Proof of concept: Malaria rapid diagnostic tests and massively parallel sequencing for surveillance of molecular markers of antimalarial resistance in Bissau, Guinea-Bissau during 2014-2017

Real-time and large-scale surveillance of molecular markers of antimalarial drug resistance is a potential method of resistance monitoring, to complement therapeutic efficacy studies in settings where the latter are logistically challenging. This study investigates whether routinely used malaria rapid diagnostic tests (RDTs) can be used for massive parallel amplicon sequencing. RDTs used for malaria diagnosis were routinely collected together with patient age and sex between 2014 and 2017, from two health centres in Bissau, Guinea-Bissau. A subset of positive RDTs (n=2,184) were tested for Plasmodium DNA content. Those containing sufficient Plasmodium DNA (n=1,390) were used for library preparation, consisting of amplification of gene fragments from pfcrt, pfmdr1, pfdhfr, pfdhps and pfK13. A total of 5532 gene fragments were successfully analysed on a single Illumina Miseq flow cell. Pre-screening of samples for Plasmodium DNA content proved necessary and the nested PCR protocol applied for library preparation varied notably in PCR-positivity from 13-87%. We found a high frequency of the pfmdr1 codon 86N at 88%-97%, a significant decrease of the pfcrt wildtype CVMNK haplotype and elevated levels of the pfdhfr/pfdhps quadruple mutant ranging from 33%-51% between 2014-2017. No polymorphisms indicating artemisinin tolerance were discovered. Lastly, the demographic data indicate a large proportion of young adults (66%, interquartile range 11-28 years) presenting with P. falciparum infections. With some caution, our findings suggest that routine collection of RDTs could facilitate large-scale molecular surveillance of antimalarial resistance.