Surveillance Of Antimalarial Resistance Research Articles

Prevalence of Plasmodium falciparum delayed clearance associated polymorphisms in adaptor protein complex 2 mu subunit (pfap2mu) and ubiquitin specific protease 1 (pfubp1) genes in Ghanaian isolates

BackgroundPlasmodium falciparum delayed clearance with the use of artemisinin-based combination therapy (ACTs) has been reported in some African countries. Single nucleotide polymorphisms (SNPs) in two genes, P. falciparum adaptor protein complex 2 mu subunit (pfap2mu) and ubiquitin specific protease 1 (pfubp1), have been linked to delayed clearance with ACT use in Kenya and recurrent imported malaria in Britain. With over 12 years of ACT use in Ghana, this study investigated the prevalence of SNPs in the pfap2mu and pfubp1 in Ghanaian clinical P. falciparum isolates to provide baseline data for antimalarial drug resistance surveillance in the country.MethodsFilter paper blood blots collected in 2015–2016 from children aged below 9 years presenting with uncomplicated malaria at hospitals in three sentinel sites Begoro, Cape Coast and Navrongo were used. Parasite DNA was extracted from 120 samples followed by nested polymerase chain reaction (nPCR). Sanger sequencing was performed to detect and identify SNPs in pfap2mu and pfubp1 genes.ResultsIn all, 11.1% (9/81) of the isolates carried the wildtype genotypes for both genes. A total of 164 pfap2mu mutations were detected in 67 isolates whilst 271 pfubp1 mutations were observed in 72 isolates. The majority of the mutations were non-synonymous (NS): 78% (128/164) for pfap2mu and 92.3% (250/271) for pfubp1. Five unique samples had a total of 215 pfap2mu SNPs, ranging between 15 and 63 SNPs per sample. Genotypes reportedly associated with ART resistance detected in this study included pfap2mu S160N (7.4%, 6/81) and pfubp1 E1528D (7.4%, 6/81) as well as D1525E (4.9%, 4/81). There was no significant difference in the prevalence of the SNPs between the three ecologically distinct study sites (pfap2mu: χ2 = 6.905, df = 2, P = 0.546; pfubp1: χ2 = 4.883, df = 2, P = 0.769).ConclusionsThe detection of pfap2mu and pfubp1 genotypes associated with ACT delayed parasite clearance is evidence of gradual nascent emergence of resistance in Ghana. The results will serve as baseline data for surveillance and the selection of the genotypes with drug pressure over time. The pfap2mu S160N, pfubp1 E1528D and D1525E must be monitored in Ghanaian isolates in ACT susceptibility studies, especially when cure rates of ACTs, particularly AL, is less than 100%.

Country-Wide Surveillance of Molecular Markers of Antimalarial Drug Resistance in Senegal by Use of Positive Malaria Rapid Diagnostic Tests.

.In Senegal, antimalarial drugs used in treatment and prevention of malaria are one of the main reasons for the current success in controlling malaria. However, the successful control of malaria is highly dependent on continued effectiveness of these drugs which may be compromised by the spread of drug resistance. Therefore, surveillance of drug resistance in the malaria parasites is essential. The objective of this pilot study was to test the feasibility of routinely sampled malaria rapid diagnostic tests (RDTs) at a national scale to assess the temporal changes in the molecular profiles of antimalarial drug resistance markers of Plasmodium falciparum parasites. Overall, 9,549 positive malaria RDTs were collected from 14 health facilities across the country. A limited random set of RDTs were analyzed regarding Pfcrt gene polymorphisms at codon 72–76. Overall, a high but varied prevalence (> 50%) of the wild-type CVMNK haplotype was observed including a higher CVMNK prevalence in the northern part (75%) compared with the southern part of the country (59%). With caution, the study provides a proof of concept that reuse of discarded P. falciparum positive RDTs can be applied in large-scale surveillance of antimalarial drug resistance.

Polymorphisms of Plasmodium falciparum k13-propeller gene among migrant workers returning to Henan Province, China from Africa

BackgroundHenan Province has been in the malaria elimination stage, with all reports of the disease being imported since 2012 and over 90% coming from Africa. Surveillance and population studies are essential for the early detection and subsequent prevention of the spread of drug resistance. The K13-propeller gene was recently identified as a proposed molecular marker of artemisinin (ART) resistance. In this study, we detected mutations of the K13-propeller gene in samples taken from imported malaria cases in Henan Province from 2012 to 2015.MethodsThere were 483 samples that were obtained from Plasmodium falciparum–infected malaria migrant workers who returned to Henan Province from Africa between 2012 and 2015. The single nucleotide polymorphisms in the K13-propeller gene were assessed by nested PCR with DNA sequencing. Frequency and geographic difference of K13-propeller gene mutant types were analyzed.ResultsOf 483 patients, 476 were cured and 7 died. There were no K13-propeller mutations in the blood samples from the 7 patients who died, but there were 23 different genotypes of the K13-propeller that were observed in 24 (4.97%) of the samples. C580Y, which was the predominant one in the resistance of ART, was not detected in the samples, but R539T and P574L which have also been associated with ART resistance, were observed in two samples from Angola and Equatorial Guinea. No mutations were detected in 11 samples from North Africa. The frequency of the K13-propeller was 6.50% (8/123) in Central Africa, followed by East Africa (1/19, 5.26%), West Africa (9/198, 4.55%) and South Africa (6/132, 4.55%). There was no significant difference among these four areas (P = 0.795).ConclusionR539T and P574L were found in migrant workers who traveled from Africa to Henan Province, although the frequency of the K13-propeller mutants was low. These data may enrich the molecular surveillance of antimalarial resistance and will be helpful for developing and updating the antimalarial policy in Henan Province.

High throughput resistance profiling of Plasmodium falciparum infections based on custom dual indexing and Illumina next generation sequencing-technology

Genetic polymorphisms in P. falciparum can be used to indicate the parasite’s susceptibility to antimalarial drugs as well as its geographical origin. Both of these factors are key to monitoring development and spread of antimalarial drug resistance. In this study, we combine multiplex PCR, custom designed dual indexing and Miseq sequencing for high throughput SNP-profiling of 457 malaria infections from Guinea-Bissau, at the cost of 10 USD per sample. By amplifying and sequencing 15 genetic fragments, we cover 20 resistance-conferring SNPs occurring in pfcrt, pfmdr1, pfdhfr, pfdhps, as well as the entire length of pfK13, and the mitochondrial barcode for parasite origin. SNPs of interest were sequenced with an average depth of 2,043 reads, and bases were called for the various SNP-positions with a p-value below 0.05, for 89.8–100% of samples. The SNP data indicates that artemisinin resistance-conferring SNPs in pfK13 are absent from the studied area of Guinea-Bissau, while the pfmdr1 86 N allele is found at a high prevalence. The mitochondrial barcodes are unanimous and accommodate a West African origin of the parasites. With this method, very reliable high throughput surveillance of antimalarial drug resistance becomes more affordable than ever before.

Assessment of copy number variation in genes related to drug resistance in Plasmodium vivax and Plasmodium falciparum isolates from the Brazilian Amazon and a systematic review of the literature

BackgroundParasite resistance to anti-malarials represents a great obstacle for malaria elimination. The majority of studies have investigated the association between single-nucleotide polymorphisms (SNPs) and drug resistance; however, it is becoming clear that the copy number variation (CNV) is also associated with this parasite phenotype. To provide a baseline for molecular surveillance of anti-malarial drug resistance in the Brazilian Amazon, the present study characterized the genetic profile of both markers in the most common genes associated with drug resistance in Plasmodium falciparum and Plasmodium vivax isolates. Additionally, these data were compared to data published elsewhere applying a systematic review of the literature published over a 20-year time period.MethodsThe genomic DNA of 67 patients infected by P. falciparum and P. vivax from three Brazilian States was obtained between 2002 and 2012. CNV in P. falciparum multidrug resistance gene-1 (pfmdr1), GTP cyclohydrolase 1 (pfgch1) and P. vivax multidrug resistance gene-1 (pvmdr1) were assessed by real-time PCR assays. SNPs in the pfmdr1 and pfcrt genes were assessed by PCR–RFLP. A literature search for studies that analysed CNP in the same genes of P. falciparum and P. vivax was conducted between May 2014 and March 2017 across four databases.ResultsAll analysed samples of P. falciparum carried only one copy of pfmdr1 or pfgch1. Although the pfcrt K76T polymorphism, a determinant of CQ resistance, was present in all samples genotyped, the pfmdr1 N86Y was absent. For P. vivax isolates, an amplification rate of 20% was found for the pvmdr1 gene. The results of the study are in agreement with the low amplification rates for pfmdr1 gene evidenced in the Americas and Africa, while higher rates have been described in Southeast Asia. For P. vivax, very low rates of amplification for pvmdr1 have been described worldwide, with exceptions in French Guiana, Cambodia, Thailand and Brazil.ConclusionsThe present study was the first to evaluate gch1 CNV in P. falciparum isolates from Brazil, showing an absence of amplification of this gene more than 20 years after the withdrawal of the Brazilian antifolates therapeutic scheme. Furthermore, the rate of pvmdr1 amplification was significantly higher than that previously reported for isolates circulating in Northern Brazil.

In Vitro and Molecular Surveillance for Antimalarial Drug Resistance in Plasmodium falciparum Parasites in Western Kenya Reveals Sustained Artemisinin Sensitivity and Increased Chloroquine Sensitivity.

Malaria control is hindered by the evolution and spread of resistance to antimalarials, necessitating multiple changes to drug policies over time. A comprehensive antimalarial drug resistance surveillance program is vital for detecting the potential emergence of resistance to antimalarials, including current artemisinin-based combination therapies. An antimalarial drug resistance surveillance study involving 203 Plasmodium falciparum malaria-positive children was conducted in western Kenya between 2010 and 2013. Specimens from enrolled children were analyzed in vitro for sensitivity to chloroquine (CQ), amodiaquine (AQ), mefloquine (MQ), lumefantrine, and artemisinin derivatives (artesunate and dihydroartemisinin) and for drug resistance allele polymorphisms in P. falciparum crt (Pfcrt), Pfmdr-1, and the K13 propeller domain (K13). We observed a significant increase in the proportion of samples with the Pfcrt wild-type (CVMNK) genotype, from 61.2% in 2010 to 93.0% in 2013 (P < 0.0001), and higher proportions of parasites with elevated sensitivity to CQ in vitro. The majority of isolates harbored the wild-type N allele in Pfmdr-1 codon 86 (93.5%), with only 7 (3.50%) samples with the N86Y mutant allele (the mutant nucleotide is underlined). Likewise, most isolates harbored the wild-type Pfmdr-1 D1246 allele (79.8%), with only 12 (6.38%) specimens with the D1246Y mutant allele and 26 (13.8%) with mixed alleles. All the samples had a single copy of the Pfmdr-1 gene (mean of 0.907 ± 0.141 copies). None of the sequenced parasites had mutations in K13. Our results suggest that artemisinin is likely to remain highly efficacious and that CQ sensitivity appears to be on the rise in western Kenya.

Increasing prevalence of a novel triple-mutant dihydropteroate synthase genotype in Plasmodium falciparum in western Kenya.

The molecular basis of sulfadoxine-pyrimethamine (SP) resistance lies in a combination of single-nucleotide polymorphisms (SNPs) in two genes coding for Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and P. falciparum dihydropteroate synthase (Pfdhps), targeted by pyrimethamine and sulfadoxine, respectively. The continued use of SP for intermittent preventive treatment in pregnant women in many African countries, despite SP's discontinuation as a first-line antimalarial treatment option due to high levels of drug resistance, may further increase the prevalence of SP-resistant parasites and/or lead to the selection of new mutations. An antimalarial drug resistance surveillance study was conducted in western Kenya between 2010 and 2013. A total of 203 clinical samples from children with uncomplicated malaria were genotyped for SNPs associated with SP resistance. The prevalence of the triple-mutant Pfdhfr C50 I51R59N108: I164 genotype and the double-mutant Pfdhps S436 G437E540: A581A613 genotype was high. Two triple-mutant Pfdhps genotypes, S436 G437E540G581: A613 and H436G437E540: A581A613, were found, with the latter thus far being uniquely found in western Kenya. The prevalence of the S436 G437E540G581: A613 genotype was low. However, a steady increase in the prevalence of the Pfdhps triple-mutant H436G437E540: A581A613 genotype has been observed since its appearance in early 2000. Isolates with these genotypes shared substantial microsatellite haplotypes with the most common double-mutant allele, suggesting that this triple-mutant allele may have evolved locally. Overall, these findings show that the prevalence of the H436G437E540: A581A613 triple mutant may be increasing in this population and could compromise the efficacy of SP for intermittent preventive treatment in pregnant women if it increases the resistance threshold further.

Surveillance of antimalarial drug resistance in China in the 1980s-1990s.

Since the successful preparation of the microplates and the medium for field application, the resistance degree and its geographical distribution of chloroquine-resistant Plasmodium falciparum, the fluctuation of the resistance degree of P. falciparum to chloroquine, and the sensitivity of the parasite to commonly used antimalarial drugs were investigated between 1980 and 2003 by the in vitro microtest and the in vivo four-week test recommended by the World Health Organization (WHO). The results indicated that chloroquine-resistant falciparum malaria was present in all eight provinces/autonomous regions endemic for falciparum malaria in China, and the resistance was high and widely distributed in the Hainan and Yunnan provinces. When the use of chloroquine was stopped or administered in a decreased quanity, the drug resistance gradually decreased. In Hainan and Yunnan, P. falciparum was still highly resistant to chloroquine, amodiaquine and piperaquine, and sensitive to pyronaridine and artemisinin derivatives, but the sensitivity was gradually reduced. Based on these results, principles and therapeutic regimens for antimalarial drug use in China were formulated, the use of the antimalarials which had already developed resistance was stopped or reduced, and recommendations to use artemisinin derivatives or compound pyronaridine to promote a rational use of antimalarials and strengthen malaria control were made. The results showed that malaria incidence had declined, and endemic areas of falciparum malaria have been gradually reducing since the mid-1980s.

Efficacy of Mobile Phone Short Message Service (SMS) Reminders on Malaria Treatment Adherence and Day 3 Post-Treatment Reviews (SMS-RES-MAL) in Kenya: A Study Protocol.

BackgroundMobile phone short messaging services (SMS) have been investigated in health information reporting, provider performance, drug and diagnostic stock management and patient adherence to treatment for chronic diseases. However, their potential role in improving patients’ adherence to malaria treatment and day 3 post treatment reviews remains unclear.Methods/DesignA “proof of concept” open label randomised controlled trial will be conducted at four sites in Western Kenya. Principal research questions are: 1) Can mobile phone SMS reminders improve patient adherence to malaria treatment? 2) Can mobile phone SMS reminders improve day 3 post treatment reviews? Eligible caregivers (n=1000 per arm) of children under five years old with uncomplicated malaria will be randomly assigned (one to one) to: a) the current standard of care (provider counselling and health education); and b) the current standard of care plus SMS reminders. Within each arm, caregivers will be further randomized to three different categories. In categories 1 and 2, 300 caregivers per arm per category will be visited at home on day 1 and 2 of follow up respectively, to measure appropriate timing and adherence of the second Artemether-Lumefantrine (AL) dose and doses 3 and 4. Further, caregivers in categories 1 and 2 will be required to come to the health facility for the day 3 post treatment reviews. Finally, in category 3, 400 caregivers per arm will be visited at home on day 3 to measure adherence for the full AL course. Each category will be visited at home only once to avoid biases in the measures of adherence as a result of home consultations. Primary outcomes will be adherence to the full AL course (category 3), as well as, the proportion of patients reporting back for day 3 post treatment reviews (categories 1 and 2). The primary analysis will be intention-to-treat. Costs of the intervention will be measured over the period of the intervention, and a cost-effectiveness ratio will be estimated.DiscussionIf successful, evidence from this trial could improve malaria treatment adherence and offer pragmatic approaches for antimalarial drug resistance surveillance and risk mitigation in Africa.Current Controlled TrialsISRCTN39512726

Surveillance of travellers: an additional tool for tracking antimalarial drug resistance in endemic countries.

IntroductionThere are growing concerns about the emergence of resistance to artemisinin-based combination therapies (ACTs). Since the widespread adoption of ACTs, there has been a decrease in the systematic surveillance of antimalarial drug resistance in many malaria-endemic countries. The aim of this work was to test whether data on travellers returning from Africa with malaria could serve as an additional surveillance system of local information sources for the emergence of drug resistance in endemic-countries.MethodologyData were collected from travellers with symptomatic Plasmodium falciparum malaria returning from Senegal (n = 1,993), Mali (n = 2,372), Cote d’Ivoire (n = 4,778) or Cameroon (n = 3,272) and recorded in the French Malaria Reference Centre during the period 1996–2011. Temporal trends of the proportion of parasite isolates that carried the mutant genotype, pfcrt 76T, a marker of resistance to chloroquine (CQ) and pfdhfr 108N, a marker of resistance to pyrimethamine, were compared for travellers and within-country surveys that were identified through a literature review in PubMed. The in vitro response to CQ was also compared between these two groups for parasites from Senegal.ResultsThe trends in the proportion of parasites that carried pfcrt 76T, and pfdhfr 108N, were compared for parasites from travellers and patients within-country using the slopes of the curves over time; no significant differences in the trends were found for any of the 4 countries. These results were supported by in vitro analysis of parasites from the field in Senegal and travellers returning to France, where the trends were also not significantly different.ConclusionThe results have not shown different trends in resistance between parasites derived from travellers or from parasites within-country. This work highlights the value of an international database of drug responses in travellers as an additional tool to assess the emergence of drug resistance in endemic areas where information is limited.

A Bayesian model for estimating with-in host P. falciparum haplotype frequencies

The necessity for effective surveillance of antimalarial resistance is all the more reinforced by the recent emergence of resistance to artemisinin derivatives. However, the accurate detection of resistant haplotypes from patient samples that are infected with multiple parasite clones is nontrivial. When the multiplicity of infection exceeds one, the allele sequences of the constituent clones at genotyped loci are convoluted. Nevertheless, statistical methods can be used to reconstruct the allele sequences, infer distinct haplotypes and ascertain their frequencies from prevalence data. We have developed a Bayesian model for estimating haplotype frequencies. The model estimates haplotype frequencies based on prevalence data collected for one or more molecular markers known to been associated with antimalarial resistance. Prior knowledge of the MOI is not required. The model uses a Metropolis-Hasting Monte Carlo Markov chain algorithm to explore the different possible haplotype compositions that are compatible with the sample observed, and calculates the likelihood of the data given the current estimate of the haplotype frequencies. For each haplotype the model returns a distribution of frequency estimates from which the mean and its credible interval are derived. For each sample the model returns a distribution, over the possible haplotype compositions with which it is compatible. The model is validated using simulated data sets for which the true haplotype estimates are known. We present results of the application of our model to estimate haplotype frequencies for a set of historic data from Africa in which the prevalence of mutations associated with sulphadoxine-pyrimethamine resistance were obtained.

Sentinel network for monitoring in vitro susceptibility of Plasmodium falciparum to antimalarial drugs in Colombia: a proof of concept

Drug resistance is one of the principal obstacles blocking worldwide malaria control. In Colombia, malaria remains a major public health concern and drug-resistant parasites have been reported. In vitro drug susceptibility assays are a useful tool for monitoring the emergence and spread of drug-resistant Plasmodium falciparum. The present study was conducted as a proof of concept for an antimalarial drug resistance surveillance network based on in vitro susceptibility testing in Colombia. Sentinel laboratories were set up in three malaria endemic areas. The enzyme linked immunosorbent assay-histidine rich protein 2 and schizont maturation methods were used to assess the susceptibility of fresh P. falciparum isolates to six antimalarial drugs. This study demonstrates that an antimalarial drug resistance surveillance network based on in vitro methods is feasible in the field with the participation of a research institute, local health institutions and universities. It could also serve as a model for a regional surveillance network. Preliminary susceptibility results showed widespread chloroquine resistance, which was consistent with previous reports for the Pacific region. However, high susceptibility to dihydroartemisinin and lumefantrine compounds, currently used for treatment in the country, was also reported. The implementation process identified critical points and opportunities for the improvement of network sustainability strategies.

Malaria in selected non-Amazonian countries of Latin America

Approximately 170 million inhabitants of the American continent live at risk of malaria transmission. Although the continent's contribution to the global malaria burden is small, at least 1–1.2 million malaria cases are reported annually. Sixty percent of the malaria cases occur in Brazil and the other 40% are distributed in 20 other countries of Central and South America. Plasmodium vivax is the predominant species (74.2%) followed by P. falciparum (25.7%) and P. malariae (0.1%), and no less than 10 Anopheles species have been identified as primary or secondary malaria vectors. Rapid deforestation and agricultural practices are directly related to increases in Anopheles species diversity and abundance, as well as in the number of malaria cases. Additionally, climate changes profoundly affect malaria transmission and are responsible for malaria epidemics in some regions of South America. Parasite drug resistance is increasing, but due to bio-geographic barriers there is extraordinary genetic differentiation of parasites with limited dispersion. Although the clinical spectrum ranges from uncomplicated to severe malaria cases, due to the generally low to middle transmission intensity, features such as severe anemia, cerebral malaria and other complications appear to be less frequent than in other endemic regions and asymptomatic infections are a common feature. Although the National Malaria Control Programs (NMCP) of different countries differ in their control activities these are all directed to reduce morbidity and mortality by using strategies like health promotion, vector control and impregnate bed nets among others. Recently, international initiatives such as the Malaria Control Program in Andean-country Border Regions (PAMAFRO) (implemented by the Andean Organism for Health (ORAS) and sponsored by The Global Fund to Fight AIDS, Tuberculosis and Malaria (GFATM)) and The Amazon Network for the Surveillance of Antimalarial Drug Resistance (RAVREDA) (sponsored by the Pan American Health Organization/World Health Organization (PAHO/WHO) and several other partners), have made great investments for malaria control in the region. We describe here the current status of malaria in a non-Amazonian region comprising several countries of South and Central America participating in the Centro Latino Americano de Investigación en Malaria (CLAIM), an International Center of Excellence for Malaria Research (ICEMR) sponsored by the National Institutes of Health (NIH) National Institute of Allergy and Infectious Diseases (NIAID).

Medikamentensensibilität von Plasmodium falciparum entlang der thailändischen Grenze bestimmt mittels des neuen HRP2-in-vitro-Feldtests

Drug resistance in Plasmodium falciparum has turned into a major problem throughout the malaria endemic regions of the world. Surveillance of antimalarial drug resistance is therefore essential for early changes in drug policies. In the course of this first major application of the new field-deployable histidine-rich protein 2 (HRP2) in vitro drug sensitivity assay, we investigated the current status of drug sensitivity to the standard antimalarial drugs dihydroartemisinin, mefloquine, quinine, and chloroquine in an area with an exceptionally high level of multidrug resistance in Thailand. The geometric mean of 50 and 90% inhibitory concentrations determined for 44 successfully tested samples by the HRP2 assay were comparable to earlier published results from the same area suggesting relatively little change in the local drug resistance pattern during the past years. The HRP2 field test was found to be highly sensitive and simple enough to be entirely performed in the field.

Increasing antimalarial drug resistance in Uganda and revision of the national drug policy.

Chloroquine (CQ) resistance was first documented in Uganda in 1988. Subsequent surveillance of antimalarial drug resistance, conducted by the Ugandan Ministry of Health and several research organizations, suggests that resistance to CQ is now widespread, reaching critical levels in many areas of the country. In June 2000, the Ministry of Health held a National Consensus Meeting to evaluate the available drug efficacy data and review the national antimalarial drug policy. After extensive debate, the combination of CQ + sulfadoxine-pyrimethamine (SP) was chosen to replace CQ as the first-line treatment of uncomplicated malaria as an interim policy. This review evaluates the in vivo drug efficacy studies conducted in Uganda since 1988 and issues confronted in revision of the drug policy. The Ugandan experience illustrates the challenges faced by sub-Saharan African countries confronted with rising CQ resistance but limited data on potential alternative options. The choice of CQ + SP as a provisional policy in the absence of prerequisite efficacy, safety and cost-effectiveness data reflects the urgency of the malaria treatment problem, and growing pressure to adopt combination therapies. Surveillance of CQ + SP treatment efficacy, collection of additional data on alternative regimens and active consensus building among key partners in the malaria community will be necessary to develop a rational long-term antimalarial treatment policy in Uganda.