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

Abstract

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.Importance (word count: 147)Continuous spread and repeated emergence of Plasmodium falciparum parasites resistant towards one or more antimalarials represents an enormous threat to current treatment efficacy levels, especially in sub-Saharan Africa, where 90% of malaria infections occur. In order to prevent substantial treatment failure, it is therefore recommended to monitor treatment efficacy every 2-3 years. Therapeutic efficacy studies, however, can present insurmountable logistical and financial challenges in some settings in sub-Saharan Africa. Molecular surveillance of antimalarial resistance is therefore an important proxy for treatment efficacy. However, the scale by which such studies can be performed depends on the development of high-throughput protocols and the accessibility of samples. If RDTs can be used in the high-throughput protocols available with Next Generation Sequencing (NGS)-technology, surveillance can be performed efficiently for any setting in which RDTs are already used for malaria diagnosis. The majority of settings in sub-Saharan Africa have access to RDTs.

Stable high frequencies of sulfadoxine-pyrimethamine resistance associated mutations and absence of K13 mutations in Plasmodium falciparum 3 and 4 years after the introduction of artesunate plus sulfadoxine-pyrimethamine in Ujjain, Madhya Pradesh, India

“…Finally, the poor PCR outcome in DNA extracted from RDTs is in line with other studies indicating the difficulty of extracting sufficient DNA [37]. Optimally blood for this type of survey should therefore be collected by other means so as not to introduce potential bias such as not being able to analyse samples with low parasite density.…”

Stable high frequencies of sulfadoxine-pyrimethamine resistance associated mutations and absence of K13 mutations in Plasmodium falciparum 3 and 4 years after the introduction of artesunate plus sulfadoxine-pyrimethamine in Ujjain, Madhya Pradesh, India

“…Finally, the poor PCR outcome in DNA extracted from RDTs is in line with other studies indicating the di culty of extracting su cient DNA [37]. Optimally blood for this type of survey should, therefore, be collected by other means so as not to introduce potential bias such as not being able to analyse samples with low parasite density.…”