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Specific IgE Antibody Responses to Somatic and Excre-tory-Secretory Antigens of Third Stage G. spinigerumLarvae in Human Gnathostomiasis

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WlLAI 8AKSIRISAMPANT, M.Sc.*,KANYARAT KRAIVICRAIN,M.D.*,

RUNGLAWAN CRA WENGKIA TTIKUL, M.Ed. **,SURANG NUCHPRA YOON, M.D., Ph.D. *

<,

Abstract

Specific IgE antibody levels in the serum of patients with proven gnathostomiasis and inthose with intermittent cutaneous migratory swelling (CMS) were determined by the enzyme-linkedimmunosorbent assay (ELlSA) using somatic extract and excretory-secretory (ES) products ofGnathostoma spinigerum infective larvae as antigens. The third stage larval used were obtainedfrom naturally infected eels. There was an increase in specific IgE antibody to both antigens inthese patients. The mean levels of these specific IgE antibodies were significantly higher than thatof the healthy control (P<O.OI). Comparison between using somatic extract and ES products inthe test showed, a positive result in the group of suspected patients with gnathostomiasis or CMSwas significantly higher when using ES products (81.81%) than somatic extract (59.09%) as theantigens (P<0.05). However, both somatic and ES antigens cross-reacted with other parasitic sera.The overall sensitivity of the ELISA for these IgE antibodies detection were 71.87 per cent and87.50 per cent with somatic and ES antigens, respectively. The specificity was 57.53 per centwhen somatic antigen was used and increased to 69.86 per cent when ES antigen was used. Thepositive and negative predictive values of the test were 42.59 per cent and 82.35 per cent by usingsomatic antigen. Both of these values, were also increased to 56.00 per cent and 92.72 per centby using the ES antigen. It is obvious that more potential components may be present in ES pro-ducts than those in the somatic extract. The ES antigen may have to be further purified and may besuitable for evaluation of the effectiveness of chemotherapy. As such, the antibody responses tosecreted products are more closely related to active infection than the anti-whole worm antibodythat may persist following the death of the parasites. However, in this disease, the effect of theIgE antibody on its pathophysiology it is still not known.

Key word: IgE Antibody, Gnathostoma spinigerum, SomaticAntigen,_Excretory-SecretoryAntigen

SAKSIRISAMPANT W, CRAWENGKIATTIKUL R,KRAIVICHAIN K, NUCHPRA YOON SJ Med Assoc Thai 2001; 84 (Suppll): 8173-S181

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~, . Depanment of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330,..Depanment of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.

8174 W. SAKSIRlSAMPANT et aL

For immunological studies, gnathostomia-sis has received less attention in antibodies studies

than other parasitic infections, for example; trichi-nellosis, toxocariasis and anisakiasis etdl,2). Manynematode infections elicit pronounced antibody res-ponses, involving all major isotypes(3,4). An in-crease in parasite-specific IgE antibody is a parti-cular feature of those having a tissue migration(3,5).

Gnathostoma spinigerum is a nematode indogs and cats but can cause severe and even fatalinfection in humans(6-8). Man is an accidental hostand acquires the infection by eating inadequatelyprocessed intermediate hosts, e.g. fresh water fish,frogs, and snakes which contain encapsulated thirdstage larvae (L3s). Following ingestion, the parasitemay migrate to any tissue of the body. The mostcommon outcome of the disease in humans is inter-

mittent cutaneous migratory swelling(9). The para-site migration within the central nervous system oreye can result in intracranial haemorrhage, eosino-philic meningoencephalitis, or blindness(7-9). Thepatients can die if they are not properly diagnosedand managed. The disease is fairly common in Thai-land and has also been reported from Japan, China,Malaysia, Indonesia,the Philippines,Israel, and manyother Pacific Islands(10-12).

The clinical symptom of intermittent cuta-neous migratory swelling may persist for manyyears. Diagnosis is currently based on a number ofclinical criteria(9,13). However, a few other parasi-tic infections may also cause migratory swelling thatcannot be readily distinguished from that caused byG. spinigerum(14,15). The host-parasite relation-ship in human gnathostomiasis, especially immune-responses, is not completely understood and thedata of serological immunoassay have shown limitedpotential use. Many studies have demonstrated anincrease in antibody titer that can, however, be usedas evidence of recent infection and also of a stronglikelihood of existing infection. This might be usedto support a clinical diagnosis of gnathostomiasis(16-24). However, like the immunodiagnosis ofother parasitic infections, the specificity of the testsleave much to be improved. This is due to the anti-gen used of crude somatic aqueous extract(25).

Generally, the most common source ofnematode immunodiagnostic antigen is the wholeworm extract, but the glandular secre,tions (excre-tory-secretory products; ES) are cufrently being

J M<d A- Thai Joo< ",.1

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considered. The development of suitable in virrJcultivation techniques for nematodes is essential f~the practical preparation of acceptably pure anti!gens. In this regard, the cultivation system has bee~~,currently developed in many nematode serologf'~(27), It was desirable to use synthetic culture mediawith unsupplemented by host factors or other un:

defined components. Furthermore, it was necess~to simplify antigen purification as well as to per.mit antigen adsorption to immunoassay solid-phaseswithout the competitive effects of other macro-molecules. Characterization of nematode secretedproducts suggests that there are usually simple mix.tures of relatively few components compared withthe multiplicity of antigens present in crude wholeworm preparation(26). We have previously shownthat G. spinigerum L3s somatic extract comprisedmore than 29 polypeptides in polyacrylamide gel'electrophoresis (SDS-PAGE)(28). By immunoblot.!ting analysis, four components which are strongly'recognized by IgE antisera of gnathostoma patientshave been identified. Of these, one antigenic bandgives a consistent reaction with gnathostoma p2antisera. The specificity of this protein band is 99'per cent. The other protein bands are cross-reacted,with IgE antibody from those with other intestinalparasitic infections. In addition, the ES productsof this G. spinigerum L3s have at least 4 bands'in SDS-PAGE. A few protein bands show crossreaction with other intestinal parasitic infection, ~whereas, one band is specific with IgE antisera of ,ttthis infection (unpublished observations). The pre-vious studies have demonstrated that among the ~

components released by the worm, major immu-nogens are present in ES material(26). Infections ~

with helminth and some arthropod parasites arecharacterized by elevated IgE antibody responses.

iI

particularly when mucous membrane or skin are in-volved at some stage of the parasite life cycle(3.5, tJ29). This associationhas made scientistsstudythe ~.'"parasite allergensand detect the specific 19B-Ab,"responses. For the parasite allergen, much atten-'tion has been focused on ES products(3,27,30,3l).These antigens are more likely to be specific thanthe crude somatic or have a high potential for diag-nositic evaluation especially for 19B-Ab detection(26,30). On the other hand, some recent evidencehas indicated that the parasite ES is also highlycross-reactive with other parasites(30-33). Thesecontroversies leave much to be proved in gnathos-

IgE ANTmODY TO SOMATIC AND ES IN HUMAN GNATHOSTOMIASIS

iasis. The data of using ES products for IgEtibody detection of human gnathostomiasis have

been reported.The aim of this study was to detect the IgE

, 8fltibodYin gnathostomiasis patients compared with

., .patients with other parasitic infections by usingcrude somatic extract and ES products of G. spini-

gtrum L3s. The informationof IgE antibodyres-ponsemay help to understand the humoral immuneresponses. thus leading to improved monitoring ofcbeserodiagnosis and management of the patients.

MATERIAL AND METHODSpecimens

Sera were obtained from patients attend-~ "'iDgthe outpatient parasitology clinic of King Chula-.~ Iongkorn Memorial Hospital, Bangkok; Thailand." Stool examinations for intestinal parasitic infections

andcomplete blood count (CBC) were routinely per-Connedin patients who had intermittent cutaneousmigratoryswelling (CMS) consistent with infectionby G. spinigerum. Twenty two patients diagnosedwith gnathostomiasis who had no intestinal para-sites and had positive anti G. spinigerum L3s IgG-antibody by Western blot analysis, were recruitedfor this study. Ten patients with parasitologicaIly

1.0

0.9

0.8

0.7

- 0.6g~ 0.5~"~ 0.4€0

~ 0.3

0.2

0.1

0CMS(22)

Pv(10)

Ss(13)

C(30)

Qv(25)

Fig.1.

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proven gnathostoma larvae recovered were alsorecruited. Healthy adults with no history of gnathos-tomiasis and negative for intestinal parasite servedas the healthy controls. There were 73 additionalpatients with other intestinal parasites also availablefor the study.

Antigens preparation crude somatic aqueousextract

Third stage larvae of G. spinigerum wereobtained by liver digestion of infected eels purchasedfrom Klong-Toey market. The livers were pooledand digested at 3i C with 1.5 per cent pepsin pH2.0 for 4 hours. G. spinigerum L3s were indivi-dually picked and separated from the digested livertissue under a dissecting microscope. Crude somaticaqueous extract of G. spinigerum L3s was preparedby homogenization in a ground-glass tissue grinderfollowed by sonication as described previously(28).The protein concentration of this L3s somatic anti-gen was determined by Lowry's method(34).

Excretory-secretory products preparationThe third stage G. spinigerum larvae ES

antigen was prepared from spent culture medium(BME, Gibco, USA) containing 100 u/ml penicillin

X+ 2 SD.

Tt(7)

Hw(7)

AI(3)

Ft(6)

GI

(12)

Optical density value (mean :t: standard error oCthe mean) oCthe ELISA reaction Cor specific IgEantibody to somatic extract (0) and ES product 03) in patient with intermittent cutaneous migratorysweUing (CMS), in patient with proven G. spinigerum inCection (Pv), compared to those of healthycontrol (C), Opisthrochis viverrini infection (Ov), strongyloides stercolaris infection (Ss), Trichuristrichiura infection (Tt), Hookworm infection (Hw), Ascaris lumbricoides infection (AI), Filariasis(FI) and Giardiasis (GI). The mean+ 2 standard deviation value Cor the normal control group( ).

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8176 W. SAKSlRISAMPANT et aL J Med Assoc ThaiJuo< ,... ,

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Table 1. Comparison of ELISA value of using G. spinigerumthird stage larva somatic extractand excretory-secretory(ES) products as antigens in gnathostomiasisand other para.sitic infected patients.

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Patients Group Number Absorbance density (492 nm)tested Somatic Ag ES Ag

Range Mean:t SE Range Mean:t: SE - 'I

Cutaneous migratory swelling 22 0.059 - 1.586 0.386 :t 0.082 0.1oo - 1.435 0.464 :t 0.099 ~Proven gnathostomiasis 10 0.316 - 1.116 0.630 :t 0.097 0.132 - 0.664 0.378 :t 0.05Healthy control 30 0.061-0.092 0.074:t0.ool 0.071-0.095 0.079:t0.013Opisthorchiasis 25 0.094 - 1.354 0.456:t O.loo 0.085 - 0.907 0.272:t 0.052Strongyl9idiasis 13 0.076 - 1.869 0.637 :t 0.167 0.090 - 1.236 0.395 :t 0.098Trichuriasis 7 0.121-0.322 0.211:t0.033 0.082-0.132 0.1O9:t0.oo8Paragonimiasis 7 0.109-0.304 0.165:t0.030 0.091-0.115 0.097:t0.oo3Ascariasis 3 0.105 - 0.1 12 0.122:t 0.013 0.093 - 0.1 17 0.350:t 0.245Filariasis 6 0.109-0.933 0.381:t0.130 0.113-0.982 0.370:t0.135Giardiasis 12 0.071-0.310 0.140:t0.oo5 0.086-0.338 O.l29:t0.oo5

Table 2~l

The number positive cases for anti Gnathostoma spinigerum thirdstage larva IgE antibody level in gnathostomiasis and other para-sitic infected patients, using somatic extract and excretory-secre-tory (ES) products as antigens.

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Patient groups

Cutaneous migratory swelling (CMS)Proven gnathostomiasisHealthy controlOpisthorchiasisStrongyloidiasisTrichuriasis

ParagonimiasisAscariasisFilariasisGiardiasis !i\!I

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G and 100 uglml streptomycin in which the wonnhad been maintained for 24 hours at 37"C under 5

per cent CO2 in air as described previously(35).After incubation, the wonns were removed and thecollected spent medium was centrifuged at 10,000g for 30 minutes at 4°c. The medium was concen-trated by ultrafiltration using Amicon UM-2 mem-brane filter (Grace CO, USA). The concentratedspent medium was then dialyzed against distilledwater containing proteinase inhibitors which were0.1 mM phenyl-methylsulphonyl fluoride, 0.1 mMtosylamide -2- phenylethyl-chloromethyl ketone, 1urn N-(N-L-3 trans carboxyoxiran-2-carbonyl-L-leucyl) - agmatine. The protein concentration wasdetennined by Lowry's method(34).

~:i<i.

IgE antibody determination ~Specific IgE antibody to G. spinigerumL3s .It'

antigen was detennined by enzyme-linked immu- '1.:1

nosorbent assay (ELISA). Wells of the microtitertplates (Immulon 11 ; Dynatech Laboratories, Inc., .~Alexandria, Va.) were coated with 50 III of 5 Ilg! .mI Ag. After incubation overnight at 4°C, the plates Jjwere washed with ELISA buffer. Serum (50 Ill) I]with dilution of 1:5 was each added and allowed t01react with the antigenat 37"C for 1 hour. Horsera-f .~

dish peroxidase-conjugated rabbit anti-human IgE .~(50 III ; Dakopatts NS, Copenhagen, Denmark) was Jjthen added. All conditions of conjugate dilution and '!IgE antibody titres for each specimen were opti. jmized and calculated using the end point criteria '\:as described by Soesatyo et al(24).

tJ

Number Number positivetesled Somalic Ag ESAg

% %

22 13 59.09 18 81.8110 10 100 10 10030 0 0 0 025 II 44.00 10 40.0013 II 84.61 8 61.537 4 57.14 0 07 I 14.28 0 03 0 0 I 33.336 3 50.00 3 50.00

12 2 16.66 I 8.33

/

Suppl 1 IgE ANTIBODY TO SOMATIC AND ES IN HUMAN GNATHOSTOMIASIS S177

Sensitivity and specificity of the ELlSA for specific anti-G. spinigerum L3s IgE antibody detectionin patients with gnathostomiasis and other parasitic infected patients, using crude somatic extractand excretory-secretory (ES) products as antigens (Ag).

Results GnathostOlmasis Other parasitic Sensitivity Specificity Predictive valueSuspected Proven patients % % Positive Negative

% %-SomaticAg + 13 10 31 (42.46%) 71.87 57.53 42.59 82.35

9 0 42Total 22 10 73

ESAg + 18 10 22(30.13%) 87.50 69.86 56.00 92.724 0 51

Total 22 10 73

RESULTS, ELISA values of anti G.spinigerum IgEantibodyusing 2 different antigens are presented inFig. I and Tables 1-3. The levels of IgE antibodyto gnathostoma antigens both somatic and ES pro-ducts in 10 patients with parasitologically provengnathostomiasis(Pv) were significantly higher thanthe background levels in the healthy controls (P<O.ol, Fig. I). The mean ELISA values of these IgEantibody were also significantly higher in sera ofsuspected gnathostomiasis patients than in healthycontrols(P<O.OI).All of these suspectedpatients hadbeen clinically diagnosed with cutaneous migratoryswelling(CMS), IgG antibody positive to G. spini-geruminfectionby Westernblot analysisand hada history of consuming improperly cooked fish. TheELISA values (optical density OD reading at 492nm of a 1:5 serum dilution) of each group in thisStudywere calculated by using Duncan' s multiplerange test. The IgE antibody levels for each speci-men were calculated using the end point criteria asdescribed by Soesatyo et al(24). The sensitivity ofthe test using somatic antigen reduced from 100 percent to 71.87 per cent when the suspected cases or

'" CMS group were included (Table 3). By using ESantigen, the sensitivity was also reduced from 100percent to 87.50 per cent when the CMS group wasincluded.

One .hundred per cent negative and posi-" live results were observed in the group of healthy~ COntrolsand a group of proven cases (Table 2). The

Dumberof positive results in suspected patients or~ CMs group was not very high. These were 59.09.. per cent and 81.81 per cent by using somatic extract!'~;~d ES products (Table 2). However, when com-

paring somatic and ES antigens, the number of

~

positives in the CMS group tended to be higherwhen ES antigen was used. This was statisticallysignificantly different (P<O.O5).In the group withother parasitoses, cross-reactivity was detected. Thepositive rate in each of these infections ranged from0 per cent to 84.61 per cent (Table 2). The highestcross-reactive was found in strongyloidiasis. Soma-tic antigen showed a higher cross-reaction (42.46%)than the ES antigen (30.13%) but it was not signifi-cantly different (Table 3). Interestingly, the cross-reactive could not be found in trichuriasis and para-gonimiasis when the ES antigen was used. For thespecificity of the test, it was 100 per cent in thegroup of proven cases but decreased when the sus-pected cases were included. These showed 57.53per cent and 69.86 per cent for somatic and ESantigen, respectively.Combining the data from para-sitoses and healthy controls, the positive and nega-tive predictive values were 42.59 per cent and 82.35per cent. Both of these values of the test also tendedto increase when ES products were used (56.00%,92.72%, Table 3) but were not statistically signifi-cantly different.

DISCUSSION

Our results have clearly demonstrated thatan infecdan caused by G. spinigerum could stimu-late specific IgE antibody. Such antibody produc-tion in man and animals has been noted in manyother helminth infections(35,36). Nevertheless, theantibodies stimulation by this small migrating tissuelarva have also been demonstrated in other iso-types including IgG antibody(l6-23) and all the IgGsubclasses (unpublished observations).

The worms which were surgically obtainedor spontaneously emerged from the patients were

S178 W. SAKSIRlSAMPANT et at

those of third-stage larvae or immature adults{12,37). The antigens used for the immunological testshould be prepared from the stage associated withthe disease. As such, this was the rationale in theselection of third-stage larvae for antigen preparationin the present study. Generally, the most commonsource of antigen is the whole worm extract. How-ever, whole worm antigens have only.a few compo-nents of functional antigens and hence, are poten-tially poor sources of specific antigens. For humangnathostomiasis, a number of recent reviews havedemonstrated that the antigen of G. spinigerumsomatic third stage larva extract has a high degreeof cross reaction with other parasitic diseases in-cluding angiostrongyliasis( 16,21). opisthorchiasis(20), paragonimiasis, taeniasis, and hook-worm in-fection(l5.22). In the present study, attention hasturned to the use of antigen preparations which con-tain a more restricted range of antigen epitopes,particularly those obtained from parasite ES pro-ducts. The ES products are usually simple mixturesof relatively few antigenic mosaic compared with themultiplicity of those present in crude somatic extract(18,36,38). The previous report of Morakote et alshowed that the ELISA values of anti-gnathostomaIgG-antibody had no significant difference for diag-nosis of gnathostomiasis when comparing larvalsomatic with adult ES antigens(22). Unfortunately.data of the third stage larva ES products were notreported, especially the anti-IgE antibody to this ESproducts.

In the present study, the levels of IgE-anti-body to somatic and ES antigens in both group ofCMS and the parasitologically proven group weresignificantly higher than those in the healthy con-trol group (P<O.OI).Comparing the use of somaticextract and ES products in the test, the positive ratesin the group of suspected patients with gnathosto-miasis or CMS were significantly higher using ESproducts (81.81%) as the antigens when comparedwith somatic extract (59.09%) (P<0.05, Table 3).The potential components may be present !n greaterproportion in ES products of the larva than those insomatic extract when the protein concentration isequal. However, the test was 100 per cent positivein the parasitologically proven group when bothkinds of antigens were used. When approachingimmunodiagnosis, most diseases are moving awayfrom techniques based on precipitation and aggluti-nation to the assay with, in general, higher sensiti-

J Med AssocThaiJune 20011

!{

vity, such as ELISA. The sensitivity of our data was'

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reduced from 100 per cent to the level of 71.87percent and 87.50 per cent by using somatic extract'and ES products, respectively, when the suspected.group was included. How to make an improvement '.)may be not due to technical advances, but is more.

likely to come from selection of a suitable target'antigen. Work is now in progress to identify and'purify this antigen and to assess its value for diag.'nosis. By Western blot analysis, the specific corn.

ponent of G. spinigerum L3s against IgE antibodyis the peptide with a molecular weight of 43 kDa(unpublished observations). The cross-reactivity orthe suboptimalspecificity is primarily a consequenceof sharing of antigenic epitopes among nematode

.1

,.

1

parasites (36,40,4 1). '.The diagnosis of suspected or CMS group

in the present study was currently based on clini. ~

cal manifestations,eosinophilia,positive IgG anti.body to 24 kDa peptide in Western blot analysisand a historyof consumingimproperlycookedfish ~

or other intermediate hosts. The specificity of the"test was also reduced when the ELISA value of thisgroup was included. These low levels of IgE anti-body in the sera may be due to T cell immuno-regulatory events. In addition. the IgE antibody canbind to the IgE receptors' on the mast cell surfacein the circulation and lose reaction with parasiteantigen in the in vitro system. However, the reasonfor higher IgE antibody level in proven cases than 1;1

that of suspected cases is still not known. The :cti!

mechanism by which parasites exert potent stimu. '1f

latory effect on the IgE antibody system in manytnematode infections is under investigation in severallaboratories(29,36.39,41). It is possible that in pro.ven gnathostomiasis, the larva has to come up and ,~actively migrate in the cutaneous tissue and. then. in "'the skin for a period of time before immediately ¥emerging from the skin(32). During the emerging .it1phase, the higher amount of exo-enzyme or allergen .~may have to be released. The principle of cutaneous ~or skin involvement is characterized by elevati!,,:g:fJthe reaginic (IgE) antibody, particulary to a higher'"level compared to the deep tissue phase or the non. .~

emerging phase(3,5,29,36,39). Even the ES anti.gen produced some cross reaction in the ELlSA ~

of the present study, it can be used for furtherstudy in evaluation of the effectiveness of chemo-therapy. Since the antibody responses to nematode.secreted products are more closely related to active m

~

'",,14 Suppl 1IgE ANTIBODY TO SOMATIC AND ES IN HUMAN GNATHOSTOMIASIS S179

J!iJDf.ecUonthan the anti-whole WOoDantibody res-[f";ponseswhich mayper~ist or even increase follow-binS death of the parasltes( 41). These ES products

fiDlybe a suitable target antigen for identification_d purification of the potential component(s). How-~ver, there is no infonnation regarding the role ofIhcse specific IgE antibodies on the pathophysio-

logy of this disease.

ACKNOWLEDGEMENTThe authors wish to thank the following for

their assistance: Yenthakam Sutin for the sera pre-parations, ChotikasoponOrapin and Saiim Kulwadeefor typing the manuscripts. This work was supportedby RajadapiseakSompoj China Medical Board grant,Faculty of Medicine, Chulalongkorn University,Bangkok 10330, Thailand.

/(Received for publication on March 21,2001)

REFERENCESI. Loukas A, OpdebeeckJ, Croese J, Prociv P. Immu-

nologic incriminationof Ancylostomacani-num asa humanenteric pathogen. Am J Trop MedHyg, 1994;50: 69-77.Radennecker M, Bekhti A, Poncelet E, SalmonJ. Serum IgE levels in protozoal and helminthicinfection. Inter Arch Allergy Appl Immunol 1974;47: 285-95.Wakelin D, Hamett W, Michocl R, Parkhouse E.Nematodes. In: Kenneth SW, eds. Immunologyand molecular biology of parasitic infection. 3rded.Massachusetts: Blackwell, 1993:517-8.Korenaga M, Nawa y, Tada I. IgE response inStrongyloides ratti-infected rats with special refe-rence to the life cycle of the parasite. ZeitschrParasitenkunde 1986;72: 213-20.O'Donnell U, Mitchell GF. An investigation ofthe antigens of Ascaris lumbricoides using aradioim'munoassay radioimmunoassay and seraof naturally infected humans. Int Arch AllergAppl Immunol1980; 61: 213-7.Suntharasamai P. Gnathostomiasis. In: WeatherallDJ, Ledingham J G G, Warrell DA, eds. OxfordText book of Medicine (vol. I) Oxford: OxfordUniversity Press 1987;5: 558-61.Miyazaki L. On the genus Gnathostoma andhuman gnathostomiasis with special reference toJapan. Exp Parasitol1960; 9: 338-70.Chitanondh H, Rosen L. Fatal eosinophilicencephalomyelitis aaused by the mematodegnathostoma spinigerum. Am J Trop Med Hyg1967; 16:638-45. .Daengsvang S. Gnathostoma spinigerum andhuman gnathostomiasis. A review. In: SucharitS, eds. The 25th Anniversary of the Faculty ofTropical Medicine, Mahidol University. Bangkok:Krung Siam Press, 1986: 124-47.Rojekittikhum W, Pubampen S, HiranyachattadaP, Chiamratana B, Setasuban P. A survery on theinfective larvae of Gnathostoma spinigerum in

-~

2.

-"

3.

4.

5.

6.

7.

8.

9.

10.

..

11.

fresh water fish sold in the local markets of

Bangkok. J Trop Med Parasitol 1989; 12:7-12.Ando K, Tanaka H, Taniguchi Y, Shimizu M,Kundo K. Two human cases of gnathostomiasisand discovery of a second intennediate host ofGnathostoma nipponicum in Japan. J Parasitol1988; 74: 623-7.Akahane H, Lamothe-Argumedo R, Martinez-Cruz JM, Osorio-Sarabia D, Garcia-Prient L. Amorphological observation of the advanced third-stage larvae of Mexican Gnathostoma. Jpn J Para-sito11994; 43: 18-22.Chanthchume N, Kulkhumthom M, JongwutiwesS. Evaluation of skin test for human gnathosto-miasis. Chula Med J 1998;32: 243-8.Sirisinha S, Techasoponmani R, Dharrnkrong-atA, Uahkowithchai V. Immunology of Angio-strongylus infection. J Sci Soc Thai 1977; 3: 157-74.Welch JS, Dobson C, Campbell GR.lmmuno-diag-nosis and seroepidemiology of Angio- strongyluscantonensiszoonoses in man.Trans R SocTrop MedHyg 1980;74: 614-23.Suntharasamai P, Desakorn V, Migasena S.Bunnag D, Harinasuta T. ELISA for immuno-diagnosis of human gnathostomiasis. SoutheastAsian J Trop Med Public Health 1985;2: 274-9.Dharmkrong-AT A, Migasena S, SuntharasamaiP, Bunnag D, Priwan R, Sirisinha S. Enzyme-linked immunosorbent assay for detection of anti-body to gnathostoma antigen in patients withintermittent cutaneous migratory swelling. JClinical Microbiol1986; 23: 847-51Maleewong W, Morakote N, Thamasonthi W,Charuchinda K, Tesana S, Khamboonruang C.Serodiagnosis of human gnathostomiasis. South-east Asian J Trop Med Public Health 1988; 19:201-5.Nopparatana C, Tapchaisri P, Setasubun P,Chaicumpa W, Dekumyoy P. Antibody responses

12.

13.

14.

15.

16.

17.

18.

19.

8180 W. SAKSIRISAMPANT et at J Med Assoc Thai June 2001 I

humans: comparison with an allergen formiceiden-tified by a passive cutaneous anaphylaxis test. AustiI Bioi Sci 1978;31: 459-67. '.Kennedy MW, Tierney I, Ye P, et al. The secreted]and somatic antigens of the third stage larva or!Anisakis simplex, and antigenic relationship withAscaris suum, Ascaris lumbricoides and Toxo-cara canis. Mol Biochem Parasitol 1988; 31: 35.46.

Kennedy MW, Qureshi F, Haswell-Elkins M,Elkins DB. Homology and heterology between thesecreted antigens of the parasitic larval stages orAscaris lumbricoides and Ascaris suum. Clin ExpImmunol1987; 67: 20-30.Lowry OH, Rousebrough NI, Farr AL, RandallRI. Protein measurement with folin phenol rea-gent. I Bioi Chem 1951; 193: 265-9.Rattanasiriwilai W, Chanthachume K, Kulkum-thorn M, Yingyourd P. Studies on production ofexcretory-secretory products from 3rdstage larvaeof Gnathostoma spinigerum by an in vitro culti-vation. ChulaMedI 1985;29: 1115-20.Pritchard DI. Antigens of gastrointestinal nema-todes. Trans R Soc Trop Med Hyg 1986; 80: 728.34.

Daengsvang S. Gnathostoma spinigerum andhuman gnathostomiasis: A brief review. In : The25thAniversary of the Faculty of Tropical Medi.cine, Mahidol University. S. Sucharit et al eds.Bangkok: Krung Siam Press, 1986; 124-47.Hogarth-Scott RS. Visceral larva migrans-animmunofluorescent examination of rabbit andhuman sera for antibodies to the ES antigen of thesecond stage larvae of Toxocara canis, Toxocarecati and Toxocaris leonina. Immunology 1966; 10:217-23.

Ganguly NK, Mahajan RC, Sehgai R, Shetty P,Dilawart 18. Role of specific immunoglobulin Eto excretory-secretory antigen in diagnosis andprognosis of hookworm infections. I Clin Micro-bioi 1988; 26: 739-42.Almond NM, Parkhouse RME, Chapa-Ruiz MR,Garcia-Ortigoza E. The response of humans to sur.face and secreted antigens of Trichinella spiralis.Trop Med Parasitol1986; 37: 381-4.Almond NM, Parkhouse RME. Nematode anti-gens. CUrTTop Microbiol Immune! 1985; 120:173-03.

in human gnathostomiasis. Southeast Asian ITrop Med Public Health 1988; 19:219-24.

20. Malinee TA. ELISA for diagnosis of gnathosto-rniasis using antigens from gnathostoma doloresi 32.

and G. spinigerum. Southeast Asian I Trop MedPublic Health 1989;20: 297-04.

21. Tuntipopipat S, Chawengkiattikul R, Witoon-panich R, Chiemchanya S, Sirisinha S. Antigens,antibodies and-immune complexs in cerebros-pinal fluid of patients with cerebral gnathosto- 33.

miasis. Southeast Asian I Trop Med Public Health1989; 20: 439-46.

22. Tapchaisri P, Nopparatana C, Chaicumpa W,Setasuban P. Specific antigen of gnathosomaspinigerum for immunodiagnosis of human 34.gnathostomiasis. Inter I Parasitol 1991;21: 315-9.

23. Morakote N, Navacharoen W, Maleewong W,Sukhavat K. Comparison of larval and adult 35.worm antigens in ELISA for serodiagnosis ofhuman gnathostomiasis.ITrop MedParasitol1991;14: 9-13.

24. Soesatyo MHNE, Rattanasiriwilai W, Suntha-rasamai P, Sirisinha S. IgE responses in human 36.

gnathostomiasis. Trans R Soc..Trop Med Hyg1987; 81: 799-01.

25. Parkhouse RME, Harrison US. Antigens of para- 37.

sitic helminths in diagnosis, protection and patho-10gy.Parasitol1989; 99: S5-S19.

26. Lightowlers MW, Richard MD. Excretory-secre-tory products of helminth parasites: effect onhost immune responses. Parasitology 1988; 96: 38.SI23-S166.

27. Stromberg BE, Soulsby ElL. Ascaris suum: immu-nization with soluble antigens in the guinea pig. IntI Parasitol1977; 7: 287-92.

28. Saksirisampant W, Bunmanavong K, KraivichianP. Gnathostoma spinigerum antigen: An analysis 39.of protein profiles. Chula Med I 1986;30: 739-42.

29. Katz DH. Recent studies on the regulation ofIgE antibody synthesis in experimental animalsand man. Immunology 1980;41: 1-24.

30. Grencis RK, Crawford C, Pritchard 01, Behnke 40.IM, Wakelin D. Immunization of mice with sur-face antigens from the muscle larvae of Trichi-nella spiralis. Parasite Immunol1986; 8: 587-96.

31. 0' Donnell 11, Mitchell GF. An investigation of 41.the allergens of Ascaris using a radioallergosor-bent test (RAST) and sera of naturally infected

Suppl 1 IgE ANTIBODY TO SOMATIC AND ES IN HUMAN GNATHOSTOMIASIS S181

-

m7"DU«UDoQ~DoQ 1D~DLLDU;\UD;itfflDL'lfJ,n;\nLL~~DLDftLLDU;\L~U'nni'1£1Du- ....r ..I t ".'1T - ....r

~tJ1D"1~fl7~tJ~'Y1tf1~U~1J1tJ~7f1~tJ1D"1~fIIII

/ 51~ f"fniFT1ffmfutI, ?n~,., ioJ~lnJru L'If?oJLff!JJ#if}~,nf'T.N,H,... ... I' ... . ~ . 1'0/ ./.

nf]/f]/JVltJ nJ!J?L"1I!JJ, W.U., '[J1oJPI 2I'IfVJ::€jJ, W,u" VJ.VI.

'i!OAmn'j::~U IgE LLDU~UD~tI'iuci4';.J'lJD~r:!tbtJhf'1wmB~1~V11un~).jyj~wtnB~1~V11'JfDDn'i1nijlmr~ LLfl::n~;.J

..I.. tI..I ..I 1; 1"-'" 1 ... ..I1~

't'l).jD1m1 lV1 1J1).j Lf'1flDU't1'lJD~'JfUV1CJ1'f1U~(CMS) V1tJ1J:iELlSA 'Jf~ ODsomatic LLfl:: excretory-secretory (ES)

LUJU~L'iU'lJD~~ltJDUWtJ1B~1~V1 G, spinigerum 1::tJ::yj~1).j ~ltJDU1::tJ::yj~1).jdLV11tJ;.J~U'i1ntlfl11'f1flyj~L:fD1V1tJJ:i11;.J'Jf1~

WU11r:!tJltJ.,.t~~D~n~;.J~1::~U IgE LLDU~UDM~~n11n~;.Jf'1utln~Dri1~j1,rm~'lfi'C)!m~~ii~(P<O,01) L~DLtl1tJUL'YitJum1

HLLDU~L'iU.,.t~~D~'JfijV1dwU11 r:!tJltJ1un~).j CMS 1~CJfl1J1n (81.81 %) nu ES LLDU~L'iU<H~;.J1nn11 CJfl1J1n'lJD~ ELlSA

rnnm,H somatic LLuU~L'iU (59.09%) (P<0.05) Dri1~hnV11;.J.,.t~ somatic LLfl:: ESLLDU~L'iU LiiV1t1ijii1m-n1;.Jn~;.J

nuci4').j'lJu~n~;.Jr:!thtJyjLuu11f'1WtJ1B'JfijV1~U "1 L~D1Lf'111::\o1f'111;.J11(sensitivity) 'lJmm1't1V1~DUm IgE LLDu&iUDMd

WU11m1H somatic LLDU~L'iu1~sensitivity 71,87 LtI!rii.jiu~ LLfl::Lyj~LUU87.50 LtlD1i.jiu~ L~D1ofES LLDU&iL'iU

f'111).j~lLW1:: (specificity) j11'i1 57.53 LtlDfL-iiu~ L~DH somatic LLuU&iL'iULLfl::Lyj;.JLUU69,86 LtlDfL.jiU~ L~DH ES

LLDU~L'iU ,hul'hm1Y11U1tJCJfl1J1nLLfl::flU'lJD~m1't1V1~DUdLyhnu 42,59 LtlDfL-iiu~ LLfl:: 82.35 LtlDfL.jiU~ L~uH

somatic LLDU~L'iU LLfl::1'i1.,.t~"m~dLyj).j~ULUU56 LtlufLW~ LLfl:: 92.72 LtlufL.jiU~ L~u1of ES LLDU~L'~U LUUY!

N~LnV111 ES LLuu&iL'iUU1'i::~u~ritl'::nuuyj~AntJ111W).j1nn11 somatic LLuU&iL'iU<H~ ES LLuU~L'iudI'l11'i::~D~~lm

purity ~DLLfl::m'i LtJULLuU~L'iUyjL'I1).j1::~;.JN1'f11Um,tl1::Li)u tl1::&\'1B111W'lJu~m11n 1:11 .,.t~dLLDu&iUu~tfyjV1uU~U u~ ~D

~~I'IV1'f1t~'lJmWtJ1B U1'i::LRtJl'!Ju~ 1V1tJV11~num,&iV1L:fu).j1nn11LLDu&iUu~tfyj~~uWtJ1B.,.t~~1 LW11::LLuU&iUD~tf'JfijV1'f1~~d

D1'if'1~D~ LL~11WtJ1BLJn~1'f11uV11tJLLlfl Dri1~hnV11;.J CJfl'IJD~ IgE LLuu~uuMyj~~D pathophysiology 'lJD~hf'1dntY~1)jLuuyjL'!J11'inu

II1W1iqj : 1u~fl LLuU~UuM. wmB~l~V1. 1'Jfu.j~n LLu'\.j~L'i'\.j.flLu('{ LLuU~L'iU

151ft ftn~~mni\ld'. i~ml"tII L~~LnrJ'~jiQft,

nqjqji'",r ni'tl1Lftl1. &fTI~.r~,h""G1

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.. tnl'1i".,~~i1ii1tJ1. 1'1C1J:;ii1tJ1f'fl,,~i~"'lii1tJ1atJ~i1~1\ nl~Li1w '1 10400...

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