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Elisa for Immunodiagnosis of Human Gnathostomiasis

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Elisa for Immunodiagnosis of Human Gnathostomiasis ELISA FOR IMMUNODIAGNOSIS OF HUMAN GNATHOSTOMIASIS 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, Thailand. 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 infection firmed or parasitologic diagnosis is rare since with oral administration of second-stage the parasite is recovered from only a small larvae in infected cyclops. 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 skin, 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-encephalitis due various helminthic infection such as filariasis, to Angiostrongy/us cantonensis based on clini­ schistosomiasis, toxocariasis (Kagan, 1984), cal criteria (Punyagupta et a/., 1970) and a and angiostrongyliasis (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 infections 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 hookworm, mum N-concentration of antigen used was Strongyloides sterco/aris, Enterobius 1'ermi­ 5 f.lg(ml. cularis, Ascaris lumbricoides, 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 Gnathostoma! 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, Opisthorchis viverrini, lungfluke, Taenia and specificity of the ELISA employing crude spp., Trichinella spiralis, Fasciolopsis buski, extract of the third-stage larvae of G. spini­ Echinostoma spp., Wuchereria bancrofti, 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
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