AptaTrich: Development of an aptamer-based test for Trichinella detection
Trichinellosis is a zoonosis caused by the consumption of raw or undercooked meat of animals (mainly pigs, wild boars, horses) infected with the nematode Trichinella spp. Over the past decades, human trichinellosis incidence has decreased in the EU due to improvement of pig farming conditions and to stringent control measures implemented by controls at the slaughterhouse. The cost of trichinellosis control in pigs is estimated at 220 millions euros per year in EU. To date, Trichinella spp. remain in the top three of prioritized foodborne parasites in Europe (Bouwknegt et al., 2018). This parasite is still of major public health and economic importance at international level (Codex Alimentarius).
EFSA identified the type of production system as a main risk factor for Trichinella infections, and that the risk of Trichinella infection in pigs from officially recognised controlled housing conditions (ORCHC) is negligible. Therefore, EU regulation 2015/1375 introduced possible derogations from the necessity of testing each slaughter pig, if specific conditions are met. In order to verify that Trichinella is truly absent in such a population and to identify changes in disease prevalence at an early stage, monitoring programmes are recommended (EU 2015/1375). To date, serological methods have been identified, but are fraught with problems concerning specificity (false positives) and detection of infection at an early stage.
Due to the very low Trichinella prevalence in pigs from ORCHC, a test specificity bordering 100% is needed, as false positive samples would need to be retested with a second serological method (e.g. Western Blot). Such tests can only be performed by specialised laboratories, making the testing logistics more complicated and expensive. Therefore, new diagnostic methods with higher specificity and earlier detection are needed for prevention at the slaughterhouse and to improve human disease detection.
When a human outbreak occurs, the importance of early onset therapy is clearly needed as this avoids complications and reduces the patient’s parasitic load. To date, human diagnosis is based on the detection of specific IgG that appear as early as 10-20 days post infection. However, antibody titers are highly variable depending on the individuals, the initial parasite load and the infecting species. Numerous tests have been described but the most efficient systems remain the ELISA based on the use of excretion / secretion antigens (Ag E/S) and Western blot performed with this antigen. The Western blot makes it possible to obtain the best specificity; however, some cross-reactions are observed with anisakiases and bilharziases. The ELISA can be positive as early as the second week post infection, but generally, the serological reactions are clearly positive only one month post infection. This is often too late for anthelminthics to effectively reach the nematodes, as tissue cyst envelop the worm in less than two weeks. Therefore, new diagnostic methods with higher specificity and earlier detection times in meats are needed for prevention and in human serum for detection of disease.
Aptamers are synthetic nucleic acids that fold into unique three-dimensional conformations capable of binding a target with remarkable affinity and specificity. These structures specifically bind to pathogen antigens, thus combining the ease of serological sampling, and the direct detection of the presence of the pathogen. Aptamers have successfully been used for the detection of parasites in fresh produce, including in aptamer-based biosensors. The development of an aptamer based detection system for Trichinella would circumvent the caveats associated with serological testing and enable specific and early detection in both human diagnostics and Trichinella monitoring programmes in pigs. Further, the technique can be combined with aptamers directed against other pig diseases (such as Toxoplasma, Salmonella etc), making a much wider future application possible.