Evaluation of the hemoculture technique by polymerase chain reaction (PCR) using primers P35/P36 and TCZ1/TCZ2 for detection of Trypanosoma cruzi in dogs and cats

Thesis: L. J. Eloy submitted this thesis for his Masters in Tropical Diseases at the Botucatu Medical School, São Paulo State University (UNESP - Univ Estadual Paulista), Botucatu, São Paulo State, Brazil, 2010.

Advisor: Professor Simone Baldini Lucheis

Abstract: Chagas' disease is a zoonosis caused by Trypanosoma cruzi (T. cruzi) which also affects household and wild animal species. Dogs and cats play an important role in this parasite transmission cycle and they are considered to be the main household reservoirs as well as sources of infection with trypanosomatids due to their closeness to humans, their good susceptibility to chagasic infection and the fact that they attract the vector. Among diagnostic methods, hemoculture represents a parasitological test whose positivity reveals the parasite presence in the blood stream. This is an important difference in relation to the polymerase chain reaction (PCR) technique, which is regarded as gold standard and capable of detecting fragments of the parasite, thus not requiring the presence of whole organisms in the blood stream. The present study aimed at evaluating the occurrence of T. cruzi in dogs and cats from Botucatu, SP, and Campo Grande, MS, as well as at evaluating the technique of hemoculture in liver infusion tryptose (LIT) medium by using the PCR technique, in addition to comparing primers P35/P36 and TCZ1/TCZ2 in the detection of T. cruzi by PCR. Blood samples were randomly collected from 50 dogs and 50 cats from Campo Grande, MS, and from 50 dogs and 50 cats from Botucatu, SP. For hemoculture, each sample was inoculated into three tubes containing 5 mL of the LIT medium each. The first reading of the cultures occurred ten days after, and they were later observed fortnightly for four months. Upon completion of such period, all the hemocultures were processed for parasitic DNA extraction. The PCR reactions were performed by using primers P35/P36 and TCZ1/TCZ2. Of the 100 samples from Botucatu, SP, ten dogs and ten cats (20%) were positive to PCR (both for primers P35/P36 and for primers TCZ1/TCZ2). Four dogs and three cats (7%) showed to be positive to hemoculture, and one cat (1%) was positive to the three techniques. As to the samples from Campo Grande, MS, all of them showed a negative result for both techniques. The results showed that the PCR technique presented higher sensitivity for diagnosing Chagas' disease in the studied animals, that primers TCZ1/TCZ2 confirmed the diagnostic positivity for T. cruzi shown by primers P35/P36 and that, in the studied samples, the dogs and cats from Botucatu, SP, are playing the role of household reservoirs of this parasite, which reveals the constant need for epidemiological surveillance for this zoonosis not only in Botucatu, SP, but also in Campo Grande, MS.

Key words: Trypanosomacruzi, Chagas' disease, polymerase chain reaction, dogs and cats.

Correspondence to: Luciano José Eloy, Departamento de Doenças Tropicais e Diagnóstico por Imagem, Faculdade de Medicina de Botucatu, UNESP, Botucatu, SP, Brasil. Phone: +55 14 3811 6372 or +55 14 9746 2217. Email: ljeloy@yahoo.com.br