Original Article

Ann Lab Med 2024; 44(2): 155-163

Published online March 1, 2024

Copyright © Korean Society for Laboratory Medicine.

Head-to-Head Comparison of Nine Assays for the Detection of Anti-Echinococcus Antibodies: A Retrospective Evaluation

Carolina Mattwich, M.D.1 , Kristina Huber, M.D.2 , Gisela Bretzel, M.D.2 , Sebastian Suerbaum, M.D.1 , Andreas Wieser, M.D.1,2,3,4,* , and Karl Dichtl, M.D.1,5,*

1Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Medizinische Fakultät, LMU München, Munich, Germany; 2Division of Infectious Diseases and Tropical Medicine, University Hospital Ludwig-Maximilian University Munich, Munich, Germany; 3German Centre for Infection Research (DZIF), Munich, Germany; 4Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Immunology, Infection and Pandemic Research, Munich, Germany; 5Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria

Correspondence to: Karl Dichtl, M.D.
Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Neue Stiftingtalstraße 6, Graz 8010, Austria

Andreas Wieser, M.D.
Division of Infectious Diseases and Tropical Medicine, University Hospital Ludwig-Maximilian University Munich, Leopoldstraße 5, Munich 80802, Germany

* These authors contributed equally to this study.

Received: May 19, 2023; Revised: July 25, 2023; Accepted: September 12, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Echinococcosis is a neglected tropical disease that is severely underdiagnosed in resource-limited settings. In developed countries, diagnosing echinococcosis is challenging, and reliable serological assays are urgently needed. In the Central European Alps, EM is more common than EG; however, data on the diagnostic performance of assays for EM cases are scarce. We evaluated the suitability of nine antibody assays for routine diagnostics.
Methods: Nine commercially available serological assays for detecting anti-Echinococcus antibodies were compared head-to-head using samples collected from 50 patients with echinococcosis and 50 age- and sex-matched control subjects. The assays are Anti-Echinococcus ELISA (IgG) (Euroimmun), Echinococcus IgG ELISA (DRG), Echinococcus IgG ELISA (IBL International), Echinococcus Western Blot IgG (LDBIO Diagnostics), EUROLINE WB (Euroimmun), Hydatidosis ELISA IgG (VirCell), Hydatidosis VIRCLIA IgG Monotest (VirCell), Ridascreen Echinococcus IgG (R-Biopharm), and Virapid Hydatidosis (VirCell). The cases were ranked according to the WHO-Informal Working Group on Echinococcosis (WHO-IWGE) criteria as confirmed, probable, or possible.
Results: The performance of the assays varied greatly, with overall sensitivities ranging between 50% and 88% and specificities between 62% and 100%. We observed a trend toward better performance with cases classified as “confirmed” using the WHO-IWGE criteria. Combined analysis with sequential screening and confirmatory testing resulted in a maximum sensitivity of 84% and specificity of 100%. Differentiation between EG and EM infections is clinically relevant but was found to be unreliable.
Conclusions: Echinococcus serological assays are highly variable in terms of sensitivity and specificity. Knowledge of the pre-test probability in the patient cohort is required to choose a suitable assay. A combined approach with screening and confirmatory assays may be the best diagnostic strategy in many situations.

Keywords: Alveolar echinococcosis, Antibodies, Comparative study, Cystic echinococcosis, Echinococcosis, Echinococcus granulosus, Echinococcus multilocularis, Hydatidosis, Serology

Echinococcosis is a zoonotic disease caused by cestodes of the genus Echinococcus. Echinococcus granulosus (EG) and Echinococcus multilocularis (EM) are the most relevant species in virtually all cases of human echinococcosis [1]. The incidence and prevalence of echinococcosis are highly dependent on the region studied, and up to 18,000 cases of EM infections may occur per year, with the vast majority occurring in China (>90%), followed by Central Europe [2-4]. The burden of EG infections is mostly associated with large stray dog populations and is increasing in endemic areas such as North and East Africa, South America, and Central Asia [5, 6]. Prevalences up to 5% have been reported in Chinese hotspots [4, 5, 7]. Underdiagnosis is suspected in many resource-limited settings worldwide [4].

When echinococcosis is left untreated or responds poorly to treatment, infection often results in liver failure and death [5, 8]. Mortality rates in patients with untreated alveolar echinococcosis (AE) exceed 90% [7]. The burden of disability-adjusted life years (DALYs) due to cystic echinococcosis (CE) is estimated to exceed one million [9], and nearly 700,000 DALYs per year are attributable to AE [10]. Furthermore, echinococcosis causes major economic damage because it affects patients in all age groups, including the most economically productive age groups; yearly global losses are estimated to exceed 760 million dollars [9].

Targeted antiparasitic therapy, surgery, and percutaneous treatment strategies for EG cysts like PAIR (puncture, aspiration, injection, re-aspiration) can reduce morbidity and mortality and even be curative [7]. However, chemotherapy and surgical treatment can cause severe side effects, complications, and permanent disabilities [5]. Therefore, all therapeutic interventions must be based on a reliable diagnosis and longitudinal surveillance. Only direct pathogen detection using microscopy or molecular testing can provide certainty [7]. However, this requires diagnostic puncture of a suspicious lesion, which is stressful for the patient and may have serious consequences. For example, puncture of EG cysts may lead to pathogen release, resulting in secondary dissemination, allergic reactions, or anaphylactic shock [5, 8, 11]. Although ultrasound imaging is the basis for diagnosing abdominal CE, an atypical presentation occurs in approximately 30% of all cases and requires an experienced examiner [7]. Therefore, the guidelines of the WHO-Informal Working Group on Echinococcosis (WHO-IWGE) recommend serology for diagnosing CE and AE [7]. Detecting specific antibodies in venous blood samples is inexpensive, non-invasive, and applicable also in resource-limited settings. However, the specificity of detection can be lowered by cross-reactions with other cestode or helminth infections, malignancies, and liver cirrhosis. Concerns also exist regarding reduced sensitivity for infections with extrahepatic lesions [7]. We evaluated and compared the performance of nine serological assays for diagnosing EG and EM infections.

Patients and samples

For this retrospective analysis, we analyzed stored clinical samples between August and November 2021 at the Max von Pettenkofer Institute (Munich, Germany). All 100 patients from whom samples were obtained were treated at LMU Klinikum, an academic medical center (Munich, Germany). The study cohort consisted of serum samples from 50 patients who were pre-characterized as positive for echinococcosis. Seventeen, 23, and 10 patients met the criteria for confirmed, probable, and possible echinococcosis, respectively, as defined by the WHO-IWGE consensus recommendations [7]. We diagnosed 24 of the 50 infections through direct pathogen detection (13 cases of EG and 11 cases of EM). However, due to a lack of clinical data concerning “epidemiological history” as requested in the WHO-IGWE guidelines, not all of these cases met the criteria for categorization as confirmed cases [7]. The CE and AE subgroups consisted of 24 and 20 patients, respectively. In the remaining six cases, definitive differentiation was not possible or the data were unavailable to us. The diagnoses and patient characteristics are summarized in Supplemental Data Table S1. The control group consisted of serum samples from 50 outpatients matched for age and sex who presented for Lyme disease assessment. This study was reviewed and approved by the ethics committee of our university hospital (Ethikkommission der Medizinischen Fakultät der LMU München; approval No. 17-244), which waived the requirement for informed consent.

Applied assays

Samples were tested using five ELISAs, two line blots, a chemiluminescence immunoassay (CLIA), and a lateral flow assay (LFA), which are summarized in Table 1, including their abbreviated names. The ELISAs were performed manually, except for the ELISA Euro, which was processed using the EUROIMMUN Analyzer I system (Euroimmun Medizinische Labordiagnostika, Lübeck, Germany). This device was used for the optical readout of all ELISAs. CLIA testing was performed using the Thunderbolt Analyzer platform (Gold Standard Diagnostics, Davis, CA, USA). The Euroimmun blot results were assessed using the manufacturer’s EUROLineScan system. The second line blot and the LFA results were visually evaluated independently by two skilled examiners. In cases of incongruency, a third independent examiner was consulted whose vote was used to obtain a definitive assessment. All assays were performed according to the manufacturer’s instructions.

Table 1 . Assays evaluated in this study

Assay typeAssay nameAbbreviationTarget*ManufacturerRequired sample volume (µL)Time to result (mins)Required equipment
Line blotEUROLINE WBBlot EuroEG, EMEuroimmun30150Blot shaker (scanner)
Line blotEchinococcus Western Blot IgGBlot LDBEG, EMLDBIO Diagnostics25170Blot shaker
CLIAHydatidosis VIRCLIA IgG MonotestCLIA VirCEGVirCell5130CLIA processor
ELISAAnti-Echinococcus ELISA (IgG)ELISA EuroEG, EMEuroimmun10130- (ELISA processor)
ELISAEchinococcus IgG ELISAELISA DRGEG, EMDRG10140- (ELISA processor)
ELISAEchinococcus IgG ELISAELISA IBLEG, EMIBL International10110- (ELISA processor)
ELISARidascreen Echinococcus IgGELISA rBioEG, EMR-Biopharm1050- (ELISA processor)
ELISAHydatidosis ELISA IgGELISA VirCEGVirCell5100- (ELISA processor)
LFAVirapid HydatidosisLFA VirCEGVirCell3030-

*The category “Target” indicates the species (EG or EM), against which the specific antibodies detected by this assay are directed.

The required equipment includes laboratory equipment other than pipettes, pipette tips, incubation trays, and tubes. Equipment in parentheses is supportive but not mandatory. The manufacturer offers a specific scanner system (EuroScanner) for the blot Euro. The CLIA VirC requires a specific CLIA processor (VirCLIA or VirCLIA Lotus). All ELISAs shown can be performed manually or with an automated ELISA processor.

Abbreviations: EG, Echinococcus granulosus; EM, Echinococcus multilocularis; CLIA, chemiluminescence immunoassay; LFA, lateral flow assay.

Statistical analysis, data protection, and ethics approval

Sample processing and data analyses were performed anonymously. Clinical information and reference standard results were not available to the performers or readers of the assay. Sensitivity and specificity were determined as the fractions of true-positive results in the case group and true-negative results in the control group, respectively. For assays that can yield borderline results, sensitivities and specificities were calculated for both scenarios with (1) borderline results interpreted as positive and (2) borderline results interpreted as negative results. We determined 95% confidence intervals (CIs) for all sensitivity and specificity calculations. Microsoft Excel (Microsoft Corporation, Redmond, WA, USA), GraphPad Prism 9 (GraphPad, San Diego, CA, USA), and the GraphPad QuickCalcs online tool were used for statistical analyses.

Sensitivity and specificity

We assessed 50 serum samples from patients with echinococcosis in nine assays. The sensitivities ranged from 50% to 88% (CI: 36–64 and 79–97; Table 2). The sensitivity varied with different WHO-IWGE categories (sensitivities: confirmed>probable >possible) and species (Supplemental Data Table S2). In seven of the nine assays, borderline results can occur. The rate of borderline results ranged from 2% (CLIA VirC and blot Euro; CI: 0–5) to 13% (ELISA IBL; CI: 6–20). When borderline results were counted as positive results, the sensitivities of all seven assays increased (sensitivities ranging from 72% to 96% [CI: 60–84 and 91–100]; Table 2). The sensitivity of the ELISA VirC assay increased most by including borderline results, with a sensitivity increase by 16% to 84% (CI: 74–94). In each assessment (positive vs. positive and borderline), the ELISA IBL demonstrated the highest sensitivity, followed by the Euroimmun assays (blot and ELISA). The LFA VirC had the lowest sensitivity (50% [CI: 36–64]). However, this assay and the other two VirCell assays are only certified for diagnosing CE (Table 1), as indicated in the manufacturer’s intended use declaration. In patients with CE, the sensitivity of LFA VirC was 67% (CI: 48–86) but was only 25% in the AE group (CI: 6–44; Supplemental Data Table S2). Neither the other two VirCell assays nor any other assays demonstrated major differences in performance between the AE and CE cases.

Table 2 . Sensitivities and specificities of the assays

AssaySensitivity Cases (N=50)Specificity Controls (N=50)
% (CI)N% (CI)N% (CI)N% (CI)N
Blot Euro84 (74–94)4288 (79–97)4496 (91–100)4896 (91–100)48
Blot LDB78 (67–89)39--100 (100–100)50--
CLIA VirC78 (67–89)3982 (71–93)41100 (100–100)50100 (100–100)50
ELISA Euro80 (69–91)4090 (82–98)4598 (94–100)4998 (94–100)49
ELISA DRG72 (60–84)3678 (67–89)3992 (85–100)46100 (100–100)50
ELISA IBL88 (79–97)4496 (91–100)4862 (49–75)3180 (69–91)40
ELISA rBio64 (51–77)3272 (60–84)36100 (100–100)50100 (100–100)50
ELISA VirC68 (55–81)3484 (74–94)42100 (100–100)50100 (100–100)50
LFA VirC50 (36–64)25--100 (100–100)50--

*Assay sensitivities after counting the borderline results among the positive samples and test specificities after counting the borderline results among the negatives are only indicated for assays for which borderline cut-off values are defined.

Abbreviations: CI, 95% confidence interval; CLIA, chemiluminescence immunoassay; LFA, lateral flow assay.

According to the manufacturers, the two line blot-based assays can differentiate between AE and CE. The blot Euro yielded species results in 58% of all cases (CI: 44–72), and the blot LDB yielded species results in 46% of all cases (CI: 32–60). For EM infection, the rates of correct species identification with the blot Euro and the blot LDB were 55% and 10% (CI: 33–77 and 0–23), respectively. Notably, the blot LDB misidentified two EM infections as EG infections, which were correctly identified by the blot Euro. For EG infection, the identification rates of the blot Euro and the blot LDB were 58% and 58% (CI: 39–78 each), respectively, with another case misclassified by the blot LDB. The assays demonstrated high specificity, except for the ELISA IBL (Table 2), with specificities of 62% and 80% (with borderline results assigned to the positive/negative results; CI: 49–75 and 69–91), respectively.

We also assessed the sensitivity and specificity of two sequential assays. The first assay was considered a screening assay, and samples yielding positive and borderline results were retested with the second assay (confirmatory assay), which rejected negative and borderline results for added specificity. The highest sensitivity was achieved by applying the ELISA Euro as a screening assay and the ELISA IBL as a confirmatory assay (86%; CI: 76–96). While the specificity of this combination was only 98% (CI: 94–100), four other combinations yielded 100% specificity (CI: 100–100) with still 84% sensitivity (CI: 74–94): blot Euro+ELISA IBL, ELISA Euro+blot Euro, ELISA IBL+blot Euro, and ELISA VirC+ELISA IBL.

Agreement and correlation

Twenty-four control samples tested as non-negative, that is, positive or borderline, in at least one assay (Fig. 1A). The two positive blot Euro results were not confirmed with any other assay. A single sample that was positive in the ELISA Euro was also detected by the ELISA IBL (positive) and ELISA DRG (borderline). Notably, all false non-negative ELISA DRG results were borderline but not positive. Finally, 18 of the 19 control samples tested positive or borderline (nine each) by the ELISA IBL were exclusively tested non-negative by this assay.

Figure 1. Venn diagrams depicting the agreement of different assay systems. (A) Agreement of all measurements for control samples that yielded positive or borderline results. Assays that did not yield false non-negative results are not represented. Intersections without numerals represent instances with zero concordant results. (B) Agreement of positive results between three anti-Echinococcus IgG assays produced by the same manufacturer (VirCell).

In the positive cohort, 22 of 50 infections were detected (positive or borderline results) in all assays (44% [CI: 30–58]). Two samples were negative in all assays, and three samples were detected with only one assay (ELISA IBL). Notably, these five cases were only categorized as probable or possible Echinococcus infections (one and four cases, respectively), which may reflect misdiagnoses. In 20 of the remaining 23 cases (87% [CI: 73–100]), five or more assays showed positive results with anti-Echinococcus antibodies. We observed a correlation between the different assays of one manufacturer: the Euroimmun assays (ELISA and blot) only had one discrepant result. All VirC LFA-positive cases were detected using two other VirCell assays: the CLIA and ELISA (Fig. 1B). By comparing the results obtained with the two blots, we observed that the Euroimmun assay detected every LDB-positive sample.

Two assays were evaluated visually by two readers, resulting in several discordant results: for the LFA VirC, discordant qualitative results (negative vs. positive) occurred with 4% of samples (CI: 0–8), and discordant results regarding the signal intensity (four degrees of positivity) occurred with another 5% of samples (CI: 1–9). The blot LDB yielded discordant results for 25% of the samples (CI: 17–33). In 3% of the samples (CI: 0–6), the readers’ disagreement concerned qualitative results. Discrepancies in species identification occurred with 21% of the samples (CI: 13–29).

Correlations between the quantitative assay results varied widely, which is exemplary depicted (CLIA VirC vs. every ELISA) in Fig. 2 and summarized in Supplemental Data Table S2. We did not observe the highest correlation between two assays from the same manufacturer, that is, the VirCell CLIA and ELISA. Instead, the highest correlation was found between the ELISA Euro and the ELISA DRG, which demonstrated a strong correlation (Pearson’s r value: 0.98). For all comparisons, except for one assay, the Pearson’s r value was ≥0.81. The ELISA rBio correlated poorly with all other assays (Pearson’s r values ranging from 0.57 to 0.64).

Figure 2. Correlation of measured results obtained with the chemiluminescence immunoassay (CLIA) from VirCell and the indicated ELISAs. The gray bars represent regions containing borderline results. The triangle represents a result above the measurement range of the ELISA rBio.

Cut-off analysis

We performed ROC curve analysis for all quantitative assays (Fig. 3; the CLIA and ELISAs). Despite notable differences in sensitivity and specificity, we found little variation in the area under the curve (AUC) values. The AUC values ranged from 0.94 (CI: 0.90–0.99; ELISA IBL) to 0.98 (CI: 0.96–1.00; ELISA VirC). As these data suggest that the discriminative power of the tests was essentially comparable, optimized cut-off values were investigated. Youden indices (YIs) ranged between 0.78 (ELISA IBL) and 0.88 (ELISA Euro and ELISA VirC). In cases where multiple cut-offs resulted in the same YI, we used the cut-off that resulted in the highest specificity. With only one assay (ELISA Euro), the optimized cut-off and the manufacturer-recommended cut-off agreed. However, our YI analysis identified already the lower manufacturer’s cut-off (negative vs. borderline) as the optimal cut-off for positivity (Table 3). Compared with the optimized cut-off values, the manufacturers’ recommendations are in some cases dramatically higher, i.e., with the CLIA VirC (YI of 0.3 vs. 0.9) or the ELISA VirC (YI of 3 vs. 9). For all but one assay, the optimized cut-off values offered increased sensitivity with little or no loss of specificity (Table 3). With only the ELISA IBL, which initially stood out with extreme values for sensitivity (highest: 96% [CI: 91–100]) and specificity (lowest: 62% [CI: 49–75]), the YI analysis resulted in a higher cut-off of 15 instead of 11. This moderate increase reduced the sensitivity to 84% (CI: 74–94) but increased the specificity by 32% to 94% (CI: 87–101). Using this modification, the ELISA IBL demonstrated a test performance comparable to that of the other quantitative assays.

Table 3 . Changes in the assay performance based on optimized co values*

AssayOriginal coOptimized coSensitivity, %Specificity, %
NegativePositiveOriginal co (CI)Optimized co (CI)Original co (CI)Optimized co (CI)
CLIA VirC<0.9>1.1Lowered to ≥0.378 (67–89)86 (76–96)100 (100–100)100 (100–100)
ELISA DRG<9>11Lowered to ≥772 (60–84)80 (69–91)100 (100–100)96 (91–100)
ELISA Euro<0.8≥1.1Lowered to ≥0.880 (69–91)90 (82–98)98 (94–100)98 (94–100)
ELISA IBL<9>11Raised to ≥1588 (79–97)84 (74–94)80 (69–91)94 (87–100)
ELISA rBio<0.9>1.1Lowered to ≥0.564 (51–77)86 (76–96)100 (100–100)98 (94–100)
ELISA VirC<9>11Lowered to ≥368 (55–81)90 (82–98)100 (100–100)98 (94–100)

*The original co values represent the manufacturers’ recommendations, and the optimized co values relied on the YI calculations. When two co values had the same YI, the co with higher specificity was selected.

Abbreviations: co, cut-off; CI, 95% confidence interval; CLIA, chemiluminescence immunoassay; YI, Youden’s index.

Figure 3. Results obtained for the case and control groups along with ROC curves for each quantitative assay. (A) The gray bars represent regions containing borderline results. The triangle represents a result above the measurement range of the ELISA rBio. (B) Sensitivity is plotted on the Y-axis and 1–specificity is plotted on the X-axis. The gray tick marks on the ROC curve represent the upper and lower cut-offs recommended by the respective manufacturer. Squares mark the cut-offs with the highest Youden’s indices.
Abbreviation: AUC, area under the curve.

Handling of the assays

The ELISAs assessed can be performed manually with no specific requirements or with standard ELISA processors. For the CLIA VirC, a specific testing device enabling random-access single-sample testing is required. The blots can be run and evaluated manually. While the standardized antigen-blotting pattern allows automated readout for the blot Euro, notable variability of the blot LDB regarding the band position and size requires manual evaluation of the results. The LFA requires only a centrifuge and pipette as laboratory equipment, and the assay can even be stored at room temperature.

In most cases, imaging is the method that initially raises suspicion of an Echinococcus infection [7, 12]. However, imaging provides low specificity, especially for AE but less so for CE [5, 12]. Extrahepatic manifestations are also challenging in imaging, especially when bone structures are affected [5, 12]. Direct pathogen detection is highly specific but has low sensitivity and requires invasive sampling [7]. Serological testing is recommended as a convenient additional diagnostic tool [7, 13]. However, ambiguity exists regarding the performance of antibody-based detection, and a recent meta-analysis showed that the sensitivity of serology testing ranges from 14% to 99% [14].

The evaluated assays proved their utility in diagnosing echinococcosis. Regarding sensitivity, we found one exception, the VirC LFA, which detected 50% of Echinococcus infections. However, this assay was only designed and certified for diagnosing CE, which was identified in 67% of the respective samples. The VirC LFA (which is virtually independent of laboratory equipment) was designed for use in resource-limited healthcare settings, like those found in developing countries. Because CE is the most prevalent in developing countries, it seems reasonable to optimize this assay for detecting CE infection. Our ROC curve analysis showed that most assays did not reach their full potential; minor cut-off modifications resulted in a major gain of performance. For example, such modifications increased the sensitivity of the ELISA VirC by 22% or the specificity of the ELISA IBL by 14%. Combining high-sensitivity screening and high-specificity confirmation assays is recommended [7]. Therefore, considering only the results of each single assay even underestimates the performance of serological testing. Combining the ELISA IBL with either the CLIA VirC or the blot LDB resulted in a sensitivity of 78% and a specificity of 100% using the manufacturer-recommended cut-off values and classifying borderline results as negative.

We included several assays for which little or no performance data are available (CLIA VirC, ELISA VirC, blot Euro, and ELISA Euro). All four assays demonstrated performances comparable to those of more widespread and established serological assays. However, we observed notable differences from previous reports showing that the sensitivity of the LFA VirC was comparable to that of ELISA testing and was as high as 97% (this study: 50%) [15-17]. This surprising finding might be explained by the applied case definitions. For instance, the diagnosis of echinococcosis relied on the ELISA DRG results in the study by Tamer, et al. [15]. In our study group, the LFA VirC assay yielded positive results for 33 of the 34 ELISA DRG-positive samples (97%). Considerably, the ELISA DRG demonstrated a sensitivity of only 72%. Regarding the ELISA rBio, two studies were published recently: one report showed higher sensitivity (88%) and one report showed lower sensitivity (47%) than that observed in this study (64%) [18, 19]. Interestingly, ELISA IBL, which demonstrated the highest sensitivity in our study, was excluded from further analysis in a recent study by Tamarozzi, et al. [20] owing to a surprisingly low sensitivity of <30%.

Several reasons might account for the observed differences. This study relied on WHO-IWGE case definitions, which could have resulted in different pre-test probabilities. Furthermore, all the aforementioned studies relied exclusively on cases of CE. While this infection plays only a minor role in developed countries, AE is endemic in a substantial portion of Central Europe. However, few studies have been conducted to investigate the role of serology in AE. Two recent studies based on a mixed AE/CE cohort included 10 and 15 cases of AE, respectively [17, 21]. However, differentiation between AE and CE relied on serological results in one study, and in the other study, only the two blot assays were compared with sera from patients preselected through serological screening, which might explain the reported high sensitivity (>90% for both blot assays). However, a noteworthy trend emerged that we also observed in our cohort: species identification was particularly challenging for AE, with sensitivities being much lower for AE than for CE. For instance, the blot LDB identified 58% of CE cases but only 10% of AE cases. Additionally, the assay yielded false species diagnoses in 7%–8% of our cases and also in a recent evaluation [21]. In contrast, the blot LDB performed best among all assays evaluated in a CE cohort [20]. Antigen selection appears to be the key to assay performance. By comparing the assays of one manufacturer, i.e., Euroimmun, we found that all assays provided concordant results except with one sample. Similarly, all VirC LFA-positive cases were detected by the two other VirCell assays (CLIA and ELISA; Fig. 1B). However, it seems that not many manufacturers take special precautions to identify specific EM antigens and to evaluate them. Overall, our results suggest that further studies are necessary to investigate the performance of serology in AE, and awareness of these species-dependent pitfalls should be raised in medical and laboratory teams.

Because of the significant proportion of AE cases and the comprehensive assay selection, we believe that our study contributes to the understanding of antibody-based detection of echinococcosis. However, this study also has certain limitations, as all our cases rely on the most recent WHO-IWGE classification guidelines that are over a decade old and are currently under revision, and broad consensus exists in the field that previous developments need to be considered [7]. However, to ensure comparability with other studies, we refrained from modifying the definitions, which were widely used in previous studies. Other limitations can be primarily attributed to the lack of clinical data such as details pertaining to the specific nature of the observed lesions, although lesion presentation can be associated with infection activity. For instance, according to the WHO-IWGE classification, lesions in stadium CE4 or CE5 are typically found when the infection is overcome [7]. Notably, antibody production decreases significantly in such situations [14, 22, 23]. Another important factor affecting the sensitivity of serology is ongoing or successful treatment, which eventually results in reduced antibody concentrations [7]. Due to the lack of clinical data, we cannot exclude the possibility that our samples were obtained from patients in whom the infection was cleared or suppressed. These factors might have led to false-negative results and, consequently, to an underestimation of the assay sensitivity. For subjects in the control cohort, the clinical data were restricted to the information that the individuals were outpatients tested for antibodies against Borrelia. It even cannot be excluded that patients suffering from echinococcosis were part of the control cohort. However, the epidemiological data and the finding of only very few positive results in this group strongly argue against this consideration.

Taken together, our data demonstrate that antibody detection is a supportive and valuable tool for diagnosing echinococcosis. Nevertheless, the performance of several assays could be improved with minor cut-off modifications. Healthcare providers should be aware of the local prevalence of AE vs. CE when choosing between the available assays. Species identification based on antibody testing must be considered with caution.

Mattwich C contributed to the methodology, formal analysis, data investigation of the study and to manuscript preparation (original draft, review, and editing). Huber K contributed to data investigation. Bretzel G contributed to data investigation, resources, and manuscript preparation (review and editing). Suerbaum S contributed to resources and manuscript preparation (review and editing). Wieser A contributed to the conception, resources, supervision, and administration of the study and manuscript preparation (review and editing). Dichtl K contributed to the conception, methodology, formal analysis, investigation, visualization, supervision, and administration of the study and manuscript preparation (original draft, review, and editing). All authors have read and approved the final manuscript.

Mattwich C, Huber K, Bretzel G, and Suerbaum S have no conflicts of interest to disclose. Wieser A received personal and grant funds from different pharmaceutical and technical companies unrelated to the project. Dichtl K received grants to the institution by Fujifilm Wako Chemicals Europe and Euroimmun Medizinische Labordiagnostika outside of this study.

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