title: Usefulness of real time PCR to quantify parasite load in serum samples from chronic Chagas disease patients.
abstract: BACKGROUND
  Inconclusive results of serological diagnosis in Chagas disease have an important impact on blood banks worldwide, reflecting in the high number of discarded bags or in an increased transmission by blood transfusion. Molecular techniques such as qPCR have been used for diagnosis and to monitor Trypanosoma cruzi load in peripheral blood samples. A promising perspective refers to the possibility of parasite DNA detection in serum, taking advantage in using the same samples collected for serological screening.
  
  
  METHODS
  In order to evaluate the effectiveness of a qPCR strategy for detecting and quantifying T. cruzi DNA in serum, we selected 40 chronic Chagas disease patients presenting different clinical manifestations: Cardiac (23), Digestive (4), Mixed form [cardiodigestive] (7), and asymptomatic (6). Twenty seronegative individuals from non-endemic areas were included as controls. Samples were extracted using QIAamp DNA mini kit (QIAGEN) and qPCR was performed in a multiplex format with TaqMan probes for the nuclear satellite DNA of T. cruzi and for the human RNase P gene. In addition, DNA migration to serum during blood coagulation was assessed using a commercial exogenous control (Exo IPC, Applied Biosystems) in a separate qPCR reaction.
  
  
  RESULTS
  The comparative duplex qPCR analysis revealed that, even with an increase in Ct values, it was possible to detect all DNA targets in serum. In addition, the same linearity range for T. cruzi quantification (from 10(5) to 0.5 par. eq./mL) between serum, blood or culture samples (T. cruzi epimastigotes - Cl Brener strain) was found. When patient samples were evaluated, no significant differences in parasite load between the distinct clinical manifestations were found for both blood and serum samples. Moreover, median values of parasite burden were 1.125 and 1.230 par. eq./mL for serum and blood, respectively. Using serology as gold standard, we found 95% sensitivity for T. cruzi detection in serum and 97.5% for blood, and 100% specificity for both samples.
  
  
  CONCLUSIONS
  Taken together, our data indicate the potential of using serum samples for molecular diagnosis and parasite load quantification by qPCR, suggesting its use in reference laboratories for the diagnosis of Chagas disease patients.
authors.affiliation: Laboratório de Biologia Molecular e Doenças Endêmicas, IOC /Fiocruz, Av. Brasil, 4365. Pavilhão Leônidas Deane, sala 209. Manguinhos, Rio de Janeiro, Brazil. myllena@ioc.fiocruz.br.|Programa Integrado de Doença de Chagas/Fiocruz, Rio de Janeiro, RJ, Brazil. myllena@ioc.fiocruz.br.
authors.forename: Myllena F
authors.initials: MF
authors.lastname: Melo
authors.affiliation: Laboratório de Biologia Molecular e Doenças Endêmicas, IOC /Fiocruz, Av. Brasil, 4365. Pavilhão Leônidas Deane, sala 209. Manguinhos, Rio de Janeiro, Brazil. otacilio@ioc.fiocruz.br.|Programa Integrado de Doença de Chagas/Fiocruz, Rio de Janeiro, RJ, Brazil. otacilio@ioc.fiocruz.br.
authors.forename: Otacilio C
authors.initials: OC
authors.lastname: Moreira
authors.affiliation: Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães-CPqAM /Fiocruz, Recife, PE, Brazil. primoutinho28@gmail.com.
authors.forename: Priscila
authors.initials: P
authors.lastname: Tenório
authors.affiliation: Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães-CPqAM /Fiocruz, Recife, PE, Brazil. lorena@cpqam.fiocruz.br.|Programa Integrado de Doença de Chagas/Fiocruz, Rio de Janeiro, RJ, Brazil. lorena@cpqam.fiocruz.br.
authors.forename: Virginia
authors.initials: V
authors.lastname: Lorena
authors.affiliation: Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães-CPqAM /Fiocruz, Recife, PE, Brazil. izaura.lorena@gmail.com.
authors.forename: Izaura
authors.initials: I
authors.lastname: Lorena-Rezende
authors.affiliation: Ambulatório de doença de Chagas e Insuficiência Cardíaca do Pronto Socorro Cardiológico de Pernambuco (PROCAPE), Universidade de Pernambuco (UPE), Recife, PE, Brazil. woliveirajr@uol.com.br.
authors.forename: Wilson Oliveira
authors.initials: WO
authors.lastname: Júnior
authors.affiliation: Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães-CPqAM /Fiocruz, Recife, PE, Brazil. yara@cpqam.fiocruz.br.|Programa Integrado de Doença de Chagas/Fiocruz, Rio de Janeiro, RJ, Brazil. yara@cpqam.fiocruz.br.
authors.forename: Yara
authors.initials: Y
authors.lastname: Gomes
authors.affiliation: Laboratório de Biologia Molecular e Doenças Endêmicas, IOC /Fiocruz, Av. Brasil, 4365. Pavilhão Leônidas Deane, sala 209. Manguinhos, Rio de Janeiro, Brazil. cbritto@ioc.fiocruz.br.|Programa Integrado de Doença de Chagas/Fiocruz, Rio de Janeiro, RJ, Brazil. cbritto@ioc.fiocruz.br.
authors.forename: Constança
authors.initials: C
authors.lastname: Britto
mesh_terms: D000293:Adolescent; D000328:Adult; D014355:Chagas Disease / Q000097:blood* / Q000469:parasitology*; D002908:Chronic Disease; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D055815:Young Adult