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A specific test for the identification of cyst fluid samples from suspected human hydatid infections
256
TRANSACTIONS OF THE ROYAL SOCIETYOF TROPICALMEDICINEAND HYCENE (1986) 80, 256-257
A specific
test
Liverpool
for the identification of cyst fluid suspected human hydatid infections
samples
P. S. CRAIG, W. BAILEY AND G. S. NELSON School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA,
from
England
Abstract
A simple and sensitivetwo-step enzyme immunoassayfor Echinococcus antigenwas developed, usingperoxidaseconjugatedIgG fractions of rabbit anti-Echinococcus granulosus cyst fluid serumor of pooledserafrom humanhydatid patients,to assaysamplesof cystic fluid taken from patientswith suspectedhydatid cyst infections.The assaywas100%positivewith nine proven humanhydatid cyst fluids; in six patientswith cystswhereserologywasnegative,there wasno evidenceof Echinococcus antigenin the cyst fluid. This simpleprocedureis thereforeof value in confirmingor excluding the diagnosisof hydatid in cystic fluid obtained by biopsy or after surgery. Introduction
Surgerystill remainsthe maintreatmentfor human hydatidosis,even though chemotherapyhasrecently becomemore promisingfor this disease(MORRISet al., 1983).Serologicaldiagnosisis extremelyusefulin most human hydatid casesthough a significant number of hydatid patients may be seronegative. Samplesof fluid from suspectedhydatid cysts are frequently taken during exploratory surgeryor before cyst removal, particularly when serologyis negative. Where protoscoleces have not been aspiratedin the fluid or are not presentin the cyst and/or histology’is not appropriatq, it would be very useful to identify reliably the flmd as being of hydatid origin or not. This paper describesa simple and sensitiveenzyme-linked immunosorbentassayto detect Echinococcllsantigens in cyst fluid from patients with suspectedhydatidosis(E. granuZosus). Materials Hydatid
cyst fluid
and Methods
samples
Samples of hydatid cyst fluid were available from nine Datients at surgerv in Britain or Kenva. In all casesfluid was irom cysts whi-ch had protoscoleces present though some had low numbers. In addition cyst fluid was obtained from sheep hydatids from local abattoirs. Samples of hydatid cyst fluid were also available from sterile sheep hydatids. fluid from suspected human hydatid infections Samples of cyst fluid obtained from six patients with suspected hydatid infections of liver,, kidney or spleen were sent to the Liverpool School of Tropical Medicine for testing during 1984. All samples were refrigerated and tested within 24 to 48 hours of receipt. All except one patient were serologically negative for anti-Echinococcus antibodies using either latex agglutination, complement fixation or ELISA tests. Cyst
conjugated specific anti-Echinococcus antisera Rabbit antisera were raised against an extract of sheep hydatid cyst fluid (SHCF) which had previously been affinity purified by absorption against rabbit anti-normal sheep serum coupled to CNBr activated seoharose (Pharmacia) as described previously (CRAIG & NELSON, 1984). A pooi of sera from high specific antibody titre hydatid patients was also used as a specific antiserum. Protein A purified IgG fractions of both rabbit and human antisera were conjugated to horseradish peroxidase (Type Perowidase
VI, Sigma) using the periodate method of WILSON 81 NAKANE
(1978).
Enzyme-linked
immunosmbent
assay (ELZSA)
Known and suspected hydatid cyst fluid samples were diluted in 0.05 M carbonate-bicarbonate buffer pH 9.6 and incubated overnight at 4°C in wells (100 ~1) of PVC microtitre trays (Flow). Wells were washed three times in 0.15 M phosphate buffered saline pH 7.4 with 0.05% Tween-20 added (PBS-Tween) then incubated with 2% bovine albumin in PBS-Tween (100 @well) for one hour at room temperature. After washing wells once in PBS-Tween, optimal dilutions of either peroxidase conjugated rabbit anti-SHCF or hyperimmune hydatid antisera were added for two hours at 4°C (or one hour at room temperature). Microtitre wells were given three final washes in PBS-Tween as above and then ‘developed’ by adding 100 @well of substrate, S-aminosalicylic acid in phosphate buffer with EDTA added as described previously (ELLENS & GIELKINS, 1980). Optimal colour change (to brown) occurred within 15 min at room temperature. Plates were easily read visually and optical density values were also measured using an automatic ELISA plate reader (Microplate ELISA, Dynatech). Results
The sensitivity of this ELISA for detectinghuman hvdatid cvst fluid antirrencs)was- 30 ng nrotein/ml. The perokdaseconju&eh iabbit anti-SkeF antisera was more reactive in ELISA than the high titre human hydatid antisera, though both gave good “signal to noise” binding ratios with positive and ‘negative’
cyst fluid
samples.
The figure shows the binding curves of the peroxidaseconjugatedhumanantiserato nine proven hydatid cyst fluid samplesfrom British or Kenyan patients, to two sheephydatid fluid samplesfrom sterile cysts and six samplesof suspectedhydatid cyst fluid from UK hospital patients. The nine samplesof human hydatid fluid and the two sheep hvdatid fluid sampleswere unequivocallypositive in the assay,though‘a range of antigen activities was auDarent. ‘No antigen’ controls in ELISA were negative. All six &ples of fluid from suspected
human hydatid cysts were negative in the assay and on subsequent trace back cysts were identified as being of non-parasite origin in the six patients. It is interesting to note that one patient had already had a previous
history of hydatid infection with the removal of a
P. s. (x41ci
et al.
257
may not be evident, The present study has shown that a simple enzyme immunoassay can overcome this problem by detecting specific Echinococcus antigen(s) in small quantities of cyst fluid. All samples of fluid from proven human hydatid cysts (from both UK and Kenya cases) gave strong positive reactions in the assay using either the rabbit peroxidase conjugated anti-cyst fluid or human hydatid antisera, and the assay was positive with cyst fluid samples from sterile (no protoscoleces) hydatids of sheep origin and from human hydatid cysts with extremely small numbers of protoscoleces. In contrast the cyst fluid samples from the six patients with “suspected” hydatids were completely negative in the assay and follow up studies in all cases showed that they were not of Echinococcus origin. It seems likely that the peroxidase labelled rabbit and human antibodies responsible for positive ELISA reactions with human hydatid cyst fluids, are recognizing the same antigen(s) which may also be present in serum from human hydatid infections as circulating antigen or associated in specific immune complexes (CRAIG
& NELSON,
1984).
Acknowledgements We would like to thank MS J. Moffit for processing some of the cyst fluid samples and Dr. Nazareth (Royal Free Hospital, London) together with Mr. I. Blenkhorn FRCS (Royal Postgraduate Medical School, London) and Dr. R. Peller (Royal Berkshire Hospital, Reading) for follow up of Fig. 1. Titration of cyst fluid samples in ELISA against peroxidase conjugated IgG from hyperimmune human hydatid sera. Known human hydatid cyst fluid samples (U), sheep hydatid fluid from sterile cysts (C&--O), cyst fluid samples (x6) from suspected human hydatids (fZZl). Binding of peroxidase conjugate in absence of cyst fluid ‘antigen’ A.
hepatic cyst. In this case, who was positive for Echinococcus antibodies, the antigen test showed that a cyst in the kidney was not of hydatid origin. Discussion
The detection of specific antibodies using various immunodiagnostic tests is extremely helpful in the diagnosis of human hydatidosis (WILLIAMS, 1982) but false negative results occur quite frequently, sometimes in patients with very large cysts, especially in Turkana in Kenya. In these cases the detection of specific antigen and immune complexes has shown potential for diagnosis (CRAIG & NELSON, 1984). When there are problems of differential diagnosis, cyst fluid is removed for microscopical examination but the characteristic protoscoleces or their hooks
patients. We are grateful to Drs. C. N. L. Macpherson and Dr. D. P. McManus for supplying hydatid cyst fluid samples from Turkana patients. PSC was supported by rhe Wellcome Trust
and the EEC.
References Craig, I’. S. & Nelson, G. S. (1984). The detection of hrculating antigen iti human hydatid disease. Annals of Tropical Medicine and Parasitology, 78, 219-227. Ellens, D. J. & Gielkins, A. L. J. (1980). A simple method for the purification of S-aminosalicylic acid. Application of the product as substrate in enzyme-linked immunosorbent assay (ELISAI. Journal of Immunological Methods, 37, 325-332. Morris, D. I,., Dykes, l’. W., Dickson, B., Marriner, S. F., Boga?, J. A. & Burrows, E. G. 0. (19833. Albendazole in hydatld disease. British Medical Journal, 286, 103-104. Wilhams, J. F. (1982). Cestode infections. In: Immunology of Parask Infections. S. Cohen & K. Warren (Editors). London: Blackwell Scientific Publications, p. 676Wilson, B. M. & Nakane, P. K. (1978). Recent develop: ments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In: ImmunofZuorescence and Related Straining Techniques. W. Knapp, K. Holubar & G. Wicks (Editors). Amsterdam: Elsevier, North Holland Biomedical Press. pp. 21 S-224. --
Accepted for publication
15th February,
1985
TRANSACTIONS OF THE ROYAL SOCIETYOF TROPICALMEDICINEAND HYCENE (1986) 80, 256-257
A specific
test
Liverpool
for the identification of cyst fluid suspected human hydatid infections
samples
P. S. CRAIG, W. BAILEY AND G. S. NELSON School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA,
from
England
Abstract
A simple and sensitivetwo-step enzyme immunoassayfor Echinococcus antigenwas developed, usingperoxidaseconjugatedIgG fractions of rabbit anti-Echinococcus granulosus cyst fluid serumor of pooledserafrom humanhydatid patients,to assaysamplesof cystic fluid taken from patientswith suspectedhydatid cyst infections.The assaywas100%positivewith nine proven humanhydatid cyst fluids; in six patientswith cystswhereserologywasnegative,there wasno evidenceof Echinococcus antigenin the cyst fluid. This simpleprocedureis thereforeof value in confirmingor excluding the diagnosisof hydatid in cystic fluid obtained by biopsy or after surgery. Introduction
Surgerystill remainsthe maintreatmentfor human hydatidosis,even though chemotherapyhasrecently becomemore promisingfor this disease(MORRISet al., 1983).Serologicaldiagnosisis extremelyusefulin most human hydatid casesthough a significant number of hydatid patients may be seronegative. Samplesof fluid from suspectedhydatid cysts are frequently taken during exploratory surgeryor before cyst removal, particularly when serologyis negative. Where protoscoleces have not been aspiratedin the fluid or are not presentin the cyst and/or histology’is not appropriatq, it would be very useful to identify reliably the flmd as being of hydatid origin or not. This paper describesa simple and sensitiveenzyme-linked immunosorbentassayto detect Echinococcllsantigens in cyst fluid from patients with suspectedhydatidosis(E. granuZosus). Materials Hydatid
cyst fluid
and Methods
samples
Samples of hydatid cyst fluid were available from nine Datients at surgerv in Britain or Kenva. In all casesfluid was irom cysts whi-ch had protoscoleces present though some had low numbers. In addition cyst fluid was obtained from sheep hydatids from local abattoirs. Samples of hydatid cyst fluid were also available from sterile sheep hydatids. fluid from suspected human hydatid infections Samples of cyst fluid obtained from six patients with suspected hydatid infections of liver,, kidney or spleen were sent to the Liverpool School of Tropical Medicine for testing during 1984. All samples were refrigerated and tested within 24 to 48 hours of receipt. All except one patient were serologically negative for anti-Echinococcus antibodies using either latex agglutination, complement fixation or ELISA tests. Cyst
conjugated specific anti-Echinococcus antisera Rabbit antisera were raised against an extract of sheep hydatid cyst fluid (SHCF) which had previously been affinity purified by absorption against rabbit anti-normal sheep serum coupled to CNBr activated seoharose (Pharmacia) as described previously (CRAIG & NELSON, 1984). A pooi of sera from high specific antibody titre hydatid patients was also used as a specific antiserum. Protein A purified IgG fractions of both rabbit and human antisera were conjugated to horseradish peroxidase (Type Perowidase
VI, Sigma) using the periodate method of WILSON 81 NAKANE
(1978).
Enzyme-linked
immunosmbent
assay (ELZSA)
Known and suspected hydatid cyst fluid samples were diluted in 0.05 M carbonate-bicarbonate buffer pH 9.6 and incubated overnight at 4°C in wells (100 ~1) of PVC microtitre trays (Flow). Wells were washed three times in 0.15 M phosphate buffered saline pH 7.4 with 0.05% Tween-20 added (PBS-Tween) then incubated with 2% bovine albumin in PBS-Tween (100 @well) for one hour at room temperature. After washing wells once in PBS-Tween, optimal dilutions of either peroxidase conjugated rabbit anti-SHCF or hyperimmune hydatid antisera were added for two hours at 4°C (or one hour at room temperature). Microtitre wells were given three final washes in PBS-Tween as above and then ‘developed’ by adding 100 @well of substrate, S-aminosalicylic acid in phosphate buffer with EDTA added as described previously (ELLENS & GIELKINS, 1980). Optimal colour change (to brown) occurred within 15 min at room temperature. Plates were easily read visually and optical density values were also measured using an automatic ELISA plate reader (Microplate ELISA, Dynatech). Results
The sensitivity of this ELISA for detectinghuman hvdatid cvst fluid antirrencs)was- 30 ng nrotein/ml. The perokdaseconju&eh iabbit anti-SkeF antisera was more reactive in ELISA than the high titre human hydatid antisera, though both gave good “signal to noise” binding ratios with positive and ‘negative’
cyst fluid
samples.
The figure shows the binding curves of the peroxidaseconjugatedhumanantiserato nine proven hydatid cyst fluid samplesfrom British or Kenyan patients, to two sheephydatid fluid samplesfrom sterile cysts and six samplesof suspectedhydatid cyst fluid from UK hospital patients. The nine samplesof human hydatid fluid and the two sheep hvdatid fluid sampleswere unequivocallypositive in the assay,though‘a range of antigen activities was auDarent. ‘No antigen’ controls in ELISA were negative. All six &ples of fluid from suspected
human hydatid cysts were negative in the assay and on subsequent trace back cysts were identified as being of non-parasite origin in the six patients. It is interesting to note that one patient had already had a previous
history of hydatid infection with the removal of a
P. s. (x41ci
et al.
257
may not be evident, The present study has shown that a simple enzyme immunoassay can overcome this problem by detecting specific Echinococcus antigen(s) in small quantities of cyst fluid. All samples of fluid from proven human hydatid cysts (from both UK and Kenya cases) gave strong positive reactions in the assay using either the rabbit peroxidase conjugated anti-cyst fluid or human hydatid antisera, and the assay was positive with cyst fluid samples from sterile (no protoscoleces) hydatids of sheep origin and from human hydatid cysts with extremely small numbers of protoscoleces. In contrast the cyst fluid samples from the six patients with “suspected” hydatids were completely negative in the assay and follow up studies in all cases showed that they were not of Echinococcus origin. It seems likely that the peroxidase labelled rabbit and human antibodies responsible for positive ELISA reactions with human hydatid cyst fluids, are recognizing the same antigen(s) which may also be present in serum from human hydatid infections as circulating antigen or associated in specific immune complexes (CRAIG
& NELSON,
1984).
Acknowledgements We would like to thank MS J. Moffit for processing some of the cyst fluid samples and Dr. Nazareth (Royal Free Hospital, London) together with Mr. I. Blenkhorn FRCS (Royal Postgraduate Medical School, London) and Dr. R. Peller (Royal Berkshire Hospital, Reading) for follow up of Fig. 1. Titration of cyst fluid samples in ELISA against peroxidase conjugated IgG from hyperimmune human hydatid sera. Known human hydatid cyst fluid samples (U), sheep hydatid fluid from sterile cysts (C&--O), cyst fluid samples (x6) from suspected human hydatids (fZZl). Binding of peroxidase conjugate in absence of cyst fluid ‘antigen’ A.
hepatic cyst. In this case, who was positive for Echinococcus antibodies, the antigen test showed that a cyst in the kidney was not of hydatid origin. Discussion
The detection of specific antibodies using various immunodiagnostic tests is extremely helpful in the diagnosis of human hydatidosis (WILLIAMS, 1982) but false negative results occur quite frequently, sometimes in patients with very large cysts, especially in Turkana in Kenya. In these cases the detection of specific antigen and immune complexes has shown potential for diagnosis (CRAIG & NELSON, 1984). When there are problems of differential diagnosis, cyst fluid is removed for microscopical examination but the characteristic protoscoleces or their hooks
patients. We are grateful to Drs. C. N. L. Macpherson and Dr. D. P. McManus for supplying hydatid cyst fluid samples from Turkana patients. PSC was supported by rhe Wellcome Trust
and the EEC.
References Craig, I’. S. & Nelson, G. S. (1984). The detection of hrculating antigen iti human hydatid disease. Annals of Tropical Medicine and Parasitology, 78, 219-227. Ellens, D. J. & Gielkins, A. L. J. (1980). A simple method for the purification of S-aminosalicylic acid. Application of the product as substrate in enzyme-linked immunosorbent assay (ELISAI. Journal of Immunological Methods, 37, 325-332. Morris, D. I,., Dykes, l’. W., Dickson, B., Marriner, S. F., Boga?, J. A. & Burrows, E. G. 0. (19833. Albendazole in hydatld disease. British Medical Journal, 286, 103-104. Wilhams, J. F. (1982). Cestode infections. In: Immunology of Parask Infections. S. Cohen & K. Warren (Editors). London: Blackwell Scientific Publications, p. 676Wilson, B. M. & Nakane, P. K. (1978). Recent develop: ments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In: ImmunofZuorescence and Related Straining Techniques. W. Knapp, K. Holubar & G. Wicks (Editors). Amsterdam: Elsevier, North Holland Biomedical Press. pp. 21 S-224. --
Accepted for publication
15th February,
1985