TOWARDS THE USE OF QUALITATIVE REAL TIME PCR AS A ROUTINE TOOL FOR NEGLECTED TROPICAL DISEASES: EVALUATION OF A COMMERCIAL KIT FOR DETECTION OF THE TRYPANOSOMA CRUZI DNA

BESUSCHIO, SUSANA A.; WEHRENDT D. P; KUHN, HANS; LONGHI, SILVIA A.; ROTTENGATTER, KARIN; SCHIJMAN A. G

Buenos Aires

Congreso; XV Congreso Argentino de Microbiología; 2019

TOWARDS THE USE OF QUALITATIVE REAL TIME PCR AS A ROUTINE TOOL FOR NEGLECTED TROPICAL DISEASES: EVALUATION OF A COMMERCIAL KIT FOR DETECTION OF THE TRYPANOSOMA CRUZI DNABESUSCHIO, Susana Alicia 1 | WEHRENDT, Diana Patricia1 | KUHN, Hans2 | LONGHI, Silvia Andrea1 | ROTTENGATTER, Karin3 | SCHIJMAN, Alejandro Gabriel1LAB. BIOLOGÍA MOLECULAR DE LA ENFERMEDAD DE CHAGAS - INGEBI- CONICET 1; ALTONA DIAGNOSTICS ARGENTINA SRL 2; ALTONA DIAGNOSTICS GMBH 3Introduction and objectives: Chagas Disease (CD) is a complex infection caused by Trypanosoma cruzi, a parasite with many Triatominae insects as vectors, diverse ways of transmission and stages, unspecific symptoms, long-term morbidity and more than 10000 deaths per year due to related complications, with only two available drugs for treatment. With an estimated 8 million people affected worldwide, this endemic disease in Latin America spread to other continents in the last decades. T. cruzi infection is curable if treatment is initiated soon after infection. In the chronic phase, antiparasitic treatment can also prevent or curb disease progression, (WHO Chagas Disease Factsheets, 2019); hence, research efforts focus in the early detection of the parasite with reliable and easy to use methods.Materials and methods: The Limit of Detection (LOD) for this qualitative kit was determined following the CLSI approved guidelines. Artificial samples spiked with T.cruzi parasites of ClBrener strain at low concentration range were prepared with Guanidine-EDTA buffer and non- infected blood (GEB). DNA from 8 replicates from 4 samples was extracted everyday for 5 days and then qPCR was run for target DNA by triplicates. Statistics involved Probit analysis, excluding IC outliers with Tukey range test. The kit was also challenged with 20 clinical samples, from congenital, chronic and reactivated CD clinical groups were tested, plus a seronegative control group, as an index test. A validated real time multiplex satellite DNA qPCR method was chosen as a reference test (Duffy, T et al. PLOS NTD 2013 Vol. 7 Issue 1 e2000), that also includes an exogenous IC. Cohen Kappa index has been used to measure interrater reliability .Results: The RealStar® Chagas detection kit and the reference test had shown comparable LODs ( 0.54 vs 0.7 p.e/mL) and Cohen Kappa index shows near perfect agreement between methods,Conclusions: The results are good enough to further evaluation.