Desenvolvimento de método para extração de COVs exalados do pelo de cães infectados com Leishmania sp. por meio da técnica TD-GC-FID

Abstract

Visceral leishmaniasis is a major public health problem, affecting thousands of people around the world every year, especially in developing countries such as Brazil. Dog is a principal host of the disease, because of its proximity to human beings and the ease to acquire and develop high parasite loads. Recent studies suggest that the infected dog with L. infantum produces physiopathological responses that changes, among other things, the profile of volatile metabolites exhaled from the animal hair and that there is a preferred demand from vector to infected dogs. The present work develops a method to extract volatiles exhaled from canine hair by TD-GC-FID technique, in order to enable studies to better understand the role of these VOCs in vector-host attraction. In order to carry out this work, samples were classified into three groups: infected dogs and with high parasite load; infected dogs, but with low parasite load and healthy dogs, but without the presence of parasite. The method developed includes solid phase extraction and sampling by purge and trap. All the factors that affect in the VOCs canine hair sampling were evaluated by multivariate designs. The huge amount of data generated by 105 samples were classified into three groups studied by Partial Least Squares Discriminant Analysis (PLS-DA). The misclassification of samples were mitigated by removing of uninformative VOCs, using techniques such as Multiple Linear Regressions Genetic Algorithm (MLR-GA-DA) and Stepwise Multiple Linear Regression (Stepwise MLR-DA). The number of samples misclassified were significantly reduced, providing a satisfactory model for classification of those three groups. Changes in the regression coefficients of the MLR-GA-DA/Stepwise MLR-DA in the selected VOCs indicate the 1-hexanol, 1-octanol and nonadecane as potential biomarkers of canine visceral leishmaniasis. A method for extraction of VOCs using TD-GC-FID, which enables to obtain an extract from canine hair volatiles and allow us to investigate the vector-host attraction was developed and used to discriminate the three sample classes.