Resistance Management for Cancer: Lessons from Farmers.

One of the main reasons we have not been able to cure cancers is that treatments select for drug-resistant cells. Pest managers face similar challenges with pesticides selecting for pesticide-resistant insects, resulting in similar mechanisms of resistance. Pest managers have developed ten principles that could be translated to controlling cancers: (1) prevent onset, (2) monitor continuously, (3) identify thresholds below which there will be no intervention, (4) change interventions in response to burden, (5) preferentially select non-chemical control methods, (6) use target-specific drugs, (7) use the lowest effective dose, (8) reduce cross-resistance, (9) evaluate success based on long-term management, and (10) forecast growth and response. These principles are general to all cancers and cancer drugs and so could be employed broadly to improve oncology. Here, we review the parallel difficulties in controlling drug resistance in pests and cancer cells. We show how the principles of resistance management in pests might be applied to cancer. Integrated pest management inspired the development of adaptive therapy in oncology to increase progression-free survival and quality of life in patients with cancers where cures are unlikely. These pest management principles have the potential to inform clinical trial design.

Repelling Aedes aegypti mosquitoes with electric fields using insulated conductor wires.

BACKGROUND: The control and prevention of mosquito-borne diseases is mostly achieved with insecticides. However, their use has led to the rapid development and spread of insecticide resistance worldwide. Health experts have called for intensified efforts to find new approaches to reduce mosquito populations and human-mosquito contact. A promising new tool is the use of electrical fields (EFs), whereby mosquitoes are repelled by charged particles in their flight path. Such particles move between two or more conductors, and the use of uninsulated copper or aluminum plates as conductors has been proven to be effective at repelling mosquitoes. Here, for the first time, we assess if EFs generated using a single row of insulated conductor wires (ICWs) can also successfully repel mosquitoes, and whether mosquitoes are equally repelled at the same EF strength when the electrodes are a) orientated differently (horizontal vs. vertical placement), and b) spaced more apart. METHODOLOGY/PRINCIPAL FINDINGS: Over a period of 23 hours, the number of host-seeking female Aedes aegypti mosquitoes that were successfully repelled by EFs, using ICWs, at EF strengths ranging from 0 kV/cm (control) to 9.15 kV/cm were quantified. Mosquitoes were released inside a 220×220×180 cm room and lured into a BG-Pro trap that was equipped with a BG-counter and baited with CO2 using dry ice. Mosquitoes had to pass through an EF window, that contained a single row of ICWs with alternating polarity, to reach the bait. The baseline interaction between EF strength and repellency was assessed first, after which the impact of different ICW orientations and ICW distances on repellency were determined. Over 50% of mosquitoes were repelled at EF strengths of ≥ 3.66 kV/cm. A linear regression model showed that a vertical ICW orientation (vertical vs. horizontal) had a small but insignificant increased impact on mosquito repellency (p = 0.059), and increasing ICW distance (while maintaining the same EF strength) significantly reduced repellency (p = 0.01). CONCLUSIONS/SIGNIFICANCE: ICWs can be used to generate EFs that partially repel host-seeking mosquitoes, which will reduce human-mosquito contact. While future studies need to assess if (i) increased repellency can be achieved, and (ii) a repellency of 50-60% is sufficient to impact disease transmission, it is encouraging that EF repellency using ICWs is higher compared to that of some spatial repellent technologies currently in development. This technology can be used in the housing improvement toolkit (i.e. preventing mosquito entry through eaves, windows, and doors). Moreover, the use of cheap, over-the-counter ICWs will mean that the technology is more accessible worldwide, and easier to manufacture and implement locally.

Non-target effects of chemical malaria vector control on other biological and mechanical infectious disease vectors.

Public health insecticides play a crucial role in malaria control and elimination programmes. Many other arthropods, including mechanical and biological vectors of infectious diseases, have similar indoor feeding or resting behaviours, or both, as malaria mosquitoes, and could be exposed to the same insecticides. In this Personal View, we show that little is known about the insecticide susceptibility status and the extent of exposure to malaria interventions of other arthropod species. We highlight that there is an urgent need to better understand the selection pressure for insecticide resistance in those vectors, to ensure current and future active ingredients remain effective in targeting a broad range of arthropod species, allowing us to prevent and control future outbreaks of infectious diseases other than malaria.

COMPARING THE EFFICIENCY OF DIFFERENT CARBON DIOXIDE SOURCES IN COLLECTING MOSQUITO VECTOR SPECIES ON THE TEMPE CAMPUS OF ARIZONA STATE UNIVERSITY.

We compared the effectiveness of 4 different carbon dioxide (CO2) sources (sugar-fermented BG-CO2, sugar-fermented Fleischmann yeast, dry ice, and compressed gas cylinders) in attracting different mosquito species in 2 separate 4 × 4 Latin square trials. The CO2 generated by dry ice and the gas cylinders collected more Culex quinquefasciatus than the sugar-fermented BG-CO2 and Fleischmann yeasts during the 1st trial (16-h surveillance periods), but there was no significant difference in Aedes aegypti numbers. There were no significant differences between the different CO2 sources in collecting Cx. quinquefasciatus and Ae. aegypti mosquitoes in the 2nd trial (24-h surveillance periods). Catches for Culiseta inornata and Cx. tarsalis were too low in both experiments for formal statistical analysis. Data can be used to inform local mosquito surveillance programs, but the selection of a CO2 source will also depend on financial and logistical constraints.

Challenges in cybersecurity: Lessons from biological defense systems.

Defending against novel, repeated, or unpredictable attacks, while avoiding attacks on the 'self', are the central problems of both mammalian immune systems and computer systems. Both systems have been studied in great detail, but with little exchange of information across the different disciplines. Here, we present a conceptual framework for structured comparisons across the fields of biological immunity and cybersecurity, by framing the context of defense, considering different (combinations of) defensive strategies, and evaluating defensive performance. Throughout this paper, we pose open questions for further exploration. We hope to spark the interdisciplinary discovery of general principles of optimal defense, which can be understood and applied in biological immunity, cybersecurity, and other defensive realms.

Fine-scale spatial distribution of deltamethrin resistance and population structure of Anopheles funestus and Anopheles arabiensis populations in Southern Mozambique.

BACKGROUND: Insecticide resistance in malaria vectors can be spatially highly heterogeneous, yet population structure analyses frequently find relatively high levels of gene flow among mosquito populations. Few studies have contemporaneously assessed phenotypic, genotypic and population structure analysis on mosquito populations and none at fine geographical scales. In this study, genetic diversity, population structure, and insecticide resistance profiles of Anopheles funestus and Anopheles arabiensis were examined across mosquito populations from and within neighbouring villages. METHODS: Mosquitoes were collected from 11 towns in southern Mozambique, as well as from different neighbourhoods within the town of Palmeira, during the peak malaria transmission season in 2016. CDC bottle bioassay and PCR assays were performed with Anopheles mosquitoes at each site to determine phenotypic and molecular insecticide resistance profiles, respectively. Microsatellite analysis was conducted on a subsample of mosquitoes to estimate genetic diversity and population structure. RESULTS: Phenotypic insecticide resistance to deltamethrin was observed in An. funestus sensu stricto (s.s.) throughout the area, though a high level of mortality variation was seen. However, 98% of An. funestus s.s. were CYP6P9a homozygous resistant. An. arabiensis was phenotypically susceptible to deltamethrin and 99% were kdr homozygous susceptible. Both Anopheles species exhibited high allelic richness and heterozygosity. Significant deviations from Hardy-Weinberg equilibrium were observed, and high linkage disequilibrium was seen for An. funestus s.s., supporting population subdivision. However, the FST values were low for both anophelines (- 0.00457 to 0.04213), Nm values were high (9.4-71.8 migrants per generation), AMOVA results showed almost 100% genetic variation among and within individuals, and Structure analysis showed no clustering of An. funestus s.s. and An. arabiensis populations. These results suggest high gene flow among mosquito populations. CONCLUSION: Despite a relatively high level of phenotypic variation in the An. funestus population, molecular analysis shows the population is admixed. These data indicate that CYP6P9a resistance markers do not capture all phenotypic variation in the area, but also that resistance genes of high impact are likely to easily spread in the area. Conversely, other strategies, such as transgenic mosquito release programmes will likely not face challenges in this locality.

Fine-scale spatial distribution of deltamethrin resistance and population structure of Anopheles funestus and Anopheles arabiensis populations in Southern Mozambique

Insecticide resistance in malaria vectors can be spatially highly heterogeneous, yet population structure analyses frequently find relatively high levels of gene flow among mosquito populations. Few studies have contemporaneously assessed phenotypic, genotypic and population structure analysis on mosquito populations and none at fine geographical scales. In this study, genetic diversity, population structure, and insecticide resistance profiles of Anopheles funestus and Anopheles arabiensis were examined across mosquito populations from and within neighbouring villages. Methods: Mosquitoes were collected from 11 towns in southern Mozambique, as well as from different neighbourhoods within the town of Palmeira, during the peak malaria transmission season in 2016. CDC bottle bioassay and PCR assays were performed with Anopheles mosquitoes at each site to determine phenotypic and molecular insecticide resistance profiles, respectively. Microsatellite analysis was conducted on a subsample of mosquitoes to estimate genetic diversity and population structure. Results: Phenotypic insecticide resistance to deltamethrin was observed in An. funestus sensu stricto (s.s.) throughout the area, though a high level of mortality variation was seen. However, 98% of An. funestus s.s. were CYP6P9a homozygous resistant. An. arabiensis was phenotypically susceptible to deltamethrin and 99% were kdr homozygous susceptible. Both Anopheles species exhibited high allelic richness and heterozygosity. Significant deviations from Hardy-Weinberg equilibrium were observed, and high linkage disequilibrium was seen for An. funestus s.s., supporting population subdivision. However, the FST values were low for both anophelines (- 0.00457 to 0.04213), Nm values were high (9.4-71.8 migrants per generation), AMOVA results showed almost 100% genetic variation among and within individuals, and Structure analysis showed no clustering of An. funestus s.s. and An. arabiensis populations. These results suggest high gene flow among mosquito populations. Conclusion: Despite a relatively high level of phenotypic variation in the An. funestus population, molecular analysis shows the population is admixed. These data indicate that CYP6P9a resistance markers do not capture all phenotypic variation in the area, but also that resistance genes of high impact are likely to easily spread in the area. Conversely, other strategies, such as transgenic mosquito release programmes will likely not face challenges in this locality.

Comparison of the variability in mortality data generated by CDC bottle bioassay, WHO tube test, and topical application bioassay using Aedes aegypti mosquitoes.

BACKGROUND: Insecticide resistance remains a major public health problem. Resistance surveillance is critical for effective vector control and resistance management planning. Commonly used insecticide susceptibility bioassays for mosquitoes are the CDC bottle bioassay and the WHO tube test. Less commonly used in the field but considered the gold standard for assessing insecticide susceptibility in the development of novel insecticides is the topical application bioassay. Each of these bioassays has critical differences in how they assess insecticide susceptibility that impacts their ability to differentiate between resistant and susceptible populations or determine different levels of resistance intensity. METHODS: We compared the CDC bottle bioassay, the WHO tube test, and the topical application bioassay in establishing the dose-response against deltamethrin (DM) using the DM-resistant Aedes aegypti strain MC1. Mosquitoes were exposed to a range of insecticide concentrations to establish a dose-response curve and assess variation around model predictions. In addition, 10 replicates of 20-25 mosquitoes were exposed to a fixed dose with intermediate mortality to assess the degree of variation in mortality. RESULTS: The topical application bioassay exhibited the lowest amount of variation in the dose-response data, followed by the WHO tube test. The CDC bottle bioassay had the highest level of variation. In the fixed-dose experiment, a higher variance was similarly found for the CDC bottle bioassay compared with the WHO tube test and topical application bioassay. CONCLUSION: These data suggest that the CDC bottle bioassay has the lowest power and the topical application bioassay the highest power to differentiate between resistant and susceptible populations and assess changes over time and between populations. This observation has significant implications for the interpretation of surveillance results from different assays. Ultimately, it will be important to discuss optimal insecticide resistance surveillance tools in terms of the surveillance objective, practicality in the field, and accuracy of the tool to reach that objective.

The realized efficacy of indoor residual spraying campaigns falls quickly below the recommended WHO threshold when coverage, pace of spraying and residual efficacy on different wall types are considered.

Indoor residual spraying (IRS) has been and remains an important malaria control intervention in southern Mozambique, South Africa and Eswatini. A better understanding of the effectiveness of IRS campaigns is critical to guide future elimination efforts. We analyze the three IRS campaigns conducted during a malaria elimination demonstration project in southern Mozambique, the "Magude project", and propose a new method to calculate the efficacy of IRS campaigns adjusting for IRS coverage, pace of house spraying and IRS residual efficacy on different wall types. Anopheles funestus sensu lato (s.l.) and An. gambiae s.l. were susceptible to pirimiphos-methyl and DDT. Anopheles funestus s.l. was resistant to pyrethroids, with 24h post-exposure mortality being lower for An. funestus sensu stricto (s.s.) than for An. parensis (collected indoors). The percentage of structures sprayed was above 90% and percentage of people covered above 86% in all three IRS campaigns. The percentage of households sprayed was above 83% in 2015 and 2016, but not assessed in 2017. Mosquito mortality 24h post-exposure stayed above 80% for 196 days after the 2016 IRS campaign and 222 days after the 2017 campaign and was 1.5 months longer on mud walls than on cement walls. This was extended by up to two months when 120h post-exposure mortality was considered. The district-level realized IRS efficacy was 113 days after the 2016 campaign. While the coverage of IRS campaigns in Magude were high, IRS protection did not remain optimal for the entire high malaria transmissions season. The use of a longer-lasting IRS product could have further supported the interruption of malaria transmission in the district. To better estimate the protection afforded by IRS campaigns, National Malaria Control Programs and partners are encouraged to adjust the calculation of IRS efficacy for IRS coverage, pace of house spraying during the campaign and IRS efficacy on different wall types combined with wall type distribution in the sprayed area.

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies.

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along a range of insecticide concentrations. This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

The need for practical insecticide-resistance guidelines to effectively inform mosquito-borne disease control programs.

Monitoring local mosquito populations for insecticide resistance is critical for effective vector-borne disease control. However, widely used phenotypic assays, which are designed to monitor the emergence and spread of insecticide resistance (technical resistance), do not translate well to the efficacy of vector control products to suppress mosquito numbers in the field (practical resistance). This is because standard testing conditions such as environmental conditions, exposure dose, and type of substrate differ dramatically from those experienced by mosquitoes under field conditions. In addition, field mosquitoes have considerably different physiological characteristics such as age and blood-feeding status. Beyond this, indirect impacts of insecticide resistance and/or exposure on mosquito longevity, pathogen development, host-seeking behavior, and blood-feeding success impact disease transmission. Given the limited number of active ingredients currently available and the observed discordance between resistance and disease transmission, we conclude that additional testing guidelines are needed to determine practical resistance-the efficacy of vector control tools under relevant local conditions- in order to obtain programmatic impact.

Effect of mass dihydroartemisinin-piperaquine administration in southern Mozambique on the carriage of molecular markers of antimalarial resistance.

BACKGROUND: Mass drug administration (MDA) can rapidly reduce the burden of Plasmodium falciparum (Pf). However, concerns remain about its contribution to select for antimalarial drug resistance. METHODS: We used Sanger sequencing and real-time PCR to determine the proportion of molecular markers associated with antimalarial resistance (k13, pfpm2, pfmdr1 and pfcrt) in Pf isolates collected before (n = 99) and after (n = 112) the implementation of two monthly MDA rounds with dihydroartemisinin-piperaquine (DHAp) for two consecutive years in Magude district of Southern Mozambique. RESULTS: None of the k13 polymorphisms associated with artemisinin resistance were observed in the Pf isolates analyzed. The proportion of Pf isolates with multiple copies of pfpm2, an amplification associated with piperaquine resistance, was similar in pre- (4.9%) and post-MDA groups (3.4%; p = 1.000). No statistically significant differences were observed between pre- and post-MDA groups in the proportion of Pf isolates neither with mutations in pfcrt and pfmdr1 genes, nor with the carriage of pfmdr1 multiple copies (p>0.05). CONCLUSIONS: This study does not show any evidence of increased frequency of molecular makers of antimalarial resistance after MDA with DHAp in southern Mozambique where markers of antimalarial resistance were absent or low at the beginning of the intervention.

Effect of mass dihydroartemisinin-piperaquine administration in southern Mozambique on the carriage of molecular markers of antimalarial resistance

Background Mass drug administration (MDA) can rapidly reduce the burden of Plasmodium falciparum (Pf). However, concerns remain about its contribution to select for antimalarial drug resistance. Methods We used Sanger sequencing and real-time PCR to determine the proportion of molecular markers associated with antimalarial resistance (k13, pfpm2, pfmdr1 and pfcrt) in Pf isolates collected before (n = 99) and after (n = 112) the implementation of two monthly MDA rounds with dihydroartemisinin­piperaquine (DHAp) for two consecutive years in Magude district of Southern Mozambique. Results None of the k13 polymorphisms associated with artemisinin resistance were observed in the Pf isolates analyzed. The proportion of Pf isolates with multiple copies of pfpm2, an amplification associated with piperaquine resistance, was similar in pre- (4.9%) and post-MDA groups (3.4%; p = 1.000). No statistically significant differences were observed between pre- and post-MDA groups in the proportion of Pf isolates neither with mutations in pfcrt and pfmdr1 genes, nor with the carriage of pfmdr1 multiple copies (p>0.05). Conclusions This study does not show any evidence of increased frequency of molecular makers of antimalarial resistance after MDA with DHAp in southern Mozambique where markers of antimalarial resistance were absent or low at the beginning of the intervention.

Long-lasting insecticidal nets and the quest for malaria eradication: a mathematical modeling approach.

Recent dramatic declines in global malaria burden and mortality can be largely attributed to the large-scale deployment of insecticidal-based measures, namely long-lasting insecticidal nets (LLINs) and indoor residual spraying. However, the sustainability of these gains, and the feasibility of global malaria eradication by 2040, may be affected by increasing insecticide resistance among the Anopheles malaria vector. We employ a new differential-equations based mathematical model, which incorporates the full, weather-dependent mosquito lifecycle, to assess the population-level impact of the large-scale use of LLINs, under different levels of Anopheles pyrethroid insecticide resistance, on malaria transmission dynamics and control in a community. Moreover, we describe the bednet-mosquito interaction using parameters that can be estimated from the large experimental hut trial literature under varying levels of effective pyrethroid resistance. An expression for the basic reproduction number, [Formula: see text], as a function of population-level bednet coverage, is derived. It is shown, owing to the phenomenon of backward bifurcation, that [Formula: see text] must be pushed appreciably below 1 to eliminate malaria in endemic areas, potentially complicating eradication efforts. Numerical simulations of the model suggest that, when the baseline [Formula: see text] is high (corresponding roughly to holoendemic malaria), very high bednet coverage with highly effective nets is necessary to approach conditions for malaria elimination. Further, while >50% bednet coverage is likely sufficient to strongly control or eliminate malaria from areas with a mesoendemic malaria baseline, pyrethroid resistance could undermine control and elimination efforts even in this setting. Our simulations show that pyrethroid resistance in mosquitoes appreciably reduces bednet effectiveness across parameter space. This modeling study also suggests that increasing pre-bloodmeal deterrence of mosquitoes (deterring them from entry into protected homes) actually hampers elimination efforts, as it may focus mosquito biting onto a smaller unprotected host subpopulation. Finally, we observe that temperature affects malaria potential independently of bednet coverage and pyrethroid-resistance levels, with both climate change and pyrethroid resistance posing future threats to malaria control.

Correction: Phenotypic insecticide resistance in arbovirus mosquito vectors in Catalonia and its capital Barcelona (Spain).

[This corrects the article DOI: 10.1371/journal.pone.0217860.].

Taking the 'I' out of LLINs: using insecticides in vector control tools other than long-lasting nets to fight malaria.

Long-lasting insecticidal nets, or LLINs, have significantly reduced malaria morbidity and mortality over the past two decades. The net provides a physical barrier that decreases human-mosquito contact and the impregnated insecticide kills susceptible mosquito vectors upon contact and may repel them. However, the future of LLINs is threatened as resistance to pyrethroids is now widespread, the chemical arsenal for LLINs is very limited, time from discovery of next-generation insecticides to market is long, and persistent transmission is frequently caused by vector populations avoiding contact with LLINs. Here we ask the question whether, given these challenges, insecticides should be incorporated in nets at all. We argue that developing long-lasting nets without insecticide(s) can still reduce vector populations and provide both personal and community protection, if combined with other approaches or technologies. Taking the insecticide out of the equation (i) allows for a faster response to the current pyrethroid resistance crisis, (ii) avoids an LLIN-treadmill aimed at replacing failing bed nets due to insecticide resistance, and (iii) permits the utilization of our current and future insecticidal arsenal for other vector control tools to target persistent malaria transmission.

Counter-Selection of Antimalarial Resistance Polymorphisms by Intermittent Preventive Treatment in Pregnancy

Background: Innovative approaches are needed to limit antimalarial resistance evolution. Understanding the role of intermittent preventive treatment in pregnancy (IPTp) on the selection for resistance and the impact such selection has on pregnancy outcomes can guide future interventions. Methods: Plasmodium falciparum isolates (n = 914) from 2 randomized clinical trials were screened for pfmdr1 copy number variation and pfcrt, pfmdr1, pfdhfr, and pfdhps resistance markers. The trials were conducted between 2010 and 2013 in Benin, Gabon, Kenya, and Mozambique to establish the efficacy of IPTp-mefloquine (MQ) compared with IPTp-sulphadoxine-pyrimethamine (SP) in human immunodeficiency virus (HIV)-uninfected and to IPTp-placebo in HIV-infected women. Results: In HIV-uninfected women, the prevalence of pfcrt mutants, pfdhfr/pfdhps quintuple mutants, and pfmdr1 copy number was similar between women receiving IPT-SP and IPTp-MQ. However, prevalence of pfmdr1 polymorphism 86Y was lower in the IPTp-MQ group than in the IPTp-SP group, and within the IPTp-MQ group it was lower at delivery compared with recruitment. No effect of IPTp-MQ on resistance markers was observed among HIV-infected women. The carriage of resistance markers was not associated with pregnancy outcomes.

Plasmodium vivax Malaria Viewed through the Lens of an Eradicated European Strain.

The protozoan Plasmodium vivax is responsible for 42% of all cases of malaria outside Africa. The parasite is currently largely restricted to tropical and subtropical latitudes in Asia, Oceania, and the Americas. Though, it was historically present in most of Europe before being finally eradicated during the second half of the 20th century. The lack of genomic information on the extinct European lineage has prevented a clear understanding of historical population structuring and past migrations of P. vivax. We used medical microscope slides prepared in 1944 from malaria-affected patients from the Ebro Delta in Spain, one of the last footholds of malaria in Europe, to generate a genome of a European P. vivax strain. Population genetics and phylogenetic analyses placed this strain basal to a cluster including samples from the Americas. This genome allowed us to calibrate a genomic mutation rate for P. vivax, and to estimate the mean age of the last common ancestor between European and American strains to the 15th century. This date points to an introduction of the parasite during the European colonization of the Americas. In addition, we found that some known variants for resistance to antimalarial drugs, including Chloroquine and Sulfadoxine, were already present in this European strain, predating their use. Our results shed light on the evolution of an important human pathogen and illustrate the value of antique medical collections as a resource for retrieving genomic information on pathogens from the past.

Counter-Selection of Antimalarial Resistance Polymorphisms by Intermittent Preventive Treatment in Pregnancy.

BACKGROUND: Innovative approaches are needed to limit antimalarial resistance evolution. Understanding the role of intermittent preventive treatment in pregnancy (IPTp) on the selection for resistance and the impact such selection has on pregnancy outcomes can guide future interventions. METHODS: Plasmodium falciparum isolates (n = 914) from 2 randomized clinical trials were screened for pfmdr1 copy number variation and pfcrt, pfmdr1, pfdhfr, and pfdhps resistance markers. The trials were conducted between 2010 and 2013 in Benin, Gabon, Kenya, and Mozambique to establish the efficacy of IPTp-mefloquine (MQ) compared with IPTp-sulphadoxine-pyrimethamine (SP) in human immunodeficiency virus (HIV)-uninfected and to IPTp-placebo in HIV-infected women. RESULTS: In HIV-uninfected women, the prevalence of pfcrt mutants, pfdhfr/pfdhps quintuple mutants, and pfmdr1 copy number was similar between women receiving IPT-SP and IPTp-MQ. However, prevalence of pfmdr1 polymorphism 86Y was lower in the IPTp-MQ group than in the IPTp-SP group, and within the IPTp-MQ group it was lower at delivery compared with recruitment. No effect of IPTp-MQ on resistance markers was observed among HIV-infected women. The carriage of resistance markers was not associated with pregnancy outcomes. CONCLUSIONS: Selection of wild-type pfmdr1 polymorphism N86 by IPTp-MQ highlights the strong selective pressure IPTp can exert and the opportunity for using negative cross-resistance in drug choice for clinical treatment and IPTp.

Mosquitoes as a feasible sentinel group for anti-malarial resistance surveillance by Next Generation Sequencing of Plasmodium falciparum.

BACKGROUND: Plasmodium falciparum drug resistance surveillance is key to successful disease control and eradication. Contemporary methods that only allow determination of prevalence of resistance are expensive, time consuming and require ethical considerations. A newer method involving Next Generation Sequencing (NGS) permits obtaining frequency of resistance while allowing to detect minority variants in mixed infections. Here, NGS was tested for P. falciparum resistance marker detection in mosquito samples as a feasible and suitable alternative for molecular resistance surveillance. Anopheles funestus were collected in southern Mozambique using CDC light traps and manual collections. DNA was extracted from either whole mosquito, head-thorax and abdomen separately or pools of five mosquitoes. These samples were screened for P. falciparum and if positive for k13, pfcrt, pfmdr1, pfdhps and pfdhfr mutations related to anti-malarial drug resistance with Sanger sequencing and NGS. RESULTS: Among the 846 samples screened for P. falciparum, 122 were positive by 18S ssrDNA qPCR with an infection rate of 23.6%. No mutations were observed for k13 and pfcrt72-76 and almost zero for pfmdr86, but quintuple pfdhfr/pfdhps mutations were near fixation and about half of the isolates contained the pfmdr184F polymorphism. Similar allele frequencies of resistance markers were estimated with NGS in comparison with the prevalence of markers obtained with the gold standard Sanger sequencing. CONCLUSIONS: Pooled deep sequencing of P. falciparum isolates extracted from mosquitoes is a promising, efficient and cost-effective method to quantify allele frequencies at population level which allows to detect known and unknown markers of resistance in single and mixed infections in a timelier manner. Using mosquitoes as sentinel group and focusing on allele frequency opposed to prevalence, permits active surveillance across a more homogeneous geographical range.