Antimalarial Resistance Markers Research Articles

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

BackgroundMass drug administration (MDA) can rapidly reduce the burden of Plasmodium falciparum (Pf). However, concerns remain about its contribution to select for antimalarial drug resistance.MethodsWe 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.ResultsNone 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).ConclusionsThis 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.

Establishing a National Molecular Surveillance Program for the Detection of Plasmodium falciparum Markers of Resistance to Antimalarial Drugs in Haiti.

.Chloroquine remains the first-line treatment for uncomplicated malaria in Haiti, and until recently, sulfadoxine–pyrimethamine was the second-line treatment. A few studies have reported the presence of molecular markers for resistance in Plasmodium falciparum parasites, and in vivo therapeutic efficacy studies (TESs) have been limited. Recognizing the history of antimalarial resistance around the globe and the challenges of implementing TESs in low-endemic areas, the Ministry of Health established a surveillance program to detect molecular markers of antimalarial resistance in Haiti. Sentinel sites were purposefully selected in each of Haiti’s 10 administrative departments; an 11th site was selected in Grand’Anse, the department with the highest number of reported cases. Factors considered for site selection included the number of malaria cases identified, observed skills of laboratory technicians conducting rapid diagnostic tests (RDTs), stock and storage conditions of RDTs, accuracy of data reporting to the national surveillance system, and motivation to participate. Epidemiologic data from 2,437 patients who tested positive for malaria from March 2016 to December 2018 and consented to provide samples for molecular sequencing are presented here. Of these, 936 (38.4%) patients reported self-treatment with any medication since the onset of their illness before diagnosis; overall, 69 (2.8%) patients reported taking an antimalarial. Ten patients (0.4%) reported travel away from their home for at least one night in the month before diagnosis. Establishing a molecular surveillance program for antimalarial drug resistance proved practical and feasible in a resource-limited setting and will provide the evidence needed to make informed treatment policy decisions at the national level.

Molecular surveillance of antimalarial partner drug resistance in sub-Saharan Africa: a spatial-temporal evidence mapping study

Molecular markers for antimalarial drug resistance can be used to rapidly monitor the emergence and spatial distribution of resistance to artemisinin-based combination therapies (ACTs). Little has been done to analyse molecular surveillance efforts or to assess surveillance coverage. This study aimed to develop an evidence map to characterise the spatial-temporal distribution and sampling methodologies of drug resistance surveillance in sub-Saharan Africa, specifically focusing on markers associated with ACT partner drugs. By use of a systematic search, we identified studies that reported data on the following mutations associated with ACT partner drug resistance: pfmdr1 Asn86Tyr, Tyr184Phe, Asp1246Tyr, and copy number variation and pfcrt Lys76Thr, with sample collection occurring in sub-Saharan Africa between Jan 1, 2004, and Dec 31, 2018, corresponding to the uptake of ACTs. For each identified study, we extracted information on its sampling and laboratory methods, author and publication affiliations, years of sampling and of publication, geographic coordinates of the study sites, and prevalence of the partner drug resistance-associated markers. We used linear models to test whether urbanicity, population density, and endemicity were predictors of drug resistance survey sites and linear regressions to identify associations between the number of resistance surveys within a given country and the at-risk malaria population in 2010, the per-capita GDP in 2010, and the mean amount of funding directed to malaria and to determine trends in marker prevalence over time. For country case studies with three or more datapoints, we assessed global spatial autocorrelation using Moran's I. Our search yielded 254 studies encompassing 492 year-specific and location-specific surveys from 35 malaria-endemic countries, the most complete set of molecular partner drug surveillance data to date. We observed a median time lag of 3·1 years (95% CI 1·0-7·7) from final sample acquisition to publication. 22 (49%) of the 44 countries in the study region conducted, on average, one or fewer studies every 3 years. The locations of surveillance sites were positively associated with urbanicity (p<0·0001), and the abundance of country-level data was associated with reported donor funding in 2004-18 (p=0·0011) and local government funding in 2004-09 (p=0·014). Nearly all molecular markers displayed significant regional trends over time and global spatial autocorrelation in space. For selected countries with more widespread coverage of surveillance data, some markers also displayed spatial heterogeneity. In most sub-Saharan countries, molecular data on antimalarial resistance might not be representative of the temporal and geographic heterogeneity of partner drug resistance, and likely do not represent the true spatially dependent distribution of partner drug resistance. Our results highlight several inefficiencies that can be improved upon to develop more accurate data landscapes, including the expansion of sentinel surveillance systems, syndemic usage of research samples, and increased participation in reporting published and unpublished data to centralised platforms.

Assessment of molecular markers of anti-malarial drug resistance among children participating in a therapeutic efficacy study in western Kenya

BackgroundAnti-malarial drug resistance remains a major threat to global malaria control efforts. In Africa, Plasmodium falciparum remains susceptible to artemisinin-based combination therapy (ACT), but the emergence of resistant parasites in multiple countries in Southeast Asia and concerns over emergence and/or spread of resistant parasites in Africa warrants continuous monitoring. The World Health Organization recommends that surveillance for molecular markers of resistance be included within therapeutic efficacy studies (TES). The current study assessed molecular markers associated with resistance to Artemether−lumefantrine (AL) and Dihydroartemisinin−piperaquine (DP) from samples collected from children aged 6–59 months enrolled in a TES conducted in Siaya County, western Kenya from 2016 to 2017.MethodsThree hundred and twenty-three samples collected pre-treatment (day-0) and 110 samples collected at the day of recurrent parasitaemia (up to day 42) were tested for the presence of drug resistance markers in the Pfk13 propeller domain, and the Pfmdr1 and Pfcrt genes by Sanger sequencing. Additionally, the Pfpm2 gene copy number was assessed by real-time polymerase chain reaction.ResultsNo mutations previously associated with artemisinin resistance were detected in the Pfk13 propeller region. However, other non-synonymous mutations in the Pfk13 propeller region were detected. The most common mutation found on day-0 and at day of recurrence in the Pfmdr1 multidrug resistance marker was at codon 184F. Very few mutations were found in the Pfcrt marker (< 5%). Within the DP arm, all recrudescent cases (8 sample pairs) that were tested for Pfpm2 gene copy number had a single gene copy. None of the associations between observed mutations and treatment outcomes were statistically significant.ConclusionThe results indicate absence of Pfk13 mutations associated with parasite resistance to artemisinin in this area and a very high proportion of wild-type parasites for Pfcrt. Although the frequency of Pfmdr1 184F mutations was high in these samples, the association with treatment failure did not reach statistical significance. As the spread of artemisinin-resistant parasites remains a possibility, continued monitoring for molecular markers of ACT resistance is needed to complement clinical data to inform treatment policy in Kenya and other malaria-endemic regions.

Molecular epidemiology of resistance to antimalarial drugs in the Greater Mekong subregion: an observational study

SummaryBackgroundThe Greater Mekong subregion is a recurrent source of antimalarial drug resistance in Plasmodium falciparum malaria. This study aimed to characterise the extent and spread of resistance across this entire region between 2007 and 2018.MethodsP falciparum isolates from Myanmar, Thailand, Laos, and Cambodia were obtained from clinical trials and epidemiological studies done between Jan 1, 2007, and Dec 31, 2018, and were genotyped for molecular markers (pfkelch, pfcrt, pfplasmepsin2, and pfmdr1) of antimalarial drug resistance. Genetic relatedness was assessed using microsatellite and single nucleotide polymorphism typing of flanking sequences around target genes.Findings10 632 isolates were genotyped. A single long pfkelch Cys580Tyr haplotype (from −50 kb to +31·5 kb) conferring artemisinin resistance (PfPailin) now dominates across the eastern Greater Mekong subregion. Piperaquine resistance associated with pfplasmepsin2 gene amplification and mutations in pfcrt downstream of the Lys76Thr chloroquine resistance locus has also developed. On the Thailand–Myanmar border a different pfkelch Cys580Tyr lineage rose to high frequencies before it was eliminated. Elsewhere in Myanmar the Cys580Tyr allele remains widespread at low allele frequencies. Meanwhile a single artemisinin-resistant pfkelch Phe446Ile haplotype has spread across Myanmar. Despite intense use of dihydroartemisinin–piperaquine in Kayin state, eastern Myanmar, both in treatment and mass drug administrations, no selection of piperaquine resistance markers was observed. pfmdr1 amplification, a marker of resistance to mefloquine, remains at low prevalence across the entire region.InterpretationArtemisinin resistance in P falciparum is now prevalent across the Greater Mekong subregion. In the eastern Greater Mekong subregion a multidrug resistant P falciparum lineage (PfPailin) dominates. In Myanmar a long pfkelch Phe446Ile haplotype has spread widely but, by contrast with the eastern Greater Mekong subregion, there is no indication of artemisinin combination therapy (ACT) partner drug resistance from genotyping known markers, and no evidence of spread of ACT resistant P falciparum from the east to the west. There is still a window of opportunity to prevent global spread of ACT resistance.FundingThailand Science Research and Innovation, Initiative 5%, Expertise France, Wellcome Trust.

Evolution of Antimalarial Drug Resistance Markers in the Reservoir of Plasmodium falciparum Infections in the Upper East Region of Ghana.

The majority of Plasmodium falciparum infections, constituting the reservoir in all ages, are asymptomatic in high-transmission settings in Africa. The role of this reservoir in the evolution and spread of drug resistance was explored. Population genetic analyses of the key drug resistance-mediating polymorphisms were analyzed in a cross-sectional survey of asymptomatic P. falciparum infections across all ages in Bongo District, Ghana. Seven years after the policy change to artemisinin-based combination therapies in 2005, the pfcrt K76 and pfmdr1 N86 wild-type alleles have nearly reached fixation and have expanded via soft selective sweeps on multiple genetic backgrounds. By constructing the pfcrt-pfmdr1-pfdhfr-pfdhps multilocus haplotypes, we found that the alleles at these loci were in linkage equilibrium and that multidrug-resistant parasites have not expanded in this reservoir. For pfk13, 32 nonsynonymous mutations were identified; however, none were associated with artemisinin-based combination therapy resistance. The prevalence and selection of alleles/haplotypes by antimalarials were similar to that observed among clinical cases in Ghana, indicating that they do not represent 2 subpopulations with respect to these markers. Thus, the P. falciparum reservoir in all ages can contribute to the maintenance and spread of antimalarial resistance.

Molecular Surveillance of Antimalarial Partner Drug Resistance in Sub-Saharan Africa: A Spatial-Temporal Evidence Mapping Study

Background: Molecular markers for antimalarial drug resistance can be used to rapidly monitor the emergence and spatial distribution of resistance to artemisinin-based combination therapies (ACTs). There has been little effort to analyze molecular surveillance efforts or to standardize data collection and management strategies. This study develops an evidence map to characterize the spatial-temporal distribution and sampling methodologies on partner drug resistance surveillance in sub-Saharan Africa. Methods: Through a systematic search, we identified studies that reported data on the following partner drug resistance-associated mutations: pfmdr1 N86Y, Y184F, D1246Y, or pfcrt K76T, with sample collection occurring in sub-Saharan Africa from 2004–2018, corresponding to the uptake of ACTs. We examined trends associated with surveillance and transmission of partner drug resistance. Findings: Our search yielded a total of 254 studies encompassing 501 year- and location-specific surveys from 35 malaria endemic countries, the most complete set of molecular partner drug surveillance data to date. We observed an average time lag of 3·2 years from sample acquisition to publication. Over half of the countries in the study region conducted only a single survey, on average, every 3–4 years. The amount of surveillance data per country was positively associated with reported donor funding. Drug resistance survey sites were 5.6 times as likely to be located in urban areas than malaria prevalence surveys. In countries where data was acquired in a more spatially representative pattern, heterogeneity in molecular marker prevalence was evident across time and space. Interpretation: In the majority of sub-Saharan countries, molecular data on antimalarial resistance remains temporally and geographically limited, likely failing to represent the true heterogeneous distribution of partner drug resistance. We discuss several inefficiencies in surveillance efforts that can be improved upon to develop more accurate data landscapes. Funding Statement: HYE was supported by the NIH Ruth L. Kirschstein National Research Service Award (F31AI150168). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Not required.

Prevalence of Molecular Markers of Antimalarial Drug Resistance across Altitudinal Transmission Zones in Highland Western Uganda.

We explored spatial variation in the prevalence of established molecular markers of antimalarial resistance across a geographically diverse, highland region of western Uganda. We identified Plasmodium falciparum CQ resistance transporter 76T mutations in all pools, but there was no evidence of spatial differences across village-based strata defined by either altitude or river valley. In contrast, we identified a significant inverse association between altitude and the prevalence of Plasmodium falciparum multidrug resistance 1 mutations with the largest proportion of Y184F mutations observed in the low-elevation, high-transmission villages. These results demonstrate the substantial heterogeneity in resistance markers observed across geographic settings, even at relatively small scales, but highlight the complex nature of these ecological relationships.

Genomic analysis informs malaria evolution.

Updated analysis of Plasmodium falciparum malaria reveals markers of antimalarial resistance

Absence of kelch13 artemisinin resistance markers but strong selection for lumefantrine-tolerance molecular markers following 18\xa0years of artemisinin-based combination therapy use in Mpumalanga Province, South Africa (2001\u20132018)

BackgroundThe ability of Plasmodium falciparum parasites to develop resistance to widely used anti-malarials threatens malaria control and elimination efforts. Regular drug efficacy monitoring is essential for ensuring effective treatment policies. In low transmission settings where therapeutic efficacy studies are often not feasible, routine surveillance for molecular markers associated with anti-malarial resistance provides an alternative for the early detection of emerging resistance. Such a longitudinal survey of changes in the prevalence of selected molecular markers of resistance was conducted in the malaria-endemic regions of Mpumalanga Province, South Africa, where malaria elimination at a district-level is being pursued.MethodsMolecular analyses to determine the prevalence of alleles associated with resistance to lumefantrine (mdr86N, crt76K and mdr1 copy number variation) and sulfadoxine–pyrimethamine (dhfr triple, dhps double, SP quintuple) were conducted between 2001 and 2018, while artemisinin resistance markers (kelch13 mutations) were assessed only in 2018.ResultsParasite DNA was successfully amplified from 1667/2393 (70%) of malaria-positive rapid diagnostic tests routinely collected at primary health care facilities. No artemisinin resistance-associated kelch13 mutations nor amplification of the mdr1 gene copy number associated with lumefantrine resistance were observed. However, prevalence of both the mdr86N and crt76K alleles increased markedly over the study period, with all isolates collected in 2018 carrying these markers. SP quintuple mutation prevalence increased steadily from 14% in 2001 to 96% in 2018. Mixed alleles at any of the codons assessed were rare by 2018.ConclusionNo kelch13 mutations confirmed or suspected to be associated with artemisinin resistance were identified in 2018. Although parasites carrying the mdr86N and crt76K alleles associated with reduced lumefantrine susceptibility were strongly selected for over the study period, nearing fixation by 2018, the marker for lumefantrine resistance, namely increased mdr1 copy number, was not observed in this study. The increase in mdr86N and crt76K allele prevalence together with intense regional artemether–lumefantrine drug pressure, raises concern regarding the sustained artemether–lumefantrine efficacy. Regular, rigorous anti-malarial resistance marker surveillance across all three South African malaria-endemic provinces to inform case management is recommended.

Molecular detection of drug resistant malaria in Southern Thailand

BackgroundDrug resistance within the major malaria parasites Plasmodium vivax and Plasmodium falciparum threatens malaria control and elimination in Southeast Asia. Plasmodium vivax first-line treatment drug is chloroquine together with primaquine, and the first-line treatment for P. falciparum malaria is artemisinin in combination with a partner drug. Plasmodium vivax and P. falciparum parasites resistant to their respective first-line therapies are now found within Southeast Asia. The resistance perimeters may include high transmission regions of Southern Thailand which are underrepresented in surveillance efforts.MethodsThis study investigated blood samples from malaria centres in Southern Thailand. Genetic loci associated with drug resistance were amplified and sequenced. Drug resistance associated genes Pvmdr1, Pvcrt-o, Pvdhfr, and Pvdhps were characterized for 145 cases of P. vivax malaria, as well as the artemisinin resistance-associated Pfkelch13 gene from 91 cases of P. falciparum malaria.ResultsPlasmodium vivax samples from Southern Thai provinces showed numerous chloroquine and antifolate resistance-associated mutations, including SNP and Pvcrt-oK10-insertion combinations suggestive of chloroquine resistant P. vivax phenotypes. A high proportion of the C580Y coding mutation (conferring artemisinin resistance) was detected in P. falciparum samples originating from Ranong and Yala (where the mutation was previously unreported).ConclusionsThe results demonstrate a risk of chloroquine and antifolate resistant P. vivax phenotypes in Southern Thailand, and artemisinin resistant P. falciparum observed as far south as the Thai–Malaysian border region. Ongoing surveillance of antimalarial drug resistance markers is called for in Southern Thailand to inform case management.

Molecular Surveillance of Pfkelch13 and Pfmdr1 Mutations in Plasmodium falciparum Isolates from Southern Thailand.

Artemisinin-based combination therapy (ACT) resistance is widespread throughout the Greater Mekong Subregion. This raises concern over the antimalarial treatment in Thailand since it shares borders with Cambodia, Laos, and Myanmar where high ACT failure rates were reported. It is crucial to have information about the spread of ACT resistance for efficient planning and treatment. This study was to identify the molecular markers for antimalarial drug resistance: Pfkelch13 and Pfmdr1 mutations from 5 provinces of southern Thailand, from 2012 to 2017, of which 2 provinces on the Thai- Myanmar border (Chumphon and Ranong), one on Thai-Malaysia border (Yala) and 2 from non-border provinces (Phang Nga and Surat Thani). The results showed that C580Y mutation of Pfkelch13 was found mainly in the province on the Thai-Myanmar border. No mutations in the PfKelch13 gene were found in Surat Thani and Yala. The Pfmdr1 gene isolated from the Thai-Malaysia border was a different pattern from those found in other areas (100% N86Y) whereas wild type strain was present in Phang Nga. Our study indicated that the molecular markers of artemisinin resistance were spread in the provinces bordering along the Thai-Myanmar, and the pattern of Pfmdr1 mutations from the areas along the international border of Thailand differed from those of the non-border provinces. The information of the molecular markers from this study highlighted the recent spread of artemisinin resistant parasites from the endemic area, and the data will be useful for optimizing antimalarial treatment based on regional differences.

Proof of concept: used malaria rapid diagnostic tests applied for parallel sequencing for surveillance of molecular markers of anti-malarial resistance in Bissau, Guinea-Bissau during 2014\u20132017

BackgroundLarge-scale surveillance of molecular markers of anti-malarial drug resistance is an attractive method of resistance monitoring, to complement therapeutic efficacy studies in settings where the latter are logistically challenging.MethodsBetween 2014 and 2017, this study sampled malaria rapid diagnostic tests (RDTs), used in routine clinical care, from two health centres in Bissau, Guinea-Bissau. In order to obtain epidemiological insights, RDTs were collected together with patient data on age and sex. A subset of positive RDTs from one of the two sites (n = 2184) were tested for Plasmodium DNA content. Those testing positive for Plasmodium DNA by PCR (n = 1390) were used for library preparation, custom designed dual indexing and next generation Miseq targeted sequencing of Plasmodium falciparum genes pfcrt, pfmdr1, pfdhfr, pfdhps and pfk13.ResultsThe study found a high frequency of the pfmdr1 codon 86N at 88–97%, a significant decrease of the pfcrt wildtype CVMNK haplotype and elevated levels of the pfdhfr/pfdhps quadruple mutant ranging from 33 to 51% between 2014 and 2017. No polymorphisms indicating artemisinin tolerance were discovered. The demographic data indicate a large proportion of young adults (66%, interquartile range 11–28 years) presenting with P. falciparum infections. While a total of 5532 gene fragments were successfully analysed on a single Illumina Miseq flow cell, PCR-positivity from the library preparation varied considerably from 13 to 87% for different amplicons. Furthermore, pre-screening of samples for Plasmodium DNA content proved necessary prior to library preparation.ConclusionsThis study serves as a proof of concept for using leftover clinical material (used RDTs) for large-scale molecular surveillance, encompassing the inherent complications regarding to methodology and analysis when doing so. Factors such as RDT storage prior to DNA extraction and parasitaemia of the infection are likely to have an effect on whether or not parasite DNA can be successfully analysed, and are considered part of the reason the data yield is suboptimal. However, given the necessity of molecular surveillance of anti-malarial resistance in settings where poor infrastructure, poor economy, lack of educated staff and even surges of political instability remain major obstacles to performing clinical studies, obtaining the necessary data from used RDTs, despite suboptimal output, becomes a feasible, affordable and hence a justifiable method.

No antimalarial resistance of Plasmodium falciparum varian West Indonesia using molecular markers screening

Artemisin-based combination therapy (ACT) were widely used in Indonesia for the treatment of Malaria Plasmodium falciparum. Previous study in East Indonesia, located in Irian showed no prevalence of resistance, suggesting the treatment still effective in the area. This study aimed to provide baseline data of antimalarial drug resistance markers on P. falciparum isolates in Lampung West Indonesia. Mutation on genes associated with ACT (PfRBP9) were assessed by PCR amplification. Mutation on the marker related with artemisin resistance were determined by DNA sequencing technique. Isolates of 14 samples revealed no mutation in the D56V codon which is usually confined. This study has demonstrated a low prevalence resistance allele in the study area. Continuous surveillance of antimalarial drug efficacy is recommended and the findings provide information for stakeholders ensuring proper policy controlling malaria.

Detection of Single-Nucleotide Polymorphism Markers of Antimalarial Drug Resistance Directly from Whole Blood

Monitoring of antimalarial resistance is important to prevent its further spread, but the available options for assessing resistance are less than ideal for field settings. Although molecular detection is perhaps the most efficient method, it is also the most complex because it requires DNA extraction and PCR instrumentation. To develop a more deployable approach, we designed new probes, which, when used in combination with an inhibitor-tolerant Taq polymerase, enable single-nucleotide polymorphism genotyping directly from whole blood. The probes feature two strategic design elements: locked nucleic acids to enhance specificity and the reporter dyes Cy5 and TEX615, which have less optical overlap with the blood absorbance spectra than other commonly used dyes. Probe performance was validated on a traditional laboratory-based instrument and then further tested on a field-deployable Adaptive PCR instrument to develop a point-of-care platform appropriate for use in malaria settings. The probes discriminated between wild-type Plasmodium falciparum and the chloroquine-resistant CRT PF3D7_0709000:c.227A>C (p.Lys76Thr) mutant in the presence of 2% blood. Additionally, in allelic discrimination plots with the new probes, samples clustered more closely to their respective axes compared with samples using minor groove binder probes with 6-FAM and VIC reporter dyes. Our strategy greatly simplifies single-nucleotide polymorphism detection and provides a more accessible alternative for antimalarial resistance surveillance in the field.

Malaria Surveillance - United States, 2016.

Malaria Surveillance - United States, 2016.

An online mapping database of molecular markers of drug resistance in Plasmodium falciparum: the ACT Partner Drug Molecular Surveyor

BackgroundPrior to this project, only a handful of online visualizations existed for exploring the published literature on molecular markers of antimalarial drug resistance, and none specifically for the markers associated with Plasmodium falciparum resistance to the partner drugs in artemisinin-based combination therapy (ACT). Molecular information is collected in studies with different designs, using a variety of molecular methodologies and data analysis strategies, making it difficult to compare across studies. The purpose of this project was to develop a free online tool, which visualizes the widely published data on molecular markers of antimalarial drug resistance, starting with the two genes pfcrt and pfmdr-1, associated with resistance to the three most common partner drugs; amodiaquine, lumefantrine and mefloquine.MethodsA literature review was conducted, and a standardized method was used to extract data from publications, and critical decisions on visualization were made. A global geospatial database was developed of specific pfmdr1 and pfcrt single nucleotide polymorphisms and pfmdr1 copy number variation. An informatics framework was developed that allowed flexibility in development of the tool over time and efficient adaptation to different source data.ResultsThe database discussed in this paper has pfmdr1 and pfcrt marker prevalence information, from 579 geographic sites in 76 different countries, including results from over 86,000 samples from 456 articles published January 2001–May 2017. The ACT Partner Drugs Molecular Surveyor was launched by the WorldWide Antimalarial Resistance Network (WWARN) in March 2015 and it has attracted over 3000 unique visitors since then. Presented here is a demonstration of how the Surveyor database can be explored to monitor local, temporal changes in the prevalence of molecular markers. Here publications up to May 2017 were included, however the online ACT partner drug Molecular Surveyor is continuously updated with new data and relevant markers.ConclusionsThe WWARN ACT Partner Drugs Molecular Surveyor summarizes data on resistance markers in the pfmdr1 and pfcrt genes. The database is fully accessible, providing users with a rich resource to explore and analyze, and thus utilize a centralized, standardized database for different purposes. This open-source software framework can be adapted to other data, as demonstrated by the subsequent launch of the Artemisinin Molecular Surveyor and the Vivax Surveyor.

Prevalence of molecular markers of antimalarial drug resistance across altitudinal transmission zones in Highland Western Uganda

Prevalence of molecular markers of antimalarial drug resistance across altitudinal transmission zones in Highland Western Uganda

Diagnostic accuracy of molecular methods for detecting markers of antimalarial drug resistance in clinical samples of Plasmodium falciparum: protocol for an update to a systematic review and meta-analysis

BackgroundEach year, infection with Plasmodium causes millions of clinical cases of malaria and hundreds of thousands of deaths. Resistance to different antimalarial medications continues to develop and spread, threatening effective prophylaxis and treatment. Surveillance of resistance is required to inform health policy and preserve effective antimalarial drugs; molecular methods can be used to surveil likely parasite resistances. However, there is no consensus on the most accurate molecular methods, and large variation exists in practice. The objective of this update to this systematic review is to improve and update identification of the sensitivity and specificity of each molecular method for detecting selected antimalarial drug resistance markers.MethodsWe will include diagnostic accuracy studies that compare at least two of any molecular methods to examine blood samples from patients diagnosed with, or suspected of having malaria, to detect at least one selected marker of antimalarial drug resistance. We will search PubMed, EMBASE, BIOSIS, and Web of Science from 2000 to present. Two reviewers will independently screen all results, extract data, consider applicability, and evaluate the methodological quality of included studies using QUADAS-2. We will carry out a meta-analysis and use statistical methods to compare results from homogenous studies. We will use narrative to synthesise and compare results of heterogeneous studies.DiscussionThis review will help to identify sub-optimal molecular methods for antimalarial marker detection which may be discontinued and identify more sensitive and specific methods which may be adopted. More sensitive and specific detection of drug resistance can be used to improve the breadth and accuracy of surveillance. This would enable the identification of previously undiscovered areas of antimalarial resistances and susceptibilities, improve the precision of estimates of the prevalence of resistances, and improve our ability to detect smaller changes in these patterns. Higher-quality evidence generated by more accurate and detailed surveillance can be used to inform guidelines on the use of antimalarial drugs, leading to better outcomes for more patients.Systematic review registrationThis systematic review protocol was registered with PROSPERO on 22 November 2017 (registration number CRD42017082101).

Prevalence of molecular markers of sulfadoxine\u2013pyrimethamine and artemisinin resistance in Plasmodium falciparum from Pakistan

BackgroundIn Pakistan, artesunate (AS) in combination with sulfadoxine–pyrimethamine (SP) is the recommended treatment for uncomplicated Plasmodium falciparum malaria. Monitoring molecular markers of anti-malarial drug resistance is crucial for early detection and containment of parasite resistance to treatment. Currently, no data are available on molecular markers of artemisinin resistance (K13 mutations) in P. falciparum isolates from Pakistan. In this study, the prevalence of mutations associated with SP and artemisinin resistance was estimated in different regions of Pakistan.MethodsA total of 845 blood samples that were positive for malaria parasites by microscopy or rapid diagnostic test were collected from January 2016 to February 2017 from 16 different sites in Pakistan. Of these samples, 300 were positive for P. falciparum by PCR. Polymorphisms in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes were identified by pyrosequencing while polymorphisms in the propeller domain of the pfk13 gene were identified by Sanger sequencing.ResultsThe prevalence of the PfDHFR 108N and 59R mutations was 100% and 98.8%, respectively, while the prevalence of PfDHFR 50R and 51I mutations was 8.6%. No mutation was observed at PfDHFR position 164. In PfDHPS, the prevalence of mutations at positions 436, 437, and 613 was 9.9%, 45.2%, and 0.4%, respectively. No mutations were found at PfDHPS positions 540 and 581. The prevalence of double PfDHFR mutants (59R + 108N) ranged from 93.8% to 100%, while the prevalence of parasites having the PfDHFR 59R + 108N mutations in addition to the PfDHPS 437G mutation ranged from 9.5% to 83.3% across different regions of Pakistan. Nine non-synonymous and four synonymous mutations were observed in the PfK13 propeller domain, none of which correspond to mutations validated to contribute to artemisinin resistance.ConclusionThe absence of the highly resistant PfDHFR/PfDHPS quintuple mutant parasites and the lack of PfK13 mutations associated with artemisinin resistance is consistent with AS + SP being effective in Pakistan.