RECENT TRENDS IN THE SERODIAGNOSIS OF HYDATID DISEASE

SC Parija

Department of Microbiology, Jawaharlal Institute of Postgraduate Medical, Education and Research,Pondicherry--605006, India.

Abstract. Hydatid disease caused by Echinococcus granulosus is a zoonotic infection ofcosmopolitan distribution. As the clinical manifestations of hydatid disease in man are variable, thediagnosis of the condition presents complex problems for clinicians. Since the parasitic diagnosisof the disease is difficult, the specific diagnosis of the condition relies heavily on immunodiagnostictests. The recent approach to the diagnosis of hydatid disease in man is primarily based on: (I) acombination of two or three more serological tests to diagnose the condition, as a single test fails todetect all the cases, (2) detection of circulating hydatid antigen (CAg) in the serum by enzyme-linked immunosorbent assay (ELISA) and other assays, as the antigen detection system is useful inmonitoring post-surgical and chemotherapeutic evaluation of the cases as well as in the prognosis ofthe condition and (3) demonstration of E. granulosus antigen in the cystic fluid to establish theetiology of the hydatid cyst. Hydatid disease is essentially a disease of poor people residing in ruralareas, hence there is need for a simple, economic diagnostic immunoassay for use at the field level orin a rural health center with inadequate facilities. Counter-current-immunoelectrophoresis (ClEF)and bacterial eo-agglutination (Co-A), have been standardized and evaluated in this laboratory for thefirst time for detection of CAg in cases of hydatid disease at the field level and rural health center.

INTRODUCTION

This paper summarizes the studies on theimmunodiagnosis of hydatid disease in Pond i-cherry in relation to the studies elsewhere.This paper also describes observations of thepresent author regarding the developmentof simple and rapid immunoassays for thedetection of the circulating hydatid antigen inthe serum at the field level and rural healthcenters.

Hydatid disease, caused by the larval stage ofEchinococcus granulosus, is a chronic zoonoticdisease having a worldwide distribution andvariable geographical incidence (Williams et al,1971). The diagnosis of hydatid disease inhumans has presented complex problems forclinicians and laboratory workers. The clinicalsymptoms are invariably non-specific and asso-ciated with the pressure of hydatid cysts inthe tissues of the host. Direct demonstration ofthe parasite or parasitic materials by aspirationbiopsy of the hydatid cyst is inadvisable becauseof possible spillage resulting in the anaphylaxisand formation of secondary cysts. The specificdiagnosis of the condition by usual methods,such as radiography, Ultrasonography and com-

puterized axial tomography, used for thedetection of space-occupying lesions is oftendifficult. This has led to widespread interest inthe use of serological tests for the diagnosisof hydatid disease (Bhatia and Pathak, 1990).Immunodiagnostic tests play an important rolein early diagnosis and treatment of the condition,thereby considerably reducing morbidity andmortality due to the disease. Serological tests arebased primarily on the demonstration of circulat-ing anti-echinococcal antibodies which occurfrequently in established cystic hydatid infections(Kagan, 1968).

Even though India is not primarily a sheeprearing country, reports of hydatid disease haveincreased from different parts of India (Roy etai, 1970), such as Patna, Madras, Vellore, Gunur,Kurnool, Ahrnedabad, Delhi, Amritsar andJamnagar. It has also been reported fromAssam, West Bengal, Orissa, and HimachalPradesh, and areas in and around Pondicherry(Parija et ai, 1987b). Since the first documentedstudy of hydatid disease in Pondicherry (Parijaet ai, 1983) in early 1980, this laboratory hasworked to develop simple and rapid serologicaltests to detect circulating antibodies and, morerecently, antigens in the serum, to use at the field

371

FOOD ~BORNE PARASITIC ZOONOSIS

level and in rural health centers for the diagnosisof hydatid disease.

DETECTION OF ANTIBODIES

Since the first attempt to devise a serologicaltest by using a complement fixation test, anumber of serological tests have been developedfor the detection of circulating anti-echinococcalantibodies (Kagan, 1968) (Table 1). However,no single test provides complete sensitivity andspecificity for hydatid disease. A combinationof two or three tests, such as immunoelectro-phoresis and double diffusion, indirect immuno-

fluorescence, indirect hemagglutination andlatex agglutination, may be necessary to obtainreliable results (Chemtai et ai, 1981). Many ofthese immunodiagnostic tests have been usedwith varying sensitivity and specificity for thedemonstration of antibodies.

Hydatid disease in humans is essentially adisease of poor people who live in rural areas.Therefore, there is a need for a simple, economicimmunoassay which would permit diagnosis ofthe disease in the field or in rural healthcenters with inadequate laboratory facilities.An attempt was made along this line in thislaboratory, which is situated in an area endemic

Table IImmunodiagnostic tests in hydatid disease.

1. Complement fixation test Lower sensitivity (36-93%).Higher non-specificity (28%).Useful in post-op evaluation.Sensitivity 66-100% (Kagan, 1968).Specific.Sensitivity 66-100%.(Kagan, 1968).Highly sensitive (100%).Employed to identify antibodyimmunoglobulin classes and tolocate antigens in hydatid cystmaterial.

Capron et al Valuable in diagnosis of persistent1967 or recurrent infection.

Lower sensitivity,need for concentratingthe serum and antigen, not suitable formass screening as it requires largeamounts of serum and antigen.

Castagnari and Sensitive and specific.Sorice, Shorter test time, small volumes1971 of antigen and serum required.Coltorti and Sensitive and specific.Varela-Diaz1978Farag 1975

2. Hemagglutination test

l Latex agglutination testI

4. Indirect fluorescent antibody test

5~ Immunoelectrophoresis test (IEP)

6. Counter current immunoelectrophoresosj

test

7. Double diffusion test

8., ELISA

9. RadioimmunoassayI

10. Lymphocyte transformation test

Ghedini1906

Garabedian1957Fischman1960Fraga deAzevedo andRombert1965

Musiani1974Miggiano1966

As sensitive as IHA.Required small quantity ofantigen, suitable for automation.Higher sensitivity and specificity.

Valuable in pulmonary hydatiddisease where circulatingantibodies are absent.

372

SERODIAGNOSIS OF HYDATID DISEASE

for hydatid disease. The indirect hemaggluti-nation (IHA) test introduced by Garabedian et al(1957) for the serodiagnosis of hydatid disease,is inexpensive, easy to perform, and can beread without errors of subjectivity (Lewis et ai,1975). However, the conventional IHA in itspresent form is not suitable for use in a poorly-equipped rural health laboratory, primarily forthe following two reasons: First, the procedureis time-consuming, requiring three successivesteps of stabilization, tanning and sensitizationof the red blood cells (RBCs) with hydatid cystfluid antigen, each step being preceded andfollowed by the washing of RBCs with salineor buffer. Secondly, the shelf-life of the antigen-coated RBCs subjected to a single aldehydestabilization and tanning treatment is short, evenwhen stored at 4° C. Invariably the RBCs haveto be prepared each time the test is performed.Hence, an effort was made by the authors tomodify the IHA assay for routine application inless equipped laboratories and in those withless technical expertise, such as a rural healthlaboratory .

The IHA test could be a relatively simple andrapid diagnostic tool if it would be possible topreserve sensitized cells stored at 4°C for a longerperiod. In an earlier study (parija and Ananthakrish-nan, 1985), we evaluated the use of sheep RBCs,stabilized in various ways, in IHA tests onthe sera of 21 surgically confirmed cases ofhydatid disease and on control sera. Testswith double aldehyde stabilized cells (DAS)treated sequentially with pyruvic aldehyde,tannic acid and glutaraldehyde were moresensitive than tests with cells treated onlywith formaldehyde, glutaraldehyde or pyruvicaldehyde and subsequently tanned. The ex-perience of these authors with filariasis (Parijaet ai, 1987a) and amebiasis (Parija et ai, 1988)has also shown that the use of DAS cells greatlyincreased the sensitivity of the IHA and obviatedthe need for frequent preparation of sensitizedcells.

Overnight delay in hemagglutination isanother disadvantage. The use of sheep orhuman "0" RBCs, which are non-nucleated,requires an overnight incubation with test serato obtain the definite settling pattern ofhemagglutination. . It makes IHA a time con-suming procedure. To increase the speed of

the reaction, the nucleated cells from chicks,instead sheep, were evaluated in the IHA inhydatid disease (Parija et ai, 1986). The testwas performed with DAS chick cells on serumfrom 26 confirmed cases of hydatid disease and45 control sera. The results were comparedwith those obtained in an IHA test with DASsheep RBC using the same batch of sera]both tests were equally sensitive. The chickcells settled quickly and the result could bedetermined within 30-45 minutes. Heterophilicantigen was not a problem. This study alsoshowed that without lyophilization, the hydatidantigen sensitized DAS prepared chick cellsremain stable when stored at 4° C for 31 days.This study also established that sensitized DA$chick cells stored at 4°C for 31 days can btused as a ready-made reagent directly in theIHA test without affecting the sensitivity of thetest. Results could be obtained within 60 to 90minutes of receipt of the sera to be tested. Alsothe sensitized DAS RBCs may be prepared ina central laboratory and sent in a cold-chainto distant hospitals and laboratories to be usedthere for quick diagnosis of hydatid disease(Parija et ai, 1986). This makes the IHA lasimple and rapid procedure for serodiagnosis Ofhydatid disease at a less equipped laboratory. I

Even though the IHA test is widely used, ,itis not as sensitive as an ELISA or RIA becau,esome of the IgG bound to RBCs fails to cross-link the cells and hemagglutination does nbtoccur. Cross-linking could be achieved by t~euse of an anti-immunoglobulin (Coombs et ~/,1953). Alternatively, Staphylococcus aureus-bearing protein A can be used because thisprotein will bind IgG and subsequently aggluti-nate antigen sensitized RBCs. In this laboratory,we have developed a modification of the IHA,the protein A-IHA, which is as sensitive asELISA for the diagnosis of hydatid disea~e(Parija and Rao, 1986). In the modified assay,the protein A-IHA, Cowan's strain of S. aureuswhich contains protein A was used to enhancehemagglutination of sensitized red cells. Thetest was performed in parallel with the IliAtest on 31 sera from surgically confirmedcases of hydatid disease and on 45 sera fromhealthy blood donors. Use of S. aureus proteinA enhanced sensitivity of the test and greatlyincreased the titers obtained with most of ~e

B73

FOOD - BORNE PARASITIC ZOONOSIS

'sera. Since Wood 46 strain of S. aureus, which.lacks protein A, did not agglutinate sensitized!DAS cells in the absence of antibody, eo-jhemagglutination appears to be mediatedttbrough protein A and occurs only in thepresence of specific antibody. None of the seratfrom healthy blood donors showed false positivereactions. The sensitivity of this test compareswell with the ELISA. Reagents for the proteinr\-IHA are inexpensive, easily available andstable, The sensitized RBCs and preparedsuspension of S. aureus can be stored at 40 Cfor a longer period without loss of activity.the test is simple, inexpensive and does notrequire much technical skill; hence, it has~he potential for wide application in the sero-cjliagnosis of hydatid disease. The test has beenJPodified further by the use of chick RBCsipstead of human "0" RBCs, which makes theq::st a rapid procedure (Parija et ai, 1987a).

I As no single test provides complete sensitivityand specificity, the combination of two or threeserological tests have been suggested to givemost reliable results in the diagnosis of hydatiddisease (Chemtai et ai, 1981), but few effortshave been made to evaluate the possible use of<l:asoni's intradermal skin test along with aserological test. Our observation from a study

f 29 surgically-confirmed cases of hydatidisease showed that the IHA test and Casoni's

t st, when used alone had a sensitivity of 75%a d 47.3%, respectively, for the diagnosis ofh datid disease; however, when used in com-b nation could establish the diagnosis in 27( 3.1%) of 29 cases. In addition, the lHA test

as able to rule out 3 false positive reactionsm Casoni' s test. The later could diagnose

5 additional cases, which were negative by theI~ test. Thus, the combined use of Casoni'st t and the IHA test is suggested for furthere aluation in the reliable diagnosis of the hydatidd· ease (Parija and Rao, 1987).

II

I Many false negative reactions is the majorproblem associated with the immunodiagnosticmethods detecting the circulating antibodies.ne location, size and fertility of hydatid cystsin \the tissues as well as low antibody responsesinl the human host (Chemtai et ai, 1981) andtht presence of immune complexes (Pini et ai,

DETECTION OF ANTIGEN

37l

1983), are possible factors which contributeto the high incidence of false negatives inhydatid serology.

New avenues for the serodiagnosis of hydatidc!isease are needed using the approach of detect-ing circulating antigen in serum and other bodyfluids. Circulating antigens have been describedin a variety of bacterial, viral, fungal and para-sitic infections (Greenwood et ai, 1971; Lalithaet ai, 1989; Prince and Burke, 1970; Remingtonet ai, 1972; Tselentis et ai, 1981). In hydatiddisease, the detection of circulating antigen(CAg) will be important in monitoring thedisease, as it may more reliably reflect thanantibody titers, the viability and quantity of theparasite in the host (Eckart and Gottstein,1983). The CAg detection will also help in thediagnosis and post-surgical and chemothera-peutic followup of hydatid disease. The demon-stration of CAg may also provide useful infor-mation on antibody negative hydatid cases andthe status of the infection whether recent orpast. The CAg was detected in mice by experi-mental infection with Mesocestoides corti andE. multilocularis (Alikhan and Siboo, 1983;Sogdandares-Bemal et ai, 1981) and in rabbitby Taenia pisiformis (Craig, 1984). In humanhydatid disease, immunodiffusion-in-gel was thefirst qualitative test to be employed for thedetection of CAg in Greek patients by Tselentiset al (1981) and in Russian patients by Leikinaet al (1982). Recently, a sensitive double anti-body sandwich-ELISA was employed to detectCAg in the case of hydatid disease in Swit-zerland by Gottstein (1984). In another study,affinity purified rabbit and human antisera toantigen prepared from E. granulosus cysts wasused to detect CAg in British and Kenyanpatients by ELISA (Craig and Nelson, 1984).Thus, an ELISA is now available as a sensitiveimmunoassay to detect the antigen in humanhydatid disease. The ELISA, though highlysensitive, is expensive and requires technicalexpertise, perishable reagents and is difficultto adapt in under equipped laboratories. Asimple immunoassay to detect the circulatingantigen in the hydatid disease is yet to bedeveloped. Counter-current-immunoelectro-phoresis (ClEF) and bacterial coagglutination(Co-A) are the widely used simple and rapidimmunoassays for detecting antigens and anti-

SERODIAGNOSIS OF HYOA TlO DISEASE

bodies in a variety of parasitic, bacterial, fungaland viral infections. These two assays have highpotential in detecting CAg in cases of hydatiddisease. No reports are currently availablesuggesting the application of the two rapid andversatile immunoassays for the detection ofhydatid antigen. In this laboratory CIEP andCo-A have been developed and evaluated in astudy for the detection of circulating antigensin hydatid patients in Pondicherry. The Co-Awas shown to be sensitive and specific. Thetest could detect CAg in the sera of surgically-and ultrasound-proven hydatid disease casesand clinically suspected hydatid disease cases.The test is simple, reliable and rapid; the resultcould be obtained within 30 to 45 minutes ofreceipt of the sera (Parija and Malini Shariff,in press). Similarly, the CIEP could detect theCAg in 55.55% surgically-proven and 100%ultrasound-proven hydatid disease cases. Thetest was highly specific, no false positive reactionswere observed with sera from disease controls orhealthy controls (Malini Shariff and Parija, inpress). Therefore, the findings of the presentstudy demonstrate that the Co-A and CIEP couldbe employed as single and rapid diagnosticimmunoassays to detect circulating antigen inthe hydatid disease at the field or in less thanwell-equipped laboratories; hence, they deserveadditional evaluation for their wide applicationin the hydatid serology.

DETECTION OF ANTIGEN IN THEHYDATID FLUID

The diagnosis of hydatid cyst is confirmed bythe demonstration of daughter cysts and scolicesin the hydatid fluid, along with the histopatho-logical evidence of the germinal layer of the cyst.The routine diagnosis is not always feasible bythese methods in conditions where protoscoliceshave not been aspirated in the cyst fluid orare not present in the cyst. In such cases, itwould be useful to identify the fluid as being ofhydatid origin. An enzyme immunoassay todetect antigen in the hydatid cyst fluid, as analternative to the direct parasitological methodshas been reported recently (Craig et ai, 1986).In this laboratory the CIEP as well as Co-Awere successful in establishing the etiologicaldiagnosis of 4 (80%) hydatid cysts by detectingE. granulosus antigen in the cyst fluid (Parija

and Malini Shariff, unpublished observation).The results of our study suggest that CIEP andCo-A tests can be employed as simple and rapiddiagnostic procedures for the detection of antigenin hydatid fluid as an alternative to parasito-logical methods. We are now evaluating thesetwo assays on additional cases of hydatid cystsfor the demonstration of antigen in the hydatidfluid.

REFERENCES

Alikhan Z, Siboo R. Immune complexes in experi-mental alveolarhydatidosis. Tropenmed Parasitol1983; 34:187-92.

Bhatia BB, Pathak KLM. Echinococcosis. In: ParijaSC,ed. Reviewof Parasitic Zoonoses.New Delhi:AITBS Publishersand Distributors, 1990; 212-9.1

Chemtai AK, Bowry TR, Ahmad Z. Evaluation offive immunodiagnostic techniques in echino-coccosispatients. Bull WHO 1981; 59:767-72. I

Coombs RRA, Howard AN, Mynors LS. A sero-logical proceduretheoriticallycapableof detectingincomplete or non-precipitating antibodies tosolubleprotein antigens. Br J Exp Parasitol 1953;34:525-34.

Craig PS, Bailey W, Nelson OS. A specific test foridentificationof cyst fluid samplesfrom suspectedhumanhydatid. Trans R Soc Trop Med Hyg 1986;80:256-7.

Craig PS, Nelson GS. The detection of circulatingantigen in human hydatid disease. Ann Trop MedParasitol1984; 78:219-27.

Craig PS. Circulating antigens, antibodies andimmune complexes in experimental Taenia pisi-formis infections of rabbits. Parasitol 1984; 89:121-31.

Eckert J, Gottstein B. Advances in diagnostic andinvestigationalprocedures for parasitic zoonoses.In: Dunsmoe JD, ed. Trop Parasit Zoonoses,WAAVP,Perth. 1983; 73-90. 1

Garabedian GA, Matossian RM, Djanian AY. Anindirecthaemagglutinationtest for hydatiddisease.J Immunol1957; 78:269-72.

Gottstein B. An immunoassay for the detection ofcirculating antigens in human echinococcosis.Am J Trop Med Hyg 1984; 33:1105-91.

Greenwood VM, Whittle HC, Dominic Rajkovic O.CIE in diagnosis of meningococcal infections.Lancet 1971; 2:579.

375

FOOD - BORNE PARASITIC ZOONOSIS

Kagan IG. A review of serological tests for thediagnosis of hydatid disease. Bull WHO 1968; 39:25-37.

Lalitha MK., Sridharan G, John M. Co-agglutinationfor diagnosis of bacterial infections. Indian JPediatr 1989; 56:327-33.

Leikina ES, Kovrova EA, Krasovekaya NN. Detectionof circulating antigens in the blood stream ofpatients with hydatid disease, alveococcosis andtrichinosis. Med Parasitol Parasit Bolezni 1982;51: 7-15 (In Rus).

Lewis JW, Koss N, Kerstein MD. A review of echino-coccal disease. J Immunol 1957; 78:269-72.

Malini Shariff G, Parija SC. Counter-current-immunoelectrophoresis for serodiagnosis ofhydatid disease by detection of circulating hydatidantigen. J Microbiol Methods (In press).

Parija SC, Ananthzkrishnan N. Evaluation of stabilizedcells in the indirect haemagglutination test forechinococcosis. J Med Microbiol1985; 19:95-8.

Parija SC, Malini Shariff G. Co-agglutination (Co-A)test: A novel immunoassay for the detection ofcirculating antigen in the diagnosis of hydatiddisease (In press).

Parija SC, Kasinathan S, Rao RS. Long shelf lifeof antigen sensitized erythrocytes by doublealdehyde stabilization for the serodiagnosis ofamoebiasis by indirect haemagglutination.J Diarrheal Dis 1988; 6:29-34.

Parija SC, Mehta RB, Rao RS. A study on use of chickcells in protein A-antibody mediated haemagglu-tination for serodiagnosis of hydatid disease.Med Sci Res 1987a; 15:1509-10.

Parija SC, Mehta RB, Rao RS. Evaluation of stabilizedand sensitized human 0 cells in indirect haemag-glutination test for filariasis. Med Sci Res 1988;16:214-5.

Parija SC, Mishra SR, Rao RS. Sensitized chick cellsin iliA for echinococcosis. J Med Microbiol1986; 22:243-5.

Parija SC, Rao RS. Enhancement of sensitivity of thehaemagglutination test for echinococcosis by use

of Staphylococcus auraus protein A. J MedMicrobiol1986; 22:241-4.

Parija SC, Rao RS. Evaluation of combined use ofindirect haemagglutination test and Casoni's skintest in diagnosis of hydatid disease. Indian JPathol Microbiol1987; 30:117-21.

Parija SC, Rao RS, Badrinath S, Sengupta ON.Hydatid disease in Pondicherry. Trop Med Hyg1983; 86: 113-5.

Parija SC, Sasikala K, Rao RS. Serological survey ofhydatid disease in Pondicherry, India. Trans RSac TropMed Hyg 1987; 81:802-3.

Prince AM, Burke K. Serum hepatitis antigen. Rapiddetection by high voltage immunoelectrophoresis.Science 1970; 169:519-22.

Pini C, Pastone R, Valesini G. Circulating immunecomplexes in sera of patients infected withEchinococcus granulosus. Clin Exp Immunol1983; 51:572-8.

Roy SC, Chakravarthy M, Das MM, Chatterjee PP.World incidence of hydatid disease in generaland pulmonary hydatid disease in particular withspecial reference to India. J Indian Med Assac1970; 55:212-7.

Remington IS, Gaines 10, Gilmer MA. Demonstrationof Candida precipitins in human sera by counterimmunoelectrophoresis. Lancet 1972; 2:519-20.

Sogandares-Bernal F, Race MC, Dennis MV, VogeM. Circulating antigens in infections of mice bytetrathyridis of Mesocestoides corti Hoeppli. ZParasitenkd 1981; 64:157-67.

Tse1entis J, Phataris J, Giannopolos A, Golematis B,Melissinos K. Ruptured echinococcal cysts andtheir immunological diagnosis. Acta MicrobialHellenica 1981; 26:337-42.

Williams IF, Porez Esandi MV, Oriol R. Prevalenceand distribution of hydatidosis with specialreference to the Americas. Am J Trop Med Hyg1971; 20:224-35.

376