cystic USE OF ENZYME LINKED IMMUNOSERBENT ASSAY ( ELISA ) FOR THE DIAGNOSIS OF CLINICALLY SUSPECTED REFERRED HUMAN CYSTIC AND ALVEOLAR HYDATIDOSIS CASES IN NEPAL

Cystic and alveolar hydatidosis is caused by the larval form of cestode parasite Echinococcus granulosus belonging to family taeniidae. Immunological investigation is a basic complement to clinical and instrumental diagnosis of this parasitic disease. Serodiagnosis is carried out by tests such as double diffusion, immunoelectrophoresis (IEP), indirect hemagglutination (IHA) and ELISA which identify antibodies against crude antigen.1 Development of imaging techniques in medical science, world health organization article of May-June 1995 issue provides an overview of today's' modern technology in radiation medicine. Imagine techniques have got the diagnostic potentiality to image the morphological features of hydatid cysts located in the lungs or in the liver. In cystic USE OF ENZYME LINKED IMMUNOSERBENT ASSAY (ELISA) FOR THE DIAGNOSIS OF CLINICALLY SUSPECTED REFERRED HUMAN CYSTIC AND ALVEOLAR HYDATIDOSIS CASES IN NEPAL ORIGINAL ARTICLE J Nep Med Assoc 2004; 43: 312-316


INTRODUCTION
Cystic and alveolar hydatidosis is caused by the larval form of cestode parasite Echinococcus granulosus belonging to family taeniidae.Immunological investigation is a basic complement to clinical and instrumental diagnosis of this parasitic disease.Serodiagnosis is carried out by tests such as double diffusion, immunoelectrophoresis (IEP), indirect hemagglutination (IHA) and ELISA which identify antibodies against crude antigen. 1 Development of imaging techniques in medical science, world health organization article of May-June 1995 issue provides an overview of today's' modern technology in radiation medicine.Imagine techniques have got the diagnostic potentiality to image the morphological features of hydatid cysts located in the lungs or in the liver.In cystic

USE OF ENZYME LINKED IMMUNOSERBENT ASSAY (ELISA) FOR THE DIAGNOSIS OF CLINICALLY SUSPECTED REFERRED HUMAN CYSTIC AND ALVEOLAR HYDATIDOSIS CASES IN NEPAL
screening technique which requires low quantities of antigen and sera.IgG is the dominant class of specific antibody in cystic hydatidosis.Enzyme immunoassay employ antibodies or antigens conjugated to enzymes in such a way that the Immunological and enzymatic activity of each moiety is maintained. 3The prevalence, incidence and risk factors of infection by E. granulosus were studied in the domestic and street dogs of Kathmandu, Nepal using an ELISA coproantigen test and the prevalence was 5.7% (5/88 dogs) in the city and 1.8% (3/171 dog) in the veterinary clinics. 4inical diagnosis of hydatidosis may be difficult and rely on detection of space occupying masses primarily with x-ray, ultrasound or CT scans, although images may also be difficult to interpret.Larval cestodes are among the few parasitic infections where the basis for laboratory diagnosis is primarily serology, surgery still remain basic form of treatment.In this study ELISA was used for the diagnosis of human cystic and alveolar hydatidosis in Nepal.Cross reactivity between echinococcosis and cysticercosis (Taenia solium infection) has been identified. 5

MATERIALS AND METHODS
All the serum samples were collected from the suspected patients referred by different hospitals, nursing homes and private clinics.
The basic principles of the ELISA is detection of antibody using enzyme -labelled antiglobulin.The antigen is coupled to a solid phase support and the sera thought to contain antibody are incubated in this sensitized carrier.Excess serum components are washed away and then the enzyme -labelled antiglobulin (conjugate) is added.The conjugate will become attached to the antigen-antibody complexes on the carrier surface and the amount of conjugate attached is measured by the amount of substrate that it degrades. 6r diagnosis of hydatidosis commercially available Echinococcus serology screening test, microwell ELISA cit.EG-8.48 test was used.Number of microwells needed (Three for controls plus number of samples) were break off and placed in the strip holder 100 µl of negative control to well # 1,100 µl of weak positive control to well # 2,100 µl of strong positive control to well # 3, and 100 µl of diluted (1:128) test samples to the remaining well were added and incubated at room temp (15 0 to 25 0 C) for 10 minutes, which was washed 3 times with diluted wash buffer then added two drops of enzyme conjugate to each well and incubated at room temperature for 5 minutes.
The contents was shacked out and again washed 3 times with wash buffer and followed by distilled water.In each well 1 drop of substrate A and 1 drop of substrate B was added, mixed well by tapping string holder and incubated for 5 minutes then two drops of stop solution was added.
The reading obtained from ELISA plate reader with 450 nm filters.It was considered that 0.0 to 0.49 OD units indicated Negative, 0.5 to 1.19 OD unit indicated weak positive and 1.2 and above OD unit indicated strong positive as given in the manual.

RESULTS
In   The comparison of the previous results with the present indicates that maximum number of clinically suspected cases were also positive by confirmative ELISA test.Since the clinical features appeared very lately and few persons can afford for expensive diagnostic techniques most of the cases remain still hidden in the communities.Dog fecal samples were tested for E. granulosus by coproantigen ELISA test and 154 (3.8%) dogs were found positive.

8
One of the study carried out on hydatidosis in water buffaloes of western part of Kathmandu revealed that out of 535 water buffaloes examined 21% were found positive, of which 11% lung, 6% liver and 4% both liver and lungs.9 In human out of 23 sera samples clinically suspected hydatidosis 17 (74%) were found positive in ELISA test.8   Imagine techniques have got the diagnostic potentiality to image the morphological features of the hydatid cysts located in the lungs or in the liver.Cysts in the lungs can be detected by x-ray examination.Ultrasonogram (USG) and computerized topography (CT scan) are the methods of choice for the diagnosis of hepatic and other abdominal cysts.In this study 75% of CT-scan suspected cases were found positive by ELISA.The image and ultrasound signs that correlates with each type of cyst will be described in the oral exposition.The different ultrasound images shown in the ecographic test, allow us to establish the different biological stages of the cyst, and the complication it may suffer as well as its evolution and growth.A good morphological and tomographical ultrasound diagnosis allows us to prescribe the suitable and appropriate surgical or medical treatment or both at a time or pair of the cystic hydatidosis.
10 Hence these imaging techniques must be used to detect the shape, size and location of the cyst but not as confirmative.ELISA tests the confirmative test for cystic and alveolar hydatidosis since it also close cross-reaction with cysticercosis.
Out of 44 sample quite a number of patients were clinically suspects as a case of neurocysticercosis, however this ELISA test gives 8-10% cross reaction results with cysticercosis patients.One of the study on differential serodiagnosis for cystic and alveolar echinococcosis using fractions of Echinococcus granulosus cyst fluid (Antigen B) and E. multilocularis protoscolex (EM18) revealed that preliminary blind tests for differentiation of AE from other diseases were carried out by Em18-immunoblot analysis using crude antigens of protoscoleces of E. multilocularis.Approximately 83% (35 of 42) of the sera from patients with AE sera were from inactive cases with calcified lesions.All other sera, including neurocysticercosis (0 of 8), trematodiases (0 of 5), or nematodiases (0 of 5), were negative.This serological result should not be used as an aid in diagnosis of hydatidosis and should not be interpreted as diagnostic for cysticercosis by itself.

11
Hydatidosis is common in those places where there is close association between man-sheep-dog, man-pig-dog or manbuffalo-dog.The sheep strain predominates in most domestic hosts and human through out the Europe.
12 But we identified three E. granulosus genotypes (G1, G5 and G6) in the mammalias host from Kathmandu.Three distinct COI and NDI sequences were identified anomgst the 25 isolates following alignment with the published E. granulosus G1 -G9 Genotypic sequences.Eighteen samples, including fourteen buffaloes isolates, two sheep isolates and two goat isolates, produced identical COI and NDI sequences to the cattle strain (G5 genotype, whereas one buffalo isolate, two sheep isolates and two goat isolates, shared identical sequences with those of the common sheep strain (G1 genotype).Notably, the two human isolates examined produced identified COI and NDI sequences to the G6 (camel strain) genotype; neither were infected with the G1 or G5 genotype, the latter being the predominant strain (18/25 isolates examined) identified in the study.13   Blood examination results Biochemical tests and stool and urine examination have basically no relation with hydatidosis although in some cases total count, neutrophyl, lymphocyte esinophyls counts were high and low, that might be due to other causes.In case of the cysticercosis, stool examination is very much essential because of its auto infection possibility but in hydatidosis cases sometimes protoscoleses may be observed in sputum test in case the cysts are rapture in lungs.

Table I : Age and Sexwise ELISA Test result
the present study diagnosis and monitoring of active clinical cases of Alveolar and cystic hydatidosis disease referred to our centre by different hospitals and private nursing homes were carried out.Early diagnosis by ELISA and imaging techniques of active cases was done and they were monitored and sent to the hospital for surgery as well as chemotherapy.Forty -four serum samples from suspected active hydatid cases as well as suspected neurocyticercosis cases were received from August 1999 to March 2001 from the hospitals, nursing homes and private clinics.There were 35 (79.54%) found positive and nine specimens testing negative.During this period 31 samples were collected from males and 13 samples were from females.years age received during the study period, the maximum positive rate i. e. (12) 27.3% was observed.In this serological diagnostic study, one sample received from the age group 0-5 years of male which in testing gave positive result.See detail in TableI.

Blood examination results of suspected patients for cystic and alveolar hydatidosis:
Out of 44 patients referred for ELISA test, only 10 patients had requested for blood examination before this test.One patient found high range of total count and neutrophyl and 3 patients had high range of esinophyls counts and 2 patients had high range of ESR, similarly one had low range of lymphocyte one had low range of hemoglobin.Rest of all posses normal range of blood parameters.The result revealed that in both alveolar and cystic hydatidosis as well as suspected neurocysticercosis cases no remarkable relation observed with blood examination (TableII).During inspection the hydatidosis suspected cases scanning usually performed were CT-scan, X-ray, MRI and USG and other imaging techniques which were comparatively necessary technique as the imaginary purpose (see tableIII).
Comparison of various imaging techniques with ELISA:Out of 44 patients referred 8 of them had performed CT-scan in which 6 of CT scan suspected cases were found positive for ELISA also, while three of them had performed USG, X-ray and MRI by each, which were found positive for ELISA test.Stool and Urine examination results:Very few patients were referred for stool/urine test before ELISA.Patients who had performed urine test revealed that in one patient -Albumin trace, 3 of them pus cells (1-8), 2 of them calcium oxalate (10-12), 1 of them amorphous urate and 5 of them had epithelial

Table IV : Biochemical examination of suspected patients for cysticercosis/hydatidosis
In stool one of them posses ova of T. trichiuris, 2 of them were infected with Giardia and Entamoeba histolytic cyst and one of them was infected with H. nana the result revealed that there is no relationship with stool and urine examination with hydatidosis.