PHARE: a bioinformatics pipeline for compositional profiling of multiclonal Plasmodium falciparum infections from long-read Nanopore sequencing data.

BACKGROUND: The emergence of drug-resistant clones of Plasmodium falciparum is a major public health concern, and the ability to detect and track the spread of these clones is crucial for effective malaria control and treatment. However, in endemic settings, malaria infected people often carry multiple P. falciparum clones simultaneously making it likely to miss drug-resistant clones using traditional molecular typing methods. OBJECTIVES: Our goal was to develop a bioinformatics pipeline for compositional profiling in multiclonal P. falciparum samples, sequenced using the Oxford Nanopore Technologies MinION platform. METHODS: We developed the 'Finding P. falciparum haplotypes with resistance mutations in polyclonal infections' (PHARE) pipeline using existing bioinformatics tools and custom scripts written in python. PHARE was validated on three control datasets containing P. falciparum DNA of four laboratory strains at varying mixing ratios. Additionally, the pipeline was tested on clinical samples from children admitted to a paediatric hospital in the Central African Republic. RESULTS: The PHARE pipeline achieved high recall and accuracy rates in all control datasets. The pipeline can be used on any gene and was tested with amplicons of the P. falciparum drug resistance marker genes pfdhps, pfdhfr and pfK13. CONCLUSIONS: The PHARE pipeline helps to provide a more complete picture of drug resistance in the circulating P. falciparum population and can help to guide treatment recommendations. PHARE is freely available under the GNU Lesser General Public License v.3.0 on GitHub: https://github.com/Fippu/PHARE.

Time to scale up molecular surveillance for anti-malarial drug resistance in sub-saharan Africa.

Artemisinin resistance has emerged and spread in the Greater Mekong Sub-region (GMS), followed by artemisinin-based combination therapy failure, due to both artemisinin and partner drug resistance. More worrying, artemisinin resistance has been recently reported and confirmed in Rwanda. Therefore, there is an urgent need to strengthen surveillance systems beyond the GMS to track the emergence or spread of artemisinin and partner drug resistance in other endemic settings. Currently, anti-malarial drug efficacy is monitored primarily through therapeutic efficacy studies (TES). Even though essential for anti-malarial drug policy change, these studies are difficult to conduct, expensive, and may not detect the early emergence of resistance. Additionally, results from TES may take years to be available to the stakeholders, jeopardizing their usefulness. Molecular markers are additional and useful tools to monitor anti-malarial drug resistance, as samples collected on dried blood spots are sufficient to monitor known and validated molecular markers of resistance, and could help detecting and monitoring the early emergence of resistance. However, molecular markers are not monitored systematically by national malaria control programmes, and are often assessed in research studies, but not in routine surveillance. The implementation of molecular markers as a routine tool for anti-malarial drug resistance surveillance could greatly improve surveillance of anti-malarial drug efficacy, making it possible to detect resistance before it translates to treatment failures. When possible, ex vivo assays should be included as their data could be useful complementary, especially when no molecular markers are validated.

Tools for surveillance of anti-malarial drug resistance: an assessment of the current landscape.

To limit the spread and impact of anti-malarial drug resistance and react accordingly, surveillance systems able to detect and track in real-time its emergence and spread need to be strengthened or in some places established. Currently, surveillance of anti-malarial drug resistance is done by any of three approaches: (1) in vivo studies to assess the efficacy of drugs in patients; (2) in vitro/ex vivo studies to evaluate parasite susceptibility to the drugs; and/or (3) molecular assays to detect validated gene mutations and/or gene copy number changes that are associated with drug resistance. These methods are complementary, as they evaluate different aspects of resistance; however, standardization of methods, especially for in vitro/ex vivo and molecular techniques, is lacking. The World Health Organization has developed a standard protocol for evaluating the efficacy of anti-malarial drugs, which is used by National Malaria Control Programmes to conduct their therapeutic efficacy studies. Regional networks, such as the East African Network for Monitoring Antimalarial Treatment and the Amazon Network for the Surveillance of Antimalarial Drug Resistance, have been set up to strengthen regional capacities for monitoring anti-malarial drug resistance. The Worldwide Antimalarial Resistance Network has been established to collate and provide global spatial and temporal trends information on the efficacy of anti-malarial drugs and resistance. While exchange of information across endemic countries is essential for monitoring anti-malarial resistance, sustainable funding for the surveillance and networking activities remains challenging. The technology landscape for molecular assays is progressing quite rapidly, and easy-to-use and affordable new techniques are becoming available. They also offer the advantage of high throughput analysis from a simple blood spots obtained from a finger prick. New technologies combined with the strengthening of national reference laboratories in malaria-endemic countries through standardized protocols and training plus the availability of a proficiency testing programme, would contribute to the improvement and sustainability of anti-malarial resistance surveillance networks worldwide.

Population Pharmacokinetic Properties of Piperaquine in Falciparum Malaria: An Individual Participant Data Meta-Analysis.

BACKGROUND: Artemisinin-based combination therapies (ACTs) are the mainstay of the current treatment of uncomplicated Plasmodium falciparum malaria, but ACT resistance is spreading across Southeast Asia. Dihydroartemisinin-piperaquine is one of the five ACTs currently recommended by the World Health Organization. Previous studies suggest that young children (<5 y) with malaria are under-dosed. This study utilised a population-based pharmacokinetic approach to optimise the antimalarial treatment regimen for piperaquine. METHODS AND FINDINGS: Published pharmacokinetic studies on piperaquine were identified through a systematic literature review of articles published between 1 January 1960 and 15 February 2013. Individual plasma piperaquine concentration-time data from 11 clinical studies (8,776 samples from 728 individuals) in adults and children with uncomplicated malaria and healthy volunteers were collated and standardised by the WorldWide Antimalarial Resistance Network. Data were pooled and analysed using nonlinear mixed-effects modelling. Piperaquine pharmacokinetics were described successfully by a three-compartment disposition model with flexible absorption. Body weight influenced clearance and volume parameters significantly, resulting in lower piperaquine exposures in small children (<25 kg) compared to larger children and adults (≥25 kg) after administration of the manufacturers' currently recommended dose regimens. Simulated median (interquartile range) day 7 plasma concentration was 29.4 (19.3-44.3) ng/ml in small children compared to 38.1 (25.8-56.3) ng/ml in larger children and adults, with the recommended dose regimen. The final model identified a mean (95% confidence interval) increase of 23.7% (15.8%-32.5%) in piperaquine bioavailability between each piperaquine dose occasion. The model also described an enzyme maturation function in very young children, resulting in 50% maturation at 0.575 (0.413-0.711) y of age. An evidence-based optimised dose regimen was constructed that would provide piperaquine exposures across all ages comparable to the exposure currently seen in a typical adult with standard treatment, without exceeding the concentration range observed with the manufacturers' recommended regimen. Limited data were available in infants and pregnant women with malaria as well as in healthy individuals. CONCLUSIONS: The derived population pharmacokinetic model was used to develop a revised dose regimen of dihydroartemisinin-piperaquine that is expected to provide equivalent piperaquine exposures safely in all patients, including in small children with malaria. Use of this dose regimen is expected to prolong the useful therapeutic life of dihydroartemisinin-piperaquine by increasing cure rates and thereby slowing resistance development. This work was part of the evidence that informed the World Health Organization technical guidelines development group in the development of the recently published treatment guidelines (2015).

Altered Plasmodium falciparum Sensitivity to the Antiretroviral Protease Inhibitor Lopinavir Associated with Polymorphisms in pfmdr1.

The HIV protease inhibitor lopinavir inhibits Plasmodium falciparum aspartic proteases (plasmepsins) and parasite development, and children receiving lopinavir-ritonavir experienced fewer episodes of malaria than those receiving other antiretroviral regimens. Resistance to lopinavir was selected in vitro over ∼9 months, with ∼4-fold decreased sensitivity. Whole-genome sequencing of resistant parasites showed a mutation and increased copy number in pfmdr1 and a mutation in a protein of unknown function, but no polymorphisms in plasmepsin genes.

Global survey of malaria rapid diagnostic test (RDT) sales, procurement and lot verification practices: assessing the use of the WHO-FIND Malaria RDT Evaluation Programme (2011-2014).

BACKGROUND: Malaria rapid diagnostic tests (RDTs) play a critical role in malaria case management, and assurance of quality is a key factor to promote good adherence to test results. Since 2007, the World Health Organization (WHO) and the Foundation for Innovative New Diagnostics (FIND) have coordinated a Malaria RDT Evaluation Programme, comprising a pre-purchase performance evaluation (product testing, PT) and a pre-distribution quality control of lots (lot testing, LT), the former being the basis of WHO recommendations for RDT procurement. Comprehensive information on malaria RDTs sold worldwide based on manufacturers' data and linked to independent performance data is currently not available, and detailed knowledge of procurement practices remains limited. METHODS: The use of the PT/LT Programme results as well as procurement and lot verification practices were assessed through a large-scale survey, gathering product-specific RDT sales and procurement data (2011-14 period) from a total of 32 manufacturers, 12 procurers and 68 National Malaria Control Programmes (NMCPs). RESULTS: Manufacturers' reports showed that RDT sales had more than doubled over the four years, and confirmed a trend towards increased compliance with the WHO procurement criteria (from 83% in 2011 to 93% in 2014). Country-level reports indicated that 74% of NMCPs procured only 'WHO-compliant' RDT products, although procurers' transactions datasets revealed a surprisingly frequent overlap of different products and even product types (e.g., Plasmodium falciparum-only and Plasmodium-pan) in the same year and country (60 and 46% of countries, respectively). Importantly, the proportion of 'non-complying' (i.e., PT low scored or not evaluated) products was found to be higher in the private health care sector than in the public sector (32% vs 5%), and increasing over time (from 22% of private sector sales in 2011 to 39% in 2014). An estimated 70% of the RDT market was covered by the LT programme. The opinion about the PT/LT Programmes was positive overall, and quality of RDTs as per the PT Programme was rated as the number one procurement criteria. CONCLUSIONS: This survey provided in-depth information on RDT sales and procurement dynamics, including the largely unstudied private sector, and demonstrated how the WHO-FIND Programme has positively influenced procurement practices in the public sector.

Antiretroviral agents and prevention of malaria in HIV-infected Ugandan children.

BACKGROUND: Human immunodeficiency virus (HIV) protease inhibitors show activity against Plasmodium falciparum in vitro. We hypothesized that the incidence of malaria in HIV-infected children would be lower among children receiving lopinavir-ritonavir-based antiretroviral therapy (ART) than among those receiving nonnucleoside reverse-transcriptase inhibitor (NNRTI)-based ART. METHODS: We conducted an open-label trial in which HIV-infected children 2 months to 5 years of age who were eligible for ART or were currently receiving NNRTI-based ART were randomly assigned to either lopinavir-ritonavir-based ART or NNRTI-based ART and were followed for 6 months to 2 years. Cases of uncomplicated malaria were treated with artemether-lumefantrine. The primary end point was the incidence of malaria. RESULTS: We enrolled 176 children, of whom 170 received the study regimen: 86 received NNRTI-based ART, and 84 lopinavir-ritonavir-based ART. The incidence of malaria was lower among children receiving the lopinavir-ritonavir-based regimen than among those receiving the NNRTI-based regimen (1.32 vs. 2.25 episodes per person-year; incidence-rate ratio, 0.59; 95% confidence interval [CI], 0.36 to 0.97; P=0.04), as was the risk of a recurrence of malaria after treatment with artemether-lumefantrine (28.1% vs. 54.2%; hazard ratio, 0.41; 95% CI, 0.22 to 0.76; P=0.004). The median lumefantrine level on day 7 after treatment for malaria was significantly higher in the lopinavir-ritonavir group than in the NNRTI group. In the lopinavir-ritonavir group, lumefantrine levels exceeding 300 ng per milliliter on day 7 were associated with a reduction of more than 85% in the 63-day risk of recurrent malaria. A greater number of serious adverse events occurred in the lopinavir-ritonavir group than in the NNRTI group (5.6% vs. 2.3%, P=0.16). Pruritus occurred significantly more frequently in the lopinavir-ritonavir group, and elevated alanine aminotransferase levels significantly more frequently in the NNRTI group. CONCLUSIONS: Lopinavir-ritonavir-based ART as compared with NNRTI-based ART reduced the incidence of malaria by 41%, with the lower incidence attributable largely to a significant reduction in the recurrence of malaria after treatment with artemether-lumefantrine. Lopinavir-ritonavir-based ART was accompanied by an increase in serious adverse events. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development; ClinicalTrials.gov number, NCT00978068.).

Clinical determinants of early parasitological response to ACTs in African patients with uncomplicated falciparum malaria: a literature review and meta-analysis of individual patient data.

BACKGROUND: Artemisinin-resistant Plasmodium falciparum has emerged in the Greater Mekong sub-region and poses a major global public health threat. Slow parasite clearance is a key clinical manifestation of reduced susceptibility to artemisinin. This study was designed to establish the baseline values for clearance in patients from Sub-Saharan African countries with uncomplicated malaria treated with artemisinin-based combination therapies (ACTs). METHODS: A literature review in PubMed was conducted in March 2013 to identify all prospective clinical trials (uncontrolled trials, controlled trials and randomized controlled trials), including ACTs conducted in Sub-Saharan Africa, between 1960 and 2012. Individual patient data from these studies were shared with the WorldWide Antimalarial Resistance Network (WWARN) and pooled using an a priori statistical analytical plan. Factors affecting early parasitological response were investigated using logistic regression with study sites fitted as a random effect. The risk of bias in included studies was evaluated based on study design, methodology and missing data. RESULTS: In total, 29,493 patients from 84 clinical trials were included in the analysis, treated with artemether-lumefantrine (n = 13,664), artesunate-amodiaquine (n = 11,337) and dihydroartemisinin-piperaquine (n = 4,492). The overall parasite clearance rate was rapid. The parasite positivity rate (PPR) decreased from 59.7 % (95 % CI: 54.5-64.9) on day 1 to 6.7 % (95 % CI: 4.8-8.7) on day 2 and 0.9 % (95 % CI: 0.5-1.2) on day 3. The 95th percentile of observed day 3 PPR was 5.3 %. Independent risk factors predictive of day 3 positivity were: high baseline parasitaemia (adjusted odds ratio (AOR) = 1.16 (95 % CI: 1.08-1.25); per 2-fold increase in parasite density, P <0.001); fever (>37.5 °C) (AOR = 1.50 (95 % CI: 1.06-2.13), P = 0.022); severe anaemia (AOR = 2.04 (95 % CI: 1.21-3.44), P = 0.008); areas of low/moderate transmission setting (AOR = 2.71 (95 % CI: 1.38-5.36), P = 0.004); and treatment with the loose formulation of artesunate-amodiaquine (AOR = 2.27 (95 % CI: 1.14-4.51), P = 0.020, compared to dihydroartemisinin-piperaquine). CONCLUSIONS: The three ACTs assessed in this analysis continue to achieve rapid early parasitological clearance across the sites assessed in Sub-Saharan Africa. A threshold of 5 % day 3 parasite positivity from a minimum sample size of 50 patients provides a more sensitive benchmark in Sub-Saharan Africa compared to the current recommended threshold of 10 % to trigger further investigation of artemisinin susceptibility.

Micro-encapsulated pirimiphos-methyl shows high insecticidal efficacy and long residual activity against pyrethroid-resistant malaria vectors in central Côte d'Ivoire.

BACKGROUND: The wide-scale implementation of insecticide-treated nets and indoor residual spraying (IRS) has contributed to a considerable decrease of malaria morbidity and mortality in sub-Saharan Africa over the last decade. Due to increasing resistance in Anopheles gambiae sensu lato mosquitoes to dichlorodiphenyl trichloroethane (DDT) and pyrethroids, alternative insecticide formulations for IRS with long-lasting residual activity are required to sustain the gains obtained in most malaria-endemic countries. METHODS: Three experimental capsule suspension (CS) formulations of the organophosphate pirimiphos-methyl were evaluated together with Actellic 50 EC, an emulsifiable concentrate (EC) of pirimiphos-methyl, and the pyrethroid ICON 10 CS, a lambda-cyhalothrin CS formulation, in an experimental hut trial. The formulations were tested on two types of surfaces: mud and cement. The study with a 12-month follow-up was carried out in Bouaké, central Côte d'Ivoire, where An. gambiae mosquitoes show high levels of resistance against pyrethroids, DDT and carbamates. Residual activity was also tested in cone bioassays with the susceptible An. gambiae KISUMU strain. RESULTS: One of the CS formulations of pirimiphos-methyl, CS BM, outperformed all other formulations tested. On cement surfaces, the odds ratios of overall insecticidal effect on An. gambiae s.l. of pirimiphos-methyl CS BM compared to Actellic 50 EC were 1.4 (95% confidence interval (CI): 1.2-1.7) for the first three months, 5.6 (95% CI: 4.4-7.2) for the second three months, and 3.6 (95% CI: 3.0-4.4) for the last six months of follow-up. On mud surfaces, the respective odds ratios were 2.5 (95% CI: 1.9-3.3), 3.5 (95% CI: 2.7-4.5), and 1.7 (95% CI: 1.4-2.2). On cement, the residual activity of pirimiphos-methyl CS BM measured using cone tests was similar to that of lambda-cyhalothrin and for both treatments, mortality of susceptible Kisumu laboratory strain was not significantly below the World Health Organization pre-set threshold of 80% for 30 weeks after spraying. Residual activity was shorter on mud surfaces, mortality falling below 80% on both pirimiphos-methyl CS BM and lambda-cyhalothrin treated surfaces at 25 weeks post-treatment. CONCLUSION: CS formulations of pirimiphos-methyl are promising alternatives for IRS, as they demonstrate prolonged insecticidal effect and residual activity against malaria mosquitoes.

Correction: Development and evaluation of PlasmoPod: A cartridge-based nucleic acid amplification test for rapid malaria diagnosis and surveillance.

[This corrects the article DOI: 10.1371/journal.pgph.0001516.].

Optimization of a ligase detection reaction-fluorescent microsphere assay for characterization of resistance-mediating polymorphisms in African samples of Plasmodium falciparum.

Genetic polymorphisms in the malaria parasite Plasmodium falciparum mediate alterations in sensitivity to important antimalarial drugs. Surveillance for these polymorphisms is helpful in assessing the prevalence of drug resistance and designing strategies for malaria control. Multiple methods are available for the assessment of P. falciparum genetic polymorphisms, but they suffer from low throughput, technical limitations, and high cost. We have optimized and tested a multiplex ligase detection reaction-fluorescent microsphere (LDR-FM) assay for the identification of important P. falciparum genetic polymorphisms. For 84 clinical samples from Kampala, Uganda, a region where both transmission intensity and infection complexity are high, DNA was extracted from dried blood spots, genes of interest were amplified, amplicons were subjected to multiplex ligase detection reactions to add bead-specific oligonucleotides and biotin, fragments were hybridized to magnetic beads, and polymorphism prevalences were assessed fluorometrically in a multiplex format. A total of 19 alleles from the pfcrt, pfmdr1, pfmrp1, pfdhfr, and pfdhps genes were analyzed by LDR-FM and restriction fragment length polymorphism (RFLP) analyses. Considering samples with results from the two assays, concordance between the assays was good, with 78 to 100% of results identical at individual alleles, most nonconcordant results differing only between a mixed and pure genotype call, and full disagreement at individual alleles in only 0 to 3% of results. We estimate that the LDR-FM assay offers much higher throughput and lower cost than RFLP. Our results suggest that the LDR-FM system offers an accurate high-throughput means of classifying genetic polymorphisms in field samples of P. falciparum.

Bridging the Gap from Molecular Surveillance to Programmatic Decisions for Malaria Control and Elimination.

An increasing number of molecular and genomic assays are available to study malaria parasite populations. However, so far they have played a marginal role in informing policy and programmatic decision-making. Currently, molecular data are mainly used for monitoring drug efficacy against Plasmodium falciparum; assessing molecular markers of drug and insecticide resistance; and assessing P. falciparum histidine-rich protein 2 and 3 genes (Pfhrp2/3) deletion. We argue that additional use cases for molecular routine surveillance could be implemented in the near future, especially in transmission settings approaching elimination. These would include using quantitative polymerase chain reaction to monitor the prevalence of sub-patent infections in asymptomatic carriers, monitoring parasite genetic diversity as transmission intensity is changing, using genomic data to determine the origin of imported infections and characterize transmission chains in settings with very low malaria transmission, and using serology to monitor recent and past exposures in low-transmission settings. Molecular surveillance could inform control programs on adapting novel strategies, such as reactive case detection or focal mass drug administration, and help evaluate the impact of interventions currently in place. To better integrate molecular and genomic data into control program decision-making, engagement of national malaria control experts is crucial. Local laboratory capacity needs to be strengthened, shortening the time from sample collection to data availability. Here, we discuss opportunities and challenges of the use of molecular and genomic data for supporting malaria control and elimination efforts, as well as the avenues to link molecular and genomic data with gold standard epidemiological measurements through mathematical modeling.

Efficacy of ICON® Maxx in the laboratory and against insecticide-resistant Anopheles gambiae in central Côte d'Ivoire.

BACKGROUND: Long-lasting treatment kits, designed to transform untreated nets into long-lasting insecticidal nets (LLINs), may facilitate high coverage with LLINs where non-treated nets are in place. In this study, the efficacy of ICON® Maxx (Syngenta) was evaluated under laboratory conditions and in an experimental hut trial in central Côte d'Ivoire, where Anopheles gambiae s.s. are resistant to pyrethroid insecticides. METHODS: In the laboratory, polyester and polyethylene net samples were treated with ICON® Maxx, washed up to 20 times and their efficacy determined in World Health Organization (WHO) cone assays against a susceptible laboratory An. gambiae s.s. colony. Over a 12-month period, the polyester nets were evaluated in a hut trial to determine mosquito deterrence, induced exophily, blood-feeding inhibition and mortality. RESULTS: In the laboratory, ICON® Maxx-treated polyethylene nets showed higher efficacy against pyrethroid-susceptible mosquitoes than polyester nets. After 20 washings, insecticidal efficacy in bioassays was 59.4% knockdown (KD) and 22.3% mortality for polyethylene, and 55.3% KD and 17.9% mortality for polyester nets. In experimental huts, treated nets showed strong deterrence, induced exophily and an over three-fold reduction in blood-fed mosquitoes. More than half (61.8%) of the mosquitoes entering the huts with treated nets were found dead the next morning despite high levels of KD resistance. After washing the treated nets, KD and mortality rates were close to or exceeded predefined WHO thresholds in cone bioassays. CONCLUSION: In contrast to previous laboratory investigation, ICON® Maxx-treated nets showed only moderate KD and mortality rates. However, under semi-field conditions, in an area where mosquitoes are resistant to pyrethroids, ICON® Maxx showed high deterrence, induced exophily and provided a significant reduction in blood-feeding rates; features that are likely to have a positive impact in reducing malaria transmission. The WHO cone test may not always be a good proxy for predicting product performance under field conditions.

In vitro activity of antiretroviral drugs against Plasmodium falciparum.

Malaria and HIV infection are both very common in many developing countries. With the increasing availability of therapy for HIV infection, it was of interest to determine whether antiretroviral drugs exert antimalarial effects. We therefore tested the in vitro activity of 19 antiretroviral drugs against the W2 and 3D7 strains of Plasmodium falciparum at concentrations up to 50 µM. None of 5 tested nucleoside reverse transcriptase inhibitors demonstrated activity. Two nonnucleoside reverse transcriptase inhibitors, efavirenz (mean 50% inhibitory concentration [IC(50)] of 22 to 30 µM against the two strains) and etravirine (3.1 to 3.4 µM), were active; nevirapine was not active. Also active were the fusion inhibitor enfuvirtide (6.2 to 7.9 µM) and the entry inhibitor maraviroc (15 to 21 µM). Raltegravir was not active. However, for all active drugs mentioned above, the IC(50)s were considerably greater than the concentrations achieved with standard dosing. The effects most likely to be clinically relevant were with HIV protease inhibitors. Of the tested compounds, activity was seen with lopinavir (2.7 to 2.9 µM), atazanavir (3.3 to 13.0 µM), saquinavir (5.0 to 12.1 µM), nelfinavir (6.5 to 12.1 µM), ritonavir (9.5 to 10.9 µM), tipranavir (15.5 to 22.3 µM), and amprenavir (28.1 to 40.8) but not darunavir. Lopinavir was active at levels well below those achieved with standard dosing of coformulated lopinavir-ritonavir. Lopinavir also demonstrated modest synergy with the antimalarial lumefantrine (mean fractional inhibitory concentration index of 0.66 for W2 and 0.53 for 3D7). Prior data showed that lopinavir-ritonavir also extends the pharmacokinetic exposure of lumefantrine. Thus, when used to treat HIV infection, lopinavir-ritonavir may have clinically relevant antimalarial activity and also enhance the activity of antimalarials.

Quantifying the evolution and impact of antimalarial drug resistance: drug use, spread of resistance, and drug failure over a 12-year period in Papua New Guinea.

BACKGROUND. Antimalarial use is a key factor driving drug resistance and reduced treatment effectiveness in Plasmodium falciparum malaria, but there are few formal, quantitative analyses of this process. METHODS. We analyzed drug usage, drug failure rates, and the frequencies of mutations and haplotypes known to be associated with drug resistance over a 12-year period (1991-2002) in a site in Papua New Guinea. This period included 2 successive treatment policies: amodiaquine (AQ) or chloroquine (CQ) from 1991 through 2000 and their subsequent replacement by sulfadoxine-pyrimethamine (SP) plus AQ or SP plus CQ. RESULTS. Drug use approximated 1 treatment per person-year and was associated with increasing frequencies of pfcrt and pfmdr1 mutations and of treatment failure. The frequency of pfdhfr mutations also increased, especially after the change in treatment policy. Treatment failure rates multiplied by 3.5 between 1996 and 2000 but then decreased dramatically after treatment policy change. CONCLUSIONS. With high levels of resistance to CQ, AQ, and SP, the deployment of the combination of both drugs appears to increase clinical effectiveness but does not decelerate growth of resistance. Our estimates of mutation and haplotype frequencies provide estimates of selection coefficients acting in this environment, which are key parameters for understanding the dynamics of resistance.

Strengthening Surveillance Systems for Malaria Elimination by Integrating Molecular and Genomic Data.

Unprecedented efforts in malaria control over the last 15 years have led to a substantial decrease in both morbidity and mortality in most endemic settings. However, these progresses have stalled over recent years, and resurgence may cause dramatic impact on both morbidity and mortality. Nevertheless, elimination efforts are currently going on with the objective of reducing malaria morbidity and mortality by 90% and malaria elimination in at least 35 countries by 2030. Strengthening surveillance systems is of paramount importance to reach those targets, and the integration of molecular and genomic techniques into routine surveillance could substantially improve the quality and robustness of data. Techniques such as polymerase chain reaction (PCR) and quantitative PCR (qPCR) are increasingly available in malaria endemic countries, whereas others such as sequencing are already available in a few laboratories. However, sequencing, especially next-generation sequencing (NGS), requires sophisticated infrastructure with adequate computing power and highly trained personnel for data analysis that require substantial investment. Different techniques will be required for different applications, and cost-effective planning must ensure the appropriate use of available resources. The development of national and sub-regional reference laboratories could help in minimizing the resources required in terms of equipment and trained staff. Concerted efforts from different stakeholders at national, sub-regional, and global level are needed to develop the required framework to establish and maintain these reference laboratories.

Resistance to Artemisinin Combination Therapies (ACTs): Do Not Forget the Partner Drug!

Artemisinin-based combination therapies (ACTs) have become the mainstay for malaria treatment in almost all malaria endemic settings. Artemisinin derivatives are highly potent and fast acting antimalarials; but they have a short half-life and need to be combined with partner drugs with a longer half-life to clear the remaining parasites after a standard 3-day ACT regimen. When introduced, ACTs were highly efficacious and contributed to the steep decrease of malaria over the last decades. However, parasites with decreased susceptibility to artemisinins have emerged in the Greater Mekong Subregion (GMS), followed by ACTs' failure, due to both decreased susceptibility to artemisinin and partner drug resistance. Therefore, there is an urgent need to strengthen and expand current resistance surveillance systems beyond the GMS to track the emergence or spread of artemisinin resistance. Great attention has been paid to the spread of artemisinin resistance over the last five years, since molecular markers of decreased susceptibility to artemisinin in the GMS have been discovered. However, resistance to partner drugs is critical, as ACTs can still be effective against parasites with decreased susceptibility to artemisinins, when the latter are combined with a highly efficacious partner drug. This review outlines the different mechanisms of resistance and molecular markers associated with resistance to partner drugs for the currently used ACTs. Strategies to improve surveillance and potential solutions to extend the useful therapeutic lifespan of the currently available malaria medicines are proposed.

Molecular assays for antimalarial drug resistance surveillance: A target product profile.

Antimalarial drug resistance is a major constraint for malaria control and elimination efforts. Artemisinin-based combination therapy is now the mainstay for malaria treatment. However, delayed parasite clearance following treatment with artemisinin derivatives has now spread in the Greater Mekong Sub region and may emerge or spread to other malaria endemic regions. This spread is of great concern for malaria control programmes, as no alternatives to artemisinin-based combination therapies are expected to be available in the near future. There is a need to strengthen surveillance systems for early detection and response to the antimalarial drug resistance threat. Current surveillance is mainly done through therapeutic efficacy studies; however these studies are complex and both time- and resource-intensive. For multiple common antimalarials, parasite drug resistance has been correlated with specific genetic mutations, and the molecular markers associated with antimalarial drug resistance offer a simple and powerful tool to monitor the emergence and spread of resistant parasites. Different techniques to analyse molecular markers associated with antimalarial drug resistance are available, each with advantages and disadvantages. However, procedures are not adequately harmonized to facilitate comparisons between sites. Here we describe the target product profiles for tests to analyse molecular markers associated with antimalarial drug resistance, discuss how use of current techniques can be standardised, and identify the requirements for an ideal product that would allow malaria endemic countries to provide useful spatial and temporal information on the spread of resistance.

New Biomarkers and Diagnostic Tools for the Management of Fever in Low- and Middle-Income Countries: An Overview of the Challenges.

A lack of simple, inexpensive, and rapid diagnostic tests for febrile illnesses other than malaria leads to overtreatment with antibiotics for those who test negative for malaria, and contributes to the global rise in antimicrobial resistance. New tests for the detection of host biomarkers provide promising tools to differentiate bacterial from non-bacterial infections in febrile patients. However, most available biomarker tests are not currently used in resource-limited settings, and very few evaluations have been performed in low- and middle-income country populations with non-severe febrile illness. As a result, our knowledge of the performance of these tests in settings with high prevalence of infectious and poverty-related diseases such as malaria, HIV, malnutrition and intestinal parasites is poor. This paper describes challenges faced during the process of getting to an approved test, including difficulties in selecting the most appropriate fever biomarkers; suitable study designs and sites for test evaluations; lack of available reference tests to evaluate the performance of new tests; and lack of clear regulatory pathways to introduce such tests. As many new biomarker assays are in development, understanding these challenges will better enable those working in this area to address them during product development.

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine.

Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 - 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36-17.97, P < 0.001 : were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine.