Counterimmunoelectrophoresis in the immunodiagnosis of amoebiasis.
MM Bapat, GG Bhave
Department of Microbiology, Seth G. S. Medical College, Bombay, Maharashtra.
M M Bapat
Department of Microbiology, Seth G. S. Medical College, Bombay, Maharashtra.
Counterimmunoelectrophoresis (CIE) detection of antiamoebic antibodies in the patients«SQ» sera, has been carried out and correlated with the routine diagnostic microscopic examination of stool and pus samples. The clinically suspected amoebiasis cases were divided into two main groups, (i) proved positive for Entamoeba histolytica as detected by microscopic examination of samples, and (ii) negative for the parasite. A total 153 cases of intestinal amoebiasis were studied. CIE was positive in 27 of the 84 proved cases, and in 12 out of 69 unproved cases showing negative microscopy. A total of 59 cases of amoebic liver abscess (ALA) were studied, of which CIE was positive in 20 of the 30 proved cases of ALA and in 4 of the 29 unproved cases. Sera from patients with non-amoebic illness (n = 48) gave negative results with CIE. Similarly sera from normal healthy controls (NHC) (n = 100) and asymptomatic cyst passers (n = 75) were negative by CIE.
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Bapat M M, Bhave G G. Counterimmunoelectrophoresis in the immunodiagnosis of amoebiasis. J Postgrad Med 1990;36:124-7
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Bapat M M, Bhave G G. Counterimmunoelectrophoresis in the immunodiagnosis of amoebiasis. J Postgrad Med [serial online] 1990 [cited 2022 Jun 28 ];36:124-7
Available from: https://www.jpgmonline.com/text.asp?1990/36/3/124/849
Immunoelectrophoresis was introduced by Prince and Burke for the detection of serum hepatitis antigen. Counterimmunoelectrophoresis (CIE) has been used by many workers as an immunodiagnostic test e.g. Krupp, Mahajan et al, Sharma et al have used it for the detection of antibodies in the cases of amoebiasis. CIE technique is rapid and easy to perform, and can be easily adapted in an average clinical laboratory. The test does not give false positive results. In the present study the CIE has been carried out for clinically suspected cases of amoebiasis and the results have been compared with routine diagnostic methods (stool and pus examination) in order to evaluate the sensitivity and specificity of CIE.
Amoebic antigen: The antigen was prepared from E. histolytica (strain NIH 200), which was cultured axenically in the medium TY-S-33. Amoebae were concentrated by centrifugation from 72 hour cultures and washed thrice in phosphate buffer saline (PBS) having PH 7.2. The suspension was then sonicated in an ultrasonic disintegrator and re-centrifuged at 4?C for 10 min at 16,000 g. The supernatent obtained was used as the antigen. The protein content of the antigen was determined by the method of Lowry et al . The antigen was stored at - 20?C, in 0.5 ml aliquots. The working dilution of the antigen was obtained by carrying out checker board titrations against the known positive control serum (antiserum raised in rabbits against axenic strain of E. histolytica: NIH 200, supplied by HAL, Pimpri). The optimum concentration was found to be 2.5 mg/ml, which gave a clear precipitin line.
Sera samples: Four hundred and fifty sera samples, from various groups consisting of normal healthy donors, patients with diseases other than amoebiasis, those having clinical signs and symptoms of amoebiasis (intestinal or hepatic) and asymptomatic cyst passers [Table:1] were obtained from different hospitals in Mumbai. The sera were stored at -20?C. prior to CIE testing. In these clinical suspects, microscopic examination of stool and pus was carried out to confirm the diagnosis.
Technique of CIE: The serum samples were inactivated at 560C for 30 min, prior to use, in order to avoid any tion- specific precipitation reactions. CIE was carried out using gel slides prepared from 1% Noble agar in 0.05M barbitone acetate buffer pH 8.6. Nine pairs of the wells 3 mm in diameter and 5 mm apart were punched in 3 parallel rows. The samples to be tested were filled in the anodal wells and the antigen in the cathodal wells of the gel slide. The positive and negative controls were included with each batch.
The electrophoresis was carried out with a constant current of 10 milli. amps. per slide for 60 min. The readings were taken immediately, and after overnight incubation at 40C. The readings were further checked by slaining the slide with amidoblack for 15 min and destaining in several changes of 2%) acetic acid. A positive result was indicated by the presence of a line of precipitation between the antigen and antiserum wells, indicating the presence of anti-amoebic antibodies in the serum.
The results are illustrated in [Table:1]. Sera from normal healthy subjects and patients having diseases other than amoeblasis showed negative results by CIE. These controls had no history indicative of amoeblasis for the last 2 years; also the stool examination did not reveal presence of E. histolytica. It was seen that only 27 of the 84 intestinal amoeblasis cases confirmed by microscopy were positive by CIE. These included dysentery and non dysentery cases, whereas 12 of the 69 intestinal amoeblasis cases showing negative microscopy could be detected by CIE. Of the 30 confirmed amoebic liver abscess cases, 20 were detected by CIE. Of the 29 unproved cases, 4 were detected by CIE. While out of 15 acute non-suppurative hepatic amoeblasis cases, 6 were detected by CIE. CIE was found to be of no use in detecting asymptomatic cyst passers, as none of them gave positive results.
Counterimmunoelectrophoresis (CIE), is rapid and easy to perform. In the present study, CIE gave no false positive results with the control group sera, and was found to be specific in detecting cases of amoebiasis. The specificity of CIE was also reported by several other workers,.
We also found that CIE was less sensitive in detecting intestinal amoebiasis cases, where the tissue invasion by amoebae is less. Our findings were similar to those of Sharma et al, who found 26.1% positivity in amoebic dysentery cases. However, in cases of amoebic liver abscess (ALA), where tissue invasion is high, the positivity is comparatively high. In the present study we obtained 66.66% positivity in confirmed amoebic liver abscess cases. Many workers, have obtained 100% positive result by CIE, in amoebic liver abscess cases. In ALA cases, where demonstration of amoebic trophozoites in pus by microscopic examination is difficult, CIE is a useful test. As seen in [Table:2], CIE could detect more number of clinically suspected symptomtic amoebiasis cases, than microscopy. However in the asymptomatic cyst passers showing positive microscopy, CIE showed negative results. In such cases, the tissue invasion by amoebae is minimum. From the results obtained in the present study we found that, negative result by CIE did not rule out amoeblasis. It is, thus advisable to use more sensitive tests like enzyme linked imunosorbent assay (ELISA) or immunofluorescent assay (IFA) for the diagnosis of amoebiasis. From the present study, we found that a commbination of routine microscopic examination of clincal material for the parasite, alongwith CIE, gave more positive results (as seen in [Table:3]). Thus, a combination of microscopy and CIE, was found to be more effective in the diagnosis of amoebiasis.
We are grateful to the Dean, Seth GS Medical College and King Edward Memorial Hospital, for permitting us to carry out this work in the Institute. We would also like to express our sincere thanks to the Research and Development Department, HAL Ltd., Pimpri, Pune and to the Director, Hindustan Ciba Geigy Research Centre, for their scientific help.
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