antigen-coated tubes. The authors wish to thank Professor Svasti J. Immun., 109 : 129. Daengsvang and Mr. Paisa! Yingyourd, of JAROONVESAMA, N., CHAROENLARP, K., Bu­ the SEATO Medical Laboratory, Bangkok RANASIN, P., ZARASPE, G.G. and CROSS, for their suggestion in the maintenance of J.H., (1985). ELISA testing in cases of the life cycle of in clinical angiostrongylosis in Thailand. the laboratory. Thanks are also due to the Southeast AsianJ. Trop. Med. Pub. Hlth., medical staff of Prasat Neurological Hospital, 16: 110. Bangkok for their kind cooperation in collec­ KAGAN, I.G., (1984). Serodiagnosis. In : ting serum samples from patients with EME­ Hunter's Tropical Medicine, 6th ed., Gs, and to Siam Medico Supply Co., Ltd., p. 1005. Strickland, G.T. (ed.) W.B. Bangkok for providing goat antihuman Saunders Company, Philadelphia. IgG. KASEMSUTH, R., PANUT-AMPON, P. and SANGHIRUN, C., (1981). Study on the REFERENCES diagnosis of Gnathostoma infection in by radioimmunoassay. Southeast BooNGIRD, P., PHUAPRADIT, P., SIRIDEJ, N., Asian J. Trop. Med. Pub. Hlth., 12:410. CHIRACHARIYAVEJ, T., CHUAHIRUN, S. LAOHAPAND, T., SoNAKUL. D., LoLEKHA, s. and VEJJAJIVA, A., (1977). Neurological and DHARAMADACH, A., (1981). Gnathos­ manifestations of gnathostomiasis. J. tomiasis of the colon simulating malig­ Neurol. Sci., 31 : 279. nancy : A case report. J. Med. Ass. CRoss, J.H., (1975). Diagnostic methods in Thailand, 64 : 192. other tissue including an­ MANSON-BARR, P.E.C. and APTED, F.I.C., giostrongyliasis, gnathostomiasis and (1982). Diseases caused by helminths. . A review. In : SEAMEO­ In: Manson's Tropical Diseases. p. 148. TROPMED Technical Meeting : Dia­ Bailliere Tindall, London. gnostic Methods for Important Hel­ MoRISITA, T., KoBAYASHI, M., NAGASE, K., minthiasis and Amoebiasis in Southeast NISHIDA, Y., IWANAGA, H. and SUMI, M., and the Far-East. p. 109. Tokyo 5-8 (1969). Non-specificity of intradermal February 1974. SEAMEO-TROPMED, test with Gnathostoma antigen. Jap. J. Bangkok. Parasit., 18 : 120.

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PuNYAGUPTA, S., BuNNAG, T., JUTTIJUDATA, fication of antigen from adult Clonorchis P. and RosEN, L., (1970). Eosinophilic sinensis for complement fixation and in Thailand. Epidemiologic precipitin test. Exp. Parasit., 17 : 340. studies of 484 typical cases and the etio­ SIRIKULCHAYANONTA, V. and CHONGCHIT­ logic role of Angiostrongylus cantonensis. NANT, N., (1979). Gnathostomiasis, a Amer. J. Trop. Med. Hyg., 19 : 950. possible etiologic agent of eosinophilic PuNYAGUPTA, JUTTIJUDATA, P., Bl1NNAG, s., granuloma of the gastointestinal tract. T. and CoMER, D.S., (1968). Two fatal Amer. J. Trop. Med. Hyg., 28 : 42. cases of eosinophilic myeloencephalitis, a newly recognized disease caused by SwANSON, V.L., (1971). Gnathostomiasis. Gnathostoma spinigerum. Trans. Roy. In: Pathology of Protozoal and Helmin­ Soc. Trop. Med. Hyg., 62:801. thic Diseases with Clinical Correlation. PuNYAGUPTA, S. and PACHECO, G., (1961). Mercial-Rojas, R.A. (ed.) p. 871. Wil­ Serological studies on experimental gna­ liams & Wilkins. Baltimore. thostomiasis. Amer. J. Trop. Med. Hyg., TADA, I., KAWASHIMA, K., NISIDMURA, K. 10 : 515. and MIYAHARA, M., (1966). Intradermal SAWADA, T., TAKEI, K., WILLIAMS, J.E. and reactions with Gnathostoma nipponicum MoosE, J.W., (1965). Isolation and puri- antigen. Jap. J. Parasit., 15 : 196.

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