Pyrimethamine Resistance Research Articles

ONT sequencing identifies a high prevalence of crt sensitive, triple mutant dhfr and single mutant dhps parasites within an ANC population in Nigeria.

Malaria in pregnancy is a major public health issue, particularly among vulnerable populations in malaria-endemic sub-Saharan African countries. To mitigate its risks, WHO recommends sulphadoxine-pyrimethamine (SP) for chemoprevention and artemisinin-based combination therapy (ACT) to treat uncomplicated Plasmodium falciparum malaria. These interventions have helped to alleviate the risk associated with malaria in pregnancy; however, in the context of the emergence of SP- and ACT-resistant P. falciparum, maintained efficacy is under threat. Molecular surveillance is a reliable tool to monitor the emergence of resistance where molecular markers are known. Thus, the objective of the study was to use a multiplexed amplicon Oxford Nanopore sequencing approach to assess the molecular markers for antimalarial resistance among pregnant women in Nigeria. Dried blood spots (DBS) were collected from pregnant women who received IPTp-SP at the enrollment and follow-up visits. P. falciparum genomic DNA was extracted by the Chelex® method and Pf18S qPCR was used to detect parasite DNA in each sample. With nested PCR assays, fragments of Pfdhps, Pfdhfr, Pfmdr1, Pfcrt, Pfk13 and Pfama1 genes were amplified and multiplexed amplicon-based sequencing was conducted on the minION Oxford Nanopore Technology. In total, 251 pregnant women were enrolled in the study and 457 DBS samples were collected. P. falciparum genomic DNA was detected in 12% (56/457) of the samples, 31 at baseline and the remaining during the follow-up visits. Pfama1, pfk13, Pfdhps, Pfdhfr, Pfmdr1 and Pfcrt were successfully sequenced in a single run. Notably, k13 artemisinin resistance mutations were absent, the frequencies of Pfdhfr and Pfdhps SP resistance haplotypes, IRN for pyrimethamine resistance and ISGKA/IAGKA associated with sulphadoxine resistance were 82% (36/44) and 64% (27/42), respectively, and the Pfcrt CVIET resistant haplotype was at approximately 22% (7/32). Here a multiplexed amplicon-based ONT assay established that triple mutant Pfdfhr-IRN, double mutant Pfdhps-SG haplotypes and the chloroquine sensitive strain were prevalent among pregnant women in Nigeria.

Prevalence of mutations associated with artemisinin partial resistance and sulfadoxine–pyrimethamine resistance in 13 regions in Tanzania in 2021: a cross-sectional survey

The emergence of the artemisinin partial resistance (ART-R) mutation in the Plasmodium falciparum kelch13 gene (k13), Arg561His, in Rwanda and the regional presence of polymorphisms affecting sulfadoxine-pyrimethamine have raised concern in neighbouring Tanzania. The goal of this study was to assess the status of antimalarial resistance in Tanzania, with a focus on the border with Rwanda, to understand the distribution of the Arg561His mutation, partner drug resistance, and resistance to chemoprevention drugs. In this cross-sectional survey, capillary dried blood spots were collected from malaria positive asymptomatic individuals in the community and symptomatic individuals in health facilities aged 6months and older, in 13regions of mainland Tanzania from Jan 31to June 26, 2021. Exclusion criteria included residence of the areas other than the target sites, presenting to the health facility for care and treatment of conditions other than malaria, and not providing informed consent. Samples were assessed for antimalarial resistance polymorphisms and genetic relatedness using molecular inversion probes targeting Pfalciparum and short-read whole-genome sequencing. The primary outcome was the prevalence of molecular markers of antimalarial resistance at the region level, as well as at the district level in Kagera, a region in the northwest of the country at the border with Rwanda. 6855 (88·1%) of 7782capillary dried blood spot samples collected were successfully genotyped. The overall prevalence of k13 Arg561His in Kagera was 7·7% (90% CI 6·0-9·4; 50of 649), with the highest prevalence in the districts near the Rwandan border (22·8% [31 of 136] in Karagwe, 14·4% [17 of 118]) in Kyerwa, and 1·4% [two of 144] in Ngara). k13 Arg561His was uncommon in the other regions. Haplotype analysis suggested that some of these parasites are related to isolates collected in Rwanda in 2015, supporting regional spread of Arg561His. However, a novel k13 Arg561His haplotype was observed, potentially indicating a second origin in the region. Other validated k13 resistance polymorphisms (one Arg622Ile and two Ala675Val isolates) were also identified. Aregion of prevalent dihydrofolate reductase Ile164Leu mutation, associated with sulfadoxine-pyrimethamine resistance, was also identified in Kagera (15·2% [12·6-17·8%]; 80of 526). The mutant crt Lys76Thr mutation, associated with chloroquine and amodiaquine resistance, was uncommon, occurring only in 75of 2861genotyped isolates, whereases the wild-type mdr1 Asn86Tyr allele, associated with reduced sensitivity to lumefantrine, was found in 99·7% (3819 of 3830) of samples countrywide. These findings show that the k13 Arg561His mutation is common in northwest Tanzania and that multiple emergences of ART-R, similar as to what was seen in southeast Asia, have occurred. Mutations associated with high levels of sulfadoxine-pyrimethamine resistance are common. These results raise concerns about the long-term efficacy of artemisinin and chemoprevention antimalarials in the region. Understanding how multiple emergences interact with drivers of regional spread is essential for combating ART-R in Africa. This study was funded by the Bill & Melinda Gates Foundation and the National Institutes of Health.

Efficacy and safety of Artemisinin Combination Therapy for the treatment of uncomplicated Plasmodium falciparum malaria across international borders of India

Background & objectives: Malaria due to Plasmodium falciparum (Pf) remains a major public threat in India. Artemisinin-based combination therapy (ACT) has been the country’s first-line drug for uncomplicated Pf malaria. In 2013-2014, Artesunate plus sulfadoxine (AS+SP) was replaced by Artemether Lumefantrine (AL) as the first- line antimalarial in North East (NE) states of the country which are endemic for Pf malaria. Regular monitoring of antimalarial drugs is of utmost importance to achieve the goal of elimination. This study aimed to assess the efficacy and safety of ACT for treating uncomplicated Pf malaria in the NE states of India. Methods: A prospective study of 28-day follow-up was conducted to monitor the efficacy and safety of AL from 2018-2019 in four districts, Udalgiri, Meghalaya, Lawngtlai, and Dhalai of NE, India. The clinical and parasitological response and the polymorphism analysis of the Pfdhps, P/dhfr, and Pfkelch 13 gene were evaluated. Results: A total of 234 patients were enrolled in the study out of 216 patients who completed the follow-up to 28 days. One-hundred percent adequate clinical and parasitological responses (ACPR) were observed with polymerase chain reaction (PCR) correction. The genotype results suggest no recrudescence in the treatment-failure patients. The classical single nucleotide polymorphisms (SNP) in the Pfdhfr gene was S108N (94.9%), followed by C59R (91.5%), whereas, in the Pfdhps gene, the common SNP was A437G (79.6%), followed by S3436A. No associated or validated mutations were found in the propeller region of the PfKelch13 gene. Interpretation & conclusion: AL was efficacious and safe in uncomplicated P. falciparum malaria in North East India. In contrast, mutations in the genes responsible for sulfadoxine and pyrimethamine resistance have been fixed in northeast India’s population.

Mutant DHFR Prevalence and Pyrimethamine Resistance among Asymptomatic Adolescents in Kwara State

Plasmodium falciparum malaria remains a leading public health problem in sub-Saharan Africa and its control is seriously challenged by drug resistance. Resistance to sulfadoxine-pyrimethamine is mediated by point mutations in genes encoding the target enzymes dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps). Blood from a total of 176 subjects aged 11-19 years from Ilorin East and Irepodun Local Government areas (LGAs) was examined for mutant alleles of the Plasmodium falciparum dihydrofolate reductase gene by polymerase chain reaction (PCR) at codon 51 and 108 respectively. Rapid Diagnostic Test kits were used for the malaria test. Of the 176 participants, 73 (41.5%) subjects tested positive for malaria parasite while 95 (54.0%) tested negative. For both N51I and S108N SNPscreening, mutant alleles were dominant over the wild type. The Iponrin community (Ilorin East LGA) recorded the highest percentage of mutant alleles 14 (33.33%) of the N51I SNP, while the Igbonla community (Irepodun LGA) had the least number of mutant alleles 8 (19.04%). On the other hand, the Alakuko community recorded the highest number of alleles 14 (31.80%) for S108N mutant while the Igbonla community (Irepodun LGA) had the least number of mutant alleles 8 (18.18%). There was widespread pyrimethamine resistance among the studied population and the malaria parasite remains persistent among the studied population. Therefore, there is a need for monitoring antimalarial drug resistance in Nigeria for prompt management of the antimalarial drug resistance menace.

Evolution of Plasmodium falciparum antimalarial drug resistance markers post-adoption of artemisinin-based combination therapies in Yaounde, Cameroon

ObjectivesThis study aimed to investigate the evolution of Plasmodium falciparum antimalarial drug resistance markers by comparing the pre- and post-adoption of artemisinin-based combination therapies (ACTs) in Yaounde, Cameroon. MethodsThe molecular characterization of known antimalarial drug resistance markers (Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, and Pfk13) in P. falciparum-positive samples collected in 2014 and 2019-2020 was achieved using nested polymerase chain reaction, followed by targeted amplicon deep sequencing on the Illumina MiSeq platform. Data derived were compared with those published during the pre-ACT adoption period from 2004 to 2006. ResultsA high prevalence of Pfmdr1 184F, Pfdhfr 51I/59R/108N, and Pfdhps 437G mutant alleles was observed during the post-ACT adoption period. The Pfcrt 76T and Pfmdr1 86Y mutant alleles significantly declined between 2004 and 2020 (P <0.0001). Conversely, the resistance markers to antifolates, Pfdhfr 51I/59R/108N and Pfdhps 437G, significantly increased during the same study period (P <0.0001). We identified nine mutations in the propeller domains of Pfk13; although they were all present in single parasite isolates, none of them are known to confer artemisinin resistance. ConclusionThis study documented a near-complete reversion to sensitive parasites for markers conferring resistance to the 4-aminoquinolines and arylamino alcohols in Yaounde. In contrast, the Pfdhfr mutations associated with pyrimethamine resistance are moving toward saturation.

Genomic characterization of Plasmodium falciparum genes associated with anti-folate drug resistance and treatment outcomes in eastern India: A molecular surveillance study from 2008 to 2017.

After being used vigorously for the previous two decades to treat P. falciparum, chloroquine and sulfadoxine-pyrimethamine were replaced in 2009 with an artemisinin-based combination therapy (artesunate-sulfadoxine-pyrimethamine) in an effort to combat multidrug-resistant parasites. We set out to assess the genetic variants of sulfadoxine-pyrimethamine resistance and the effectiveness of its treatment in eastern India prior to, during, and 6 to 8 years following the introduction of the new pharmacological regime. In 2008-2009, 318 P. falciparum-positive patients got the recommended doses of sulfadoxine-pyrimethamine. We used 379 additional isolates from 2015 to 2017 in addition to the 106 isolates from 2010. All 803 isolates from two study sites underwent in vitro sulfadoxine-pyrimethamine sensitivity testing and genomic characterisation of sulfadoxine-pyrimethamine resistance (pfdhfr and pfdhps). In Kolkata and Purulia, we observed early treatment failure in 30.7 and 14.4% of patients, respectively, whereas recrudescence was found in 8.1 and 13.4% of patients, respectively, in 2008-2009. In 2017, the proportion of in vitro pyrimethamine and sulfadoxine resistance steadily grew in Kolkata and Purulia despite a single use of sulfadoxine-pyrimethamine. Treatment failures with sulfadoxine-pyrimethamine were linked to quintuple or quadruple pfdhfr- pfdhps mutations (AICII-AGKAT, AICII-AGKAA, AICII-SGKGT, AICII-AGKAA, AICNI-AGKAA) in 2008-2009 (p < 0.001). The subsequent spread of mutant-haplotypes with higher in vitro sulfadoxine-pyrimethamine resistance (p < 0.001), such as the sextuple (dhfr-AIRNI+dhps-AGEAA, dhfr-ANRNL+dhps-AGEAA) and septuple (dhfr-AIRNI+dhps-AGEAT), mutations were observed in 2015-2017. This successive spread of mutations with high in vitro sulfadoxine-pyrimethamine resistance confirmed the progressive increase in antifolate resistance even after an 8-year withdrawal of sulfadoxine-pyrimethamine.

Single- and duplex TaqMan-quantitative PCR for determining the copy numbers of integrated selection markers during site-specific mutagenesis in Toxoplasma gondii by CRISPR-Cas9.

Herein, we developed a single and a duplex TaqMan quantitative PCR (qPCR) for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wtdhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics.

Prevalence of Mutations in the Pfdhfr, Pfdhps, and Pfmdr1 Genes of Malarial Parasites Isolated from Symptomatic Patients in Dogondoutchi, Niger.

The effectiveness of artemisinin-based combination therapies (ACTs) depends not only on that of artemisinin but also on that of partner molecules. This study aims to evaluate the prevalence of mutations in the Pfdhfr, Pfdhps, and Pfmdr1 genes from isolates collected during a clinical study. Plasmodium genomic DNA samples extracted from symptomatic malaria patients from Dogondoutchi, Niger, were sequenced by the Sanger method to determine mutations in the Pfdhfr (codons 51, 59, 108, and 164), Pfdhps (codons 436, 437, 540, 581, and 613), and Pfmdr1 (codons 86, 184, 1034, and 1246) genes. One hundred fifty-five (155) pre-treatment samples were sequenced for the Pfdhfr, Pfdhps, and Pfmdr1 genes. A high prevalence of mutations in the Pfdhfr gene was observed at the level of the N51I (84.97%), C59R (92.62%), and S108N (97.39%) codons. The key K540E mutation in the Pfdhps gene was not observed. Only one isolate was found to harbor a mutation at codon I431V. The most common mutation on the Pfmdr1 gene was Y184F in 71.43% of the mutations found, followed by N86Y in 10.20%. The triple-mutant haplotype N51I/C59R/S108N (IRN) was detected in 97% of the samples. Single-mutant (ICS and NCN) and double-mutant (IRS, NRN, and ICN) haplotypes were prevalent at 97% and 95%, respectively. Double-mutant haplotypes of the Pfdhps (581 and 613) and Pfmdr (86 and 184) were found in 3% and 25.45% of the isolates studied, respectively. The study focused on the molecular analysis of the sequencing of the Pfdhfr, Pfdhps, and Pfmdr1 genes. Although a high prevalence of mutations in the Pfdhfr gene have been observed, there is a lack of sulfadoxine pyrimethamine resistance. There is a high prevalence of mutation in the Pfmdr184 codon associated with resistance to amodiaquine. These data will be used by Niger’s National Malaria Control Program to better monitor the resistance of Plasmodium to partner molecules in artemisinin-based combination therapies.

Prevalence of Molecular Markers of Plasmodium Falciparum Resistance to Proguanil and Pyrimethamine in Children with Haemoglobin Phenotypes SS and AA in Benin City, Nigeria

Background: Chemoprophylaxis against Plasmodium falciparum (Pf) is advocated in children with sickle cell anaemia (SCA). Among them, antifolates: proguanil and pyrimethamine had replaced initial chemoprophylactic drugs because of widespread resistance. In recent past, efficacy of these antifolates has also come under scrutiny due to increasing level of drug resistance. Specific point mutations on Plasmodium falciparum dihydrofolate reductase gene (pfdhfr) have been linked with resistance to proguanil and pyrimethamine and they can be used as markers in monitoring prevalence and level of resistance to the drugs.&#x0D; Objectives: To determine the prevalence of molecular markers of Plasmodium falciparum resistance to proguanil and pyrimethamine in children with SCA.&#x0D; Methods: A total of 146 Plasmodium falciparum isolates (71 from children with SCA and 75 from those with Haemoglobin AA: HbAA) were evaluated for point mutations and mutant haplotypes on the pfdhfr gene using nested polymerase chain reaction amplification followed by direct sequencing.&#x0D; Results: The triple (S108N+N51I+C59N) mutant haplotype was present in 100.0% and 96.0% of samples from children with SCA and HbAA respectively. S108T, A16V and 1164L mutationswere not present in both groups.&#x0D; Conclusion: High prevalence of triple mutant haplotype mediates significant resistance to pyrimethamine and implies that pyrimethamine resistance is fixed in the study locale. However, the absence of pfdhfr S108T and A16V mutations, which indicate specific resistance to proguanil but not to pyrimethamine, suggests that proguanil is still useful even in the face of pyrimethamine resistance. The threat of proguanil resistance is however real due to high prevalence of the triple mutant pfdhfr haplotype.&#x0D; Keywords: Drug-resistance; Molecular markers; Plasmodium falciparum; Proguanil; Pyrimethamine.

Genetic population of Plasmodium knowlesi during pre-malaria elimination in Thailand

BackgroundThailand is committed to eliminating malaria by 2024. From 2013 to 2020, the total number of malaria cases have decreased, from 37,741 to 4474 (an 88.1% reduction). However, infections with Plasmodium knowlesi, a monkey malarial pathogen that can also infect humans, have been increasingly observed. This study focused on the molecular analysis of P. knowlesi parasites causing malaria in Thailand.MethodsUnder Thailand’s integrated Drug Efficacy Surveillance (iDES), which includes drug-resistance monitoring as part of routine case-based surveillance and responses, specimens were collected from malaria patients (n = 966) between 2018 and 2020. Thirty-one mono P. knowlesi infections (3.1%), most of which were from eastern and southern Thailand, were observed and confirmed by nested PCR assay and DNA sequencing. To evaluate whether these pathogens were from different lineages, cluster analysis based on seven microsatellite genotyping markers and the merozoite surface protein 1 (pkmsp1) gene was carried out. The P. knowlesi pyrimethamine resistance gene dihydrofolate reductase (pkdhfr) was sequenced and homology modelling was constructed.ResultsThe results of analysing the seven microsatellite markers and pkmsp1 sequence demonstrated that P. knowlesi parasites from eastern Thailand were of the same lineage as those isolated in Cambodia, while the parasites causing malaria in southern Thailand were the same lineage as those isolated from Malaysia. The sequencing results for the pkdhfr genes indicated the presence of two mutations, Arg34Leu and a deletion at position 105. On analysis with homology modelling, the two mutations were not associated with anti-malarial drug resistance.ConclusionsThis report compared the genetic populations of P. knowlesi parasites in Thailand from 2018 to 2020 and have shown similar lineages as those isolated in Cambodia and Malaysia of P. knowlesi infection in Thailand and demonstrated that the P. knowlesi parasites were of the same lineages as those isolated in Cambodia and Malaysia. The parasites were also shown to be sensitive to pyrimethamine.

Evaluation of Dihydrofolate Reductase (Dhfr) Gene, Related to Plasmodium Falciparum Pyrimethamine Resistance in Imported Malaria Cases in Iran

Background: Antimalarial drug resistance is one of the important challenges for governments in the fight against malaria. Molecular surveillance of antimalarial drug resistance supports early detection of how the recommended treatments work. This allows immediate action to reduce any threat and prevent it from spreading. Therefore, the aim of this study was to evaluate the frequency of dihydrofolate reductase (dhfr) mutants in Plasmodium falciparum resistance to pyrimethamine in Iranian malaria patients. Materials and Methods: In 2020, 27 patients (21 males and 5 females) with imported P. falciparum cases were studied. The nested-PCR technique first confirmed the species in all samples and then amplification was done by the semi-nested-PCR method in order to detect single nucleotide polymorphisms (SNPs) in dhfr gene related to pyrimethamine resistance. Results: All samples in the 18S rRNA gene had species-specific bands for P. falciparum strains. In the sequence analysis of pfdhfr gene amplification after comparison with the standard strain (wild type), 21 patients had a double mutation (C59R+S108N) and six patients had a triple mutation (N51I+C59R+S108N) of pyrimethamine resistance. Conclusion: The results of this study showed that the susceptibility of P. falciparum to pyrimethamine in the treatment of malaria is significantly reducing. These findings can raise concerns about pyrimethamine resistance in P. falciparum. Due to the high emergence of double and triple mutants related to pyrimethamine resistance, the malaria surveillance and treatment systems in Iran, the use of pyrimethamine should be considered.

Transgenic pyrimethamine-resistant plasmodium falciparum reveals transmission-blocking potency of P218, a novel antifolate candidate drug

Antimalarial drugs capable of targeting multiple parasite stages, particularly the transmissible stages, can be valuable tools for advancing the malaria elimination agenda. Current antifolate drugs such as pyrimethamine can inhibit replicative parasite stages in both humans and mosquitoes, but antifolate resistance remains a challenge. The lack of reliable gametocyte-producing, antifolate-resistant Plasmodium falciparum laboratory strain hinders the study of new antifolate compounds that can overcome antifolate resistance including development stages in the mosquito. We used clustered regularly interspaced short palindromic repeats-Cas9 genome editing to develop a transgenic gametocyte-producing strain of P. falciparum with quadruple mutations (N51I, C59R, S108N, I164L) in the dihydrofolate reductase (dhfr) gene, using NF54 as a parental strain. The transgenic parasites exhibited pyrimethamine resistance while maintaining their gametocyte-producing activity. We then demonstrated that pyrimethamine could no longer inhibit male gametocyte exflagellation in the transgenic parasite. In contrast, P218, the novel antifolate, designed to overcome antifolate resistance, potently inhibited exflagellation. The exflagellation IC50 of P218 was five times lower than the asexual stage half maximal inhibitory concentration (IC50), suggesting a strong barrier for transmission of P218-resistant parasites. The transgenic gametocyte-producing, pyrimethamine-resistant parasite is a robust system for evaluating novel antifolate compounds against non-asexual stage development.

Multiple Single-Nucleotide Polymorphism Detection for Antimalarial Pyrimethamine Resistance via Allele-Specific PCR Coupled with Gold Nanoparticle-Based Lateral Flow Biosensor.

Molecular genotyping holds tremendous potential to detect antimalarial drug resistance (ADR) related to single nucleotide polymorphisms (SNPs). However, it relies on the use of complicated procedures and expensive instruments. Thus, rapid point-of-care testing (POCT) molecular tools are urgently needed for field survey and clinical use. Herein, a POCT platform consisting of multiple-allele-specific PCR (AS-PCR) and a gold nanoparticle (AuNP)-based lateral flow biosensor was designed and developed for SNP detection of the Plasmodium falciparum dihydrofolate reductase (pfdhfr) gene related to pyrimethamine resistance. The multiple-AS-PCR utilized 3' terminal artificial antepenultimate mismatch and double phosphorothioate-modified allele-specific primers. The duplex PCR amplicons with 5' terminal labeled with biotin and digoxin are recognized by streptavidin (SA)-AuNPs on the conjugate pad and then captured by anti-digoxin antibody through immunoreactions on the test line to produce a golden red line for detection. The system was applied to analyze SNPs in Pfdhfr N51I, C59R, and S108N of 98 clinical isolates from uncomplicated P. falciparum malaria patients. Compared with the results from nested PCR followed by Sanger DNA sequencing, the sensitivity was 97.96% (96/98) for N51I, C59R, and S108N. For specificity, the values were 100% (98/98), 95.92% (94/98), and 100% (98/98) for N51I, C59R, and S108N, respectively. The limit of detection is approximately 200 fg/μl for plasmid DNA as the template and 100 parasites/μl for blood filter paper. The established platform not only offers a powerful tool for molecular surveillance of ADR but also is easily extended to interrelated SNP profiles for infectious diseases and genetic diseases.

Assessment of antimalarial drug resistant markers in asymptomatic Plasmodium falciparum infections after 4 years of indoor residual spraying in Northern Ghana.

BackgroundDrug resistance remains a concern for malaria control and elimination. The effect of interventions on its prevalence needs to be monitored to pre-empt further selection. We assessed the prevalence of Plasmodium falciparum gene mutations associated with resistance to the antimalarial drugs: sulfadoxine-pyrimethamine (SP), chloroquine (CQ) and artemisinin combination therapy (ACTs) after the scale-up of a vector control activity that reduced transmission.MethodsA total of 400 P. falciparum isolates from children under five years were genotyped for seventeen single nucleotide polymorphisms (SNPs) in pfcrt, pfmdr1, pfdhfr, pfdhps and pfk13 genes using polymerase chain reaction (PCR) and high resolution melting (HRM) analysis. These included 80 isolates, each randomly selected from cross-sectional surveys of asymptomatic infections across 2010 (baseline), 2011, 2012, 2013 (midline: post-IRS) and 2014 (endline: post-IRS) during the peak transmission season, when IRS intervention was rolled out in Bunkpurugu Yunyoo (BY) District, Ghana. The proportions of isolates with drug resistant alleles were assessed over this period.ResultsThere were significant decreases in the prevalence of pfdhfr- I51R59N108 haplotype from 2010 to 2014, while the decline in pfdhfr/pfdhps- I51R59N108G437 during the same period was not significant. The prevalence of lumefantrine (LM), mefloquine (MQ) and amodiaquine (AQ) resistance-associated haplotypes pfmdr1-N86F184D1246 and pfmdr1-Y86Y184Y1246 showed decreasing trends (z = -2.86, P = 0.004 and z = -2.71, P = 0.007, respectively). Each of pfcrt-T76 and pfmdr1-Y86 mutant alleles also showed a declining trend in the asymptomatic reservoir, after the IRS rollout in 2014 (z = -2.87, P = 0.004 and z = -2.65, P = 0.008, respectively). Similarly, Pyrimethamine resistance mediating polymorphisms pfdhfr-N108, pfdhfr-I51 and pfdhfr-R59 also declined (z = -2.03, P = 0.042, z = -3.54, P<0.001 and z = -4.63, P<0.001, respectively), but not the sulphadoxine resistance mediating pfdhps-G437 and pfdhps-F436 (z = -0.36, P = 0.715 and z = 0.41, P = 0.684, respectively). No mutant pfk13-Y580 were detected during the study period.ConclusionThe study demonstrated declining trends in the prevalence of drug resistant mutations in asymptomatic P. falciparum infections following transmission reduction after an enhanced IRS intervention in Northern Ghana.

Validation of PfSNP-LAMP-Lateral Flow Dipstick for Detection of Single Nucleotide Polymorphism Associated with Pyrimethamine Resistance in Plasmodium falciparum.

The loop-mediated isothermal amplification coupled with lateral flow dipstick (PfSNP-LAMP-LFD) was recently developed to detect single nucleotide polymorphism (AAT → ATT), corresponding to substitution of asparagine to isoleucine at amino acid position 51 in the P. falciparum dhfr-ts gene associated with antifolate resistance. In this present study, the PfSNP-LAMP-LFD was validated on 128 clinical malaria samples of broad ranged parasite densities (10 to 87,634 parasites per microliter of blood). The results showed 100% accuracy for the detection of single nucleotide polymorphism for N51I mutation. Indeed, the high prevalence of N51I in the Pfdhfr-ts gene detected in the clinical samples is in line with reports of widespread antifolate resistant P. falciparum in Thailand. The relationship between enzyme choice and reaction time was observed to have an effect on PfSNP-LAMP-LFD specificity; however, the method yielded consistent results once the conditions have been optimized. The results demonstrate that PfSNP-LAMP-LFD is a simple method with sufficient sensitivity and specificity to be deployed in routine surveillance of antifolate resistance molecular marker and inform antimalarial management policy.

Cost-effectiveness of intermittent preventive treatment with dihydroartemisinin–piperaquine for malaria during pregnancy: an analysis using efficacy results from Uganda and Kenya, and pooled data

SummaryBackgroundPrevention of malaria infection during pregnancy in HIV-negative women currently relies on the use of long-lasting insecticidal nets together with intermittent preventive treatment in pregnancy with sulfadoxine–pyrimethamine (IPTp-SP). Increasing sulfadoxine–pyrimethamine resistance in Africa threatens current prevention of malaria during pregnancy. Thus, a replacement for IPTp-SP is urgently needed, especially for locations with high sulfadoxine–pyrimethamine resistance. Dihydroartemisinin–piperaquine is a promising candidate. We aimed to estimate the cost-effectiveness of intermittent preventive treatment in pregnancy with dihydroartemisinin–piperaquine (IPTp-DP) versus IPTp-SP to prevent clinical malaria infection (and its sequelae) during pregnancy.MethodsWe did a cost-effectiveness analysis using meta-analysis and individual trial results from three clinical trials done in Kenya and Uganda. We calculated disability-adjusted life-years (DALYs) arising from stillbirths, neonatal death, low birthweight, mild and moderate maternal anaemia, and clinical malaria infection, associated with malaria during pregnancy. Cost estimates were obtained from data collected in observational studies, health-facility costings, and from international drug procurement databases. The cost-effectiveness analyses were done from a health-care provider perspective using a decision tree model with a lifetime horizon. Deterministic and probabilistic sensitivity analyses using appropriate parameter ranges and distributions were also done. Results are presented as the incremental cost per DALY averted and the likelihood that an intervention is cost-effective for different cost-effectiveness thresholds.FindingsCompared with three doses of sulfadoxine–pyrimethamine, three doses of dihydroartemisinin–piperaquine, delivered to a hypothetical cohort of 1000 pregnant women, averted 892 DALYs (95% credibility interval 274 to 1517) at an incremental cost of US$7051 (2653 to 13 038) generating an incremental cost-effectiveness ratio (ICER) of $8 (2 to 29) per DALY averted. Compared with monthly doses of sulfadoxine–pyrimethamine, monthly doses of dihydroartemisinin–piperaquine averted 534 DALYS (−141 to 1233) at a cost of $13 427 (4994 to 22 895), resulting in an ICER of $25 (−151 to 224) per DALY averted. Both results were highly robust to most or all variations in the deterministic sensitivity analysis.InterpretationOur findings suggest that among HIV-negative pregnant women with high uptake of long-lasting insecticidal nets, IPTp-DP is cost-effective in areas with high malaria transmission and high sulfadoxine–pyrimethamine resistance. These data provide a comprehensive overview of the current evidence on the cost-effectiveness of IPTp-DP. Nevertheless, before a policy change is advocated, we recommend further research into the effectiveness and costs of different regimens of IPTp-DP in settings with different underlying sulfadoxine–pyrimethamine resistance.FundingMalaria in Pregnancy Consortium, which is funded through a grant from the Bill & Melinda Gates Foundation to the Liverpool School of Hygiene and Tropical Medicine.

Genetic analysis of the orthologous crt and mdr1 genes in Plasmodium malariae from Thailand and Myanmar

BackgroundPlasmodium malariae is a widely spread but neglected human malaria parasite, which causes chronic infections. Studies on genetic polymorphisms of anti-malarial drug target genes in P. malariae are limited. Previous reports have shown polymorphisms in the P. malariae dihydrofolate reductase gene associated with pyrimethamine resistance and linked to pyrimethamine drug pressure. This study investigated polymorphisms of the P. malariae homologous genes, chloroquine resistant transporter and multidrug resistant 1, associated with chloroquine and mefloquine resistance in Plasmodium falciparum.MethodsThe orthologous P. malariae crt and mdr1 genes were studied in 95 patients with P. malariae infection between 2002 and 2016 from Thailand (N = 51) and Myanmar (N = 44). Gene sequences were analysed using BioEdit, MEGA7, and DnaSP programs. Mutations and gene amplifications were compared with P. falciparum and Plasmodium vivax orthologous genes. Protein topology models derived from the observed pmcrt and pmmdr1 haplotypes were constructed and analysed using Phyre2, SWISS MODEL and Discovery Studio Visualization V 17.2.ResultsTwo non-synonymous mutations were observed in exon 2 (H53P, 40%) and exon 8 (E278D, 44%) of pmcrt. The topology model indicated that H53P and E278D were located outside of the transmembrane domain and were unlikely to affect protein function. Pmmdr1 was more diverse than pmcrt, with 10 non-synonymous and 3 synonymous mutations observed. Non-synonymous mutations were located in the parasite cytoplasmic site, transmembrane 11 and nucleotide binding domains 1 and 2. Polymorphisms conferring amino acid changes in the transmembrane and nucleotide binding domains were predicted to have some effect on PmMDR1 conformation, but were unlikely to affect protein function. All P. malariae parasites in this study contained a single copy of the mdr1 gene.ConclusionsThe observed polymorphisms in pmcrt and pmmdr1 genes are unlikely to affect protein function and unlikely related to chloroquine drug pressure. Similarly, the absence of pmmdr1 copy number variation suggests limited mefloquine drug pressure on the P. malariae parasite population, despite its long time use in Thailand for the treatment of falciparum malaria.

Polymorphisms in Plasmodium vivax antifolate resistance markers in Afghanistan between 2007 and 2017

BackgroundPlasmodium vivax is the predominant Plasmodium species in Afghanistan. National guidelines recommend the combination of chloroquine and primaquine (CQ-PQ) for radical treatment of P. vivax malaria. Artesunate in combination with the antifolates sulfadoxine-pyrimethamine (SP) has been first-line treatment for uncomplicated falciparum malaria until 2016. Although SP has been the recommended treatment for falciparum and not vivax malaria, exposure of the P. vivax parasite population to SP might still have been quite extensive because of community based management of malaria. The change in the P. vivax antifolate resistance markers between 2007 and 2017 were investigated.MethodsDried blood spots were collected (n = 185) from confirmed P. vivax patients in five malaria-endemic areas of Afghanistan bordering Tajikistan, Turkmenistan and Pakistan, including Takhar, Faryab, Laghman, Nangarhar, and Kunar, in 2007, 2010 and 2017. Semi-nested PCR, RFLP and nucleotide sequencing were used to assess the pyrimethamine resistant related mutations in P. vivax dihydrofolate reductase (pvdhfr I13L, P33L, N50I, F57L, S58R, T61I, S93H, S117N, I173L) and the sulfonamide resistance related mutations in P. vivax dihydropteroate synthase (pvdhps A383G, A553G).ResultsIn the 185 samples genotyped for pvdhfr and pvdhps mutations, 11 distinct haplotypes were observed, which evolved over time. In 2007, wild type pvdhfr and pvdhps were the most frequent haplotype in all study sites (81%, 80/99). However, in 2017, the frequency of the wild-type was reduced to 36%, (21/58; p value ≤ 0.001), with an increase in frequency of the double mutant pvdhfr and pvdhps haplotype S58RS117N (21%, 12/58), and the single pvdhfr mutant haplotype S117N (14%, 8/58). Triple and quadruple mutations were not found. In addition, pvdhfr mutations at position N50I (7%, 13/185) and the novel mutation S93H (6%, 11/185) were observed. Based on in silico protein modelling and molecular docking, the pvdhfr N50I mutation is expected to affect only moderately pyrimethamine binding, whereas the S93H mutation does not.ConclusionsIn the course of ten years, there has been a strong increase in the frequency pyrimethamine resistance related mutations in pvdhfr in the P. vivax population in Afghanistan, although triple and quadruple mutations conferring high grade resistance were not observed. This suggests relatively low drug pressure from SP on the P. vivax parasite population in the study areas. The impact of two newly identified mutations in the pvdhfr gene on pyrimethamine resistance needs further investigation.

Molecular variation of Plasmodium vivax dehydrofolate reductase in Mexico and Nicaragua contrasts with that occurring in South America.

To research mutations associated to pyrimethamine resistance in dihydrofolate reductase (pvdhfr) of Plasmodium vivax from Mexico and Nicaragua and compare it to that reported in the rest of America. Genomic DNA was obtained from P. vivax-infected blood samples. A pvdhfr gene fragment was amplified and sequenced. The identified gene variations were compared to those observed in other affected sites of America. No mutations in pvdhfr were detected in P. vivax from Mexico and Nicaragua. One synonymous change and variation in the repeat domain was detected in Nicaraguan parasites. In South America, a high frequency of variant residues 58R and 117N associated to pyrimethamine resistance was reported. The lack of polymorphisms associated with pyrimethamine resistance suggests that drug-resistant P. vivax has not penetrated Mesoamerica, nor have local parasites been under selective pressure. These data contribute to establish the basis for the epidemiological surveillance of drug resistance.

Development and Evaluation of a Novel HNB Based Isothermal Amplification Assay for Fast Detection of Pyrimethamine Resistance (S108N) in Plasmodium falciparum.

Sulphadoxine and pyrimethamine (SP) have been used as long-acting partner antimalarial drugs in artemisinin combination therapy (ACT) for falciparum malaria. The emergence and increasing spread of SP resistance in malaria-endemic areas have become a challenge for the control of malaria. Therefore, regular monitoring of the mutation status of partner drugs is important for the better management of drug policy. There are limitations with traditional molecular methods and there is an urgent need for an easy method for diagnosis of drug resistance. In this study we have introduced and developed a novel single nucleotide polymorphism loop-mediated isothermal amplification (SNP–LAMP) approach based on a hydroxynaphthol blue (HNB) indicator for the easier and quicker detection of pyrimethamine resistance in Plasmodium falciparum malaria. To implement this novel approach, many sets of LAMP primers were designed and tested. Finally, one set of forward inner primer M1 (FIPM1) of LAMP primer was selected that specifically distinguishes pyrimethamine-resistant P. falciparum malaria. The LAMP reactions were optimized at 60–66 °C for 45 min. High sensitivity (7 parasites/µL) was observed with 10−4 fold dilutions (<2 ng DNA) of genomic DNA. Moreover, this approach has the potential to be applied even in laboratories unfamiliar with PCR or other molecular methods, and in future, this can be helpful for the better management of anti-malarial policy.