the diagnosis of human hydatidosis by measurement of specific ige antibody by enzyme immunoassay

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Scand J Infect Dis 21: 213-218, 1989 The Diagnosis of Human Hydatidosis by Measurement of Specific IgE Antibody by Enzyme lmmunoassay ANDERS SJOLANDER,' JORGE A. GUISANTES,' JOSEP M. TORRES-RODRIGUEZ3 and HASSE SCHRODER' From 'Pharmacia Diagnostics AB, Uppsala, Sweden. the 'Department of Microbiology and Parasitology, University of Nauarra. Pamplona, and 'Hospital Ntra. Sra. del Mar, Barcelona, Spain The sensitivity and specificity of an enzyme immunosorbent assay (EIA) for measurement of anti-Echinococcus granulosus specific IgE antibody using microtiter strips was evaluated. The sensitivity was 92.2 % for 90 patients with cysts in the liver or other body sites but excluding the patients with cysts in the lungs or bones. In this latter group (n=26) a sensitivity of 61.5 % was recorded. The specificity for control groups comprising 89 blood bank sera from Spain and 48 sera from atopic Swedes was 100 %. Patients infected with other parasites (n=78) were usually negative (81 %). 11 of the 15 false positives were found in patients with total IgE SO00 kUh. The microtiter EIA method employed can be considered as a very convenient method to be included in the range of immunodiagnostic tests for human hydatid disease. H. Schroder, PhD, Pharmacia Diagnostics AB, S-75182 Uppsala, Sweden INTRODUCTION Human hydatid disease caused by Echinococcus granulosus is an important zoonosis spread in wide areas of the world (1). In many countries it is a considerable public health problem. Several techniques based on the measurement of antibodies of mainly IgG and/or IgM classes have shown their value for the diagnosis of the disease. The current status of the immunodiagnosis of hydatid disease has been reviewed by several authors in the past 10 years (2, 3, 4). Application of the enzyme-linked immunosorbent assay to human hydatidosis has further improved the sensitivity, economy, and convenience of serological diagnosis of the disease (5, 6). Elevated levels of serum IgE and IgE antibodies have been reported in various helminthic infections of man including human hydatidosis (7-1 1). In the present paper we describe an enzyme immunosorbent assay (EIA) for measure- ment of anti-E. granulosus specific IgE antibody. The sensitivity and specificity of the assay was evaluated. MATERIAL AND METHODS Hydafid antigen. Antigen was obtained from the hydatid fluid of fertile cysts in sheep. After sedimentation of protoscolices, membranes and larger particles the supernatant was decantated and centrifuged at 1000 g for 30 min. The resulting supernatant was dialysed against distilled water, lyophilized, standardized by immunoelectrophoresis analysis and stored in a dry place at f4"C before use (12). Patient sera. Sera from 116 Spanish patients (56 males and 60 females, aged 7-84 years, median 50) with surgically confirmed hydatid disease were tested. The localization of the cysts was recorded for each patient (Table I). Negative controls comprised 89 blood bank sera from Spain. 48 sera of atopic patients from Sweden and 78 sera of patients with other parasite infections. The latter group included cases with cysticercosis (n= 19), schistosomiasis (22). fascioliasis (lo), ascariasis (12). onchocerciasis (9). loiasis (3), malaria (2) and invasive amoebiasis (I). Scand J Infect Dis Downloaded from informahealthcare.com by Mcgill University on 11/04/14 For personal use only.

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Page 1: The Diagnosis of Human Hydatidosis by Measurement of Specific IgE Antibody by Enzyme Immunoassay

Scand J Infect Dis 21: 213-218, 1989

The Diagnosis of Human Hydatidosis by Measurement of Specific IgE Antibody by Enzyme lmmunoassay

ANDERS SJOLANDER,' JORGE A. GUISANTES,' JOSEP M. TORRES-RODRIGUEZ3 and HASSE SCHRODER' From 'Pharmacia Diagnostics A B , Uppsala, Sweden. the 'Department of Microbiology and Parasitology, University of Nauarra. Pamplona, and 'Hospital N t ra . Sra. del Mar , Barcelona, Spain

The sensitivity and specificity of an enzyme immunosorbent assay (EIA) for measurement of anti-Echinococcus granulosus specific IgE antibody using microtiter strips was evaluated. The sensitivity was 92.2 % for 90 patients with cysts in the liver or other body sites but excluding the patients with cysts in the lungs or bones. In this latter group (n=26) a sensitivity of 61.5 % was recorded. The specificity for control groups comprising 89 blood bank sera from Spain and 48 sera from atopic Swedes was 100 %. Patients infected with other parasites (n=78) were usually negative (81 %). 11 of the 15 false positives were found in patients with total IgE S O 0 0 kUh. The microtiter EIA method employed can be considered as a very convenient method to be included in the range of immunodiagnostic tests for human hydatid disease.

H . Schroder, PhD, Pharmacia Diagnostics AB, S-75182 Uppsala, Sweden

INTRODUCTION

Human hydatid disease caused by Echinococcus granulosus is an important zoonosis spread in wide areas of the world (1). In many countries it is a considerable public health problem. Several techniques based on the measurement of antibodies of mainly IgG and/or IgM classes have shown their value for the diagnosis of the disease. The current status of the immunodiagnosis of hydatid disease has been reviewed by several authors in the past 10 years (2, 3, 4).

Application of the enzyme-linked immunosorbent assay to human hydatidosis has further improved the sensitivity, economy, and convenience of serological diagnosis of the disease ( 5 , 6). Elevated levels of serum IgE and IgE antibodies have been reported in various helminthic infections of man including human hydatidosis (7-1 1).

In the present paper we describe an enzyme immunosorbent assay (EIA) for measure- ment of anti-E. granulosus specific IgE antibody. The sensitivity and specificity of the assay was evaluated.

MATERIAL AND METHODS Hydafid antigen. Antigen was obtained from the hydatid fluid of fertile cysts in sheep. After sedimentation of protoscolices, membranes and larger particles the supernatant was decantated and centrifuged at 1000 g for 30 min. The resulting supernatant was dialysed against distilled water, lyophilized, standardized by immunoelectrophoresis analysis and stored in a dry place at f 4 " C before use (12).

Patient sera. Sera from 116 Spanish patients (56 males and 60 females, aged 7-84 years, median 50) with surgically confirmed hydatid disease were tested. The localization of the cysts was recorded for each patient (Table I). Negative controls comprised 89 blood bank sera from Spain. 48 sera of atopic patients from Sweden and 78 sera of patients with other parasite infections. The latter group included cases with cysticercosis (n= 19), schistosomiasis (22). fascioliasis ( lo) , ascariasis (12). onchocerciasis (9). loiasis (3) , malaria (2) and invasive amoebiasis ( I ) .

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Page 2: The Diagnosis of Human Hydatidosis by Measurement of Specific IgE Antibody by Enzyme Immunoassay

214 A . Sjolander et al. Scand J Infect Dis 21 (1989)

A

2.0

1.5

3

:: 1.0

- -1 .e c P

0.5

0

4u

c83 0

0 0 0

0 3

0 w 0 0

0

Fig. 1. Relative concentrations of serum IgE anti- bodies to hydatid cyst fluid antigens determined by Pharmacia Echinococcus IgE EIA.

All sera were stored for varying lengths of time at -20°C until tested. Assay o f I g E antibodies. IgE antibody specific for hydatid cyst fluid antigens was assayed using

Pharmacia Echinococcus IgE EIA test kit (Pharmacia Diagnostics AB, Uppsala) based on strips of flat-bottomed polystyrene wells with covalently coupled antigen. The wells were washed once with phosphate-buffered saline, pH 7.2, 0.5% Tween 20, before addition of 50 ~1 of undiluted patient sera and incubation for 3 h at room temperature. After washing (3x 10 min) the wells were left overnight at room temperature with 50 p1 beta-galactosidase labelled anti-IgE, washed 3x10 min and then incubated with 100 p1 substrate (0-nitrophenyl-beta-d-galactoside) for 60 rnin at +37"C. The enzyme substrate reaction was stopped by addition of 100 p1 of 0.4 M Na2C03 and the resulting yellow colour was measured at 410 nm. Variations between assays were compensated for by inclusion of a standard curve made up from 4 dilutions of a positive serum pool in each run. The results were expressed as absorbance values. The cut-off for positive scores was calculated from the mean absorbance value obtained for the 89 Spanish blood donors plus 5 standard deviations.

Table I . Localization of hydatid cysts in the patients

Localization No. of pat.

Hepatic Hepaticlpulmonar y Multiple Othef Pulmonary Bone

Total

66 6 8

10 20 6

116

' Includes kidney, peritoneum, spleen, muscle and diaphragm.

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Page 3: The Diagnosis of Human Hydatidosis by Measurement of Specific IgE Antibody by Enzyme Immunoassay

Scand J Infect Dis 21 (1989) Specific IgE in human hydatidosis 215

Determinations of tofu1 IgE. Total IgE was measured using Phadezym IgE PRIST@ (Pharmacia Diagnostics AB, Uppsala). The test was performed according to the instructions from the manufactur- er.

RESULTS

The calculated cut-off for positive values was 0.040. The anti-E. granulosus IgE antibody levels were strongly influenced by the localization

of the cyst (Table 11). The sensitivity of the test was 92.2 % for the group with cysts in the liver or other body sites but excluding the patients with cysts in the lung or bones. In this latter group a sensitivity of 61 .S % was recorded. The distribution of the absorbance values obtained in the patient sera is shown in Fig. 1.

The specificity was 100% for the control groups comprising Spanish blood donors and Swedish atopic patients. Most of the absorbance values obtained for sera from the 78 patients infected with other parasites were negative (Table 111). In this control group a specificity of 81 % was recorded (Table 111). The false positives were mostly encountered among patients with schistosomiasis (6122) and onchocerciasis (519). 1 1/15 false positives were encountered among patients with total IgE >SO00 kU/l (Fig. 2). The remaining 3

Table 11. Sensitivity as a function of cyst localization

Positive1 Sensitivity" Localization total

92.2

90

Hepatic 61166 Hepatic/pulmonary 516 Multiple 818 Other 9110

Pulmonary Bone

13/20 316

Cut-off was calculated from mean absorbance value of 89 Spanish blood donors + 5 standard deviations. Fisher's exact test showed a statistically significant difference in proportions of patients above cut-off (JKO.05) between hepatic and pulmonary cysts.

Table 111. Results obtained with the Echinococcus IgE EIA in the 78 sera from putients with other parasitic diseases

Disease No. of No. of positive Specificity" pat. EIA (Z)

C ysticercosis Schistosomiasis Ascariasis Onchocerciasis Other parasitic

Total

diseases'

19 2 22 6 12 2 9 S

16 0

78 1s

89.5 73 83 44

100

81

" Cut-off was calculated from mean absorbance value of 89 Spanish blood donors + S standard deviations.

This group includes cases of fascioliasis, loiasis. malaria and invasive amoebiasis.

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Page 4: The Diagnosis of Human Hydatidosis by Measurement of Specific IgE Antibody by Enzyme Immunoassay

216 A. Sjolander et a / . Scand J Infect Dis 21 (1989)

2 A4 10

I 2 . 0 C

0 0 0

0 8

Total IgE

k 10000

I000

w M

r: 0 b

c - v

100

10

0000

000

00

10

Fig. 2 . Correlation between total serum IgE levels and relative concentrations of serum IgE antibodies to hydatid cyst fluid antigens in patients with other parasitic infections.

Fig. 3. Total serum IgE levels in patients with hydatid disease and in non-infected control subjects.

positive sera came from patients with cysticercosis (absorbancy 0.230), schistosomiasis (absorbancy 0.050 and 0.060) and ascariasis (absorbancy 0.170), respectively.

The total IgE levels of the patients ranged from below 10 kU/1 up to 10000 kU/1 (Fig. 3). No clear difference could be found between the patient groups with hepatic and pulmonary cysts, respectively. The Spanish blood donors had clearly lower levels of total IgE than Swedish atopic patients.

There appeared to be a dependence between hydatid antigen specific IgE antibody and total IgE but a linear model does not seem appropriate (Fig. 4).

DISCUSSION

The assay of IgE antibody used in this study proved to be a sensitive and specific method for the diagnosis of hydatid disease, thus confirming results reported by others using tests

2 Fig. 4 . Correlation between total se- rum IgE levels and relative concen- trations of serum IgE antibodies to hydatid fluid antigens in patients with hydatid disease.

.- - .- a Y -n -

0.1

Total IgE

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Page 5: The Diagnosis of Human Hydatidosis by Measurement of Specific IgE Antibody by Enzyme Immunoassay

Scand J Infect Dis 21 (1989) Specific IRE in human hydutidosis 217

with the antigen covalently coupled to cellulose discs or passively coated to microtiter wells (13-19).

By using hydatid antigen covalently coupled to microtiter wells we have combined the technical convenience of microtiter technique with the high binding capacity of covalent antigen coupling. Binding of high concentrations of antigen is a crucial criterion in IgE antibody determination in order to decrease the interference of competitive IgG antibod- ies. Covalent coupling of antigen also reduces the influence of the chemical composition of the antigen mixture (hydrophobicity, PI etc.), thus allowing a more uniform binding of the components in the antigen mixture. Furthermore, the strong covalent bond makes leakage of antigen less probable during storage of the microtiter plates.

The main objective of a diagnostic test should be to discriminate between infected and healthy subjects. In accordance with this general idea the cut-off absorbance value for this study was chosen to give negative results for the 89 healthy Spanish blood donors and the 48 atopic Swedes (100 % specificity) without sacrificing sensitivity. We found a sensitivity of 92% in patients with cysts in the liver or other body sites except the lung and bone. For disease in those organs 61.5% of the patients scored positive. Similar differences in immune response have been published earlier (2, 4). Our study shows that anti-E. granulosus IgE levels were strongly influenced by the location of the cysts.

Of even greater interest were the results obtained from subjects infected with other parasites. We found that most sera from patients suffering from cysticercosis were negative for IgE antibodies against hydatid cyst antigen (17119). This is surprising in view of the close taxonomic relationship between the 2 parasites as both are cestodes with tissue encystation as part of their life cycles. The antigens from the tissue forms of both parasites are at least partly similar and cross-reactivity between cysticercosis and hydati- dosis has been reported when methods for assay of IgG antibodies are used. The presence of specific IgE that reacts with hydatid antigen in cases of human cysticercosis has been reported (20). Nevertheless, the relatively low cross-reactivity observed (10.5 %) in human cases of cysticercosis with the EIA assay for IgE antibody will allow the use of it in areas where both diseases are present.

The specificity in relation to other parasites was also good. None of the 10 patients infected with Fasciola hepatica were positive. For patients with fascioliasis, positive results have earlier been reported for an EIA measuring specific total human immuno- globulins (5). Torres et al. (17) have observed false positive reactions in fascioliasis employing another EIA for specific E. granulosus IgE. Low positive scores were, how- ever, recorded for some African patients with schistosomiasis as well as for onchocerciasis and ascariasis from Venezuela. Most of those patients showed total IgE concentrations in their sera >5000 kU11. Including all the 215 sera of the different control groups a specificity of 93 % (2001215) was obtained.

The microtiter EIA method employed promises to be a specific and useful diagnostic tool and can be considered as a very convenient method to be included in the range of irnmunodiagnostic tests for human hydatid disease.

REFERENCES 1. Matossian RM, Rickard MD, Smyth JD. Hydatidosis: A global problem of increasing importance.

2. Torres-Rodrigues JM. Valoracion de 10s metodos para el diagn6stico inmunologico de la hidatido-

3. Rickard MD. The immunological diagnosis of hydatid disease. Aust Vet J 55: 99-104, 1979. 4. Schantz PM, Kagan IG. Echinococcosis (hydatidosis). In: Houba V , ed. Immunological investi-

Bull WHO 55: 499-507, 1977.

sis humana. Med Clin (Barc) 66: 289-302, 1976.

gation of tropical parasitic diseases. Edinburgh: Churchill Livingstone, 106129, 1980.

15-898552

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218 A . Sjolander et al. Scand J Infect Dis 21 (1989)

5 . Farag H, Bout D, Capron A. Specific immunodiagnosis of human hydatidosis by the enzyme- linked immunosorbent assay (ELISA). Biomedicine 23: 276-278, 1975.

6. Guisantes JA. Aportacion de 10s mttodos inmunoenzimiticos al diagnostic0 de la hidatidosis humana. In: Comunidad de Madrid, ed. Proceedings of the XI11 International Congress of Hydatidosis, Madrid, 13C134, 1985.

7. Johansson SGO, Mellbin T, Vahlqvist B. Immunoglobulin levels in Ethiopian preschool children with special reference to high concentration of immunoglobulin E (ND). Lancet I : 1118, 1968.

8. Kojima S, Yokogawa M, Tada T. Raised levels of serum IgE in human helminthiasis. Am J Trop Med Hyg 21: 913-918, 1972.

9. Radermecker M, Bekthi A, Poncelet E , Salmon J. Serum IgE levels in protozoal and helminthic infections. Int Arch Allergy Appl Immunol47: 285-295, 1974.

10. Huldt G, Johansson SGO, Lanto S . Echinococcosis in Northern Scandinavia. Immune reactions to Echinococcus granulosus in Kautokeino Lapps. Arch Environ Health 26: 3 U 0 , 1973.

1 1 . Dessaint JP, Bout D, Wattre P, Capron A. Quantitative determination of specific IgE antibodies to Echinococcus granulosus and IgE levels in sera from patients with hydatid disease. Immu- nology 29: 813-823, 1975.

12. Varela-Diaz VM, Coltorti EA. Techniques for the immunodiagnosis of human hydatid disease. Scientific and Technical Monograph No. 7. Pan American Zoonoses Center, PAHOIWHO, Ramos Mejia, Buenos Aires, 1976.

13. Sorice F, Delia S, Vullo V, Aceti A, Ferone U. Valore e limiti del RAST (radioallergosorbent test) nella diagnosi biologica dell’ idatidosi. Ann Sclavo 71: 800-815, 1979.

14. Witassek F. Nachweis von totalem und spezifischem IgE bei Patienten mit Echinokokkose. Tagung der Deutschen Tropenmedizinischen Gesellschaft. Abstract 78, 1983.

15. Loscher T. Der Radioallergosorbenttest (RAST) in der Diagnostik und Therapiekontrolle der Echinokokkose. Tagung der Deutschen Tropenmedizinischen Gesellschaft. Abstract 80, 1983.

16. Ben-Ismael R, Mogahed A, Boiteau A, Sainte-Laudy J, Carme B, Dank M, Gentilini M. Evaluation des immunoglobulines E dans I’hydatidose. Confrontation aux donnees anatomo- cliniques. Med Ma1 Infect 12: 492496, 1982.

17. Torres JM, Sanchez MC, Llambi JM. Reacciones de hipersensibilidad inmediata en la hidatidosis humana. Comparacion entre la IgE total y especifica y la prueba intradermica. Immunologica 4: 119-125, 1985.

18. Villaroya G, Garcia R , Lopez A, Selma L, Gomez A. Valor diagnostic0 de las determinaciones de IgE total y especifica en la hidatidosis hepitica. Rev Esp Enferrn Apar Dig 70: 33-36, 1986.

19. Afferni C, Pini C, Misiti-Dorello P, Bernardini L, Conchedda M, Vicari G. Detection of specific IgE antibodies in sera from patients with hydatidosis. Clin Exp Immunol 55: 587-592, 1984.

20. Torres J. Reacciones cruzadas en pacientes afectos de cisticercosis frente a antigenos hidatidicos y de Taenia saginata. Med Clin (Barc) 81: 55-56, 1983.

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