Rapid, Field-Deployable Method for Genotyping and Discovery of Single-Nucleotide Polymorphisms Associated with Drug Resistance in Plasmodium falciparum

Abstract

f Despite efforts to reduce malaria morbidity and mortality, drug-resistant parasites continue to evade control strategies. Recently, emphasis has shifted away from control and toward regional elimination and global eradication of malaria. Such a campaign requires tools to monitor genetic changes in the parasite that could compromise the effectiveness of antimalarial drugs and undermine eradication programs. These tools must be fast, sensitive, unambiguous, and cost-effective to offer real-time reports of parasite drug susceptibility status across the globe. We have developed and validated a set of genotyping assays using high-resolution melting (HRM) analysis to detect molecular biomarkers associated with drug resistance across six genes in Plasmodium falciparum. We improved on existing technical approaches by developing refinements and extensions of HRM, including the use of blocked probes (LunaProbes) and the mutant allele amplification bias (MAAB) technique. To validate the sensitivity and accuracy of our assays, we compared our findings to sequencing results in both culture-adapted lines and clinical isolates from Senegal. We demonstrate that our assays (i) identify both known and novel polymorphisms, (ii) detect multiple genotypes indicative of mixed infections, and (iii) distinguish between variants when multiple copies of a locus are present. These rapid and inexpensive assays can track drug resistance and detect emerging mutations in targeted genetic loci in P. falciparum. They provide tools for monitoring molecular changes associated with changes in drug response across populations and for determining whether parasites present after drug treatment are the result of recrudescence or reinfection in clinical settings. The Malaria Eradication Research Agenda (malERA) initiative recently reported (19) that development of new tools for surveillance and detection of drug-resistant parasites is imperative for any successful eradication effort. Rapidly expanding genetic data sets for malaria can be leveraged to identify molecular biomarkers related to important parasite characteristics, which could be key for surveillance.Arguably, the most well studied and clinically important sets of molecular biomarkers are genetic loci associated with resistance to antimalarial compounds. Resistance to aminoquinolines, such as chloroquine, and antifolates, such as pyrimethamine and sulfadoxine, is widespread, and molecular markers associated with these resistant phenotypes have been identified. Specifically, researchers have identified genetic changes in the pfcrt locus for chloroquine resistance (12), the dhfr locus for pyrimethamine and proguanil resistance (23, 24), and the dhps locus for sulfadoxine resistance (5). Other candidate molecular biomarkers for drug resistance are pfmdr1 (13, 27), associated with resistance to amodiaquine and other aminoquinolines; cytb, associated with atovaquone resistance (15, 17); and PfATPase6, implicated in artemisinin resistance (6,16,25). New discovery efforts, including genome-wide a...

Relevance of Undetectably Rare Resistant Malaria Parasites in Treatment Failure: Experimental Evidence from Plasmodium chabaudi

“…The standard RFLP-based assay 1,7,11,12,26 can detect parasites clones at frequencies down to around 10%. There are several more sensitive assays, including capillary electrophoresis, 27 heteroduplex tracking assays, 28-30 molecular barcoding using high resolution melting assays, 6,9 and ultra-deep sequencing, 10,31 but for none of these methods have frequencies lower than 1% reported.…”

“…[1][2][3] Detecting resistant parasites is important for monitoring and surveillance and to determine the health burden of resistance. 1 Resistant parasites frequently occur in infections with sensitive parasites, [4][5][6] a discovery first made with isozymes and then early polymerase chain reaction (PCR) methods. 7 In recent years, more sensitive molecular marker detection assays have been developed such as ligase detection reaction fluorescent microspheres, 5,8 high-resolution melting assays, 6,9 and ultra-deep sequencing.…”

“…1 Resistant parasites frequently occur in infections with sensitive parasites, [4][5][6] a discovery first made with isozymes and then early polymerase chain reaction (PCR) methods. 7 In recent years, more sensitive molecular marker detection assays have been developed such as ligase detection reaction fluorescent microspheres, 5,8 high-resolution melting assays, 6,9 and ultra-deep sequencing. 10 These newer methods have reduced genotype detection thresholds from the frequencies of 10% to 20% achievable with traditional restriction fragment length polymorphism (RFLP) assays 11,12 down to 1%.…”

High Resolution Melting Method in Molecular Diagnostics and Their Clinical Importance

“…HRM yöntemi antimikrobiyal direnç mekanizmalarını göstermek için de kullanılmaktadır ve Plasmodium falciparum'da ilaç direnç mutasyonları HRM analizi ile saptanmıştır. Yapılmış başka bir çalışmada ise, 6 gendeki çoklu mutasyonların bu mikroorganizmanın ilaç direnciyle ilişkili olduğu gösterilmiştir (39). Ayrıca HRM yöntemi enfeksiyon hastalıkların tedavisiyle ilgili araştırmalarda da kullanılmaktadır.…”

Malaria Diagnostics in Clinical Trials

“…Additional molecular assay types that may be useful for some trials also include those for determining the multiplicity of infection, 95 genotyping to determine recrudescence versus re-infection, 96 and those for detection of mutations that confer drug resistance 97 ; such assays are beyond the scope of this report. Considerations for use of molecular assays in clinical trials.…”