DEVELOPMENT OF DUPLEX TAQMAN PCR ASSAYS FOR DETECTION AND QUANTIFICATION OF TRYPANOSOMA CRUZI INFECTION IN WILD AND DOMESTIC RESERVOIRS

DIANA P. WEHRENDT; JANINE M. RAMSEY; MARCELO ABRIL ; 1, ANDREA GÓMEZ-BRAVO; CAROLINA CURA; FELIPE GUHL; ANGÉLICA PECH-MAY; M. DE LOS ÁNGELES CURTO; ALEJANDRO G. SCHIJMAN,

Resistencia

Congreso; XXX Reunión Anual de la Sociedad Argentina de Protozoologia y Enfermedades Parasitarias; 2018

Sociedad Argentina de Protozoología y Enfermedades Parasitarias

A major question of epidemiological relevance in Chagas disease studies is to understand the dynamics of T.cruzi infection in sylvatic and domestic transmission cycles and to trace the origins of (re)emerging cases in areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity and molecular approaches, such as the polymerase chain reaction may fill in this gap, provided that a standardized method can be developed and validated. We have developed two duplex Real Time PCR assays for sensitive detection of T.cruzi satellite or minicircle repetitive sequences in blood samples from mammal reservoirs, incorporating an internal amplification standard that allows distinction of false negative PCR findings due to inadequate conditions of storage and transport of samples, DNA degradation during nucleic acid purification and/or inhibition of PCR by interfering substances present in the sample. The housekeeping gene that encodes the interphotoreceptor retinoid-binding protein (IRBP) has been selected as internal standard, because it is highly conserved among all mammal species and its usefulness as a DNA integrity control was previously reported in a conventional PCR protocol. Based on the alignment of the IRBP sequence available for several domestic and wild reservoir species, we designed primers and a TaqMan probe to a highly conserved region. The analytical sensitivity was 0.01 par. eq/mL and 0.1 par.eq/mL for satDNA/IRBP duplex and kDNA/IRBP duplex, respectively, as tested with two series of canine blood spiked with known concentrations of Silvio X10 (Tc I) and CL-Brener (Tc VI) cultured epimastigotes. The assays were evaluated in DNA extracts from blood samples of 87 wild and 147 domestic animals. Our DNA integrity control worked well for wild reservoir species including small rodents (Akodon toba, Galea leucoblephara, Rattus rattus), opossums (Didelphis virginiana, D. marsupialis), bats (Tadarida brasiliensis, Promops nasutus, Desmodus rotundus) and other mammals such as the skunk, viscacha, wildcat, brown brocket deer, hare and Pampas fox. Mean Ct values for IRBP amplification varied among species between 24 and 33. For domestic reservoirs, IRBP amplified in dog, cat, cow, sheep, goat, horse and mule specimens with mean Ct values between 23 and 25. In T.cruzi infected cases the assays allowed quantification of parasitic loads.Our results promote the use of these methods for rapid and accurate screening of T.cruzi infection in different species of reservoirs.