Introduction
Human cestodiases, primarily cystic echinococcosis (CE) and neurocysticercosis (NCC), remain among the most significant neglected tropical diseases (NTDs) worldwide, causing a substantial socioeconomic and public health burden. While CE, caused by the larval stage of Echinococcus granulosus, typically affects the liver and lungs, NCC, caused by Taenia solium larvae, is a leading cause of preventable epilepsy globally (Garcia et al. Reference Garcia, Nash and Del Brutto2014).
Despite their different clinical management and therapeutic requirements, the diagnosis of these two infections often converges into a complex dilemma. In many endemic regions, patients may present with non-specific radiological findings where a calcified or cystic lesion in the brain or visceral organs cannot be definitively attributed to one parasite or the other. This diagnostic uncertainty is exacerbated by the poor performance of traditional serological methods based on crude parasite extracts, such as hydatid fluid (HF) or cysticerci homogenates. These native preparations are notorious for high rates of cross-reactivity due to the presence of phylogenetically conserved epitopes shared among different helminth species, a major diagnostic hurdle extensively reviewed by Deckers and Dorny (Reference Deckers and Dorny2010).
To overcome these limitations, the field has transitioned toward the use of purified and recombinant antigens. For CE, the development of chimeric proteins such as DIPOL and specific subunits like 2B2t has been associated with improved diagnostic sensitivity and specificity compared to native antigens (Sánchez-Ovejero et al. Reference Sánchez-Ovejero, Akdur, Manzano-Román, Hernández-González, González-Sánchez, Becerro-Recio and Siles-Lucas2020). Similarly, for NCC, the identification of specific glycoproteins (LLGP) and the recombinant T24H antigen has provided a robust framework for detecting antibodies with minimal cross-reactivity (Handali et al. Reference Handali, Klarman, Gaspard, Dong, Laborde, Noh, Lee, Pratt, Tsang and Wilkins2010).
However, the current diagnostic paradigm still relies mostly on ‘single-disease’ assays, which require multiple separate tests, increasing both cost and the volume of serum needed. The emergence of bead-based multiplex technologies, such as Luminex, offers a potential alternative approach. By coupling specific antigens from both E. granulosus and T. solium onto different fluorescently coded beads, it is now possible to generate a comprehensive ‘immunological profile’ in a single reaction (Hernández-González et al. Reference Hernández-González, González-Bertolín, Urrea, Fleury, Ferrer, Siles-Lucas and Perteguer2022).
This review aims to explore the clinical utility of this multiplex approach, analysing how the integration of precision-engineered antigens can solve the long-standing challenge of differential diagnosis in cestodiases, ultimately leading to more accurate clinical decision-making and improved patient outcomes.
Antigen selection for precision serology
The success of a multiplex platform depends entirely on the ‘building blocks’ (antigens) used to coat the beads. A dual-panel for the differential diagnosis of CE and NCC must include targets that are not only highly sensitive but, most importantly, exclusive to each genus.
The hydatidosis panel: DIPOL and 2B2t
As previously demonstrated, the chimeric antigen DIPOL (comprising epitopes from AgB8/1, AgB8/2, and Ag5) offers a multi-epitope approach that captures a wider range of the host’s antibody repertoire than single subunits. When combined with the 2B2t recombinant protein, the panel achieves a diagnostic sensitivity that rivals native HF while eliminating the risk of cross-reactivity with other helminths (Ozturk et al. Reference Ozturk, Manzano-Román, Sánchez-Ovejero, Caner, Angın, Gunduz and Unver2022; Sánchez-Ovejero et al. Reference Sánchez-Ovejero, Akdur, Manzano-Román, Hernández-González, González-Sánchez, Becerro-Recio and Siles-Lucas2020).
The neurocysticercosis panel: T24H and LLGP
For Taenia solium, the focus shifts to membrane proteins and glycoproteins. The T24H recombinant antigen, derived from the 24 kDa diagnostic protein, has been successfully adapted to multiplex formats, showing very high specificity in selected clinical settings in patients with multiple viable cysts (Levine et al. Reference Levine, Lewis, Rodriguez, Jimenez, Khan, Lin and Tsang2007). Complementing this, the lentil lectin-adherent glycoproteins (LLGP) remain the gold standard for detecting antibodies in NCC, particularly in cases with high parasitic loads (Garcia et al. Reference Garcia, Nash and Del Brutto2014; Handali et al. Reference Handali, Klarman, Gaspard, Dong, Laborde, Noh, Lee, Pratt, Tsang and Wilkins2010).
The multiplex advantage: creating the ‘serological fingerprint’
The technical transition from traditional monovalent assays to multiplexed systems represents a notable methodological shift in the parasitology laboratory. Beyond the mere identification of a pathogen, these platforms allow for a comprehensive ‘immunoprofiling’ of the host’s response.
As summarised in Table 1, the multiplex approach offers several critical advantages over the conventional enzyme-linked immunosorbent assay (ELISA). By utilising microspheres coated with precision-engineered antigens such as DIPOL and T24H, the assay requires a minimal sample volume while providing a simultaneous differential diagnosis. This ‘serological fingerprint’ not only increases laboratory throughput but also may enhance diagnostic accuracy by providing a comparative view of antibody levels against multiple species-specific targets in a single reaction (Hernández-González et al. Reference Hernández-González, González-Bertolín, Urrea, Fleury, Ferrer, Siles-Lucas and Perteguer2022).
Comparative analysis of traditional ELISA versus recombinant-based multiplex bead assays for the differential diagnosis of human cestodiases
Table 1. Long description
The table consists of three columns: Feature, Traditional E L I S A (single target), and Multiplex bead-based assay (D I P O L plus T 24 H).
* Sample volume: Traditional E L I S A requires high volume with separate wells for each test, while the multiplex assay requires low volume (less than 5 micro-L of serum).
* Throughput: Traditional E L I S A is sequential and slow, whereas the multiplex assay is simultaneous and rapid.
* Cross-reactivity: Traditional E L I S A shows high cross-reactivity common with native extracts, while the multiplex assay has minimal cross-reactivity due to precision recombinants.
* Diagnostic output: Traditional E L I S A provides binary positive or negative results, while the multiplex assay provides immunoprofiling with specific fingerprints.
* Co-infection detection: Traditional E L I S A is difficult to interpret, whereas the multiplex assay provides clear, distinct signals for each species.
This review explores the transformative impact of bead-based multiplex technologies in the parasitology laboratory. As shown in Figure 1, by coupling microspheres with a precision dual-panel of recombinant antigens (DIPOL/2B2t for CE and T24H/LLGP for NCC), a single micro-sample of patient serum can be used to generate a comprehensive ‘serological fingerprint’ based on median fluorescence intensity (MFI).
Conceptual workflow for the multiplex bead-based differential diagnosis of human cestodiases. The diagram illustrates the paradigm shift from single-analyte ELISA to multi-parameter immunoprofiling. (A) Patient sample: Patients presenting with clinical suspicion (e.g., SOLs, epilepsy) provide a minimal serum sample containing a range of antibodies against potential pathogens. (B) Multiplex assay process: Different sets of MagPlex® beads, each identified by a unique fluorescent code, are coupled with precision-engineered recombinant antigens: DIPOL/2B2t (highly specific for CE, shown in turquoise) and T24H/LLGP (highly specific for NCC, shown in deep blue). A third control bead set supports assay validity. The beads are incubated with the patient’s serum and a secondary anti-human IgG reporter antibody conjugated to phycoerythrin (PE). (C) Results and interpretation: Data are acquired on a Luminex-based platform to determine the MFI for each bead set. Representative comparative bar graphs show how the resulting ‘serological fingerprint’ more clearly discriminates between a hydatidosis profile (CE+) (left graph, dominant turquoise signal) and a cysticercosis profile (NCC+) (right graph, dominant deep blue signal), supporting a more confident differential diagnosis in complex clinical cases.
Figure 1. Long description
Conceptual workflow for the multiplex bead-based differential diagnosis of human cestodiases. The diagram illustrates the paradigm shift from single-analyte ELISA to multi-parameter immunoprofiling.Panel A, Patient Sample, shows a silhouette of a patient with icons for brain, heart, liver, and lungs. An arrow points to a vial labeled Serum Sample containing Patient Antibodies (Anti-Cestode Abs).
Panel B, Multiplex Assay Process (Luminex), shows the serum added to a microcentrifuge tube containing three MagPlex bead sets. Bead Set 1 (Turquoise) is coupled with D I P O L / 2 B 2 t (C E Antigens). Bead Set 2 (Deep Blue) is coupled with T 24 H / L L G P (N C C Antigens). Bead Set 3 (Grey) contains Control Antigen. An arrow labeled Incubation points to a cross-section of a bead. At the core is a spherical bead with Y-shaped antigens on its surface. These are bound to patient antibodies, which are in turn bound to a Detection Antibody: Anti-Human I g G - P E Conjugate. This creates an Antigen-Antibody Complex for Fluorescence Readout.
Panel C, Results and Interpretation, shows the sample being processed by a Luminex machine. Two bar graphs display Data Acquisition: Median Fluorescence Intensity (M F I). The Hydatidosis Profile (C E plus) graph shows high turquoise bars for D I P O L and 2 B 2 t and low blue bars for T 24 H / L L G P. The Cysticercosis Profile (N C C plus) graph shows low turquoise bars and high blue bars. An arrow from these graphs points to the final outcome: Definitive Differential Diagnosis.
Clinical interpretation and diagnostic decision-making
The implementation of a multiplex ‘Cestode Profile’ allows for a more nuanced interpretation of serological results, moving beyond a simple ‘positive/negative’ report. This is particularly relevant in patients presenting with space-occupying lesions (SOLs) in the central nervous system or visceral organs.
Decoding the serological fingerprint
In a standardised multiplex panel using DIPOL/2B2t (for CE) and T24H/LLGP (for NCC), the assay primary outcomes align with three clinical reactivity profiles:
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• Result 1 [CE+/NCC−]: High probability of cystic echinococcosis. The high specificity of chimeric antigens like DIPOL makes cross-reactivity unlikely with T. solium.
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• Result 2 [CE−/NCC+]: High probability of neurocysticercosis. A positive signal for T24H in the absence of CE-specific markers supports the diagnosis of NCC, even in cases with single-enhancing lesions where imaging may be ambiguous.
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• Result 3 [CE+/NCC+]: Suggests either a co-infection (relevant in hyper-endemic areas) or potential residual cross-reactivity. In these cases, the MFI values provide a quantitative clue, where the dominant signal points to the primary pathogen. Additionally, a completely seronegative result [CE−/NCC−] lowers the probability of active infection, although seronegative cases (e.g., inactive calcified cysts or CE4/CE5 stages) must still be considered within the clinical context.
To facilitate the translation of laboratory data into clinical action, we propose a standardised decision tree. As illustrated in Figure 2, the integration of MFI values from both CE and NCC-specific beads allows for a rapid differentiation between these infections, guiding the clinician toward the appropriate surgical or pharmacological protocol.
Clinical diagnostic algorithm for discriminating human cestodiases using multiplex immunoprofiling. The flowchart illustrates the proposed clinical decision-making process based on the ‘Cestode Profile’ assay. (Step 1) A patient with clinical suspicion of cestodiasis (e.g., space-occupying lesions, epilepsy, or visceral cysts) provides a serum sample for a multiplex bead-based assay. The platform utilises microspheres coupled with precision recombinant antigens: DIPOL/2B2t (highly specific for Echinococcus granulosus) and T24H/LLGP (highly specific for Taenia solium). Based on the results, three primary clinical scenarios are identified:
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• Result 1 (CE+/NCC−): Supports a probable diagnosis of cystic echinococcosis, guiding the clinician toward visceral cyst management protocols (e.g., Surgery, PAIR, or Watch and Wait).
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• Result 2 (CE-/NCC+): Supports a probable diagnosis of neurocysticercosis, directing treatment toward intracranial cyst protocols involving corticosteroids and antiparasitic drugs (albendazole/praziquantel).
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• Result 3 (CE+/NCC+): Suggests potential co-infection or residual cross-reactivity; management requires evaluation of the dominant MFI signal and mandatory correlation with advanced neuroimaging or biopsy.
Figure 2. Long description
Clinical diagnostic algorithm for discriminating human cestodiases using multiplex immunoprofiling. The flowchart illustrates the proposed clinical decision-making process based on the ‘Cestode Profile’ assay. (Step 1) A patient with clinical suspicion of cestodiasis (e.g., space-occupying lesions, epilepsy, or visceral cysts) provides a serum sample for a multiplex bead-based assay. The platform utilises microspheres coupled with precision recombinant antigens: DIPOL/2B2t (highly specific for Echinococcus granulosus) and T24H/LLGP (highly specific for Taenia solium). Based on the results, three primary clinical scenarios are identified:
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• Result 1 (CE+/NCC−): Supports a probable diagnosis of cystic echinococcosis, guiding the clinician toward visceral cyst management protocols (e.g., Surgery, PAIR, or Watch and Wait).
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• Result 2 (CE-/NCC+): Supports a probable diagnosis of neurocysticercosis, directing treatment toward intracranial cyst protocols involving corticosteroids and antiparasitic drugs (albendazole/praziquantel).
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• Result 3 (CE+/NCC+): Suggests potential co-infection or residual cross-reactivity; management requires evaluation of the dominant MFI signal and mandatory correlation with advanced neuroimaging or biopsy.
Impact on patient management
The ability to differentiate these two helminthiases in a single test has direct implications for treatment. While NCC often requires corticosteroids and specific antiparasitic cycles (albendazole/praziquantel) tailored to the inflammatory response in the brain (Garcia et al. Reference Garcia, Nash and Del Brutto2014), the management of CE ranges from ‘watch and wait’ to complex surgical interventions or puncture, aspiration, injection and re-aspiration (PAIR).
By providing a more robust differential diagnosis, the ‘Cestode Profile’ prevents the administration of potentially harmful treatments and reduces the need for invasive diagnostic procedures.
The clinical management of CE, according to international consensus, ranges from pharmacological treatment to invasive procedures depending on the cyst stage (Brunetti et al. Reference Brunetti, Kern and Vuitton2010).
Concluding remarks and future perspectives
The transition from serological assays based on crude parasite extracts to approaches incorporating recombinant antigens such as DIPOL, 2B2t, and T24H represents an important step toward improving the diagnostic accuracy of human cestodiases. As outlined in this review, the integration of these targets into multiplex platforms enables the simultaneous assessment of antibody responses against multiple species-specific antigens, offering a more comprehensive serological profile than conventional single-analyte assays.
This strategy may be particularly valuable in clinical scenarios where imaging findings are inconclusive or where co-endemicity complicates interpretation. By providing complementary information to radiological and clinical data, multiplex immunoprofiling has the potential to support more informed diagnostic and therapeutic decisions, and to reduce reliance on sequential testing strategies.
Nevertheless, several challenges remain. The variability of host immune responses, the influence of geographical differences in parasite strains, and the limited accessibility of multiplex technologies in resource-constrained settings must be carefully considered. In addition, the lack of standardised protocols and validated cut-off values continues to limit comparability across studies and laboratories.
Future efforts should focus on the harmonisation of assay design, large-scale validation in diverse populations, and the development of cost-effective and accessible platforms. In this context, multiplex serology may represent a valuable component of integrated diagnostic frameworks aimed at improving the management and control of neglected cestodiases.
Limitations of multiplex serology
Despite its advantages, multiplex serology presents several limitations. Its performance depends on the host immune response, and false-negative results may occur in early, inactive, or calcified infections, as well as in immunocompromised patients. Although recombinant antigens such as DIPOL, 2B2t, and T24H substantially reduce cross-reactivity, residual overlap cannot be completely excluded, particularly in co-endemic regions.
In addition, geographical variability in parasite strains and host immune profiles may affect diagnostic accuracy, limiting the generalisability of current findings. Technical and economic barriers also persist, as platforms such as Luminex require specialised infrastructure and may not be readily accessible in resource-limited settings.
Finally, the lack of standardised protocols and cut-off values remains a challenge, potentially leading to inter-laboratory variability. Therefore, multiplex results should be interpreted in conjunction with clinical, radiological, and epidemiological data.
Financial support
No specific funding from commercial diagnostic companies was received for the preparation of this review.
Competing interests
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Ethical approval
Not applicable. This manuscript is a review article and does not involve direct research on human participants or animals performed by any of the authors.
