Development of a simplified molecular test of congenital Chagas disease for neonatal screening

LAROCCA L; STOLOWICZ FG; MACALLINI G; DIMA E; ARANDA C; VOJNOV AA; ONETO A; CARRILLO C

Mar del Plata

Congreso; LXI Reunión Anual, Sociedad Argentina de Investigación Clínica SAIC; 2016

LXI Reunión Anual, Sociedad Argentina de Investigación Clínica SAIC

DEVELOPMENT OF A SIMPLIFIED MOLECULAR TEST OF CONGENITAL CHAGAS DISEASE FOR NEONATAL SCREENINGLarocca L1, Stolowicz F1, Macallini G2, Dima E2, Aranda C2, Vojnov A1, Oneto A2, Carrillo C1.1 Instituto de Ciencias y Tecnología Dr. César Milstein - CONICET.2 División Laboratorio - Hospital General de Agudos Carlos G. Durand INTRODUCTION. Chagas disease is an American endemic caused by the protozoan parasite Trypanosoma cruzi. The estimated number of annual cases of congenital T. cruzi infection in Argentina is 1,200, but less than 400 are officially reported due to methodological limitations for early detection.OBJECTIVES. To develop a simple test for congenital Chagas detection based on a molecular isothermal amplification (AMI) adapted to simplified neonatal screening practices.MATERIALS & METHODS. Samples of negative or artificially inoculated blood were dropped onto Guthrie test cards and used as substrate for AMI reaction at predefined conditions (65°C - 40 min). It was determined: a- Sensitivity, compared with a Quantitative Polymerase chain reaction (Q-PCR); and b- Specificity, challenging the reaction with other trypanosomatids, human and yeast DNA.The reaction efficiency pipetting the reagents separately vs a pre-prepared mix, and reading the result by lateral flow dipsticks chromatography (LFD) (field method) vs electrophoresis (analytical method) were also tested. Finally, AMI tests were performed with blood samples of newborn babies delivered from not infected and infected mothers, in order to challenge the simplified AMI test in field conditions.RESULTS. a- Samples artificially inoculated with T. cruzi, showed a detection sensitivity by AMI of 1-10 parasites/sample, 1 to 10 times greater than with Q-PCR. The sensitivity was similar in LFD than electrophoresis and in this kind of simplified test, dispensed in mix reaction.b- In all cases, heterologous DNA samples were negative by AMI and Q-PCR.c- 1 of the 20 blood samples of newborn babies delivered from not infected mothers resulted positive by AMI (5%) and 95% resulted negative.CONCLUSIONS. Our results indicate that AMI can yield a high specificity and sensitivity using a dried drop of blood in a Guthrie card and pre-prepared mix, with a remarkably simpler trial without expert human resources or complex equipment.KEY WORDS: Congenital Chagas disease; newborn screening; isothermal amplification; field conditions.