Development and analytical validation of a new kit prototype for molecular diagnosis of congenital Chagas disease

RAMÍREZ, JUAN CARLOS; SALDAÑA, GUSTAVO; BORTOLOTTI, SANTIAGO; BALEANI, MARIÁNGELES; BESUSCHIO, SUSANA; LONGHI, SILVIA A; BUA, JACQUELINE; CURTO, MARÍA ANGELES; CAFFERATA, MARÍA LUISA; ALTHABE, FERNANDO; ESTEVA, MÓNICA; CAPPRIOTTI, GUSTAVO; SOSA-ESTANI, SERGIO; SCHIJMAN, ALEJANDRO

Ciudad de México

Congreso; 13 th INTERNATIONAL CONGRESS OF PARASITOLOGY; 2014

BACKGROUND: Every year around 15000 babies are born infected with Trypanosoma cruzi remaining high percentage of them undiagnosed until chronic disease symptoms appears. Therefore, early diagnosis is a priority in management of vertical transmission. Accordingly, we developed and validated a kit prototype based on Real-Time PCR intended for molecular diagnosis of congenital Chagas disease. METHODS: Methodology included DNA extraction from 1 mL of blood treated with a DNA stabilizer using magnetic beads and glass fiber columns and a multiplex Real-Time PCR based on TaqMan technology, aiming to amplify T. cruzi DNA and an internal amplification control simultaneously. Primers and probes sequences were designed against conservative regions within satellite repeats of all parasite DTUs. RESULTS: The prototype was selected based on the combination of primers, probes, master mixes and UDGs that reached the best analytical sensitivity (0.2 parasite equivalents/mL). Specificity was 100% in seronegative panels and DNA from T. rangeli and Leishmania sp. Finally, the prototype was validated following CCLS guidelines and field validation for early diagnosis of congenital Chagas disease is currently undergone in mother-newborns from health centers of endemic argentinean regions. CONCLUSIONS: The performing parameters of this prototype encourage its application to early assessment of T. cruzi infection in other scenarios such as oral transmission, organs transplantation from seropositive donors and monitoring patients under trypanocidal treatment.