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Serological diagnosis and post-operative surveillance of human hydatid disease. l. latex agglutination and immunoelectrophoresis using crude cyst fluid antigen
Pathology (1984), 16, pp. 207-210
SEROLOGICAL DIAGNOSIS AND POST-OPERATIVE SURVEILLANCE OF HUMAN HYDATID DISEASE. 1. LATEX AGGLUTINATION AND IMMUNOELECTROPHORESIS USING CRUDE CYST FLUID ANTIGEN MICHAELD. RICKARD Veterinary Clinical Centre, University of Melbourne
Summary The latex agglutination (LA) test and immunoelectrophoresis(IEP) test for the diagnosis of hydatid disease due to Echinococcus granulosus were carried out on 3600 serum samples submitted to this laboratory during the period mid-1976 to March 1981. Of the 2280 samples submitted for primary diagnosis (no known past history of hydatid disease) there were 108 cases in which infection was confirmed, 99 of them with fertile cysts. The ‘Arc 5’ was demonstrated by IEP in the serum of 65.7% of cases with fertile cysts, and using the additional criterion of 3 or more arcs with no ‘Arc 5’,a further 13.1% of cases were diagnosed apparently without ‘false positive’ results. Antibody was less readily detected in the serum of patients with calcified or sterile cysts. The LA test was positive in 70.7% of patients with fertile cysts, but this test gave an unacceptably high rate of 17.6% ‘false positive’ results. The IEP and LA tests had a combined sensitivity of 87.9% for primary diagnosis. There were 65 confirmed cases of persistent or recurrent infection, of which 64 were positive with IEP. Forty-four of 48 cases where surgical cure was effected became IEP negative within 2 yr. Among 42 patients remaining IEP-positive beyond 2 yr after surgery, recurrent disease was confirmed in 31 cases, but it was not possible to obtain confirmation in the remaining 11 patients. The LA test was unsuitable for post-operative surveillance. Key words: Hydatid disease, latex agglutination, immunoelectrophoresis, diagnosis Accepted October 21, 1983
INTRODUCTION Immunological methods for diagnosis of hydatid disease due to Echinococcus granulosus infection in man have provided a powerful and practical tool for the clinician and surgeon, and several comprehensive reviews are available concerning the merits of the various tests.’-’ Many variables can significantly affect the reliability of a particular serological test; in additionto operator and equipment factors, the source and quality of antigen as well as the immunological relationship between the parasite and a particular host population will be critical. For example, different ‘strains’ of E. granulosus are known to exist which vary in their biochemical characteristics and the host range for which they are Hydatid disease is particularly common and severe amongst the Turkana people of Kenya, yet
serological tests, especially IEP, are of limited value as a diagnostic tool.’ Routine serological diagnostic tests for hydatid disease of man using latex agglutination and immunoelectrophoresis (IEP)12-15have been carried out in this laboratory since mid-1 976 and this paper analyses the results of these tests. MATERIALS AND METHODS Antigens Hydatid cyst fluid was aspirated from fertile hydatid cysts in the livers and lungs of sheep slaughtered in the abattoir and was stored at - 20°C. Crude sheep hydatid cyst fluid antigen (CSHCF) was prepared by filtering cyst fluid through filter paper (Whatman No. 1) followed by passage through a 0.2 pm membrane filter (Millipore) and dialysis (Visking cellulose membrane) against 3 changes of lOOOx volume of distilled water at 4°C for a total of 72 h. The CSHCF was lyophilized, stored at 4°C and reconstituted with distilled water immediately prior to its use. Sera Sera for primary diagnosis of hydatid infection (i.e. diagnosis in patients with no known past history of hydatid disease) or for determination of recurrence of disease in known previously treated patients were sent to this laboratory for routine testing by LA and IEP between mid-1976 and March 1981. Samples came from all parts of Australia, from Australian born patients as well as from immigrants. Sera were stored at -20°C and tested within 4 d of arriving at the laboratory. Sufficient sera for evaluating the use of LA and IEP for post-operative surveillance were available from 90 patients. Several of these patients had received a course of mebendazole treatment in addition to surgical removal of cysts. Serological tests The IEP and LA tests were carried out according to the methods described in detail by Varela-Diaz & Coltorti.’6 For IEP, lyophilization to concentrate sera was found to be more satisfactory than 3x replenishment of the trough with neat serum. Criteria for a positive IEP were either the presence of ‘Arc 5’13 irrespective of the total number of arcs, or the presence of 3 or more arcs in the absence of ‘Arc 5’.16”’ A standard reference antiserum was prepared by hyperimmunization of a sheep using CSHCF antigen.I6 This serum was used in parallel with each human serum sample tested as a control for both the test and the antigen. Antigen was not used unless there were at least 6 precipitation arcs with the sheep serum including ‘Arc 5 ’ . Electrophoresis was carried out using a Gelman Deluxe Electrophoresis Chamber and power pack with 6-slide immunoframes (Gelman, Michigan). The only variation from the LA test described16was that 0. I pn diameter latex particles (Sigma) were used instead of 0.22 pm.
I
If--, Positive IEP Fertile cy
Site of infection Liter
Lung
Other single organs
Multiple organs
j,
56/69 (814’0) 28! (?5u’o)
71 1 1 ( 6 4 9 ) nil
9/13 (69.2’70) O / I (0%)
6/6 (100%) nil
78/99 (78.8%) 219 (22%)
-50169 (73O;o) 518 (62.5ob)
8/ I 1 (73070) nil
7/13 (53.870) 011 (0%)
5 / 6 (83%) nil
70/99 (70.7%) 519 (56%)
RESULTS Primary diagnosis A total of 3600 sera was received during the period mid-1976 to March 1981, 2280 of which were submitted for primary diagnosis of hydatid disease (i.e. patients with no known past history). Of the 108 confirmed cases, 99 had fertile cysts and 9 had sterile or calcified cysts. The serological results obtaind with these samples are shown in Tables 1 and 2 . IEP had an overall sensitivity (positive tests in known cases of disease) of 78.8% for detection of fertile cysts (Table I). Pulmonary cysts were diagnosed by IEP at the lowest rate (64%) and 100% of multiple infections were positive, although the number of samples was not large. Sterile cysts were less readily detected (22%) by IEP. Thirteen of the 99 cases with fertile cysts were IEP positive using the criterion of 3 or more arcs in the absence of ‘Arc 5’ i.e. 13.1’70, so that the ‘Arc 5’ was present in 65.7%of patients. Of the remaining 2172 primary diagnosis sera, 8 cases had positive IEP (all with ‘Arc 5’) and LA tests, but confirmation of hydatid disease was not available. This gave a possible ‘false positive’ rate of 0.37% although the patients were almost certainly infected. The LA test had overall sensitivity of 70.7% in confirmed hydatid patients with fertile cysts and showed fairly consistent detection rates across all sites of infection (Table 1). The detection rate in cases with sterile or calcified cysts (56%) was higher than with IEP which could be expected because of the superior ability of LA to detect low levels of antibody where cysts may have recently died, or where antigenic stimulation was minimal. 1 \iii
I
2
Three hundred and eighty eight of 2172 serum samples gave positive LA tests in patients where no confirmation of hydatid disease was received. Even if the 8 IEP positive (probably hydatid infected) cases are taken out of this total, it leaves a possible ‘false positive’ rate of 380/2164 or 17.6%. The various combinations of LA and IEP results in patients with fertile or sterile cysts in different organs are shown in Table 2. A number of patients with liver cysts were LA - ve with a positive IEP test i.e. ‘false negative’ LA tests. Only 12 of 99 cases with fertile cysts (12.1%) were serologically negative for both LA and IEP tests i.e. a combined sensitivity of 87.9%.
Diagnosis of persistent or recurrent infection A total of 227 serum samples were submitted from patients with a past history ( > 2 yr ago) of hydatid disease. Infection was subsequently confirmed in 65 of these patients, only one of which gave a negative IEP result, In 14 other cases which had positive IEP tests, it was not possible to obtain assured confirmation of infection or freedom from it i.e. possible ‘false positives’. Excluding these 14 non-confirmed cases, the IEP test diagnosed persistent or recurrent infection in 64 of 65 patients, a sensitivity of 98.5%. Post-operative surveillance Of the 90 patients studied, 3 1 became IEP - ve within 12 mth of surgery, and a further 13 between 1 and 2 yr. Three patients became IEP negative between 2 and 3 yr and a single patient between 3 and 4 yr. Of the 42 patients
The tarious combinations of L.4 and IEP results in primary diagnosis of hydatid disease i n patient, with confirmed infections
Serological results
IEP + \ e LA
Liter Lung Other a bdorri i 11a1 organ Peritoneal c:i\itv I\luxle Bone CNS
Multiple infection
Totals
i
+be
Fertile Sterile Fertile
44
terrile Fertile Sterile Fertile t-ertile Fertile Fertile
1 3
2 7
-
2
-
5
IEP + v e LA - b e
IEP -ve LA + v e
IEP -ve LA - v e
DIAGNOSIS OF HUMAN HYDATID DISEASE-I
remaining IEP positive beyond 2 yr, 31 were subsequently confirmed as still having hydatid infection. In the remaining 11 cases it was not possible to obtain absolute confirmation of their infection status. LA tests remained positive in most patients for long periods after IEP had become negative.
209
cysts may be due to previous surgical intervention, since it has been found (unpublished results) that the majority of patients who are seronegative prior to surgery become serologically positive soon afterwards. The LA test was of little value in diagnosing persistent or recurrent infection. An important role for serology is surveillance of the DISCUSSION progress of patients with hydatid disease following surgery. Tests which have been recommended for this The results obtained using IEP to examine serum samples purpose are the complement fixation test,25measurement submitted for primary diagnosis of hydatid disease reinof specific IgE26 and IEP.l6 The present force conclusions reached by other ~ ~ r k e rcon~ ~ ~ - ~ ~ anti-parasite - ~ ~ results showed clearly that IEP, but not LA was useful cerning the specificity of this test. The overall sensitivity for this purpose. The majority of IEP tests became of 65.7% for detecting fertile hydatid cysts using the negative within 12 mth after successful cure, but a signifipresence of ‘Arc 5’ as the criterion for a positive test is cant number remained positive for up to 2 yr. Four somewhat lower than that reported by European patients who were positive beyond this time became w0rke1-s‘~. 1 4 * 1 8 but similar to results reported with prenegative within 4 yr. It is possible that viable cyst material operative sera from South A m e r i ~ a . ” , ’ ~ The value of had persisted in these patients for some time after surgery ‘Arc 5’ is reduced where E. multilocularis infection and and that this subsequently degenerated spontaneously. Taenia solium cysticercosis are common,19-21 and Thirty-one of the patients who remained positive for > 2 although this does not pose a significant problem in yr were confirmed to be still infected but because followAustralia, it should be borne in mind when other up data were impossible to obtain a question mark diagnostic aids suggest that either of these parasites might remains over the infection status of the other 11 patients be present, especially in migrants. When sera which gave who remained positive beyond this time. 3 precipitation arcs without the diagnostic ‘Arc 5’ were of the unsatisfactory results obtained here Because included as sensitivity for fertile hydatids using the LA test, it was decided to evaluate the ELISA was increased to 78.8% without any confirmed ‘false test as an alternative for screening purposes and as a backpositive’ results. The absence of ‘Arc 5’ in these samples up for IEP. The results of this work are reported in may reflect the inherent inability of IEP to detect low another paper.27 levels of antibody, and the more sensitive DD5 test” or ELISA using purified ‘antigen 5’23might have detected ACKNOWLEDGEMENTS The author gratefully antibody in some of these patients. However, there are acknowledges the assistance of the many surgeons and a number of infected persons who do not produce detecpathologists who assisted by providing information table antibody to ‘antigen 5’ and consideration of other concerning hydatid infection in the patients. Ms L. precipitation arcs present is a useful aid to diagnosis when Kenner, Ms J. Singer, Mr P. Squires and Mr R. considered along with clinical findings. l6 Kozlowski assisted with the serological tests. The The results suggest that IEP is not effective in detecting assistance of Dr V. M. Varela-Diaz of the Pan American low levels of antibody present when cysts are sterile or Health Organization Zoonosis Center in Buenos Aires is calcified, and is also less effective in detecting pulmonary gratefully acknowledged. infection than hepatic cysts or multiple infection. Several workers have described the problem of detecting serological responses in patients with hydatid cysts in the Addressfor correspondence: M.D.R., University of Melbourne, Veterinary I~ng.’~*~~ Clinical Centre, Princes Highway, Werribee, Victoria, Australia 3030 The IEP test is not suitable for mass screening of serum samples because of the large amount of serum and antigen References required, and also because of the technical intricacy of 1. Matossian RM. The immunological diagnosis of human hydatid the test. Furthermore, IEP, at best fails t o diagnose disease. Trans Roy SOCTrop Med Hyg 1977; 71: 101-4. infection in 20% of hydatid-infected patients. Previous workers have clearly shown the advantages of using 2. Kagan IG. Recent advances in the diagnosis of hydatidosis (1970-1976). Parasitol Hung 1978; 1 1 : 31-50. multiple tests5 for primary diagnosis. By using LA and 3. Rickard MD. The immunological diagnosis of hydatid disease. Aust IEP tests, the sensitivity for fertile hydatids in the preVet J 1979; 5 5 : 99-104. sent study was increased to 87.9%. However, the LA test 4. Schantz PM, Kagan IG. Echinococcosis (Hydatidosis). In: Houba generally did not perform as well as has been described. l 2 V, ed. Immunologic investigation of tropical parasitic diseases. The sensitivity for fertile cysts was 70.7%, and the proEdinburgh, London, New York: Churchill Livingstone. 1980. bable ‘false positive’ rate of 17.6% was unacceptably 5. Schantz PM. Echinococcosis (Hydatidosis). In: Walls KF, ed. high. Schantz5 has pointed out that reports concerning Immunoserology of parasitic diseases (in press). New York: Marcel the efficacy of the LA test have varied widely, and that Decker Inc. modification of the method, especially with respect to the 6. Thompson RCA. Aspects of speciation in Echinococcus granulosus. source and size of the latex particles can have a signifiVet Parasitol 1978; 4: 121-5. cant influence on the results achieved. 7. McManus DP, Smyth JD. Differences in the chemical composiThe IEP test was valuable in diagnosing persistent or tion and carbohydrate metabolism of Echinococcus granulosus recurrent infection. The higher sensitivity for these cases (horse and sheep strains) and E. rnultilocularis. Parasitology 1978; 17: 103-9. by comparison with that for primary diagnosis of fertile
210
KIC‘KAKI)
8. Thompson RCA, Kumaratilake LM. lntraspecific variation in Echinoroccus grunulosus: The Australian situation and perspectives for the future. Trans Roy Soc Trop Med Hyg 1982; 76: 13-16. 9. Fischman A. A ranid latex test for hvdatid disease. NZ Med J 1960: 59: 485-7. 10 Szyfres B, Kagan IG. A modified slide latex screening test for
hydatid disease. J Parasitol 1963; 49: 69-72.
I 1 Williams JF, Prezioso U . Latex agglutination test for hydatid disease
using Boerner slides. J Parasitol 1970; 56: 1253-4.
12. Varela-Diaz VM, Coltorti EA, Prezioso U er al. Evaluation of three immunodiagnostic tests for human hydatid disease. Am J Trop Med Hyg 1975; 24: 312-9. 13. Capron A, Vernes A, Biguet J . The immunoelectrophoretic
diagnosis of hydatidosis (Fr). Journ Lyonnaises d’Hydatidol, SlMEP eds, Lyon 1967; 27-40.
14. Capron A. Yarzabal L, Vernes A, Fruit J . The immunological diagnosis of human echinococcosis (Fr). Pathol Biol (Paris) 1970; 18: 357-365. 15. Yarzabal LA, Leiton J , Lopez-Lemes MH. The diagnosis of human pulmonary hydatidosis by the immunoelectrophoresis test. Am J Trop Med Hyg 1974; 23: 662-6. 16. Varela-Diaz VM, Coltorti EA. Human hydatidosis.
Immunodiagnostic techniques. Series of Scientific and Technical Monographs, C.P.Z.-7, 1976. Pan Am Health Organization.
17 Yarzabal LA, Schanrz PM, Lopez-Lemes MH. Comparative sensitivity and specificity of the Casoni intradermal and the immunoelectrophoresis tests for the diagnosis of hydatid disease. Am J Trop Med Hyg 1975: 2: 843-8. 18 Picardo NGA, Guisantes JA. Comparison of three immunological
tests for seroepidemiological purposes in human echinococcosis. Parasite Immunology 1981; 3: 191-9.
Pathology (1984), 16, April
19. Varela-Diaz VM, Eckert J , Rausch RL et al. Detection of the Echinococcusgrunulosus diagnostic arc 5 in sera from patients with surgically-confirmed E. mrtltiloculuris infection. Z. Parasitenkd 1977; 53: 183-8. 20. Varela-Diaz VM, Coltorti EA, D’Alessandro A. Immunoelectrophoresis tests showing Echinococcus grunulosus arc 5 in human cases of Echinococcus vogeli and cysticercosis-multiple myeloma. Am J Trop Med Hyg 1978; 27: 554-7. 21. Schantz PM, Shanks D, Wilson M. Serologic cross-reactions with sera from patients with echinococcosis and cysticercosis. Am J Trop Med Hyg 1980; 29: 609-12. 22. Coltorti EA, Varela-Diaz VM. Detection of antibodies against Echinococcus grunulosus arc 5 antigens by double diffusion test. Trans Roy Soc Trop Med Hyg 1978; 72: 226-9. 23 Farag H , Bout D, Capron A. Specific immunodiagnosis of human hydatidosis by the enzyme linked immunosorbent assay (ELISA). Biomedicine 1975; 23: 276-8. 24. Todorov I . RaiEev 1. Tenev S. Kosturkova M et al. Immunoreac~ ~ tivity in pulmonary echinococcosis 2 . Evaluation of antibody response. Bull W H O 1979; 57: 741-50. ~
25. Dighero MW, Bradstreet CMP. The serodiagnosis of human hydatid disease: 1. The routine use of latex-agglutination and complement fixation in diagnosis. J Helminthol 1979; 53: 283-6. 26. Bekhti A, Schaaps J-P, Capron M et al. Treatment of hepatic hydatid disease with mebendazole: Preliminary results in four cases. Br Med J 1977; 2: 1047-5 1 . 27. Rickard MD, Honey RD, Brumley J L , Mitchell GF. Serological diagnosis and post-operative surveillance of human hydatid disease. 11. The enzyme-linked immunosorbent assay (ELISA) using various antigen preparations. Pathology 1984; 16: 21 1-20.
~
~
.
.
SEROLOGICAL DIAGNOSIS AND POST-OPERATIVE SURVEILLANCE OF HUMAN HYDATID DISEASE. 1. LATEX AGGLUTINATION AND IMMUNOELECTROPHORESIS USING CRUDE CYST FLUID ANTIGEN MICHAELD. RICKARD Veterinary Clinical Centre, University of Melbourne
Summary The latex agglutination (LA) test and immunoelectrophoresis(IEP) test for the diagnosis of hydatid disease due to Echinococcus granulosus were carried out on 3600 serum samples submitted to this laboratory during the period mid-1976 to March 1981. Of the 2280 samples submitted for primary diagnosis (no known past history of hydatid disease) there were 108 cases in which infection was confirmed, 99 of them with fertile cysts. The ‘Arc 5’ was demonstrated by IEP in the serum of 65.7% of cases with fertile cysts, and using the additional criterion of 3 or more arcs with no ‘Arc 5’,a further 13.1% of cases were diagnosed apparently without ‘false positive’ results. Antibody was less readily detected in the serum of patients with calcified or sterile cysts. The LA test was positive in 70.7% of patients with fertile cysts, but this test gave an unacceptably high rate of 17.6% ‘false positive’ results. The IEP and LA tests had a combined sensitivity of 87.9% for primary diagnosis. There were 65 confirmed cases of persistent or recurrent infection, of which 64 were positive with IEP. Forty-four of 48 cases where surgical cure was effected became IEP negative within 2 yr. Among 42 patients remaining IEP-positive beyond 2 yr after surgery, recurrent disease was confirmed in 31 cases, but it was not possible to obtain confirmation in the remaining 11 patients. The LA test was unsuitable for post-operative surveillance. Key words: Hydatid disease, latex agglutination, immunoelectrophoresis, diagnosis Accepted October 21, 1983
INTRODUCTION Immunological methods for diagnosis of hydatid disease due to Echinococcus granulosus infection in man have provided a powerful and practical tool for the clinician and surgeon, and several comprehensive reviews are available concerning the merits of the various tests.’-’ Many variables can significantly affect the reliability of a particular serological test; in additionto operator and equipment factors, the source and quality of antigen as well as the immunological relationship between the parasite and a particular host population will be critical. For example, different ‘strains’ of E. granulosus are known to exist which vary in their biochemical characteristics and the host range for which they are Hydatid disease is particularly common and severe amongst the Turkana people of Kenya, yet
serological tests, especially IEP, are of limited value as a diagnostic tool.’ Routine serological diagnostic tests for hydatid disease of man using latex agglutination and immunoelectrophoresis (IEP)12-15have been carried out in this laboratory since mid-1 976 and this paper analyses the results of these tests. MATERIALS AND METHODS Antigens Hydatid cyst fluid was aspirated from fertile hydatid cysts in the livers and lungs of sheep slaughtered in the abattoir and was stored at - 20°C. Crude sheep hydatid cyst fluid antigen (CSHCF) was prepared by filtering cyst fluid through filter paper (Whatman No. 1) followed by passage through a 0.2 pm membrane filter (Millipore) and dialysis (Visking cellulose membrane) against 3 changes of lOOOx volume of distilled water at 4°C for a total of 72 h. The CSHCF was lyophilized, stored at 4°C and reconstituted with distilled water immediately prior to its use. Sera Sera for primary diagnosis of hydatid infection (i.e. diagnosis in patients with no known past history of hydatid disease) or for determination of recurrence of disease in known previously treated patients were sent to this laboratory for routine testing by LA and IEP between mid-1976 and March 1981. Samples came from all parts of Australia, from Australian born patients as well as from immigrants. Sera were stored at -20°C and tested within 4 d of arriving at the laboratory. Sufficient sera for evaluating the use of LA and IEP for post-operative surveillance were available from 90 patients. Several of these patients had received a course of mebendazole treatment in addition to surgical removal of cysts. Serological tests The IEP and LA tests were carried out according to the methods described in detail by Varela-Diaz & Coltorti.’6 For IEP, lyophilization to concentrate sera was found to be more satisfactory than 3x replenishment of the trough with neat serum. Criteria for a positive IEP were either the presence of ‘Arc 5’13 irrespective of the total number of arcs, or the presence of 3 or more arcs in the absence of ‘Arc 5’.16”’ A standard reference antiserum was prepared by hyperimmunization of a sheep using CSHCF antigen.I6 This serum was used in parallel with each human serum sample tested as a control for both the test and the antigen. Antigen was not used unless there were at least 6 precipitation arcs with the sheep serum including ‘Arc 5 ’ . Electrophoresis was carried out using a Gelman Deluxe Electrophoresis Chamber and power pack with 6-slide immunoframes (Gelman, Michigan). The only variation from the LA test described16was that 0. I pn diameter latex particles (Sigma) were used instead of 0.22 pm.
I
If--, Positive IEP Fertile cy
Site of infection Liter
Lung
Other single organs
Multiple organs
j,
56/69 (814’0) 28! (?5u’o)
71 1 1 ( 6 4 9 ) nil
9/13 (69.2’70) O / I (0%)
6/6 (100%) nil
78/99 (78.8%) 219 (22%)
-50169 (73O;o) 518 (62.5ob)
8/ I 1 (73070) nil
7/13 (53.870) 011 (0%)
5 / 6 (83%) nil
70/99 (70.7%) 519 (56%)
RESULTS Primary diagnosis A total of 3600 sera was received during the period mid-1976 to March 1981, 2280 of which were submitted for primary diagnosis of hydatid disease (i.e. patients with no known past history). Of the 108 confirmed cases, 99 had fertile cysts and 9 had sterile or calcified cysts. The serological results obtaind with these samples are shown in Tables 1 and 2 . IEP had an overall sensitivity (positive tests in known cases of disease) of 78.8% for detection of fertile cysts (Table I). Pulmonary cysts were diagnosed by IEP at the lowest rate (64%) and 100% of multiple infections were positive, although the number of samples was not large. Sterile cysts were less readily detected (22%) by IEP. Thirteen of the 99 cases with fertile cysts were IEP positive using the criterion of 3 or more arcs in the absence of ‘Arc 5’ i.e. 13.1’70, so that the ‘Arc 5’ was present in 65.7%of patients. Of the remaining 2172 primary diagnosis sera, 8 cases had positive IEP (all with ‘Arc 5’) and LA tests, but confirmation of hydatid disease was not available. This gave a possible ‘false positive’ rate of 0.37% although the patients were almost certainly infected. The LA test had overall sensitivity of 70.7% in confirmed hydatid patients with fertile cysts and showed fairly consistent detection rates across all sites of infection (Table 1). The detection rate in cases with sterile or calcified cysts (56%) was higher than with IEP which could be expected because of the superior ability of LA to detect low levels of antibody where cysts may have recently died, or where antigenic stimulation was minimal. 1 \iii
I
2
Three hundred and eighty eight of 2172 serum samples gave positive LA tests in patients where no confirmation of hydatid disease was received. Even if the 8 IEP positive (probably hydatid infected) cases are taken out of this total, it leaves a possible ‘false positive’ rate of 380/2164 or 17.6%. The various combinations of LA and IEP results in patients with fertile or sterile cysts in different organs are shown in Table 2. A number of patients with liver cysts were LA - ve with a positive IEP test i.e. ‘false negative’ LA tests. Only 12 of 99 cases with fertile cysts (12.1%) were serologically negative for both LA and IEP tests i.e. a combined sensitivity of 87.9%.
Diagnosis of persistent or recurrent infection A total of 227 serum samples were submitted from patients with a past history ( > 2 yr ago) of hydatid disease. Infection was subsequently confirmed in 65 of these patients, only one of which gave a negative IEP result, In 14 other cases which had positive IEP tests, it was not possible to obtain assured confirmation of infection or freedom from it i.e. possible ‘false positives’. Excluding these 14 non-confirmed cases, the IEP test diagnosed persistent or recurrent infection in 64 of 65 patients, a sensitivity of 98.5%. Post-operative surveillance Of the 90 patients studied, 3 1 became IEP - ve within 12 mth of surgery, and a further 13 between 1 and 2 yr. Three patients became IEP negative between 2 and 3 yr and a single patient between 3 and 4 yr. Of the 42 patients
The tarious combinations of L.4 and IEP results in primary diagnosis of hydatid disease i n patient, with confirmed infections
Serological results
IEP + \ e LA
Liter Lung Other a bdorri i 11a1 organ Peritoneal c:i\itv I\luxle Bone CNS
Multiple infection
Totals
i
+be
Fertile Sterile Fertile
44
terrile Fertile Sterile Fertile t-ertile Fertile Fertile
1 3
2 7
-
2
-
5
IEP + v e LA - b e
IEP -ve LA + v e
IEP -ve LA - v e
DIAGNOSIS OF HUMAN HYDATID DISEASE-I
remaining IEP positive beyond 2 yr, 31 were subsequently confirmed as still having hydatid infection. In the remaining 11 cases it was not possible to obtain absolute confirmation of their infection status. LA tests remained positive in most patients for long periods after IEP had become negative.
209
cysts may be due to previous surgical intervention, since it has been found (unpublished results) that the majority of patients who are seronegative prior to surgery become serologically positive soon afterwards. The LA test was of little value in diagnosing persistent or recurrent infection. An important role for serology is surveillance of the DISCUSSION progress of patients with hydatid disease following surgery. Tests which have been recommended for this The results obtained using IEP to examine serum samples purpose are the complement fixation test,25measurement submitted for primary diagnosis of hydatid disease reinof specific IgE26 and IEP.l6 The present force conclusions reached by other ~ ~ r k e rcon~ ~ ~ - ~ ~ anti-parasite - ~ ~ results showed clearly that IEP, but not LA was useful cerning the specificity of this test. The overall sensitivity for this purpose. The majority of IEP tests became of 65.7% for detecting fertile hydatid cysts using the negative within 12 mth after successful cure, but a signifipresence of ‘Arc 5’ as the criterion for a positive test is cant number remained positive for up to 2 yr. Four somewhat lower than that reported by European patients who were positive beyond this time became w0rke1-s‘~. 1 4 * 1 8 but similar to results reported with prenegative within 4 yr. It is possible that viable cyst material operative sera from South A m e r i ~ a . ” , ’ ~ The value of had persisted in these patients for some time after surgery ‘Arc 5’ is reduced where E. multilocularis infection and and that this subsequently degenerated spontaneously. Taenia solium cysticercosis are common,19-21 and Thirty-one of the patients who remained positive for > 2 although this does not pose a significant problem in yr were confirmed to be still infected but because followAustralia, it should be borne in mind when other up data were impossible to obtain a question mark diagnostic aids suggest that either of these parasites might remains over the infection status of the other 11 patients be present, especially in migrants. When sera which gave who remained positive beyond this time. 3 precipitation arcs without the diagnostic ‘Arc 5’ were of the unsatisfactory results obtained here Because included as sensitivity for fertile hydatids using the LA test, it was decided to evaluate the ELISA was increased to 78.8% without any confirmed ‘false test as an alternative for screening purposes and as a backpositive’ results. The absence of ‘Arc 5’ in these samples up for IEP. The results of this work are reported in may reflect the inherent inability of IEP to detect low another paper.27 levels of antibody, and the more sensitive DD5 test” or ELISA using purified ‘antigen 5’23might have detected ACKNOWLEDGEMENTS The author gratefully antibody in some of these patients. However, there are acknowledges the assistance of the many surgeons and a number of infected persons who do not produce detecpathologists who assisted by providing information table antibody to ‘antigen 5’ and consideration of other concerning hydatid infection in the patients. Ms L. precipitation arcs present is a useful aid to diagnosis when Kenner, Ms J. Singer, Mr P. Squires and Mr R. considered along with clinical findings. l6 Kozlowski assisted with the serological tests. The The results suggest that IEP is not effective in detecting assistance of Dr V. M. Varela-Diaz of the Pan American low levels of antibody present when cysts are sterile or Health Organization Zoonosis Center in Buenos Aires is calcified, and is also less effective in detecting pulmonary gratefully acknowledged. infection than hepatic cysts or multiple infection. Several workers have described the problem of detecting serological responses in patients with hydatid cysts in the Addressfor correspondence: M.D.R., University of Melbourne, Veterinary I~ng.’~*~~ Clinical Centre, Princes Highway, Werribee, Victoria, Australia 3030 The IEP test is not suitable for mass screening of serum samples because of the large amount of serum and antigen References required, and also because of the technical intricacy of 1. Matossian RM. The immunological diagnosis of human hydatid the test. Furthermore, IEP, at best fails t o diagnose disease. Trans Roy SOCTrop Med Hyg 1977; 71: 101-4. infection in 20% of hydatid-infected patients. Previous workers have clearly shown the advantages of using 2. Kagan IG. Recent advances in the diagnosis of hydatidosis (1970-1976). Parasitol Hung 1978; 1 1 : 31-50. multiple tests5 for primary diagnosis. By using LA and 3. Rickard MD. The immunological diagnosis of hydatid disease. Aust IEP tests, the sensitivity for fertile hydatids in the preVet J 1979; 5 5 : 99-104. sent study was increased to 87.9%. However, the LA test 4. Schantz PM, Kagan IG. Echinococcosis (Hydatidosis). In: Houba generally did not perform as well as has been described. l 2 V, ed. Immunologic investigation of tropical parasitic diseases. The sensitivity for fertile cysts was 70.7%, and the proEdinburgh, London, New York: Churchill Livingstone. 1980. bable ‘false positive’ rate of 17.6% was unacceptably 5. Schantz PM. Echinococcosis (Hydatidosis). In: Walls KF, ed. high. Schantz5 has pointed out that reports concerning Immunoserology of parasitic diseases (in press). New York: Marcel the efficacy of the LA test have varied widely, and that Decker Inc. modification of the method, especially with respect to the 6. Thompson RCA. Aspects of speciation in Echinococcus granulosus. source and size of the latex particles can have a signifiVet Parasitol 1978; 4: 121-5. cant influence on the results achieved. 7. McManus DP, Smyth JD. Differences in the chemical composiThe IEP test was valuable in diagnosing persistent or tion and carbohydrate metabolism of Echinococcus granulosus recurrent infection. The higher sensitivity for these cases (horse and sheep strains) and E. rnultilocularis. Parasitology 1978; 17: 103-9. by comparison with that for primary diagnosis of fertile
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25. Dighero MW, Bradstreet CMP. The serodiagnosis of human hydatid disease: 1. The routine use of latex-agglutination and complement fixation in diagnosis. J Helminthol 1979; 53: 283-6. 26. Bekhti A, Schaaps J-P, Capron M et al. Treatment of hepatic hydatid disease with mebendazole: Preliminary results in four cases. Br Med J 1977; 2: 1047-5 1 . 27. Rickard MD, Honey RD, Brumley J L , Mitchell GF. Serological diagnosis and post-operative surveillance of human hydatid disease. 11. The enzyme-linked immunosorbent assay (ELISA) using various antigen preparations. Pathology 1984; 16: 21 1-20.
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