Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

DUFFY, T; CURA, CI; RAMÍREZ, JC; ABATE, T; CAYO, NM; PARRADO, R; BELLO, ZD; VELAZQUEZ, E; MUÑOZ, A; JUIZ, NA; BASILE, J; GARCIA, L; RIARTE, A; NASSER, JR; OCAMPO, B; YADON, ZE; TORRICO, F; NOYA, BA; RIBEIRO, I; SCHIJMAN, AG

PLOS NEGLECTED TROPICAL DISEASES

PUBLIC LIBRARY SCIENCE

Lugar: San Francisco; Año: 2013 vol. 7 p. 1 - 11

1935-2735

Background:The analytical validation of sensitive, accurate and standardized Real-Time PCR methods forTrypanosoma cruziquantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoringtreatment efficacy.Methods/Principal Findings:We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR)based on TaqMan technology, aiming to quantifyT. cruzisatellite DNA as well as an internal amplification control (IAC) in asingle-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNAduring sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit ofquantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked withT. cruziCL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing differentclinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixtythree Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4-Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with acadaveric kidney explanted from an infected subject.Conclusions/Significance:The performing parameters of this assay encourage its application to early assessment ofT. cruziinfection in cases in which serological methods are not informative, such as recent infections by oral contamination orcongenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagasdisease patients under etiological treatment.