Crude protoscolex antigens of echinococcus granulosus as serological markers for human hydatidosis detection

Abstract

Cystic echinococcosis (CE) or hydatidosis is a parasitic disease affecting both humans and animals. The present study aimed to evaluate the efficacy of the ELISA test in diagnosis of human hydatidosis using crude protoscolices antigens. Echinococcus granulosus protoscolices were isolated from the human liver hydatid cyst (local strain G1). Their crude antigen was prepared by the sonication technique. One hundred and eight sera samples were collected from patients with hydatid cyst (n = 21), individuals with other infections (n = 27) and sheep breeders (n = 60). These sera were evaluated using the ELISA assay. The predictable rate of sensitivity for the ELISA using the crude antigens was 100% depending on the cut-off. Additionally, the estimated values of specificity, positive and negative predictive values and accuracy were 97.4%; 87.3%; 100% and 97.8%, respectively. Crud E. granulosus protoscolices antigens are considered suitable candidates for the sero-diagnosis of human CE. To identify a single antigen and increase its effectiveness in serologic diagnostic testing, more research on these antigens is needed.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-17798-1.

Introduction

Cystic echinococcosis (CE) or hydatidosis caused by Echinococcus granulosus, is a neglected parasitic disease with worldwide distribution, mostly affecting all countries of the Middle East^1,2. An adult worm of E. granulosus lives in the small intestine of the carnivores as a definitive host. However, the metacestodes develop as hydatid cysts, in the internal viscera (e.g., liver, lungs…etc.) in herbivores and humans (intermediate host) after consuming the food contaminated with worms’ eggs. These cysts contain daughter cysts, protoscolices and antigens belonging to the parasite and host³.

Imaging techniques are still one of the most useful tools to identify the cyst in the body, but they are not able to differentiate the cyst from benign or malignant tumors^4,5. Different immunological methods, including an indirect hemagglutination test (IHA), a complement fixation test (CFT), an immunofluorescence assay (IFA), western blotting, and an enzyme-linked immunosorbent assay (ELISA), are the most common methods used for the specific diagnosis of CE^6,7.

E. granulosus antigens, such as crude antigen (CA), protoscolex antigens (PA), native and recombinant B antigen, have been utilized for the diagnosis of CE⁸. Antigen B is considered the most important antigen, which is produced by protoscolex coat cells and is one of the most abundant parasitic antigens present in hydatid cyst fluid⁹. Consequently, WHO recommended the preparation of native B antigens for use in the diagnosis of this disease and reliance on antigens derived from protoscolices antigen instead of hydatid fluid antigen in diagnosis mainly by ELISA¹⁰.

The present study aimed to evaluate the efficacy of the ELISA, using Echinococcus protoscolices antigens, for the diagnosis of human CE.

Materials and methods

Ethics approval and informed consent

The study was approved by the Institutional Review Board and Ethics Committee on Human Research of Damascus University (#2656/2023) before its commencement. A written informed consent was obtained from all patients participating in the study, or their parents or legal guardians.

Protoscolex antigen Preparation

Antigen was prepared from protoscolices derived from human hydatid cyst in the liver, previously sequenced and deposited in GenBank under accession number (PP385946.1). Under the aseptic conditions, the aspirated protoscolices from the hydatid cyst fluid were washed three times with PBS buffer and examined by a light microscope and 0.1% eosin (Sigma-Aldrich, Louis, MO, USA) staining to assess their viability. A crude antigen was prepared as the following: protoscolices were lysed in 500 µl lysis buffer (150 mM NaCl, 10 mM TrisHcl, 1 mM EDTA, 0.1 mM Nonidet p40) after adding the protease kinase inhibitor and serine protease inhibitor, then sonicating (8 cycles/15 sec) using a 200w ultrasonic disintegrator (Hielscher UP200St; Germany) until no intact protoscolices was visible in microscope. The sample was standing for one hour then centrifuged at 14,000 g for 10 min at 4 °C. After filtering the supernatant, protoscolices total proteins (PSCsTP) concentration was estimated by Bradford method¹¹. Antigen was aliquoted into 0.5 ml microtubes and stored at −20 C until further processing.

Human serum samples

Forty-eight blood samples were collected from National University Hospital patients for digestive disturbances, abdominal pain and fever. The blood samples were divided into two groups:

• Gr 1: Twenty-one samples (7 males and 14 females), used as positive control, were collected from hydatidosis (13 single/8 multiple cysts in liver “n = 18” and lungs “n = 3”) surgically proven patients (the cysts size varied between 6.5 and 15 cm).

• Gr 2: Twenty-seven samples (14 males and 13 females) were collected from patients with other diseases: visceral leishmaniasis (n = 2), toxoplasmosis (n = 2), blastocystosis (n = 2) and colorectal cancer patients (n = 21). The samples were used as negative controls after CT scans were performed on these patients to rule out hydatid disease.

Sixty sporadic samples (43 males and 17 females) were collected from sheep breeders and their families who are in direct contact with dogs, from eastern Damascus Suburb.

The age of the participants in this study ranged between 14 and 65 years old. All blood samples were allowed to clot for serum separation, aliquoted and stored at −20 C until used.

ELISA test

Each serum sample was tested for anti-Echinococcus antibodies in an ELISA. In Brief, an ELISA plate was sensitized with PSCsTP antigen (0.1 µg/well) diluted in coating buffer (PBS, pH = 6) and incubated overnight at 4 °C. They were washed three times by.

PBS-T₂₀ buffer and dried completely. Two hundred microliters of blocking solution (5% skimmed milk in PBS, pH = 6) were added to each well and the microplates were incubated at RT for 1 to 2 h. After three washes, 100 µl of diluted serum samples were loaded into each well (at a dilution of 1:10 in PBS containing 1% skimmed milk) and incubated at RT for 1 h. After washing, Alkaline Phosphatase rabbit anti-human was added at a dilution of 1:2000 into all the wells and incubated for 1 h at RT. After three washes to remove the excess conjugate, 50 µl per well AP substrate (p-Nitrophenyl Phosphate (pNPP) & 0.02%, hydrogen peroxide) was added and incubated at RT for 45 min in the dark. Finally, the absorbance density (OD) was registered at 405 nm by ELISA reader (Thermo Scientific™ Multiskan™ GO, USA) after the addition of 50 µl per well of stop solution (0.2 M H₂SO₄). The cutoff value was calculated by an average of negative control OD value + 3 standard deviation (SD).

Statistical analysis

The input data of samples and ELISA results were analyzed by SPSS (version 26 software) to evaluate the descriptive study’s Chi-square and ROC tests. MedCalc software Ltd was used to verify the accuracy of the assay. GraphPad Prism (version 8.0.1) was used to present the data.

Results

The viability of protoscolices, from local strain of human-cyst origin, using the light microscope (Olympus CX21 Japan), reached 95% (Fig. 1, a). Quality analysis of the prepared E. granulosus protoscolices antigen using SDS-PAGE (Fig. 1, b) showed that the extracted protein (with concentration ranging between 1 and 5 µg/µL) was of high quality and could be used in an ELISA assay.

Fig. 1.

Human protoscolices hydatid cyst. a. High number of live protoscolices under microscope (magnification x10), dead protoscolex (red). b. Electrophoresis of protoscolices proteins using 12% SDS-PAGE gel stained with Coomassie blue dye. M: molecular weight standard, PSCsTP: crude protoscolices protein.

When the crude E. granulosus protoscolices protein (PSCsTP) was used in an ELISA test to detect anti-Echinococcus antibodies, taking into consideration the calculated cut off (0.213), a total of 21 (100%) serum samples from group 1, were positive (OD = 0.386 ± 0.015). No significant relation between OD intensity and the number of hydatid cysts or its site (P = 0.496; P = 0.489 respectively) were observed. However, this test was not specific enough, because cross reaction was observed with sera pertaining to blastocystosis (2/27; 7.4%) while no cross reactions occurred with serum samples from patients infected with colon tumors (OD = 0.177 ± 0.003), leishmaniasis and toxoplasmosis (OD = 0.189 ± 0.004) (Fig. 2, a). However, only 9/60 (15%) of serum samples from group 3 (6 males and 3 females) revealed positive reaction (Fig. 2, b).

Fig. 2.

a: PSCs-ELISA sensitivity and specificity optimization depending on group 1 as positive control (red above the cut off line) and group 2 as negative control (blue below the cut off line). Cut off = 0.213. b: PSCs-ELISA screening test for all serum groups. Positive (red), other parasite/cancer patients’ serum (Negative: blue; cross reaction: green), and sheep breeder sera (purple). Cut off = 0.339.

The age of these seropositive cases ranged between 15 and 34 and 35–64 years old (Table 1). No significant relation was observed between the infected cases and the demographic features.

Table 1.

Demographic features of the studied groups.

Features	Gr 1		Gr 2		Gr 3		Total		P value
Gender
Female N (%)	Neg	Pos	Neg	Pos	Neg	Pos	Neg	Pos	0.089
	0	14 (66.7%)	13 (48.1%)	0	14 (23.3%)	3 (5%)	27 (25%)	17 (15.7%)
Male N (%)	Neg	Pos	Neg	Pos	Neg	Pos	Neg	Pos
	0	7 (33.3%)	12 (44.4%)	2 (7.4%)	37 (61.7%)	6 (10%)	49 (45.4%)	15 (13.9%)
Age (Years)
< 14 N (%)	Neg	Pos	Neg	Pos	Neg	Pos	Neg	Pos	0.976
	0	3 (14.3%)	0	0	7 (11.7%)	0	7 (6.5%)	3 (2.8%)
15–34 N (%)	Neg	Pos	Neg	Pos	Neg	Pos	Neg	Pos
	0	4 (19.0%)	0	0	12 (20%)	1 (1.7%)	12 (11.1%)	5 (4.6%)
35–64 N (%)	Neg	Pos	Neg	Pos	Neg	Pos	Neg	Pos
	0	12 (57.1%)	15 (55.6%)	0	30 (50%)	8 (13.3%)	45 (41.7%)	20 (18.5%)
> 65 N (%)	Neg	Pos	Neg	Pos	Neg	Pos	Neg	Pos
	0	2 (9.5%)	10 (37%)	2 (7.4%)	2 (3.3%)	0	12 (11.1%)	4 (3.7%)

The estimated values of sensitivity and specificity for the protoscolices antigens in the ELISA were 100% (95% CI: 88.43–100.00%), 97.44% (95% CI: 91.04–99.7%), respectively. As well, its accuracy was 97.82% (Table 2). ROC test illustrated a good value for AUC = 0.896 (81.7–97.5%).

Table 2.

The sensitivity, specificity, PPV, NPV and accuracy of ELISA using protoscolices antigens.

Statistic	Value	95% CI
Sensitivity	100.00%	88.43–100.00%
Specificity	97.44%	91.04–99.69%
Positive Predictive Value (PPV)	87.31%	63.67–96.43%
Negative Predictive Value (NPV)	100.00%	95.26–100.00%
Accuracy	97.82%	92.97–99.66%

Discussion

Human CE is one of the endemic neglected diseases in our region which spreads in rural areas where individuals are in direct contact with dogs and livestock^12,13. Imaging techniques are the most reliable method for diagnosis of CE. Serological tests are still needed to approve positive cases, but their efficacy is insufficient. Therefore, the development of the suitable serological method is needed for diagnosing this disease.

Although there are differences between antigens extracted from adult worms and from protoscolex, it has been demonstrated that these antigens, which have similar sensitivity and specificity, can be used in diagnosis of hydatid disease in different hosts^7,14. Many previous studies showed the importance of using the antigen extracted from E. granulosus protoscolices obtained from the sheep hydatid cyst in serological tests such as ELISA, IFA and latex agglutination test for the diagnosis of human CE⁸.

Our results revealed a high sensitivity of protoscolices antigen, extracted from human local strain, reaching 100% by using ELISA for diagnosing the human hydatidosis. This finding is comparable to ELISA and WB data (100% for each) previously obtained^14,15. Nevertheless, it disagrees with others that reported low sensitivity by using protoscolices for the primary sero-diagnosis of CE^16,17.

The analysis of sera from patients infected with parasitic diseases or with colorectal cancer showed an overall specificity of 97.4%, due to cross-reactivity with sera from patients infected with blastocystosis. This result is slightly higher than the earliest findings that have shown cross-reactivity with sera from patients infected with non-CE parasitic diseases (such as fascioliasis and toxoplasmosis) or autoimmune diseases^8,16. Our data may be attributed to the origin of used strain (human or animal) and to the method of preparing the protoscolices antigens.

Additionally, our study revealed 9 out of 60 (15%) of asymptomatic individuals who were seropositive for anti-HC-IgG-antibodies (anti-HC-Abs). This finding is in line with studies conducted on random human serum samples in Egypt, Iran, and Iraq (54.8%, 18.4%, 5.1%, respectively)^14,18,19. This can be likely caused by contamination either with Echinococcus eggs when contacting dogs or by consuming raw meat when slaughtering sheep in people’s residences¹⁷.

Moreover, our data revealed a high rate of infection with CE in females than in males. This result is in line with previous findings in neighboring countries^17,20–22. This result may be associated with the lifestyle of women in rural areas and to the effect of female hormones on the immune response to Echinococcus infection^23,24. Furthermore, two age groups (15–34 years and 35–64 years) of patients showed a high rate of infection with CE. This finding is comparable to many earlier studies, which showed a relationship between age and the incidence of hydatid disease and reported a high rate among individuals aged between 12 and 50 years^20,21,25. Finally, in this study, there is a limitation concerning other factors about CE (such as: stage, age, location of cyst) which have an impact on the variability of ELISA results.

Conclusion

Findings of the current study demonstrate that the E. granulosus protoscolices antigens, from local human origin strain (G1), are an excellent candidate for the sero-diagnosis of human cystic echinococcosis. Future studies are needed to identify a single specific antigen among the protoscolices antigens to improve the diagnostic performance of it.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

D. S. : sample collection, performing lab experiments and data analysis. H. Ch.: resection of hydatid cysts. AQ. A. and S. Al N. : project design, data analysis, writing and revising the manuscript. All authors reviewed the manuscript.

Funding

The study was conducted with financial assistance from Damascus University (funder No. 501100020595).

Data availability

All results obtained during the current study are included in this article and are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.