101 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 71, No. 2,1977.
The immunological Department of Bacteriology
diagnosis of human hydatid disease ROBERT M. MATOSSIAN,* and Virology, American University, Beirut, Lebanon
Summary There is evidence that the yearly incidence rate of hydatid disease may be increasing in endemic areas and in regions of the world hitherto free of the infection. The availability of an accurate method of detection in both man and other animals would facilitate understanding of the global epidemiology. The diagnostic procedures in use in human hydatidosis have often given inconclusive results in animal infections. The following tests are discussed and their usefulness appraised: intradermal, complement fixation, haemagglutination, latex agglutination, indirect fluorescent antibody, immunoelectrophoresis, counter immunoelectrophoresis, enzyme linked immunosorbent assay, radio-immuno assay and lymphocyte transformation. Introduction “It is unwise to dismiss hydatid disease as a curiosity.” Lancet, 1976, ii, 352. Echinococcosis is a cyclozoonotic infection having a world-wide distribution and a variable geographic incidence. The larval cystic states of Echinococcusgranulosus and E. multilocularis, two closely related species of cestodes, are responsible for most of the known cases of the disease. Infections with the first are endemic in South America, most of Europe, the Mediterranean countries and the Middle East, and have also occurred in Australia, New Zealand, East Africa, South Africa and India (WILLIAMS et al., 1971). Multilocular echinococcosis coexists with hydatid disease in the U.S.S.R., Central Europe, Turkey, Japan, Alaska and Canada (LUKASHENKO, 1971). Recent observations confirm the impression that the disease constitutes a serious health hazard and that the yearly incidence rate may be increasing in endemic areas as well as in regions of the world hitherto free from the infection (WILLIAMS et al., 1971). The increased prevalence of equine hydatidosis in England (THOMPSON & SMYTH, 1975) and the high incidence of E. granulosus adults in Kuwaity dogs (HASSOUNAH& BEHBEHANI, 1976) are actual indicators of such a threat. Estimates of human hydatid infections have been hampered by the difficulties involved in the collection of accurate data. Exposure rates cannot be assessedbecause the percentage of people who develop overt illness or have a reactive serology following infection is unknown. Furthermore, variations in clinical manifestations make a presumptive diagnosis uncertain. Surgical cases, though providing reliable information, may constitute a fraction of the total infected population. In most countries there is also a paucity of autopsy material. Knowledge about the prevalence of hydatid cysts in slaughtered domestic *At present, Senior Visitor, Department of Medical Helminthology, London School of Hygiene and Tropical Medicine, London, England.
animals may provide sufficient background. However, cultural, economic and other factors have often influenced the accuracy of such data. The prevalence of the adult cestodes in the definitive host indicates the endemicity of the infection but may not relate to the extent of human infections. Sero-epidemiological surveys have been conducted to determine the magnitude of the infection in different populations. The lack of sensitivity and specificity of the methods employed have often made the interpretation of results difficult. The availability of an accurate method of detection would facilitate understanding of the global epidemiology of echinococcosis. E. granulosus requires two hosts to complete its full cycle of development. Having gained access into the susceptible mammalian intermediate host through the ingestion of viable ova, the taeniid larva undergoes successive phase changes to attain the final cystic form, the fertile stage infective to the dog, its definitive host. These activities are regulated by excretory and/or secretory (ES) substances produced under the genetic influence of the parasite DNA. The functional (ES) antigens of the transforming oncosphere, the developing germinal membrane and the metabolizing protoscoleces may thus incite the production of a wide spectrum of antibodies (ARAJ et al., 1977). The recognition of a specific immune response is the raison d’Ctre of a sensitive and accurate diagnostic procedure. This is dependent on factors inherent in the technique adopted and on the outcome of the relationship between the parasite and the host. Thus, animals with rapidly growing cysts show a greater reactivity than those with immature, dead or infected ones. Ideally, the test with the highest sensitivity and accuracy would be considered as the method of choice. A positive reaction should provide diagnostic evidence for exposure to an infective agent. It should also differentiate an active infection from a healed condition. The proper interpretation of a result isessential to theadequate management of a patient. Diagnostic procedures and their evaluation The procedures described herewith apply to human hydatidosis. Their use in animal infections has often been inconclusive (PAULIJZZI & CASTAGNARI,1965; KAGAN, 1968). The diagnosis of alveolar echinococcosis presents an additional challenge. Similar methods, using common or specific multilocularis antigens, have given promising results (CAPRON et al., 1970). Inconsistencies in results have often been ascribed to variations in antigenic material. Hydatid cyst fluid (HF) is a repository of somatic and functional antigens of parasite origin. It also contains variable amounts of host proteins. Dialysed HF antigen has been used by most workers. Others have employed affinity chromatography and polyacrylamide gel electrophoresis to concentrate and separate individual reactive components (POZZIJOLI et a/., 1975). Viable or dead protoscoleces (P) provide
102
SYMPOSIUM
ON HYDATID
potent antigens. HF and P have common and independent reactive fractions. Phase specific antigens, obtained by in vitro culture of P, may be of value in early diagnosis. (i) The Intradermal
(ID) Test
An immediate hypersensitivity reaction, produced by the intradermal injection of minimal amounts of hydatid antigen, has been used for diagnostic and epidemiologic purposes. The test has a high sensitivity, but its low specificity has made the interpretation of results impossible (KAGAN, 1968; CAPRONer al., 1976). (ii) The Complement-Fixation
(CF) Test
The CF test has met with a mixed reception. Variations in its sensitivity, ranging from 36 to 93 “/” of the known infected cases, have often discouraged its use (KAGAN, 1968). Standardized, potent antigens and adequate controls have improved the accuracy of the method (BRADSTREET,1969). The CF test is of value in detecting recurrent illness (MATOSSIAN& ARAJ, 1975). (iii)
The Haemagglutination
(HA) Test
The HA test is based on the principle that adsorption of hydatid antigen on tanned erythrocytes causes their subsequent agglutination upon contact with specific antibodies. The test has proved to be a sensitive and specific diagnostic method for human hydatidosis (GARABEDIANet al., 1957; KAGAN, 1968; MATOSSIAN& ARAJ, 1975). The use of formalinized (KAGAN, 1968) or pyruvic-aldehyde treated erythrocytes (MATOSSIAN & KANE, 1971) has prolonged the storage time of sensitized cells. A slide HA test, using chromic-chloride treated erythrocytes, sensitized by exposure to formalinized hydatid antigen has produced results comparable to the tube test (MATOSSIAN et a/., 1976). (iv) The Latex-Agglufinafion
(LA) Test
Latex particles, coated with hydatid antigen, are agglutinated upon contact with corresponding antibody. Introduced by FISCHMAN (1960) the LA test has been found to be an easy, specific and sensitive slide agglutination test, useful in hydatid detection and in population surveys (VARELA-DIAZ et al., 1976). Confirmation of the diagnosis is obtained by immunoelectrophoresis (IEP). (v) The Indirect-Fluorescent-Antibody
(IFA) Test
The use of particulate antigens made visible through immunofluorescence has helped in the diagnosis, therapeutic follow-up and sero-epidemiological surveys of toxoplasmosis, malaria, schistosomiasis, hydatidosis and others (AMBROISE-THOMAS, 1976). The antigen, when absorbed on a glass surface, forms a complex with specific antibody. Addition of an anti-human globulin conjugate induces fluorescence. MATTOSIAN et al. (1972) described IgG, IgM and IgA antibodies detectable by this method. Further studies revealed the inconsistent detection of IgM and IgA hydatid antibodies. The IFA test is sensitive, but occasional reactivity of samples in patients with hepatic disorders may hinder its interpretation. (vi) The Immunoelectrophoresis
(fEP) Test
In this test there is an initial separation, by electrophoresis, of the fractions present in an antigenic complex. The separated antigens are subsequently made to react with serum to determine the formation of precipitation bands, caused by antigen-antibody reactions. CAPRON et a/. (1967) reported that the diagnosis of human hydati-
DISEASE
dosis was possible by IEP through the demonstration of an arc of precipitation that was specific for E. granulosus. The antigen corresponding to this arc, described as “Fraction 5”, was subsequently purified from hydatid fluid (BOUT et al., 1974). The formation of E. granulosus arc 5 by a serum is considered as the only specific diagnostic sign for the disease (VARELA-DIAZ & COLTORTI, 1976). The need for concentrated serum and antigen has limited the widescale use of the procedure. (vii)
The Counter-Immunoelectrophoresis
(CIE)
Test
CIE has been adopted in the diagnosis of viral, bacterial, mycotic, protozoa1 and helminth infections (DRAPER, 1976). It produces counter migration, through a gel, of antigens and immunoglobulins, in an electric field, and on a narrow front, so that maximal amounts are driven towards each other and form a line of precipi& SORICE (1971) adapted CIE to tate. CASTAGNARI hydatid disease and described it as a sensitive and specific method. Technical shortcomings have made the interpretation of the test difficult (LOPEZ-LEMES & VARELA-DIAZ. 1975). P~NON(1976). using a celluloseacetate mem&ane, ‘obtained ‘spe&c res& with 400 hydatid and 6,500 miscellaneous samples. (,viii) The Enzyme Linked Immunosorbent Assay (ELISA) ELISA, pioneered by ENGVALL et al., 1971, 1972,
has been successfully applied to the diagnosis of viral, bacterial and parasitic infections (VOLLER et al., 1976). The test is conducted by coupling an antigen to a solid surface in a tube. Serum, when incubated with the sensitized carrier, forms an antigen-antibody complex. An enzyme-labelled antiglobulin (i.e. alkaline phosphatase-labelled anti-IgG), when added, combines with the complex and cannot be washed away. The amount of residual conjugate is measured photometrically by the quantity of substrate it degrades. FARAG et al. (1975) applied ELISA to the diagnosis of human hydatidosis. When used with a purified antigen (Fraction 5), ELISA was found to be a simple, specific and sensitive diagnostic procedure. (ix) The Radio-immuno
Assay (RIA)
RIA uses tracer amounts of labelled antigen to determine the percentage of unlabelled antigen bound by specific antibodies. The percentage binding of the labelled antigen by the serum is compared with that of the standard sample, making the calculation of the unknown serum possible (SELFet al., 1976). MUSIANI et al. (1974) described the use of solid-phase RIA in the diagnosis of human hydatidosis. DESSAINT et al. (1975) isolated specific hydatid antibodies by the use of an immusorbent. These were subsequently measured by RIA to evaluate the IgE class of antibodies. Elevated levels of IgE antibodies were observed in 60 % of the patients. (x) Lymphocyte
Transformation
(LT)
Lymphocytes collected from animals previously sensitized to a wide variety of antigens undergo proliferation when in contact with the relevant antigen in vitro (GREAVES et al., 1974). MIGGIANO et al. (1966) produced blastic transformation and mitosis of blood lymphocytes from hydatid patients, upon exposure to E. granulosusantigens. YUSUFef al. (1975) observed a significant increase in the stimulatory indices of lymphocytes of hydatid patients incubated with HF and P antigens. ARAJ et al. (1977) described the time-related increase in thymidine uptake of spleen cells from syngeneic mice with secondary
ROBERT
M.
hydatidosis. The LT assay requires additional supportive data before its routine application. It may be of value in pulmonary hydatidosis where circulating antibodies are often absent. Conclusions
Data available about the overt manifestations of hydatidosis suggest that echinococcal infections are increasing in both human and animal populations of the world. However, evidence about the exact distribution and geographic incidence can only be obtained through extensive studies based on the determination of a specific immune response to the invasive agent. The procedures used for hydatid diagnosis have varying degrees of sensitivity and specificity. For sero-epidemiological surveys, a simple sensitive test can be readily used. Whenevernecessary,reactivesamplesshouId beconfirmed by tests having greater specificity. References
Ambroise-Thomas, P. (I 976). Immunofluorescence in the diagnosis, therapeutic follow-up and sero-epidemiological studies of some parasitic diseases. Transactions of the Ro.val Society of Tropical Medicine and Hygiene,
70, 107-l 12. Araj, G. F., Matossian, R. M. & Frayha, Cl. J. (1977). The host response in secondary hydatidosis of mice. I. Circulating antibodies. Zeitschrift ftr Parasitenkunde (submitted). Araj, G. F., Matossian, R. M. & Malakian, A. H. (1977). The host response to secondary hydatidosis of mice. II. Cell mediated immunity. Zeitschrif fir Parasitenkunde (,submitted). Bout, D., Fruit, J. & Capron, A. (1974). Purification d’une antigbne specifique de liquide hydatique. Annales d’lmmunologie
(Institut
Pasteur), 125C, 755-788.
Bradstreet, C. M. P. (1969). A study of two immunological tests in the diagnosis and prognosis of hydatid disease. Journal of Medical Microbiology, 2,419-433. Capron, A., Vernes, A. & Fruit, J. (1970). Diagnostic immunologique de I’echinococcose alveolaire. Revue Mhdicai% Franraise, 45, 307-3 IO. Capron, A., Vernes, A., Dessaint, J. P. & Capron, M. (1976). Contribution a l’etude de I’hvoersensibilite dans i’echinococcose hydatique: A prop& de 111observations. Revue Franraise d’Allergologie. 16, 9-15. Castagnari, L. & Sorice, F. (1971). Crossed-over electrophoresisin the serological diagnosis of hydatid disease. Preliminary note. Giornale di Malatti Itlfettive e Parassitarie,
23, 225-227.
Dessaint, J. P., Bout, D., Wattre, P. & Capron, A. (1975). Quantitative determination of specific IgE antibodies to Echinococcus granulosus and IgE levels in sera from patients with hydatid disease. Immunology, 29, 813-823. Draper, C. C. (1976). The use of counter-immunoelectrophoresis in immunodiagnosis. Transactions of the Roval Societv of‘ Tropical Medicine and Hvaiene. _70,93:97. . . Engvall, E. & Perlmann. P. (1971). ELISA. Quantitative assay of immunoglobulin G. Immunochemi.stry, 8, 871-874. Engvall, E., Johnsson, K. & Perlmann, P. (1971). ELISA II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme labelled antigen and antibody coated tubes. Biochimiea et Biophysics Acta. 251,427434.
IO3
MATOSSIAN
Engvall, E. & Perlmann, P. (1972). ELISA III. Quantitation of specific antibodies by enzyme-labelled antiimmunoglobulin in antigen coated tubes. Journal of Immunology, 109, 129. Farag, H., Bout, D. & Capron, A. (1975). Specific immunodiagnosis of human hydatidosis by the enzymelinked immunosorbent assay (ELISA). Biomedicine, 23, 276-278. Fischman, A. (1960). Flocculation tests in hydatid disease. Journal of Clinical Pathology, 13,72-75. Garabedian, Cl. A., Matossian, R. M. & Djanian. A. Y. (1957). An indirect haemagglutination test for hydatid disease. Journal of Immunologic, 78, 269-272. Greaves, M. F., Owen, J. J. T. & Raff, M. C. ( 19741. “T and B lymphoc~~tes: Origins, properties und roles in immune responses.” Excerpta Medica, Amsterdam.
Hassounah, 0. & Behbehani, K. (1976). The epidemiology of Echinococcus infection in Kuwait. Journal of’ Helmintholog)l,
50, 65-73.
Kagan, 1. G. (I 968). A review of serological tests for the diagnosis of hydatid disease. Bulletin of the World Health Orrranization. .,
39. 25-37.
Lancet (1976). The invisible worm. (Editorial) Lam-et,ii, 5522553. Lopez-Lemes, M. H. & Varela-Diaz, V. M. (1975). Evaluation of the crossed-over electrophoresis test for the immunodiagnosis of human hydatid disease. Tropical and Geographical
Medicine,
27, 2955300.
Lopez-Lemes, M. H. & Varela-Diaz, V. M. (1975). Application of the immunoelectrophoresis test for hydatidosis in patients with a presumptive diagnosis of the disease. Tropical und Geographical Medicine, 27, 301-304. Lukashenko, N. P. (1971). Problems ofepidemiology and prophylaxis of alveococcosis (multilocular echinococcosis): A general review - with particular reference to the U.S.S.R. Intwnational Journal jiw Parasitology, I, 125-134. Matossian, R. M. & Kane,G. J. (1971). The use of stable freeze-dried erythrocytes as antigens in hydatid diagnosis. Lebanese Medical Journal, 24, 227-230. Matossian, R. M., Kane, G. J., Chantler, S. M., Batty, I. & Sarhadian, H. (1972). The specific immunoglobulin in hydatid disease. Immunolog.l~, 22, 423-430. Matossian. R. M. & Araj, G. F. (1975). Serologic evidence of the post-operative persistence of hydatid cysts in man. Journal of H)agie/le, 75, 333-340. Matossian, R. M., Mamo, A. J. & Dakroub, R. (1976). Slide haemagglutination test in hydatid disease: a correlative study of diagnostic procedures. Journal of Clinicul Pathology,, 29, 3941. Miggiano, V. C., Ferrari, S. & Ingrao. F. (1966). Echinococcus granulosus and lymphocyte stimulation. Lancer, ii, p. 7422. Musiani, P., Piantelli, M., Arru, E. & Pozzuoli, R. (1975). A solid phase radioimmunoassay for the diagnosis of human hydatidosis. Journal of’ Immunologic, 112. 167441679. Pinon, J. M. (1976). Counter immunoelectrophoresis in the diagnosis of hydatid disease. Lancer, ii, p. 310. Pozzuoli, R., Piantelli, M., Perucci, C., Arru, E. & Musiani. P. (1975). Isolation of the most immunoreactive antigens of Echino~occusgranul~~srrs from sheep hydatid fluid. Journal o/‘Immunolog.l~, 115, 1459-1463. Self, M., Rees, H. & Landon, J. (1976). An introduction to radioimmunoassay. Medical Laborator,, Sciences, 33, 221-228.
104
SYMPOSIUM
ON HYDATID
Thompson, R. C. A. & Smyth, J. D. (1975). Equine hydatidosis: A review of the current status in Great Britain and the results of an epidemiological survey. Veterinary Parasitology, 1, 107-127. Varela-Diaz, V. M. & Coltorti, E. A. (1976). Techniques for the Immunodiagnosis
of Human
Hydatid
Disease,
Pan-American Zoonoses Center, Buenos Aires, Argentina. Varela-Diaz. V. M.. Coltorti. E. A.. Ricardes. M. I.. Prezioso, ‘U., Schantz, P. k. & barcia, R.’ (1976): Evaluation of immunodiagnostic techniques for the detection of human hydatid cyst carriers in field studies, American Journal of Tropical Medicine and
DISEASE
Hygiene, 25, 617-622. Voller, A., Bidwell, D. E. & Bartlett, A. (1976). Enzyme immunoassays in diagnostic medicine. Theory and practice. Bulletin of the World Health Organization, 53, 55-65.
Williams, J. F., Adaros, H. L. & Trejos, A. (1971). Current prevalence and distribution of hydatidosis, with special reference to the Americas. American Journal of Trooical Medicine and Hvaiene. 20.224-235.
Yusuf, J. N., Malakian, A. H. & F;ayha,‘G.‘J. (1975). Echinococcus grunulosus: the stimulation of host lymphocytes by parasite antigens. Experimental Parusitology, 38,30-37.
The immunological Department of Bacteriology
diagnosis of human hydatid disease ROBERT M. MATOSSIAN,* and Virology, American University, Beirut, Lebanon
Summary There is evidence that the yearly incidence rate of hydatid disease may be increasing in endemic areas and in regions of the world hitherto free of the infection. The availability of an accurate method of detection in both man and other animals would facilitate understanding of the global epidemiology. The diagnostic procedures in use in human hydatidosis have often given inconclusive results in animal infections. The following tests are discussed and their usefulness appraised: intradermal, complement fixation, haemagglutination, latex agglutination, indirect fluorescent antibody, immunoelectrophoresis, counter immunoelectrophoresis, enzyme linked immunosorbent assay, radio-immuno assay and lymphocyte transformation. Introduction “It is unwise to dismiss hydatid disease as a curiosity.” Lancet, 1976, ii, 352. Echinococcosis is a cyclozoonotic infection having a world-wide distribution and a variable geographic incidence. The larval cystic states of Echinococcusgranulosus and E. multilocularis, two closely related species of cestodes, are responsible for most of the known cases of the disease. Infections with the first are endemic in South America, most of Europe, the Mediterranean countries and the Middle East, and have also occurred in Australia, New Zealand, East Africa, South Africa and India (WILLIAMS et al., 1971). Multilocular echinococcosis coexists with hydatid disease in the U.S.S.R., Central Europe, Turkey, Japan, Alaska and Canada (LUKASHENKO, 1971). Recent observations confirm the impression that the disease constitutes a serious health hazard and that the yearly incidence rate may be increasing in endemic areas as well as in regions of the world hitherto free from the infection (WILLIAMS et al., 1971). The increased prevalence of equine hydatidosis in England (THOMPSON & SMYTH, 1975) and the high incidence of E. granulosus adults in Kuwaity dogs (HASSOUNAH& BEHBEHANI, 1976) are actual indicators of such a threat. Estimates of human hydatid infections have been hampered by the difficulties involved in the collection of accurate data. Exposure rates cannot be assessedbecause the percentage of people who develop overt illness or have a reactive serology following infection is unknown. Furthermore, variations in clinical manifestations make a presumptive diagnosis uncertain. Surgical cases, though providing reliable information, may constitute a fraction of the total infected population. In most countries there is also a paucity of autopsy material. Knowledge about the prevalence of hydatid cysts in slaughtered domestic *At present, Senior Visitor, Department of Medical Helminthology, London School of Hygiene and Tropical Medicine, London, England.
animals may provide sufficient background. However, cultural, economic and other factors have often influenced the accuracy of such data. The prevalence of the adult cestodes in the definitive host indicates the endemicity of the infection but may not relate to the extent of human infections. Sero-epidemiological surveys have been conducted to determine the magnitude of the infection in different populations. The lack of sensitivity and specificity of the methods employed have often made the interpretation of results difficult. The availability of an accurate method of detection would facilitate understanding of the global epidemiology of echinococcosis. E. granulosus requires two hosts to complete its full cycle of development. Having gained access into the susceptible mammalian intermediate host through the ingestion of viable ova, the taeniid larva undergoes successive phase changes to attain the final cystic form, the fertile stage infective to the dog, its definitive host. These activities are regulated by excretory and/or secretory (ES) substances produced under the genetic influence of the parasite DNA. The functional (ES) antigens of the transforming oncosphere, the developing germinal membrane and the metabolizing protoscoleces may thus incite the production of a wide spectrum of antibodies (ARAJ et al., 1977). The recognition of a specific immune response is the raison d’Ctre of a sensitive and accurate diagnostic procedure. This is dependent on factors inherent in the technique adopted and on the outcome of the relationship between the parasite and the host. Thus, animals with rapidly growing cysts show a greater reactivity than those with immature, dead or infected ones. Ideally, the test with the highest sensitivity and accuracy would be considered as the method of choice. A positive reaction should provide diagnostic evidence for exposure to an infective agent. It should also differentiate an active infection from a healed condition. The proper interpretation of a result isessential to theadequate management of a patient. Diagnostic procedures and their evaluation The procedures described herewith apply to human hydatidosis. Their use in animal infections has often been inconclusive (PAULIJZZI & CASTAGNARI,1965; KAGAN, 1968). The diagnosis of alveolar echinococcosis presents an additional challenge. Similar methods, using common or specific multilocularis antigens, have given promising results (CAPRON et al., 1970). Inconsistencies in results have often been ascribed to variations in antigenic material. Hydatid cyst fluid (HF) is a repository of somatic and functional antigens of parasite origin. It also contains variable amounts of host proteins. Dialysed HF antigen has been used by most workers. Others have employed affinity chromatography and polyacrylamide gel electrophoresis to concentrate and separate individual reactive components (POZZIJOLI et a/., 1975). Viable or dead protoscoleces (P) provide
102
SYMPOSIUM
ON HYDATID
potent antigens. HF and P have common and independent reactive fractions. Phase specific antigens, obtained by in vitro culture of P, may be of value in early diagnosis. (i) The Intradermal
(ID) Test
An immediate hypersensitivity reaction, produced by the intradermal injection of minimal amounts of hydatid antigen, has been used for diagnostic and epidemiologic purposes. The test has a high sensitivity, but its low specificity has made the interpretation of results impossible (KAGAN, 1968; CAPRONer al., 1976). (ii) The Complement-Fixation
(CF) Test
The CF test has met with a mixed reception. Variations in its sensitivity, ranging from 36 to 93 “/” of the known infected cases, have often discouraged its use (KAGAN, 1968). Standardized, potent antigens and adequate controls have improved the accuracy of the method (BRADSTREET,1969). The CF test is of value in detecting recurrent illness (MATOSSIAN& ARAJ, 1975). (iii)
The Haemagglutination
(HA) Test
The HA test is based on the principle that adsorption of hydatid antigen on tanned erythrocytes causes their subsequent agglutination upon contact with specific antibodies. The test has proved to be a sensitive and specific diagnostic method for human hydatidosis (GARABEDIANet al., 1957; KAGAN, 1968; MATOSSIAN& ARAJ, 1975). The use of formalinized (KAGAN, 1968) or pyruvic-aldehyde treated erythrocytes (MATOSSIAN & KANE, 1971) has prolonged the storage time of sensitized cells. A slide HA test, using chromic-chloride treated erythrocytes, sensitized by exposure to formalinized hydatid antigen has produced results comparable to the tube test (MATOSSIAN et a/., 1976). (iv) The Latex-Agglufinafion
(LA) Test
Latex particles, coated with hydatid antigen, are agglutinated upon contact with corresponding antibody. Introduced by FISCHMAN (1960) the LA test has been found to be an easy, specific and sensitive slide agglutination test, useful in hydatid detection and in population surveys (VARELA-DIAZ et al., 1976). Confirmation of the diagnosis is obtained by immunoelectrophoresis (IEP). (v) The Indirect-Fluorescent-Antibody
(IFA) Test
The use of particulate antigens made visible through immunofluorescence has helped in the diagnosis, therapeutic follow-up and sero-epidemiological surveys of toxoplasmosis, malaria, schistosomiasis, hydatidosis and others (AMBROISE-THOMAS, 1976). The antigen, when absorbed on a glass surface, forms a complex with specific antibody. Addition of an anti-human globulin conjugate induces fluorescence. MATTOSIAN et al. (1972) described IgG, IgM and IgA antibodies detectable by this method. Further studies revealed the inconsistent detection of IgM and IgA hydatid antibodies. The IFA test is sensitive, but occasional reactivity of samples in patients with hepatic disorders may hinder its interpretation. (vi) The Immunoelectrophoresis
(fEP) Test
In this test there is an initial separation, by electrophoresis, of the fractions present in an antigenic complex. The separated antigens are subsequently made to react with serum to determine the formation of precipitation bands, caused by antigen-antibody reactions. CAPRON et a/. (1967) reported that the diagnosis of human hydati-
DISEASE
dosis was possible by IEP through the demonstration of an arc of precipitation that was specific for E. granulosus. The antigen corresponding to this arc, described as “Fraction 5”, was subsequently purified from hydatid fluid (BOUT et al., 1974). The formation of E. granulosus arc 5 by a serum is considered as the only specific diagnostic sign for the disease (VARELA-DIAZ & COLTORTI, 1976). The need for concentrated serum and antigen has limited the widescale use of the procedure. (vii)
The Counter-Immunoelectrophoresis
(CIE)
Test
CIE has been adopted in the diagnosis of viral, bacterial, mycotic, protozoa1 and helminth infections (DRAPER, 1976). It produces counter migration, through a gel, of antigens and immunoglobulins, in an electric field, and on a narrow front, so that maximal amounts are driven towards each other and form a line of precipi& SORICE (1971) adapted CIE to tate. CASTAGNARI hydatid disease and described it as a sensitive and specific method. Technical shortcomings have made the interpretation of the test difficult (LOPEZ-LEMES & VARELA-DIAZ. 1975). P~NON(1976). using a celluloseacetate mem&ane, ‘obtained ‘spe&c res& with 400 hydatid and 6,500 miscellaneous samples. (,viii) The Enzyme Linked Immunosorbent Assay (ELISA) ELISA, pioneered by ENGVALL et al., 1971, 1972,
has been successfully applied to the diagnosis of viral, bacterial and parasitic infections (VOLLER et al., 1976). The test is conducted by coupling an antigen to a solid surface in a tube. Serum, when incubated with the sensitized carrier, forms an antigen-antibody complex. An enzyme-labelled antiglobulin (i.e. alkaline phosphatase-labelled anti-IgG), when added, combines with the complex and cannot be washed away. The amount of residual conjugate is measured photometrically by the quantity of substrate it degrades. FARAG et al. (1975) applied ELISA to the diagnosis of human hydatidosis. When used with a purified antigen (Fraction 5), ELISA was found to be a simple, specific and sensitive diagnostic procedure. (ix) The Radio-immuno
Assay (RIA)
RIA uses tracer amounts of labelled antigen to determine the percentage of unlabelled antigen bound by specific antibodies. The percentage binding of the labelled antigen by the serum is compared with that of the standard sample, making the calculation of the unknown serum possible (SELFet al., 1976). MUSIANI et al. (1974) described the use of solid-phase RIA in the diagnosis of human hydatidosis. DESSAINT et al. (1975) isolated specific hydatid antibodies by the use of an immusorbent. These were subsequently measured by RIA to evaluate the IgE class of antibodies. Elevated levels of IgE antibodies were observed in 60 % of the patients. (x) Lymphocyte
Transformation
(LT)
Lymphocytes collected from animals previously sensitized to a wide variety of antigens undergo proliferation when in contact with the relevant antigen in vitro (GREAVES et al., 1974). MIGGIANO et al. (1966) produced blastic transformation and mitosis of blood lymphocytes from hydatid patients, upon exposure to E. granulosusantigens. YUSUFef al. (1975) observed a significant increase in the stimulatory indices of lymphocytes of hydatid patients incubated with HF and P antigens. ARAJ et al. (1977) described the time-related increase in thymidine uptake of spleen cells from syngeneic mice with secondary
ROBERT
M.
hydatidosis. The LT assay requires additional supportive data before its routine application. It may be of value in pulmonary hydatidosis where circulating antibodies are often absent. Conclusions
Data available about the overt manifestations of hydatidosis suggest that echinococcal infections are increasing in both human and animal populations of the world. However, evidence about the exact distribution and geographic incidence can only be obtained through extensive studies based on the determination of a specific immune response to the invasive agent. The procedures used for hydatid diagnosis have varying degrees of sensitivity and specificity. For sero-epidemiological surveys, a simple sensitive test can be readily used. Whenevernecessary,reactivesamplesshouId beconfirmed by tests having greater specificity. References
Ambroise-Thomas, P. (I 976). Immunofluorescence in the diagnosis, therapeutic follow-up and sero-epidemiological studies of some parasitic diseases. Transactions of the Ro.val Society of Tropical Medicine and Hygiene,
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