DNA isolation and amplification of 
recrudescence
RECURRENCE, REINFECTION
Paired samples of blood spots on filter paper collected from patients with parasite recurrence on or before D28 (D0 and Day of parasite recurrence) were analyzed using nested PCR techniques to distinguish recrudescence from reinfection. Genomic DNA extraction was performed with QIAamp DNA Mini kit blood and tissue (QIAGEN Hilden Germany) kit, according to manufacturer’s instructions. Eluted genomic DNA was stored at − 20 °C until further PCR analysis. The small sub-unit ribosomal RNA (ssrRNA) gene of In a 20 µL reaction volume, PCR mixture containing 5X PCR Master Mix (Solis Biodyne, Estonia), 0.5 mM each of forward and reverse primers, nuclease-free PCR grade water, and 5 µL of extracted genomic DNA as template was prepared for the primary reaction. In the Nested reaction, 2 µL of the primary PCR product was used as a template in 18 µL PCR mixture, containing the same reagents as the primary reaction except with a species-specific forward and reverse primers. The PCR thermal cycling conditions were previously described [