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RESEARCH ARTICLE

Diagnostic Accuracy of Antigen 5-Based ELISAs for Human Cystic Echinococcosis Daniela Pagnozzi1*, Maria Filippa Addis1, Grazia Biosa1, Anna Maria Roggio1, Vittorio Tedde1, Mara Mariconti2,3, Francesca Tamarozzi2,3, Valeria Meroni4, Gabriella Masu5, Giovanna Masala5, Enrico Brunetti2,3,6, Sergio Uzzau1 1 Porto Conte Ricerche Srl, Tramariglio, Alghero (Sassari), Italy, 2 Department of Clinical, Surgical, Diagnostic and Pediatric Sciences, University of Pavia, Pavia, Italy, 3 WHO Collaborating Centre for the Clinical Management of Cystic Echinococcosis, Pavia, Italy, 4 Department of Microbiology and Virology, IRCCS San Matteo Hospital Foundation and Department of Internal Medicine and Clinical Therapy, University of Pavia, Pavia, Italy, 5 Centro Nazionale di Riferimento per l’Echinococcosi, IZS “G. Pegreffi”, Sassari, Italy, 6 Division of Infectious and Tropical Diseases, IRCCS San Matteo Hospital Foundation, Pavia, Italy * [email protected]

Abstract OPEN ACCESS Citation: Pagnozzi D, Addis MF, Biosa G, Roggio AM, Tedde V, Mariconti M, et al. (2016) Diagnostic Accuracy of Antigen 5-Based ELISAs for Human Cystic Echinococcosis. PLoS Negl Trop Dis 10(3): e0004585. doi:10.1371/journal.pntd.0004585 Editor: Paul Robert Torgerson, University of Zurich, SWITZERLAND Received: November 24, 2015 Accepted: March 8, 2016 Published: March 29, 2016 Copyright: © 2016 Pagnozzi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Regione Autonoma della Sardegna and MM was partly funded by HERACLES—Human cystic Echinococcosis ReseArch in CentraL and Eastern Societies an FP7 Project funded by the EU (grant number 602051). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.

Background Clinical diagnosis and follow up of cystic echinococcosis (CE) are based on imaging complemented by serology. Several immunodiagnostic tests are commercially available, but the development of new tools is still needed to overcome the lack of standardization of the target antigen, generally consisting of a crude extract of Echinococcus granulosus hydatid cyst fluid. In a previous work, we described a chromatographic method for the preparation of a highly enriched Antigen 5 fraction from hydatid cyst fluid. The high reactivity of patient sera against this preparation prompted us to evaluate further this antigen for the serodiagnosis of CE on a larger cohort of samples.

Methodology/Principal Findings A total of 327 sera from CE patients with heterogeneous conditions for cyst stage, cyst number, organ localization, drug therapy, and surgical intervention, together with 253 sera from healthy controls, were first analyzed by an ELISA based on the Ag5 preparation in two different experimental setups and, in parallel, by a commercial ELISA routinely used in clinical laboratories for CE serodiagnosis. The Ag5 ELISAs revealed different sensitivity (88.3% vs 95.3%) without significant differences in specificity (94.1% vs 92.5%), for the two setups, respectively. Moreover, possible relationships between the Ag5 ELISA absorbance results and clinical variables were investigated. Chi squared test, bivariate logistic regression and multiple regression analyses highlighted differences in the serology reactivity according to pharmacological treatment, cyst activity, and cyst number.

Conclusions/Significance The two Ag5 ELISAs revealed different performances depending on the setup. The good diagnostic sensitivity and the high reliability of the Ag5 preparation method make this

PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0004585

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Evaluation of Ag5 ELISAs for CE Diagnosis

antigen a promising candidate for the serodiagnosis of CE. Further studies will be needed to evaluate the ability of our test to provide useful information on specific CE clinical traits.

Author Summary Cystic echinococcosis is a neglected disease caused by the larval stage of the tapeworm Echinococcus granulosus complex affecting both humans and livestock. The disease is considered one of the world’s major zoonoses, and represents a public health problem. Clinical diagnosis and follow-up is mainly based on imaging, while serology should complement imaging-based diagnosis when imaging features are unclear. However, current commercial immunoassays lack satisfactory sensitivity and specificity. They are mostly based on crude antigen preparations of E. granulosus hydatid cyst fluid, a heterogeneous mixture containing molecules of both parasite and host origin, thus standardization is also an issue. Ag5 is one of the most immunogenic proteins present in the hydatid cyst fluid. In a previous work, we described a method enabling the preparation of a highly enriched Ag5 fraction. Here, we present the evaluation of the diagnostic performances of this preparation in two ELISA setups, using a large number of human sera. The influence of several clinical variables on the performance of the tests was also assessed. The results obtained by the Ag5 ELISAs, combined with the robustness of the Ag5 preparation method, make this antigen a promising candidate for the serodiagnosis of CE.

Introduction Cystic echinococcosis (CE) is a neglected zoonotic disease caused by the larval form of the tapeworm Echinococcus granulosus complex. The definitive hosts are dogs and other canids, while sheep and other livestock are the natural intermediate hosts; humans are occasional intermediate hosts. Intermediate hosts can be infected by ingestion of food and water contaminated with the parasite eggs eliminated with the feces of infected dogs. The early phase of infection is generally asymptomatic. Small, well encapsulated, viable cysts or old cysts with pseudosolid content typically do not induce major pathology, and patients may remain asymptomatic for years or even permanently. This is likely the reason why almost 50% of CE patients recorded in the Italian Hospital Discharge Records have been diagnosed accidentally during investigations for other diseases, and 57% of cases are people over 60 years old [1]. CE has many important economic effects, the most evident and tangible of which is the cost of expensive medical treatment for human cases; moreover, there is also a strong negative impact on the economy due to the large diffusion of CE among livestock [2–3]. Currently, CE can be treated according to four different approaches: surgery, percutaneous techniques, chemotherapy with benzimidazoles, and with a “watch and wait” approach for inactive cysts. Unfortunately, 20%–40% of the patients respond only temporarily to chemotherapy, and revert to their previous stage (mainly CE2 and CE3b) after the end of treatment [4]. The most affected regions include the Mediterranean, Eastern Europe, parts of South America, parts of Africa, and Central Asia/Western China. In Italy, the average annual incidence rate of hospital cases (AIh) between 2001 and 2012 was 1.6/105 inhabitants [1]. At present, no marker of cyst viability and therapy efficacy exists, and serology may remain positive for years even after successful therapy. As a consequence, long-term follow-up with imaging is required for the clinical management of patients.


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It is therefore important to invest in innovative technologies that facilitate the monitoring and control of this infectious disease in humans and in farm animals. Currently, CE diagnosis in humans is mostly based on imaging techniques [5], and the clinical approach is based on the WHO international classification of ultrasound images according to the stage of the cyst: CE1, CE2, CE3b (active), CE3a (transitional), and CE4 and CE5 (inactive) [6–8]. Ultrasonography (US), due to the relatively low cost and size of the equipment, is easily transportable in remote resource-poor areas, provides a useful tool for screening, clinical diagnosis, and cyst monitoring [9–11]. However, serology has an important role in supporting the diagnosis of CE, since serological tests are generally cheap, quick, and require less trained and specialized personnel for result interpretation. This is particularly true in areas where US expertise in the diagnosis of CE is scant and/or not easily accessible, and when lesions do not show pathognomonic signs of a parasitic origin, such as young CE1 cysts or inactive CE4–CE5 cysts. Unfortunately, these stages are also those with a broader differential diagnosis (e.g. with simple cysts, neoplastic lesions) whose serology results are also difficult to interpret [12–13]. Commercially available immunoassays are mostly based on hydatid cyst fluid (HCF), collected from infected animals. However, the heterogeneity of this preparation negatively impacts on the sensitivity and specificity of the tests. Many purified native, recombinant, or synthetic antigen preparations have been tested in the last decade, although with controversial results [14–16]. This is most likely due to the poor inter-laboratory reproducibility of antigenic preparations that often rely on outdated methodologies, improperly defined as “purifications”. This adds to the use of different panels of sera, as well as to the lack of clinical characterization and appropriate classification/grouping of sera, used for validation [17–19]. Among HCF antigens, Antigen 5 (Ag5) and Antigen B (AgB) are the most abundant and immunogenic proteins, whose role in the life cycle of the cestode has been assessed only partially [20]. In a recent work [21], our research group reported a straightforward, robust and reproducible chromatography method that enables the preparation of a HCF fraction highly enriched in native Ag5. This highly enriched antigen demonstrated a strong reactivity, both in western blotting and ELISA formats, when tested on a limited panel of sera from CE patients, encouraging a more extensive examination of its diagnostic potential. Here we present a large-scale study (327 cases and 253 controls) aimed to evaluate the diagnostic accuracy of two different Ag5 ELISA setups compared with that of a commercially available ELISA widely employed for routine diagnostic. We also investigated the association between readings of the Ag5-based ELISAs with selected clinical variables of the patients. When the three assays were compared for sensitivity and specificity, the Ag5 ELISA had significantly higher sensitivity. Moreover, the performances of both Ag5 ELISA setups were statistically associated with clinical variables known to influence serology results. These data support the use of this Ag5-based preparation in highly sensitive diagnostic tests and prompt further investigation on its use in the follow-up of CE patients.

Methods Ethics statement A written informed consent to use of leftover serum after routine serology for research was obtained from patients at the time of sample collection. The study was approved by the ethics committees of IRCCS San Matteo Hospital Foundation, Pavia, Italy, Prot N. 20150004877, for sera from CE patients, and of the local health authority of Sassari (ASL N. 1, Sassari), Prot N. 1123/L, for sera from healthy controls.


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Patients and samples A prospective study was performed on sera from patients with hepatic and extra-hepatic CE, collected at the Department of Infectious Diseases of the IRCCS San Matteo Hospital Foundation, Pavia, Italy, where the WHO Collaborating Centre for Clinical Management of Cystic Echinococcosis is based. Sera from healthy subjects, collected at the Sassari Hospital Blood Donor Center, Sassari, Italy, were used as a control group. At the time of serum collection, all patients with abdominal cysts were diagnosed by US and CE cysts were classified according to the World Health Organization–Informal Working Group on Echinococcosis (WHO-IWGE) standardized US classification, by a clinician with long standing experience in the US diagnosis of CE, as part of the routine diagnostic procedures. This classification groups cysts in six stages based on a biological/dynamic approach: active (CE1 and CE2), transitional (CE3a and CE3b) and inactive (CE4 and CE5) cysts. However, CE3b are actually biologically active [8]. On the other hand, from a serological point of view, CE3 cysts of both stages often show comparable results, indicating that biological activity at the time of serum collection may not immediately influence serological responses in these stages [13]. Therefore, for the purpose of analysis, sera from patients with active (CE1, CE2 and CE3b) and transitional (CE3a) cysts were grouped together. Patients having multiple cysts in different stages were assigned to the group of the most active cyst, independently from its hepatic or extra-hepatic localization.

Sheep HCF HCF crude samples were collected in two different Sardinian slaughterhouses (CE/IT2383M, Tula, Sassari and CE/IT2078M, Lula, Nuoro). Fluid was aspirated from liver and lung cysts found in infected sheep. The hydatid fluid was centrifuged at 1000 g at 4°C and the supernatant stored at -80°C.

Antigen 5 preparation Enriched Ag5 was obtained as described previously [21]. Briefly, after desalting and concentration, aliquots of sheep HCF were fractionated by Fast Protein Liquid Chromatography (FPLC) on a Superdex-200 column (10/300 GL, GE Healthcare, Uppsala, Sweden). The fractions of interest were pooled and their protein content was evaluated by tandem mass spectrometry on a Q-TOF hybrid mass spectrometer equipped with a nano lock Z-spray source and coupled on-line with a nanoAcquity chromatography system (Waters, Manchester, UK) to verify the quality of the preparation.

Commercial ELISA test Sera were tested in duplicate in the parasitology diagnostic laboratory of the IRCCS San Matteo Hospital Foundation, Pavia, Italy, by laboratory personnel with long standing experience in diagnostic parasitology, using a commercial ELISA test (RIDASCREEN Echinococcus IgG, R-Biopharm, Darmstadt, Germany), for the detection of Echinococcus specific total IgG, according to manufacturer’s instructions. Tests were read at 450nm in a spectrophotometer, and a Sample Index (SI) was calculated and interpreted for each serum according to manufacturer’s instructions; ELISA was considered positive for SI >1.1, negative for SI