Toxocara diagnosis and epidemiology

Human Toxocariasis (HT) is a zoonosis caused by infection with the nematode Toxocara canis, the intestinal roundworm of dogs. The disease is spread worldwide and even though it is more prevalent in tropical countries as well as strongly associated to poor sanitary conditions, sporadic transmission of the disease is possible in temperate to cold environments or even in human settlement with adequate sanitary conditions . When dogs are infested, they release together with the stools a huge amount of eggs that contaminate the soil and can remain viable even for years. Infection occurs when humans accidentally ingest these eggs containing Toxocara larvae. Upon infection, the larvae are released from the eggs and migrate through the body. Infection can be associated with a wide clinical spectrum varying from asymptomatic to severe organ injury. Clinical manifestations reflect the inflammatory reaction caused by the migrating larvae, and include visceral, neural or ocular syndromes, depending on the organ infected. Severe disease may issue from infection with one single Toxocara larva. Diagnosis of HT is challenging. Definitive diagnosis of HT implies the detection of Toxocara larvae from biopsies, but this test is difficult to perform and not always feasible. Diagnosis is commonly based on clinical and epidemiological suspicion supported by laboratory tests that include quantification of eosinophils in peripheral blood and the detection of specific immunoglobulins against a complex mix of glycoproteins that the larvae release in the blood stream during their passage towards the central nervous system, the eye or the liver, the excretory-secretory antigen (TES). Major limitations of this test are its low specificity due to cross-reactions with other helminths, especially in developing countries, where polyparasitism prevails. Moreover, serology does not allow discrimination between past and current infections, an important differentiation to guide treatment decisions at the patient level and essential to assess the frequency of occurrence of infection at population level. Nevertheless, serological tests remain the least invasive and most sensitive approach to detect these parasite antigens, but require novel approaches if we want to develop highly sensitive and specific assays that deliver results of clinical and/or epidemiological relevance. In this project, we exploit the advantages of two powerful techniques, nanobodies and the Proximity Ligation Assay (PLA), to address these needs. Specificity will be addressed by generating Nbs as TEs detection reagents with high affinity and specificity for the development of an antigen detection ELISA. Nanobody technology devised as the result of the discovery in 1993 at Vrije Universiteit Brussel of a unique type of antibodies which devoid of light chains and the first constant domain of the heavy chain. These antibodies were named heavy chain antibodies (HCAbs), they are present in camelids (lamas, camels and dromedaries) and their variable domain at the N-terminal (VHH) serves to binding the cognate antigen. Recombinant VHH’s obtained after immunization of a camelid, cloned and screened by phage display is known as Nanobodies (Nbs). The most important feature of Nbs are their small size (15kDa) compared to classical immunoglobulins (150kDa), a feature that allow them to access epitopes that normally are not recognized by classic antibodies. Improved sensitivity will be attained by coupling Nbs to the PLA, a cutting-edge methodology that combine the specificity of ELISA sandwich assays together to high degree of sensitivity through the detection of DNA amplified from two oligonucleotide probes attached to each binder (Nbs in this case), creating a primer which is able to start Rolling Circle amplification (RCA). The DNA amplified can be detected by small oligonucleotides labelled with a fluorescent dye. After development, the next step is to carry out a validation of the test in human blood and vitreous humour samples, and to evaluate the application of these assays as diagnostic tools for confirmation of clinical HT-cases, and as epidemiological tools for an improved understanding of the distribution and dynamics of toxocariasis. Eventually, the aim is to arrive at a low cost, robust, highly specific and sensitive test for the detection of active HT infections that can be used in basic laboratory conditions.

Trefwoorden:B780-tropische-geneeskunde