Evaluation of Usefulness of Three Serological Tests Using Native Crude Antigen in Diagnosis of Hepatic Cystic Echinococcosis Patients

Objective: To evaluate three different serological tests [Indirect Hemaglutination (IHA), Enzyme Linked Immunosorbent Assay (ELISA) and Western Blotting (WB)] using native crude antigen for diagnosis of hepatic cystic echinococcosis (HCE) patients. Materials and Methods: Sheep hydatid fluid (HF) was collected from fertile cysts obtained from a slaughterhouse and used as an antigen. Forty patients who were attended the Dr. Ersin Arslan Training and Research Hospital in Gaziantep, Turkey, were investigated. Serum samples were obtained from surgically confirmed CE patients. Healthy Turkish people and 16 patients with other helminthic infections were included as a control group. Results: Of the 40 analyzed patients, 10 (25%) were men and 30 (75%) were female. The average age was 46.97 years (s.d.; 18.95). The majority of the patients had a single cystic lesion situated in one lobe of the liver (usually in the right lobe) (55%), 32.5% of patients had two cystic lesions and but low specificity. Western blotting showed low sensitivity but high specificity.


Introduction
Cystic echinococcosis (CE) is a parasitic disease caused by the ingestion of Echinococcus granulosus egg which is found in dog feces. The life cycle of this parasite involves carnivores (such as dogs) as definitive hosts and herbivores (such as sheep and cattle) and humans (which is accidental) as intermediate hosts [1]. Humans are infected by the larval stage of tapeworm and hydatid cysts exist mostly in the liver (65% -70%) and lungs (20% -25%) but also other organs (kidney 2%, spleen 2% and brain less than 2%, etc.) [2] [3]. Cystic echinococcosis is one of the most important parasitic diseases and it is common almost all over the world. Turkey is also one of the endemic countries and CE poses a problem in terms of public health and the economy. In Turkey, it was stated that approximately 2663 patients every year had operations because of CE [4]. According to the data of the Ministry of Health; 408 annual cases were reported in 2008, and this number reached 1.867 by the end of 2019. The morbidity rate reported as 0.57 per 100.000 in 2008 was reported as 2.08 in 2019 [5].
Diagnostic methods of CE depend on the facilities in hospitals and laboratories therefore it is complicated. Although there are various imaging modalities such as ultrasonography (US), computed tomography (CT) and magnetic resonance imaging (MRI), especially US is the main modality for hepatic cystic echinococcosis (HCE). US has been used to detect a variety of pathologies, including parasitic infections, since the 1970s, with the use of portable US scanners in rural communities starting in the 1980s [6]. The current international WHO-IWGE (Informal Working Group on Echinococcosis) classification of CE cyst stages is based on the pathognomonic features of cysts on USG, and guides their clinical management. For the analysis, CE cysts were grouped into active [CL (cystic lesion, if active) CE1, CE2, CE3a and CE3b] and inactive (CE4 and CE5). Effective serological tests for CE diagnosis would be of great help to define and support cyst status [7] [8] [9] [10].
There is currently no standard, highly sensitive and specific serological test for CE antibody detection [11]. The main serological methods used for human CE diagnosis are based on the detection of specific IgG antibodies. Serological tools support imaging techniques. The available tests are based on antibodies against crude antigens and low or no usefulness for the follow-up of patients during the treatment. Specific recombinant antigens have good potential as diagnostic and follow-up tools for CE, but progress in this field is hampered by a lack of stan-Open Journal of Medical Microbiology dardization. Many authors have focused their research both on recombinant proteins and on synthetic peptides, to develop more sensitive and specific tests.
Numerous recombinant proteins and related peptides, mainly derived from antigen B and antigen 5, have been tested for the detection and follow-up of antibodies in correlation with US findings. But, available data were generated from small and underpowered clinical studies that have shown dissimilar Se and Sp for the same recombinant antigen [12].
Serologic methods based on the search for specific antibodies in the patient's serum are also needed in the diagnosis of HCE. Different antigens have been used for serodiagnosis of CE until now [13] [14]. Serological testing is usually based upon the use of crude antigens prepared from the metacestodes of E. granulosus. Immunofluorescence antibody tests (IFAT), based on protoscoleces of E. granulosus, have been in use since 1967 [15] and can have diagnostic sensitivities of >95% in hepatic CE but suffer from relatively poor specificities [16]. A number of recombinant antigens have also been developed, but their use in standard diagnostic laboratories is limited [17]. On the other hand, the use of purified antigens improves the specificity of serological assays but may lead to a loss of sensitivity [2]. Combination of imaging and serological tests such as Indirect Hemaglutination Assay (IHA), Enzyme Linked Immunosorbent Assay (ELISA) and Western-Blotting (WB) provides diagnostic confirmation.
In this study, we used the sera of HCE patients which were found positive by IHA, ELISA, WB tests and liver ultrasonographic images. The aim of this study was to demonstrate the usefulness of hydatid cyst fluid (HCF) collected from cysts obtained from infected sheep in Turkey and to evaluate three different serological tests (IHA, ELISA and WB) using native crude antigen. Also, to evaluate the clinical findings regarding cyst localized in the liver and individual serological responses in patients with CE. And to present the results of diagnostic performance of the WB test compared with IHA and ELISA tests in patients with liver CE.

Crude Antigen Preparation
The fertile cysts in a sheep were obtained from a slaughterhouse of city Izmir, Turkey. Hydatid cyst fluid (HCF) was aspirated using an aseptic injector and protoscoleces were detected in microscopic examination for fertility. Hydatid cyst fluid was centrifuged at 10,000 g for 20 minutes at 4˚C. The supernatant was separated and its protein concentration was calculated using the Bradford protein assay kit (Bio-Rad) and bovine plasma gamma globulin as a standard. Protein concentration was found 2.65 mg/ml [18]. Then supernatant stored at −20˚C as a hydatid crude antigen until used.

Serum Samples
Forty patients who were hospitalized in Dr. Ersin Arslan Training and Research Open Journal of Medical Microbiology Hospital in Gaziantep, Turkey were included in this study. All patients had cystic structures by the US examination and were investigated by commercial IHA test (Fumouze diagnostic, France) and in-house ELISA. We used WB analysis as a confirmation test for retesting the seropositive serum samples detected by both IHA and ELISA tests. Sera from healthy volunteers and from 16 patients with other parasitic diseases such as Fascioliasis (n: 5), Trichinellosis (n: 3), Toxocariasis (n: 4), Leishmaniasis (n: 3) and Giardiasis (n: 1) were included as a control group. These sera were divided into aliquots and kept at −20˚C until use.

Statistical Analysis
Statistical analysis was performed using Microsoft Excel (Excel 2010; Microsoft Corp., Redmond, WA) and mean, standard deviation, sensitivity, specificity values of tests were calculated.

Ethical Approval and/or Informed Consent
Patients were informed about diseases and procedure of this study. Informed written consent was obtained from each participant. Following to be given informed consent, they participated in the study. The study was approved by the local Clinical Research Ethical Committee.

Discussion
The clinical management of CE in Turkey is based on a patient's anamnesis, clinical symptoms, morphological changes identified by imaging techniques and confirmed serologically. Ultrasound is the imaging technique of choice for the diagnosis of abdominal CE. Liver cysts appear to grow at a lower rate than lung cysts [19]. Clinical symptoms usually occur when the cyst compresses or ruptures into neighbouring structures. The serological diagnosis plays a key role not only in early detection of the infection as well as follow-up of the patients and usually, but liver cysts also produce a higher antibody response than the other locations of the cysts. The same results were detected in our HCE patients.
Seropositivity in females in this study was higher than in males. This finding could be explained by women are exposed to contact with sources of infection such as dogs, soil and raw vegetable. A similar observation was made by some authors in Turkey [20] [21] [22], in Iran [23], in western Azerbaijan [24] and in China [25].
Different antigens have been used for serodiagnosis of CE. It is known that HCF is a useful antigen source for serodiagnosis of human CE [26]. Lorenzo et al. [27] attributed the best diagnostic performance obtained with HCF antigen instead of recombinant AgB1 and B2 with sera from CE patients. Similarly, our results showed that crude HCF antigen using IgG ELISA and IHA provided better diagnostic performance. In this study, we also evaluated the diagnostic performance of crude native E. granulosus antigen that is easy to produce, cost-efficient tools for the serological diagnosis of CE and to assess its value in defined liver CE patients.

Enzyme-Linked Immunosorbent Assay (ELISA) and the commercial Indirect
Hemagglutination Assay (IHA) techniques are frequently used in serodiagnosis of CE due to their ease of application and low cost. The most commonly used tests for the diagnosis of CE are based on hydatid fluid antigens of E. granulosus.
In conclusion, our study showed that based on the tests evaluated here, an efficient approach to the serological diagnosis of cystic echninococcosis is primary testing with the crude E. granulosus. We also showed that it is easy-to-prepare and inexpensive metacestode-derived native antigens of E. granulosus are valuable tools for the diagnosis of CE in clinical settings. From a diagnostic perspective, 8 kDa and 12 kDa bands are sufficient to determine the specific diagnosis of HCE patients, too.

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.