Proof of concept: used malaria rapid diagnostic tests applied for parallel sequencing for surveillance of molecular markers of anti-malarial resistance in Bissau, Guinea-Bissau during 2014–2017

• Published in: Malaria Journal
• Main Authors: Sidsel Nag, Johan Ursing, Amabelia Rodrigues, Marina Crespo, Camilla Krogsgaard, Ole Lund, Frank M. Aarestrup, Michael Alifrangis, PouL-Erik Kofoed
• Format: Article in Journal/Newspaper
• Language: English
• Published: BMC 2019
• Subjects: Plasmodium falciparum; Guinea-Bissau; Molecular markers of antimalarial resistance; Rapid diagnostic tests; Next-generation sequencing; Amplicon sequencing; Arctic medicine. Tropical medicine; RC955-962; Infectious and parasitic diseases; RC109-216; Arctic
• Online Access: https://doi.org/10.1186/s12936-019-2894-8; https://doaj.org/article/f11ca47285c84172a188555f9e8e9497

Abstract Background Large-scale surveillance of molecular markers of anti-malarial drug resistance is an attractive method of resistance monitoring, to complement therapeutic efficacy studies in settings where the latter are logistically challenging. Methods Between 2014 and 2017, this study sampled malaria rapid diagnostic tests (RDTs), used in routine clinical care, from two health centres in Bissau, Guinea-Bissau. In order to obtain epidemiological insights, RDTs were collected together with patient data on age and sex. A subset of positive RDTs from one of the two sites (n = 2184) were tested for Plasmodium DNA content. Those testing positive for Plasmodium DNA by PCR (n = 1390) were used for library preparation, custom designed dual indexing and next generation Miseq targeted sequencing of Plasmodium falciparum genes pfcrt, pfmdr1, pfdhfr, pfdhps and pfk13. Results The study 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 to 51% between 2014 and 2017. No polymorphisms indicating artemisinin tolerance were discovered. The demographic data indicate a large proportion of young adults (66%, interquartile range 11–28 years) presenting with P. falciparum infections. While a total of 5532 gene fragments were successfully analysed on a single Illumina Miseq flow cell, PCR-positivity from the library preparation varied considerably from 13 to 87% for different amplicons. Furthermore, pre-screening of samples for Plasmodium DNA content proved necessary prior to library preparation. Conclusions This study serves as a proof of concept for using leftover clinical material (used RDTs) for large-scale molecular surveillance, encompassing the inherent complications regarding to methodology and analysis when doing so. Factors such as RDT storage prior to DNA extraction and parasitaemia of the infection are likely to have an effect on whether or not parasite DNA can be successfully ...