Pre-amplification methods for tracking low-grade Plasmodium falciparum populations during scaled-up interventions in Southern Zambia

Background: Malaria is receding in many endemic countries with intervention scale -up against the disease. However, this resilient scourge may persist in low-grade submicroscopic infections among semi-immune members of the population, and be poised for possible resurgence, creating challenges for detection and assessment of intervention impact. Parasite genotyping methods, such as the molecular barcode, can identify specific malaria parasite types being transmitted and allow tracking and evaluation of parasite population structure changes as interventions are applied. This current study demonstrates application of pre-amplification methods for successful detection and genotyping of residual Plasmodium falciparum infections during a dramatic malarial decline. Methods: The study was a prospective cross-sectional design and based on a 2,000 sq km vicinity of Macha Mission Hospital in southern Zambia. Willing and predominantly asymptomatic residents of all ages were screened for malaria by microscopy during the 2005 and 2008 transmission seasons, with simultaneous collection of dried blood spots (DBS) on filter paper, and extraction of Plasmodium falciparum DNA was performed. Plasmodium falciparum infections were genotyped using a 24 SNP-based molecular barcode assay using real-time PCR. Submicroscopic parasitaemia samples were subjected to pre-amplification using TaqMan PreAmp Master Mix following the manufacturer’s instructions before SNP barcode analysis. Results: There was a dramatic decline of malaria between 2005 and 2008, and the geometric mean parasite density (95% CI) fell from 704/μL (390–1,271) in 2005 to 39/μL (23–68) in 2008, culminating in a large proportion of submicroscopic infections of which 90% failed to yield ample DNA for standard molecular characterization among 2008 samples. Pre-amplification enabled successful detection and genotyping of 74% of these low-grade reservoir infections, overall, compared to 54% that were detectable before pre-amplification (p <0.0005, n = 84). Furthermore, nine samples negative for parasites by microscopy and standard quantitative PCR amplification were positive after pre-amplification. Conclusions: Pre-amplification allows analysis of an otherwise undetectable parasite population and may be instrumental for parasites identification, tracking and assessing the impact of interventions on parasite populations during malaria control and elimination programmes when parasitaemia is expected to decline to submicroscopic levels

An Adjustable Gas-Mixing Device to Increase Feasibility of In Vitro Culture of Plasmodium falciparum Parasites in the Field

A challenge to conducting high-impact and reproducible studies of the mechanisms of P. falciparum drug resistance, invasion, virulence, and immunity is the lack of robust and sustainable in vitro culture in the field. While the technology exists and is routinely utilized in developed countries, various factors–from cost, to supply, to quality–make it hard to implement in malaria endemic countries. Here, we design and rigorously evaluate an adjustable gas-mixing device for the in vitro culture of P. falciparum parasites in the field to circumvent this challenge. The device accurately replicates the gas concentrations needed to culture laboratory isolates, short-term adapted field isolates, cryopreserved previously non-adapted isolates, as well as to adapt ex vivo isolates to in vitro culture in the field. We also show an advantage over existing alternatives both in cost and in supply. Furthermore, the adjustable nature of the device makes it an ideal tool for many applications in which varied gas concentrations could be critical to culture success. This adjustable gas-mixing device will dramatically improve the feasibility of in vitro culture of Plasmodium falciparum parasites in malaria endemic countries given its numerous advantages

Genetic evidence that the Makira region in northeastern Madagascar is a hotspot of malaria transmission

Additional file 3: Figure S1. Contains the bootstrap re-sample statistics for comparing genetic relatedness

Altered drug susceptibility during host adaptation of a <i>Plasmodium falciparum</i> strain in a non-human primate model

Infections with Plasmodium falciparum, the most pathogenic of the Plasmodium species affecting man, have been reduced in part due to artemisinin-based combination therapies. However, artemisinin resistant parasites have recently emerged in South-East Asia. Novel intervention strategies are therefore urgently needed to maintain the current momentum for control and elimination of this disease. In the present study we characterize the phenotypic and genetic properties of the multi drug resistant (MDR) P. falciparum Thai C2A parasite strain in the non-human Aotus primate model, and across multiple passages. Aotus infections with C2A failed to clear upon oral artesunate and mefloquine treatment alone or in combination, and ex vivo drug assays demonstrated reduction in drug susceptibility profiles in later Aotus passages. Further analysis revealed mutations in the pfcrt and pfdhfr loci and increased parasite multiplication rate (PMR) across passages, despite elevated pfmdr1 copy number. Altogether our experiments suggest alterations in parasite population structure and increased fitness during Aotus adaptation. We also present data of early treatment failures with an oral artemisinin combination therapy in a pre-artemisinin resistant P. falciparum Thai isolate in this animal model

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

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 pro- vide 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.Organismic and Evolutionary Biolog

Polymorphism in dhfr/dhps genes, parasite density and ex vivo response to pyrimethamine in Plasmodium falciparum malaria parasites in Thies, Senegal☆

Resistance to sulfadoxine–pyrimethamine (SP) in Plasmodium falciparum malaria parasites is associated with mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes, and these mutations have spread resistance worldwide. SP, used for several years in Senegal, has been recommended for intermittent preventive treatment for malaria in pregnancy (IPTp) and has been widely implemented since 2003 in this country. There is currently limited data on SP resistance from molecular marker genotyping, and no data on pyrimethamine ex vivo sensitivity in Senegal. Molecular markers of SP resistance and pyrimethamine ex vivo sensitivity were investigated in 416 parasite samples collected from the general population, from the Thies region between 2003 and 2011. The prevalence of the N51I/C59R/S108N triple mutation in dhfr increased from 40% in 2003 to 93% in 2011. Furthermore, the prevalence of the dhfr N51I/C59R/S108N and dhps A437G quadruple mutation increased, from 20% to 66% over the same time frame, then down to 44% by 2011. There was a significant increase in the prevalence of the dhfr triple mutation, as well as an association between dhfr genotypes and pyrimethamine response. Conversely, dhps mutations in codons 436 and 437 did not show consistent variation between 2003 and 2011. These findings suggest that regular screening for molecular markers of antifolate resistance and ex vivo drug response monitoring should be incorporated with ongoing in vivo efficacy monitoring in areas where IPTp-SP is implemented and where pyrimethamine and sulfa drugs are still widely administered in the general population

Analysis of pfhrp2 genetic diversity in Senegal and implications for use of rapid diagnostic tests

Background: The Senegalese National Malaria Control Programme has recommended use of rapid diagnostic tests (RDTs) that target the histidine-rich protein 2 (HRP2), specific to Plasmodium falciparum, to diagnose malaria cases. The target antigen has been shown to be polymorphic, which may explain the variability in HRP2-based RDT results reported in field studies. The genetic diversity of the pfhrp2 gene has not been investigated in depth in many African countries. The goal of this study is to determine the extent of polymorphism in pfhrp2 among Senegal, Mali and Uganda parasite populations, and discuss the implications of these findings on the utility of RDTs that are based on HRP2 detection. Methods: Sequencing data from the pfhrp2 locus were used to analyze the genetic diversity of this gene among three populations, with different transmission dynamics and malaria parasite ecologies. Nucleotide diversity (π) and non-synonymous nucleotide diversity (πNS) were studied in the pfhrp2 gene from isolates obtained in Senegal. Amino acid repeat length polymorphisms in the PfHRP2 antigen were characterized and parameters of genetic diversity, such as frequency and correlation between repeats in these populations, were assessed. Results: The diversity survey of the pfhrp2 gene identified 29 SNPs as well as insertion and deletion polymorphisms within a 918 bp region. The Senegal pfhrp2 exhibited a substantial level of diversity [π = 0.00559 and πNS = 0.014111 (πS = 0.0291627)], similar to several polymorphic genes, such as msp1, involved in immune responses, and the gene encoding the SURFIN polymorphic antigen, which are surface exposed parasite proteins. Extensive repeat length polymorphisms in PfHRP2, as well as similar patterns in the number, organization and the type of predicted amino acid repeats were observed among the three populations, characterized by an occurrence of Type 2, Type 4 and Type 7 repeats. Conclusions: These results warrant deeper monitoring of the RDT target antigen diversity and emphasize that development of other essential genes as a target for diagnostic tools is critical

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs. One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures. SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status. While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele. In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs. This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples

Changes in drug sensitivity and anti-malarial drug resistance mutations over time among Plasmodium falciparum parasites in Senegal

Background: Malaria treatment efforts are hindered by the rapid emergence and spread of drug resistant parasites. Simple assays to monitor parasite drug response in direct patient samples (ex vivo) can detect drug resistance before it becomes clinically apparent, and can inform changes in treatment policy to prevent the spread of resistance. Methods: Parasite drug responses to amodiaquine, artemisinin, chloroquine and mefloquine were tested in approximately 400 Plasmodium falciparum malaria infections in Thiès, Senegal between 2008 and 2011 using a DAPI-based ex vivo drug resistance assay. Drug resistance-associated mutations were also genotyped in pfcrt and pfmdr1. Results: Parasite drug responses changed between 2008 and 2011, as parasites became less sensitive to amodiaquine, artemisinin and chloroquine over time. The prevalence of known resistance-associated mutations also changed over time. Decreased amodiaquine sensitivity was associated with sustained, highly prevalent mutations in pfcrt, and one mutation in pfmdr1 – Y184F – was associated with decreased parasite sensitivity to artemisinin. Conclusions: Directly measuring ex vivo parasite drug response and resistance mutation genotyping over time are useful tools for monitoring parasite drug responses in field samples. Furthermore, these data suggest that the use of amodiaquine and artemisinin derivatives in combination therapies is selecting for increased drug tolerance within this population