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 ~  Abstract
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  Acknowledgements
 ~  References

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ORIGINAL ARTICLE
Year : 2002  |  Volume : 20  |  Issue : 2  |  Page : 79-82
 

Combined role of casoni test and indirect haemagglutination test in the diagnosis of hydatid disease


Dept. of Medical Microbiology and Parasitology, University College of Medicine, Kolkata - 700 020, India

Correspondence Address:
Dept. of Medical Microbiology and Parasitology, University College of Medicine, Kolkata - 700 020, India

 ~ Abstract 

PURPOSE: To study the combined role of Casoni test and indirect haemagglutination (IHA) test in the diagnosis of hydatid disease. METHODS: Twenty eight suspected cases of hydatid disease were subjected to Casoni intradermal test using commercially available antigen (Span Diagnostics, India) after collecting pre-test serum samples. The serum samples were tested by IHA using an indigenously developed IHA test. RESULTS: Considering the clinical diagnosis of hydatid disease as the gold standard, the specificity of both Casoni test and IHA was 47%, however, the sensitivity of IHA was higher (81.8%) than Casoni test (63.8%). With the two tests considered together the sensitivity was 90.9%. CONCLUSIONS: Combined use of Casoni test and IHA test could establish presumptive and cost effective diagnosis in upto 90.9% of clinically suspected cases of hydatid disease.

How to cite this article:
Ray R, De P K, Karak K. Combined role of casoni test and indirect haemagglutination test in the diagnosis of hydatid disease. Indian J Med Microbiol 2002;20:79-82


How to cite this URL:
Ray R, De P K, Karak K. Combined role of casoni test and indirect haemagglutination test in the diagnosis of hydatid disease. Indian J Med Microbiol [serial online] 2002 [cited 2014 Nov 1];20:79-82. Available from: http://www.ijmm.org/text.asp?2002/20/2/79/8352


Even by this millennium, hydatid disease poses a world-wide problem. Although it is not a new disease, its diagnosis, treatment and overall containment possibilities remain important public health tasks for almost all nations. It is a parasitic zoonotic disease caused by infection by the metacystode stage (Infective larvae) of the dog tapeworm, Echinococcus granulosus, which has a sustaining sheep-dog epizootic cycle, difficult to break off, having man as the accidental intermediate host. It is enzootic in sheep which may serve as the reservoir and amplification vector and generate human cases.[1]
The clinical diagnosis of hydatid disease (HD) in man may be quite devious and thus in any HD endemic area, every space-occupying lesion of the liver, lung, etc. warrants a clinical suspicion calling firstly for radiological evidences. However, serodiagnosis plays an important role in the early diagnosis, though, presumptive. This is based primarily on the demonstration of circulating hydatid antibodies which occur frequently in cases of established hydatid disease. Casoni test detects an in vivo hypersensitivity which can be both of immediate and delayed type. The specific diagnosis, however, is based on finding scolices ('hydatid sand') in the cyst fluid, despite the potential hazards that the technique of demonstration poses which can be minimised by full containment strategies.[2] In this study, the combined role of Casoni test and IHA test has been evaluated in the diagnosis of clinically suspected cases of hydatid disease.

 ~ Materials and Methods Top

Antigen for Casoni Test:
Commercially prepared antigen marketed by Span Diagnostics,® India (Code No. 18403) was used. The antigen was standardised by immunoprecipitation test and was preserved in 0.01% thiomersal, and refrigerated at 2°-8°C for the study period.
Casoni Intradermal Test:
Casoni antigen (0.1 mL) was injected intradermally at a suitable site on the vein-free volar surface of the right fore-arm using a sterile tuberculin syringe fitted with a 26 gauge needle; 0.1 mL of sterile normal saline was injected into the left fore-arm as control. Readings were taken after 15 minutes, as well as, after 30 minutes for immediate reaction, and if both were negative, these were read again at the end of 72 hours (for delayed response).
A wheal measuring >24 mm in one direction or >22 mm in both the directions within 15-30 minutes of the test with or without formation of psueudopodia was considered to be positive. The test was considered as borderline if the wheal measured 22 mm or 23 mm in the long axis and 21 mm or less in the other axis. Any wheal measuring < 22 mm in both axes was interpreted as negative.[3],[4]
Serum samples for IHA Test:
Pre-test samples (collected prior to the performance of Casoni test) were obtained from inpatients/outpatients of the departments of chest and cardiothoracic surgery of Seth Sukhlal Karnani Memorial Hospital and The Institute of Post Graduate Medical Education and Research, Calcutta. These were clinically suspected to be cases of hydatid disease with a history of cough, haemoptysis, chest pain, chronic ill-health or undiagnosed abdominal lump with symptoms of dyspnoea where chest skiagram and ultrasonography revealed space-occupying lesions. The clear samples of sera were stored in stoppered vials at -20°C until use and inactivated at 56°C for 30 minutes before use.
Antigen used for IHA Test:
Fluid aspirated from a hydatid cyst removed by operation from a case of hydatid cyst of liver of a sheep from a local abbatoir was used as a source of antigen. The fluid was clear, not anti-complimentary, and contained many scolices. 5 mm of phenylmethylsulphonyl fluoride was added to the aspirated hydatid fluid. It was then centrifuged at 3000 g for 20 minutes to settle the protoscolices and the supernatant was taken as antigen and was stored at -20°C till further use. The antigen protein concentration was estimated by the method of Lowry et al with bovine serum albumin as the reference standard. The antigen was filtered against a known positive serum control collected from a patient that was diagnosed clinically and confirmed by surgical intervention as having hydatid cyst of the liver.
Preparing optimum dilution of antigen:
To obtain optimum dilution of the antigen, we titrated four dilutions, 1:10, 1:20, 1:40 and 1:80. We found 1 in 10 dilution to be the most sensitive against a known positive serum control. The highest dilution of antigen yielding the highest serum titre was considered the optimum.
The indirect haemagglutination test:
Indirect haemagglutination test with 'tanned' red blood cells as the inert carrier particles of antigen, was performed adopting the technique of Boyden.7 For uniform results, the antigen was standardised prior to use in the IHA Test. Appropriate cell controls and serum control (with unsensitized tanned RBC) were used.
A positive IHA test was indicated by formation of a “mat” or a “carpet” of cells at the bottom of the well. A negative reaction was indicated if the cells settled to form a compact button or ring at the centre of the well. Suitable cell and serum controls were set up simultaneously to assess sensitivity and validity of the IHA test.[5],[7]

 ~ Results Top

Correlation of serological findings with surgical data:
Of the 28 suspected cases of hydatid disease studied, 11 were confirmed to belong to hydatid disease by surgical and histopathological parameters. In the remaining cases, a straightforward diagnosis of hydatid disease could not be made either because surgery was not done or results of surgery were not available. [Table - 1] shows that a positive indirect haemagglutination test was obtained at a diagnostic titre of 1/128 in 18 cases tested. A positive Casoni test was obtained in 16 of the 28 cases. However, when combined, these two serological tests could clinch the diagnosis in 22 of the 28 clinically suspected cases. The results have been evaluated on the basis of their statistically valid relevance.
The principle of validity refers to what extent a test can accurately measure what it purports to do. Thus, validity expresses the ability of a test to distinguish those who have the disease from those who do not have it. The degree of accuracy refers to the closeness with which measured values agree with “true” values. Thus,validity has two components; sensitivity and specificity. When assessing the accuracy of a diagnostic test, one considers both these components and both these values are expressed in percentages. Sensitivity and specificity are usually determined by applying the test to one group of persons having the disease and to a reference group of persons not having it [Table - 2]. Sensitivity and specificity are inherent properties of screening tests.[9]
Statistical evaluation of clinical and laboratory data:

Combined IHA and Casoni test helped diagnose 22 out of 28 cases, thus showing a total positivity of 90.91% in the clinically suspect cases in our series. This combination had also been suggested by Parija and Rao for a reliable diagnosis of hydatid disease.[6]

 ~ Discussion Top

The indirect hemagglutination (IHA) test has been preferred since its introduction by Garabedian and associates by virtue of its simplicity and sensitivity and is a valuable method for the serodiagnosis of HD. Sensitivity of the test ranged from 60-100% with an average of 83% according to their study.[5] On the other hand, Casoni intradermal test is still widely used for its simplicity. The sensitivity of Casoni test varied from 60-80% in previous studies.[2] Results of our study indicate that IHA test when used alone showed a sensitivity of 81.81% and likewise Casoni test alone showed a sensitivity of 63.64% in the diagnosis of HD; however, these two tests when combined could establish the diagnosis in 22 out of the 28 clinically suspected cases showing a total positivity of 90.91%.
Though various factors like immune-complexes and other inhibitory factors in the sera might be responsible for false negative reactions of IHA observed in this study, effective modifications of IHA test may increase the sensitivity of the test several folds. Thus 90.91% sensitivity in detecting the disease by a combined IHA and Casoni tests observed in our study may be further increased by improving upon the technique in current use, as well as, by the use of processed or purified hydatid antigen with optimum nitrogen content and use of suitable, stabilized cells in IHA as mentioned by Parija and Ananthanarayan.[8]
Hydatid cyst fluid antigen (HCF) has been found to be a good source of antigen for the serodiagnosis of hydatid disease. Use of purified antigenic fraction in highly sensitive techniques has obviated cross reactions with most of the other infections.[13],[14] Rapid ELISA, standard ELISA and IHA techniques have been compared in the diagnosis of hydatidoses, and all three methods have been found to be equally sensitive (100%). The prime role of ELISA is to eliminate cross reactivity with samples from cysticercosis, amoebiasis patients, thereby indicating its high specificity compared to IHA.[15]
False positive reaction may arise due to serological cross reactions among sera of patients of Echinococcus granulosus, Echinococcus multilocularis, Cysticercus cellulosae, as well as various carcinomas, in which it is attributed to interaction of blood group P substance appearing in hydatid fluid with anti-P antibody in infected and sensitized patients and also in mixed parasitic infections due to hookworm, Trichuris trichiura, Ascaris lumbricoides, Fasciola hepatica etc. False negative reactions are found in calcified lung cysts which may be due to absence of precipitating antibodies in the infected host. A detectable immune response is more frequent among patients with liver cysts than in those with lung cysts; in the former, the recently exposed cysts were associated consistently with a detectable immune response.[4]

 ~ Acknowledgements Top

We gratefully acknowledge the help of the Vice Chancellor of Calcutta University and the Director, Institute of Post Graduate Medical Education and Research, Calcutta for the necessary facilities to carry out this work. We thank Professor AN Chakrabarty for his valuable suggestions. 

 ~ References Top

1.Park JE, Park K. Hydatid Disease. Park's Text Book of Preventive and Social Medicine 1997; 15th Edn. 231.  Back to cited text no. 1    
2.Schantz PVMD. Casoni's Test for Hydatid Disease. New England Journal of Med 1975; 293:408.  Back to cited text no. 2    
3.Bhatia BB, Pathak KML, Parija SC. Echinococcosis. In: Review of Parasitic Zoonoses. (All India Publishers and Distributors, Madras) 1990; 268:280.  Back to cited text no. 3    
4.Lambria S. Non-specific Reactions with the Intradermal test for Hydatidosis in persons with other Helminthic Infections. American Journal of Tropical Med Hygiene 1975; 24:849.  Back to cited text no. 4    
5.Garabedian GA, Matossan RM, Djanian AY. An Indirect Hemagglutination test for Hydatid Disease. Journal of Immunol 1957; 78:269.  Back to cited text no. 5    
6.Parija SC, Rao RS. Evaluation of the combined use of Indirect Haemagglutination Test and Casoni's skin test in diagnosis of Hydatid disease. Indian Journal of Path Microbiol 1987; 30:117-121.  Back to cited text no. 6    
7.Boyden SV. The Adsorption of protein on erythrocytes treated with tannic acid and subsequent haemagglutination by antiprotein sera. Journal of Experimental Medicine 1961; 93:107-120.  Back to cited text no. 7    
8.Parija SC, Ananthanarayan R. Evaluation of stabilised cells in the indirect haemagglutination test for echinococcosis. Journal Med Microbiol 1985; 19:95-98.  Back to cited text no. 8    
9.Park JE, Park K. Screening for Disease. Park's Text Book of Preventive and Social Medicine 1991; 13th Edn. 110-112.  Back to cited text no. 9    
10.Yerushalmy J 1947; Pub. Health. Rep. 62:1432.  Back to cited text no. 10    
11.WHO 1979; Tech Rep Ser, No. 637.  Back to cited text no. 11    
12.Wattal C, Malla N, Khan IA, Agarwal SC. Comparative evaluation of enzyme linked immunosorbent assay for the diagnosis of pulmonary Echinococcosis. J Clin Microbiol 1986; 24:41-6.  Back to cited text no. 12  [PUBMED]  
13.Maddison SE, Slemenda SB, Schantz PM., Fried JA., Wilson M, Tsang VC. A specific diagnostic antigen of Echinococcus granulosus with an apparent molecular wt. of 8 kDA. Am J Trop Med Hyg 1989; 40:377-83.  Back to cited text no. 13    
14.Kanwar JR, Kaushik SP, Sawhney IMS, Kamboy MS, Mehta SK, Vinayak VK. Specific antibodies in serum of patients with hydatidoses recognised by immunoblotting J Med Microbiol 1992; 36:46-51.  Back to cited text no. 14    
15.Kaur M, Mahajan RC, Malla N. Diagnostic accuracy of rapid enzyme linked immuno sorbent assay for the diagnosis of human hydatidoses. Indian J Med Res 1999; 110:18-21.  Back to cited text no. 15  [PUBMED]  
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