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Immunodiagnosis of Ecchinococcus granulosus infestation.
Immunological techniques in diagnosis of E. granulosus infestation have been applied since last three decades. Immuno diffusion studies carried out by many have evolved varied results. Countercurrent immunoelectrophoresis reported by Kelkar and Kotwal[5] has showed encouraging results in recent years. This study was conducted to correlate the clinical and immunological features of E. granulosus infestation and to evaluate the diagnostic accuracy of counter-current immunoelectrophoresis test, agar gel diffusion test and intradermal Casoni's test. It also enabled the author to compare the efficacy of all the three tests in individual organs affected with E. granulosus cyst.
Thirty patients of E. granulosus cyst disease, proved histopathologically, seen at the Government Medical College, Aurangabad between January 1974 and December 1976 are included in the present study. Patients, after having suspected clinically and radiologically to be suffering from hydatid cyst disease underwent immunological studies to support the diagnosis. Subsequently, surgical exploration was done to confirm the diagnosis. Technique of Immunological Tests Antigen: Ecchinoccus cyst fluid was obtained by sterile puncture of the cyst. The fluid was then filtered and lyophilised and stored at 4°C. This fluid was used as antigen in all the three tests. Sera: 10 ml blood was collected from an antecubital vein of the patient and the serum was separated. These sera were stored at 20°C. after adding sodium azide in 1;10,000 dilution. Counter-current immunoelectrophoresis This test is based on the principle of detecting the precipitating antibodies in the patient's serum by micro electrophoresis. Electric current when passed through micro immunoelectrophoresis chamber enhanced the movement of the antigen and antibody towards each other producing a precipitate (Kelkar and Kotwal[5]). The gel was prepared by boiling 0.85 gm of Difco Bacto agar in 100 ml of Barbitone acetate buffer (pH 8.4 and ionic strength 0.05). 1.5 ml. of the hot gel was poured on a microscope slide. 4 mm diameter wells were punched at 3 mm distance. The patient's serum was placed in the anodal well and sterile E. granulosus fluid in the cathodal well. Electrophoresis was set up at room temperature for one hour at a constant current of 15 mA. Slides were visually examined; positive reaction was seen when semilunar precipitate appeared between the wells [Fig. 1]. Agar gel diffusion test was done as described by Abrantes and Avila[2] while intradermal Casoni's test was done by the standard technique.
Maximum number of patients in the present study were between 11 and 40 years of age. Male to female distribution was equal. Clinically, 12 patients presented with cough with expectoration while one patient presented with hemoptysis only. Hepatomegaly was seen in 10 patients and hydatids at other sites like orbit, peritoneum and subcutaneous tissue presented with local complaints. Immunological study [Table 1] Overall results of immunological reactions showed positive CCIEP test in 24 patients (80%) while ID and AGD tests were positive in 16 (53.3%) and 3 (10%) patients respectively [Table 1]. Hepatic cysts showed positive CCIEP, ID and AGD reactions in 10, 7 and 2 patients while in pulmonary cysts these tests were positive in 12, 9 and 1 patients respectively [Table 1]. Hydatids at rare sites like orbit, intraperitoneal and subcutaneous tissue showed only two positive reactions with CCIEP while ID and AGD were negative in all patients [Table 1]. Preoperative diagnosis was made in all patients with hepatic cysts, in 12 patients with pulmonary cysts and in one each with orbital and intraperitoneal cysts by using all three tests. Six patients (20%) showed false negative results [Table 2].
Paucity of the symptoms and relatively silent course of the disease leads to difficulty in the diagnosis of E. granulosus infestation in non-infested areas. In India, ecchinococcosis is quite common. The infestation in human occurs through dogs, who acquire it from sheep, cattle, hogs etc. Maximally affected age group between 11 and 40 years in the present series supports the Barret's[3] hypothesis that infestation occurs in early childhood and becomes evident in adults. Symptomatology of E. granulosus disease varies with the organ affected and could be missed for the other common conditions pertaining to that organ Talib[6]. Immunological study Review of the literature on E. granulosus immunology shows various types of tests being used in the diagnosis of ecchinococcus cysts over the years.[1], [2], [4], [5] The probable explanation for so many tests appears to be the lack of specificity and high percentage of false positive and false negative reactions and difficulties in performing the tests. Micro-immunoelectrophoresis by virtue of its property to enhance the movement of the antigen and antibody towards each other and thus producing precipitation reaction has been found to be more sensitive and has been used in serodiagnosis in many clinical conditions (Kelkar and Kotwal[5]). This prospective study was undertaken to compare the three immunologic studies as far as their efficacy in pre-operative diagnosis was concerned. Countercurrent immunoelectrophoresis gave excellent correlation with 8070 positivity while intradermal (Casoni) and agar gel diffusion tests showed only 53% and 10% positivity respectively. As this study is small and comprises of only 30 patients no definite claim could be made but it has pointed towards the superiority of micro-immunoelectrophoresis over the other two tests in the serodiagnosis of E. granulosus cyst. Looking at the diagnosis of primary and secondary sites (Liver and Lungs) of E. granulosus disease, CCIEP has shown correlation upto 100% and 92% respectively [Table 2]. This probably proves the efficacy of the test by its no or minimal false negative reactions. Comparatively less positive reactions by other two tests indicate their limitations.[1], [2] Echinoccus cysts at rare sites pose a difficult problem and more false negative responses are obtained in all immunological tests; probably the antigen produced in these instances is not as high as in the primary or secondary cysts (Kagan et al[4]). Further study of CCIEP in the line of recurrent and residual E. Granulosus cysts is needed for the establishment of its efficacy in diagnosis. This study was limited to only suspected cases and no comments can be made on this line.
I am grateful to Dr. Prof. S. S. Kelkar, Prof. and Head of Bacteriology Department, Government Medical College, Aurangabad for allowing me to conduct this study. I am thankful to Dr. J. V. Deshmane, Associate Prof. of Surgery for his invaluable guidance during the study. I wish my deep gratitude to Dr. P. C. Bansal, Dean, Medical College, Aurangabad for allowing me to use the hospital records.
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