title: Quantification by real-time PCR of Trypanosoma cruzi DNA in samples of Triatoma infestans used in xenodiagnosis of chronic Chagas disease patients.
abstract: Trypanosoma cruzi multiplies and differentiates in the digestive tract of triatomine insects. Xenodiagnosis (XD) is a parasitological tool in which the insect vectors acts as a biological culture medium to amplify and detect T. cruzi infection in mammals. The sensitivity of XD has been overcome by the application of PCR in fecal samples (FS) of XD (PCR-XD). In this study, T. cruzi amplified in Triatoma infestans fed by XD on individuals with chronic Chagas disease (CChD) is quantified by real-time PCR (qPCR-XD).
  
  
  
  Under informed consent, 100 individuals were evaluated. In 21 of them XD, PCR-XD and qPCR-XD were positive. For the contrary, 79 were negative XD. In 58 (73.4 %) and 66 cases (83.5 %) of them, PCR-XD (Fisher's exact test P = 0.005) and qPCR-XD (Fisher's exact test: P = 0.037) respectively, were positive. In cases with positive XD, qPCR-XD allowed to establish that in 9/21 cases (42.9 %) the parasite burden fluctuated between 100 and 1,000 par. eq./ml. Otherwise, in 32/79 (40.5 %) cases with negative XD, a parasite burden between 1 and 10 par. eq./ml was determined. All samples showed amplification of exogenous internal control (X12, Ct average: 31.8), so problems in the DNA extraction (excess or loss of genetic material), unspecific amplification and/or inhibition in qPCR-XD reactions were ruled out. Additionally, in all the patients qPCR in blood (qPCR-B) was performed. In the cases with positive XD, the concordance between the positivity of qPCR-XD and qPCR-B was 100 %, nevertheless, the parasite burden in blood was lower and different than XD (Chi-square test: χ (2)  = 91.82, df = 5, P = 0.0001). In the cases with negative XD the ranges of qPCR-XD and qPCR-B were similar (Chi-square test: χ (2)  = 6.71, df = 5, P = 0.1520).
  
  
  
  This study allowed the detection and quantification of T. cruzi by qPCR-XD in FS of Tr. infestans fed on patients with CChD. The highest parasite burden was observed in positive XD cases. qPCR-XD could be used in different studies related with the complex T. cruzi-vector-host interactions.
authors.affiliation: Laboratorio de Parasitología Básico-Clínico, Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Miguel
authors.initials: M
authors.lastname: Saavedra
authors.affiliation: Laboratorio de Parasitología Básico-Clínico, Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile. izulanta@med.uchile.cl.
authors.forename: Inés
authors.initials: I
authors.lastname: Zulantay
authors.affiliation: Laboratorio de Parasitología Básico-Clínico, Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Werner
authors.initials: W
authors.lastname: Apt
authors.affiliation: Laboratorio de Parasitología Básico-Clínico, Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Juan
authors.initials: J
authors.lastname: Castillo
authors.affiliation: Laboratorio de Parasitología Básico-Clínico, Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Araya
authors.affiliation: Laboratorio de Parasitología Básico-Clínico, Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Martínez
authors.affiliation: Escuela de Salud Pública, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Jorge
authors.initials: J
authors.lastname: Rodríguez
mesh_terms: D000818:Animals; D014355:Chagas Disease / Q000175:diagnosis* / Q000469:parasitology; D002908:Chronic Disease; D016054:DNA, Protozoan / Q000235:genetics; D005243:Feces / Q000469:parasitology; D006801:Humans; D007303:Insect Vectors / Q000469:parasitology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D014224:Triatoma / Q000469:parasitology*; D014349:Trypanosoma cruzi / Q000235:genetics / Q000302:isolation & purification*; D020525:Xenodiagnosis
chemical_list: D016054:DNA, Protozoan
keywords: Chronic Chagas disease; Parasite burden; Real-time PCR; Triatoma infestans; Trypanosoma cruzi; Xenodiagnosis