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

BESUSCHIO, SUSANA; CAFFERATA, MARÍA LUISA; SALDAÑA, GUSTAVO; BORTOLOTTI, SANTIAGO; RAMIREZ, JUAN CARLOS; BALEANI, MARIÁNGELES; SCOLLO KARENINA; CURTO, MARÍA ANGELES; DANESI EMMARIA; LARA LAURA; AGOLTI GUSTAVO; BARTHE, MANUEL; ABOSLAIMAN,RICARDO; CIGANDA, ALVARO; CORMICK, GABRIELA; LONGHI SILVIA A; BUA, JACQUELINE; ESTEVA, MÓNICA; ALTHABE, FERNANDO; CAPRIOTTI GUSTAVO; SOSA ESTANI SERGIO; SCHIJMAN ALEJANDRO G

Mar del Plata

Congreso; X Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2014

Sociedad Argentina de Protozoologia

Early diagnosis of Congenital Chagas disease (CCD) is a public health priority. In the context of FONARSEC FITS SALUD CHAGAS, a public-private consortium was conformed to develop a TaqMan Real-Time PCR (qPCR) kit prototype for CCD diagnosis. One mL of blood collected in DNA stabilizer solution is processed in glass fiber columns. Duplex qPCR uses primers and TaqMan probes targeting satellite DNA repeats conserved in all T.cruzi lineages and an internal standard. External Quality Control panels have been designed and reagents stability tested for 6 months. To compare the accuracy of the kit vs current diagnostics (micromethod in cord or peripheral blood within the first 15 days and at 4-8 weeks of life, plus serodiagnosis at 10 months), a double-blinded prospective field study with two follow-up events has started upon approval of CEMIC Ethical Committee and local IRBs. Activities at fellow centers involve screening, recruitment and consent processes, data management (filling the CRFs, data entry), sample inventory, weekly monitoring and reporting. Each center processes the micromethod and serodiagnosis, recruits and refers samples for qPCR and serologic tests to INGEBI and INP, respectively. The logistics guidelines ensure traceability and proper transport for barcoded samples. Database will be disclosed at the end of study. Prototype analytical validation followed Clinical Laboratory Standard Institute guidelines, rendered a Limit of detection of 0.2 parasite equivalents/mL in spiked blood and 100% specificity in seronegative panels and T. rangeli and Leishmania spp DNAs. Field validation is currently undergone in 100 out of 500 mother-newborns binomials to be recruited in Buenos Aires, Tucumán, Santiago del Estero and Chaco. The performing parameters of the kit encourage its application to early diagnosis of T. cruzi infection not only by vertical transmission but also in oral outbreaks, organ transplantation and monitoring of trypanocidal therapy.