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- Title
Development of a Fluorescent Based Immunosensor for the Serodiagnosis of Canine Leishmaniasis Combining Immunomagnetic Separation and Flow Cytometry.
- Authors
Sousa, Susana; Cardoso, Luís; Reed, Steven G.; Reis, Alexandre B.; Martins-Filho, Olindo A.; Silvestre, Ricardo; Cordeiro da Silva, Anabela
- Abstract
Background: An accurate diagnosis is essential for the control of infectious diseases. In the search for effective and efficient tests, biosensors have increasingly been exploited for the development of new and highly sensitive diagnostic methods. Here, we describe a new fluorescent based immunosensor comprising magnetic polymer microspheres coated with recombinant antigens to improve the detection of specific antibodies generated during an infectious disease. As a challenging model, we used canine leishmaniasis due to the unsatisfactory sensitivity associated with the detection of infection in asymptomatic animals where the levels of pathogen-specific antibodies are scarce. Methodology: Ni-NTA magnetic microspheres with 1,7 µm and 8,07 µm were coated with the Leishmania recombinant proteins LicTXNPx and rK39, respectively. A mixture of equal proportions of both recombinant protein-coated microspheres was used to recognize and specifically bind anti-rK39 and anti-LicTNXPx antibodies present in serum samples of infected dogs. The microspheres were recovered by magnetic separation and the percentage of fluorescent positive microspheres was quantified by flow cytometry. Principal Findings: A clinical evaluation carried out with 129 dog serum samples using the antigen combination demonstrated a sensitivity of 98,8% with a specificity of 94,4%. rK39 antigen alone demonstrated a higher sensitivity for symptomatic dogs (96,9%), while LicTXNPx antigen showed a higher sensitivity for asymptomatic (94,4%). Conclusions: Overall, our results demonstrated the potential of a magnetic microsphere associated flow cytometry methodology as a viable tool for highly sensitive laboratorial serodiagnosis of both clinical and subclinical forms of canine leishmaniasis. Author Summary: Dogs are the most important domestic reservoirs of the parasite Leishmania infantum. Some infected animals develop a subclinical infection, without the classical symptoms characteristics of this disease. One of the major challenges in the serodiagnosis of canine leishmaniasis is the detection of actively infected animals that are already able to transmit the parasite to the vector, despite the fact they did not yet show external signs of the disease. In the present work, we have developed a new tool for the laboratorial diagnosis of canine leishmaniasis that clearly improves the performance of canine leishmaniasis serodiagnosis. An immunofluorescence assay was developed combining Leishmania recombinant protein-coated magnetic microspheres and flow cytometry. The antigen-coated microspheres were used to separate anti-Leishmania specific antibodies present in the serum of infected dogs. Flow cytometry allowed the specific quantification of the antibodies. The clinical evaluation of a panel of serum samples from natural infected dogs clearly demonstrates that this method detects with high specificity and sensitivity both clinical and subclinical forms of the disease.
- Subjects
IMMUNOMAGNETIC separation; FLOW separation; LEISHMANIASIS; FLOW cytometry; SERODIAGNOSIS; COAT proteins (Viruses); EMERGING infectious diseases
- Publication
PLoS Neglected Tropical Diseases, 2013, Vol 7, Issue 8, p1
- ISSN
1935-2727
- Publication type
Article
- DOI
10.1371/journal.pntd.0002371