María Elizabeth Marquez Contreras*
Parasite Laboratory of Enzymology, University of the Andes, Merida, VenezuelaFulltext PDF
Canine Visceral Leishmaniasis (CVL) is an infection of veterinary importance, involving a complex interaction between trypanosomatid protozoa of the Leishmania donovani complex, sand fly vectors, environmental conditions that influence the distribution of the vector, dogs that are the major urban reservoirs and humans that are hosts that harbor these parasites. Dogs are a key piece in the epidemiological chain of VL because it has been shown that the high prevalence of canine infection is related to a high risk of disease in humans. Detection of the infection in dogs is essential to proceed to the chemotherapeutic treatment in infected individuals, in epidemiological studies and control programs, for this reason, the implementation of rapid, sensitive and specific diagnostic tests is essential. The diagnosis of CVL commonly uses extracts from the promastigote stage of Leishmania, which are complex and heterogeneous mixtures that may have values of low specificity (false positives) and percentages of minimal sensitivity (false negatives). In order to achieve the improvement of diagnostic tests, numerous investigations based on Molecular Biology techniques have been carried out over several years, which have allowed the obtaining of recombinant antigens from Leishmania donovani complex cDNA libraries. In this way excellent antigens have been obtained, such as rk39 and rk28, which are very specific, sensitive, obtained in large quantities at a relatively low cost through an easy and fast purification process. The recombinant protein rK39 is a 39 amino acid repetitive immunodominant B cell epitope that is part of a 230 kDa kinesinrelated protein expressed predominantly in the amastigotes of viscerotropic L. chagasi, has proved to be an exceptionally strong marker of disease, allows the detection of canine VL in asymptomatic animals, as well as in those that present clinical symptoms. The levels of anti-rK39 antibodies are highly correlated with active disease can be used to monitor the effectiveness of chemotherapeutic compounds and clinical monitoring of patients. The incorporation of this antigen in a dipstick format allows the production of a Rapid Diagnostic Test (RDT), easy to use, which provides reliable results, without the need for equipment or specialized personnel, all these advantages allow its implementation in the field; this is very important for handling in rural areas where humans and dogs of low socioeconomic conditions live. Subsequent studios have allowed to express rK9 and rK26, two related hydrophilic antigens of L. chagasi that differ for the presence of 11 copies of a 14-amino-acid repeat in the open reading frame of K26. Some evaluations have shown that the sensitivity of the rK26 antigen is only 20% to 40% while rK9 yields 78% sensitivity. The antigenicity of K9, K39, and K26 was determined in multiple-well ELISA using infected dog sera, these antigens showed independent and complementary immunoreactivities, which led to the idea of producing an antigen formed by the fusion of said proteins. Thus to solve these drawbacks, the technological advances of the last few years have been used to develop new generation of recombinant chimeric protein, resulting from fusion of L. infantum genes: k9, k39 and k26, denominated rK28 chimeric protein with multiple tandem repeat sequences, increasing antigen epitope density, with a sensitivity of 92% to 100% in Sudan. Due to the multiple advantages of rK28 has been used in the development of RDT, for example, DPP CVL a screening method established by the Brazilian government. this test shows many benefits: i) high levels of sensitivity and specificity for canine VL, ii) shows results in 15 minutes, which allows to evaluate a large number of samples, iii) in association with the confirmatory test EIE CVL give results within 15 days. Their incorporation in the current protocol accelerates the implementation of the control measures in endemic areas endemics. In conclusion, this assay constitute an approximation to the ideal test because it employs a combination of relevant epitopes in a single recombinant antigen in the form of quimera, more specific than crude antigen preparation and more sensitive than single epitope-based ELISA.
Contreras MEM. Serological Diagnosis of Canine Visceral Leishmaniasis. Am J Clin Microbiol Antimicrob. 2018; 1(3): 1015.