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

MACALLINI G; LAROCCA LUCIANA; VOJNOV ADRIAN; DIMA E; ONETO ADRIANA; STOLOWICZ FABIANA; ARANDA C; CARRILLO CAROLINA

Mar del Plata

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

LXI Reunión Anual, Sociedad Argentina de Investigación Clínica SAIC

INTRODUCTION. Chagas disease is an American endemic caused by the protozoan parasite​Trypanosoma cruzi​ . The estimatednumberofannualcasesofcongenital​T.cruziinfectioninArgentinais1,200,butlessthan 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 bloodweredroppedontoGuthrietest 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 wereperformedwithblood 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 adrieddropof bloodinaGuthriecardandpre-preparedmix,witharemarkablysimplertrialwithoutexperthumanresourcesor complex equipment.