While the power of next-generation sequencing technologies to inform and guide malaria control programs has become broadly recognized, the integration of genomic data for operational incorporation into malaria surveillance remains a challenge in most countries where malaria is endemic. The main obstacles include limited infrastructure, limited access to high-throughput sequencing facilities, and the need for local capacity to run an in-country analysis of genomes at a large-enough scale to be informative for surveillance.

Background Point-of-care diagnosis of malaria is currently based on microscopy and rapid diagnostic tests. However, both techniques have their constraints, including poor sensitivity for low parasitaemias. Hence, more accurate diagnostic tests for field use and routine clinical settings are warranted. The miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative, easy-to-use molecular assay for diagnosis of malaria in resource-limited settings. Unlike traditional molecular methods, mini-dbPCR-NALFIA does not require DNA extraction and makes use of a handheld, portable thermal cycler that can run on a solar-charged power pack. Result read-out is done using a rapid lateral flow strip enabling differentiation of Plasmodium falciparum and non-falciparum malaria infections. A laboratory evaluation was performed to assess the performance of the mini-dbPCR-NALFIA for diagnosis of pan-Plasmodium and P. falciparum infections in whole blood. Methods Diagnostic accuracy of the mini-dbPCR-NALFIA was determined by testing a set of Plasmodium-positive blood samples from returned travellers (n = 29), and Plasmodium-negative blood samples from travellers with suspected malaria (n = 23), the Dutch Blood Bank (n = 19) and intensive care patients at the Amsterdam University Medical Centers (n = 16). Alethia Malaria (LAMP) with microscopy for species differentiation were used as reference. Limit of detection for P. falciparum was determined by 23 measurements of a dilution series of a P. falciparum culture. A fixed sample set was tested three times by the same operator to evaluate the repeatability, and once by five different operators to assess the reproducibility. Results Overall sensitivity and specificity of the mini-dbPCR-NALFIA were 96.6% (95% CI, 82.2%–99.9%) and 98.3% (95% CI, 90.8%–100%). Limit of detection for P. falciparum was 10 parasites per microlitre of blood. The repeatability of the assay was 93.7% (95% CI, 89.5%–97.8%) and reproducibility was 84.6% (95% CI, 79.5%–89.6%). Conclusions Mini-dbPCR-NALFIA is a sensitive, specific and robust method for molecular diagnosis of Plasmodium infections in whole blood and differentiation of P. falciparum. Incorporation of a miniature thermal cycler makes the assay well-adapted to resource-limited settings. A phase-3 field trial is currently being conducted to evaluate the potential implementation of this tool in different malaria transmission areas.

BackgroundMalaria molecular surveillance has great potential to support local national malaria control programs (NMCPs) to inform policy for malaria control and elimination. Molecular markers associated with drug resistance are good predictors of treatment responses. In addition, molecular detection of deletions in hrp2 and hrp3 genes are indicative of potential failure of HRP2-based rapid diagnostic tests. However, there is an urgent need for feasible, cost-effective and fast molecular surveillance tools that NMCPs can implement.MethodsHere we present a new 3-day workflow for targeted resequencing of markers in 13 resistance-associated genes, hrp2&3, a country-specific 28 SNP-barcode for population genetic analysis, and ama1. The assay was applied to control isolates and retrospective samples collected between 2003-2018 in the Loreto region (n = 254) in Peru. Pf AmpliSeq libraries were prepared using a multiplex PCR simultaneously amplifying a high number of targets from dried blood spots and sequenced at high coverage (median 1336, range 20-43795).ResultsThere was no evidence suggesting the emergence of artemisinin resistance in Peru. However, alleles in ubp1 and coronin contributed to recent genetic differentiation of the parasite population. After 2008, predominant parasite lineages in Peru are resistant to sulfadoxine-pyrimethamine (sextuple dhfr/dhps mutant) and chloroquine (SVMNT in crt and NDFCDY in mdr1) and can escape HRP2 based RDTs.ConclusionsThese findings indicate a parasite population under drug pressure, and demonstrates the added value of molecular surveillance systems and offers a highly multiplexed surveillance tool. The targets in the assay can be easily adjusted to suit the needs of other settings.FundingThis work was funded by the Belgium Development Cooperation (DGD) under the Framework Agreement Program between DGD and ITM (FA4 Peru, 2017-2021) and the sample collections in 2018 were supported by VLIR-UOS (project PE2018TEA470A102; University of Antwerp). Funding for the sample collections lead by the U.S. Naval Medical Research Unit 6 (NAMRU-6) in 2011 and 2012 was provided by the Armed Forces Health Surveillance Division (AFHSD) and its Global Emerging Infections Surveillance and Response (GEIS) Section (P0144_20_N6_01, 2020-2021).