K13 Polymorphisms Research Articles

Indigenous emergence and spread of kelch13 C469Y artemisinin-resistant Plasmodium falciparum in Uganda.

Artemisinin-based combination therapies (ACTs) were introduced as the standard of care for uncomplicated malaria in Africa almost two decades ago. Recent studies in East Africa have reported a gradual increase in kelch13 (k13) mutant parasites associated with reduced artesunate efficacy. As part of the Community Access to Rectal Artesunate for Malaria project, we collected blood samples from 697 children with signs of severe malaria in northern Uganda between 2018 and 2020, before and after the introduction of rectal artesunate (RAS) in 2019. K13 polymorphisms were assessed, and parasite editing and phenotyping were performed to assess the impact of mutations on parasite resistance. Whole-genome sequencing was performed, and haplotype networks were constructed to determine the geographic origin of k13 mutations. Of the 697 children, 540 were positive for Plasmodium falciparum malaria by PCR and were treated with either RAS or injectable artesunate monotherapy followed in most cases by ACT. The most common k13 mutation was C469Y (6.7%), which was detected more frequently in samples collected after RAS introduction. Genome editing confirmed reduced in vitro susceptibility to artemisinin in C469Y-harboring parasites compared to wild-type controls (P < 0.001). The haplotypic network showed that flanking regions of the C469Y mutation shared the same African genetic background, suggesting a single and indigenous origin of the mutation. Our data provide evidence of selection for the artemisinin-resistant C469Y mutation. The realistic threat of multiresistant parasites emerging in Africa should encourage careful monitoring of the efficacy of artemisinin derivatives and strict adherence to ACT treatment regimens.

Molecular surveillance of Kelch 13 polymorphisms in Plasmodium falciparum isolates from Kenya and Ethiopia

BackgroundTimely molecular surveillance of Plasmodium falciparum kelch 13 (k13) gene mutations is essential for monitoring the emergence and stemming the spread of artemisinin resistance. Widespread artemisinin resistance, as observed in Southeast Asia, would reverse significant gains that have been made against the malaria burden in Africa. The purpose of this study was to assess the prevalence of k13 polymorphisms in western Kenya and Ethiopia at sites representing varying transmission intensities between 2018 and 2022.MethodsDried blood spot samples collected through ongoing passive surveillance and malaria epidemiological studies, respectively, were investigated. The k13 gene was genotyped in P. falciparum isolates with high parasitaemia: 775 isolates from four sites in western Kenya (Homa Bay, Kakamega, Kisii, and Kombewa) and 319 isolates from five sites across Ethiopia (Arjo, Awash, Gambella, Dire Dawa, and Semera). DNA sequence variation and neutrality were analysed within each study site where mutant alleles were detected.ResultsSixteen Kelch13 haplotypes were detected in this study. Prevalence of nonsynonymous k13 mutations was low in both western Kenya (25/783, 3.19%) and Ethiopia (5/319, 1.57%) across the study period. Two WHO-validated mutations were detected: A675V in three isolates from Kenya and R622I in four isolates from Ethiopia. Seventeen samples from Kenya carried synonymous mutations (2.17%). No synonymous mutations were detected in Ethiopia. Genetic variation analyses and tests of neutrality further suggest an excess of low frequency polymorphisms in each study site. Fu and Li’s F test statistic in Semera was 0.48 (P > 0.05), suggesting potential population selection of R622I, which appeared at a relatively high frequency (3/22, 13.04%).ConclusionsThis study presents an updated report on the low frequency of k13 mutations in western Kenya and Ethiopia. The WHO-validated R622I mutation, which has previously only been reported along the north-west border of Ethiopia, appeared in four isolates collected from eastern Ethiopia. The rapid expansion of R622I across Ethiopia signals the need for enhanced monitoring of the spread of drug-resistant P. falciparum parasites in East Africa. Although ACT remains currently efficacious in the study areas, continued surveillance is necessary to detect early indicators of artemisinin partial resistance.

Molecular markers associated with drug resistance in Plasmodium falciparum parasites in central Africa between 2016 and 2021.

The widespread occurrence of anti-malarial drug resistance threatens the current efforts to control malaria in African regions. Molecular marker surveillance helps to track the emergence and spread of drug-resistant malaria cases. A total of 237 Plasmodium falciparum infections imported from central Africa to Zhejiang Province, China, between 2016 and 2021, were investigated. Genomic DNA was extracted from blood samples of each patient and nested PCRs was used to detect molecular markers in k13, Pfcrt, and Pfmdr1 genes. The spatial and temporal distributions of the molecular markers were analyzed. A limited polymorphism of k13 was observed, including two nonsynonymous (D464E and K503E) and five synonymous mutations. Wild-type CVMNK of Pfcrt predominated (78.5%), whereas 19.5% of the samples harbored the mutant haplotype, CVIET. The point mutation Y184F and the single mutant haplotype NF of Pfmdr1 were the most frequently observed. The geographical distributions of the Pfcrt and Pfmdr1 haplotypes displayed distinct patterns, with the mutant haplotype of Pfcrt more common in Gabon (53.9%) and Congo (50.0%), and wild haplotypes of Pfmdr1 more frequently found in Cameroon, Angola, and Congo. The prevalence of wild-type CVMNK of Pfcrt increased from 68.5-74.6% in 2016-2017 to 81.8-87.5% in 2018-2021. The proportion of wild-type Pfmdr1 also increased from 27.1% in 2016 to 38.5% in 2019. The geographical and temporal distribution of k13, Pfcrt, and Pfmdr1 polymorphisms in P. falciparum parasites imported from central Africa between 2016 and 2021 are demonstrated. Our data provide updated evidence that can be used to adjust anti-malarial drug policies in central Africa and China.

Therapeutic efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in four malaria endemic states of India

BackgroundMalaria is a major public health problem in India and accounts for about 88% of malaria burden in South-East Asia. India alone accounted for 2% of total malaria cases globally. Anti-malarial drug resistance is one of the major problems for malaria control and elimination programme. Artemether-lumefantrine (AL) is the first-line treatment of uncomplicated Plasmodium falciparum in north eastern states of India since 2013 after confirming the resistance against sulfadoxine-pyrimethamine. In the present study, therapeutic efficacy of artemether-lumefantrine and k13 polymorphism was assessed in uncomplicated P. falciparum malaria.MethodsThis study was conducted at four community health centres located in Koraput district of Odisha, Bastar district of Chhattisgarh, Balaghat district of Madhya Pradesh and Gondia district of Maharashtra state. Patients with uncomplicated P. falciparum malaria were administered with fixed dose combination (6 doses) of artemether-lumefantrine for 3 days and clinical and parasitological response was recorded up to 28 days as per World Health Organization protocol. Nucleotide sequencing of msp1 and msp2 gene was performed to differentiate between recrudescence and reinfection. Amplification and sequencing of k13 propeller gene region covering codon 450–680 was also carried out to identify the polymorphism.ResultsA total 376 malaria patients who fulfilled the enrolment criteria as well as consented for the study were enrolled. Total 356 patients were followed up successfully up to 28 days. Overall, the adequate clinical and parasitological response was 98.9% and 99.4% with and without PCR correction respectively. No case of early treatment failure was observed. However, four cases (1.1%) of late parasitological failure were found from the Bastar district of Chhattisgarh. Genotyping of msp1 and msp2 confirmed 2 cases each of recrudescence and reinfection, respectively. Mutation analysis of k13 propeller gene showed one non-synonymous mutation Q613H in one isolate from Bastar.ConclusionsThe study results showed that artemether-lumefantrine is highly effective in the treatment of uncomplicated P. falciparum malaria among all age groups. No functional mutation in k13 was found in the study area. The data from this study will be helpful in implementation of artemether-lumefantrine in case of treatment failure by artesunate plus sulfadoxine-pyrimethamine.

Molecular surveillance of drug resistance: Plasmodium falciparum artemisinin resistance single nucleotide polymorphisms in Kelch protein propeller (K13) domain from Southern Pakistan

BackgroundK13 propeller (k13) polymorphism are useful molecular markers for tracking the emergence and spread of artemisinin resistance in Plasmodium falciparum. Polymorphisms are reported from Cambodia with rapid invasion of the population and almost near fixation in south East Asia. The study describes single nucleotide polymorphisms in Kelch protein propeller domain of P. falciparum associated with artemisinin resistance from Southern Pakistan.MethodsTwo hundred and forty-nine samples were collected from patients with microscopy confirmed P. falciparum malaria attending Aga Khan University Hospital during September 2015-April 2018. DNA was isolated using the whole blood protocol for the QIAmp DNA Blood Kit. The k13 propeller gene (k13) was amplified using nested PCR. Double-strand sequencing of PCR products was performed using Sanger sequencing methodology. Sequences were analysed with MEGA 6 and Bio edit software to identify specific SNP combinations.ResultsAll isolates analysed for k13 propeller allele were observed as wild-type in samples collected post implementation of ACT in Pakistan. C580Y, A675V, Y493H and R539T variants associated with reduced susceptibility to artemisinin-based combination therapy (ACT) were not found. Low frequency of M476I and C469Y polymorphisms was found, which is significantly associated with artemisinin resistance.ConclusionLow frequencies of both nonsynonymous and synonymous polymorphisms were observed in P. falciparum isolates circulating in Southern Pakistan. The absence of known molecular markers of artemisinin resistance in this region is favourable for anti-malarial efficacy of ACT. Surveillance of anti-malarial drug resistance to detect its emergence and spread need to be strengthened in Pakistan.

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.

A Review: Artemisinin-Based Combination Therapies [ACTs] and K13 Polymorphism

A review: artemisinin-based combination therapies [acts] and k13 polymorphism - Malaria is caused by four plasmodium species

Stable high frequencies of sulfadoxine\u2013pyrimethamine resistance associated mutations and absence of K13 mutations in Plasmodium falciparum 3 and 4\xa0years after the introduction of artesunate plus sulfadoxine\u2013pyrimethamine in Ujjain, Madhya Pradesh, India

BackgroundArtesunate plus sulfadoxine–pyrimethamine (ASP) is first-line treatment for uncomplicated Plasmodium falciparum malaria in most of India, except for six North-eastern provinces where treatment failure rates were high. In Ujjain, central India, the frequency of mutations associated with increased drug tolerance, but not overt resistance to sulfadoxine and pyrimethamine were 9% and > 80%, respectively, in 2009 and 2010, just prior to the introduction of ASP. The frequency of drug resistance associated mutations in Ujjain in 2015–2016 after 3–4 years of ASP use, are reported.MethodsBlood samples from patients with P. falciparum mono-infection verified by microscopy were collected on filter-paper at all nine major pathology laboratories in Ujjain city. Codons pfdhfr 16–185, pfdhps 436–632 and K13 407–689 were identified by sequencing. Pfcrt K76T and pfmdr1 N86Y were identified by restriction fragment length polymorphism.ResultsSulfadoxine–pyrimethamine resistance-associated pfdhfr 108 N and 59R alleles were found in 100/104 (96%) and 87/91 (96%) samples, respectively. Pfdhps 437G was found in 10/105 (10%) samples. Double mutant pfdhfr 59R + 108 N were found in 75/81 (93%) samples. Triple mutant pfdhfr 59R + 108 N and pfdhps 437G were found in 6/78 (8%) samples. Chloroquine-resistance-associated pfcrt 76T was found in 102/102 (100%). Pfmdr1 N86 and 86Y were identified in 83/115 (72%) and 32/115 (28%) samples, respectively.ConclusionThe frequency of P. falciparum with reduced susceptibility to sulfadoxine–pyrimethamine remained high, but did not appear to have increased significantly since the introduction of ASP. No polymorphisms in K13 associated with decreased artemisinin susceptibility were found. ASP probably remained effective, supporting continued ASP use.

A Review: Artemisinin-Based Combination Therapies [ACTs] and K13 Polymorphism

Malaria is caused by four plasmodium species in humans (P. falciparum, P. vivax, P. malariae and P. ovale) which spread from one person to another via the bite of female Anopheles mosquito. P. falciparum causes most deaths from malaria [1] and is most prevalent on the African continent whereas P. vivax has a wider geographical distribution [2]. According to the latest WHO estimates, released in December 2015, there were 214 million cases of malaria in 2015 and 438,000 deaths [3]. Existing strategies to control malaria include vector control, chemoprevention and case management [4]. Without a fruitful antibody that would offer security against malaria, we have to depend on anti-malarial prescription to treat just as lessen the odds of getting the disease [5-8]. Artemisinin in mix with other moderate acting medications is suggested for the treatment of P. falciparum [9,10].

Kelch 13 propeller gene polymorphism among Plasmodium falciparum isolates in Lagos, Nigeria: Molecular Epidemiologic Study.

To assess polymorphism in Kelch 13 gene of Plasmodium falciparum isolates in Lagos, Nigeria. 195 Plasmodium falciparum-positive dried blood spots collected from individuals that accessed diagnostic care at some health facilities and during community surveys across several Local Government Areas of Lagos State, Nigeria, were investigated for the presence of mutations in the K13 gene by nested polymerase chain reaction (PCR) using haplotype-specific probes and sequencing. Three mutant genotypes of K13 gene were observed: A578S in 0.5%, D464N in 0.5% and Q613H in 1.5%. The frequency of K13 polymorphism was 3.1%, while the remaining parasite population had the wild K13 propeller genes. No validated Kelch 13 polymorphism associated with artemisinin resistance was seen among P.falciparum isolates from Lagos, Nigeria. As no clinical study was done, this could not be correlated with artemisinin sensitivity.

In vitro susceptibility of Plasmodium falciparum isolates from the China-Myanmar border area to artemisinins and correlation with K13 mutations

Mutations in the Kelch domain of the K13 gene (PF3D7_1343700) were previously associated with artemisinin resistance in Plasmodium falciparum. This study followed the dynamics of the K13 polymorphisms in P. falciparum parasites from the China-Myanmar border area obtained in 2007–2016, and their in vitro sensitivities to artesunate (AS) and dihydroartemisinin (DHA). The 50% effective concentration (EC5072h) values of 133 culture-adapted field isolates to AS and DHA, measured by the conventional 72 h SYBR Green I-based assay, varied significantly among the parasites from different years; all were significantly higher than that of the reference strain 3D7. Compared with parasites from 2007 to 2008, ring survival rates almost doubled in parasites obtained in later years. Sequencing the full-length K13 genes identified 11 point mutations present in 85 (63.9%) parasite isolates. F446I was the predominant (55/133) variant, and its frequency was increased from 17.6% (3/17) in 2007 to 55.9% (19/34) in 2014–2016. No wild-type (WT) Kelch domain sequences were found in the 34 samples obtained from 2014 to 2016. In the 2014–2016 samples, a new mutation (G533S) appeared and reached 44.1% (15/34). Collectively, parasites with the Kelch domain mutations (after amino acid 440) had significantly higher ring survival rates than the WT parasites. Individually, F446I, G533S and A676D showed significantly higher ring survival rates than the WT. Although the drug sensitivity phenotypes measured by the RSA6h and EC5072h assays may be intrinsically linked to the in vivo clinical efficacy data, the values determined by these two assays were not significantly correlated. This study identified the trend of K13 mutations in parasite populations from the China-Myanmar border area, confirmed an overall correlation of Kelch domain mutations with elevated ring-stage survival rates, and emphasized the importance of monitoring the evolution and spread of parasites with reduced artemisinin sensitivity along the malaria elimination course.

No polymorphisms in K13-propeller gene associated with artemisinin resistance in Plasmodium falciparum isolated from Brazzaville, Republic of Congo

BackgroundIn the Republic of Congo, artemisinin-based combinations have been recommended for the treatment of uncomplicated malaria since 2006. However, the emergence of resistant parasites again these combinations in Southeast Asia is a threat for the control of this disease, especially in sub-Saharan Africa where the weight of the disease is important. Indeed, polymorphisms in Plasmodium falciparum K13-propeller gene have been involved in variations of drug sensitivity of Plasmodium falciparum to artemisinin-based combinations. The aim of the current study is to determine the prevalence of mutations of this gene in isolates collected in three health centers in Brazzaville.MethodsFrom May 2015 to May 2016, a total of 131, 259 and 416 samples from patients with suspected malaria were collected at the Laboratoire National de Santé Publique, Hôpital de Mfilou, and the CSI «Maman Mboualé» respectively. After DNA isolation, genotyping and sequencing of Plasmodium falciparum K13-propeller were performed in positive Plasmodium falciparum isolates identified after msp-2 gene genotyping.ResultsAll 806 samples collected were msp-2 genotyped and Plasmodium falciparum infections were confirmed in 287 samples with 43, 85, 159 samples from Laboratoire National de Santé Publique, Hôpital de Mfilou, and the CSI «Maman Mboualé» respectively. Of these 287 msp-2 positives samples, K13-propeller nested PCR products were successfully obtained from 145 (50.52%) isolates and sequences were generated from 127(87.58%) nested products. None of mutations that were associated with ACTs resistance in Southeast Asia were detected on the samples from three different study sites from Brazzaville. However, one mutation type was observed at position 578, where alanine was substituted by serine (A578S) in two isolates (1.57%, 2/127), those from the Hôpital de Mfilou. No mutation was found in isolates from the two other sites.ConclusionThe current study shows a very limited polymorphism in the K13-propeller gene in isolates from the Republic of Congo and K13 polymorphisms associate with ACT resistance are not present in this country. However, permanent and large surveillance of resistant parasite population using K13-propeller gene is recommended.

Absence of K13 Polymorphism in Plasmodium falciparum from Brazilian Areas Where the Parasite Is Endemic.

Plasmodium falciparum artemisinin-resistant parasites can be evaluated by examining polymorphisms in the kelch (PfK13) domain. A total of 69 samples from patients with falciparum malaria were analyzed. All samples were from areas in states in Brazil where the parasite was endemic: Acre (n = 14), Amapá (n = 15), Amazonas (n = 30), and Pará (n = 10). After DNA alignment with the 3D7 reference sequence, all samples were found to be wild type. These data provide a baseline for PfK13 and reinforce the pertinence of artemisinin combination therapy in Brazilian areas.

Evaluation of Plasmodium falciparum K13 gene polymorphism and susceptibility to dihydroartemisinin in an endemic area

Introduction: Plasmodium falciparum has developed resistance to artemisinin drugs in Southeast Asia, and its reduced sensitivity has been reported in other regions. This study aims to determine parasite susceptibility to the bioactive form of artemisinin derivatives- dihydroartemisinin (DHA)-, and to detect the K13 polymorphism in isolates from an endemic area of Nigeria.&#x0D; Methods: Ex-vivo response in 55 parasites isolates obtained from malaria-positive patients were exposed to pulse DHA concentration and cultured for 66 hours ex-vivo. Parasite ring stage survival (RSAex-vivo) relative to unexposed matched control was determined by microscopy, and parasite growth was compared using Mann-Whitney U-test at a significance level of P&lt;0.05. The Kelch propeller gene was amplified using specific primers, then sequenced and analyzed for single nucleotide polymorphisms (SNPs), which were compared to reference PF3D7_1343700.&#x0D; Results: Overall, 151 of 375 (40.2%) individuals were positive during the study period. In 55 selected isolates, there was increased growth in unexposed wells but growth was inhibited in DHA-exposed wells, with growth rate between 14.9 – 96.7%. The mean RSAex-vivo value was 0.18 ± 0.09%, 95% CI (0.15-0.20). There was no significant mutation of the K13 gene in the parasite isolates evaluated.&#x0D; Conclusions: Plasmodium falciparum isolates from this endemic area show high sensitivity to dihydroartemisinin ex-vivo, with no mutations conferring artemisinin resistance. Continuous monitoring of parasite susceptibility to artemisinin combination drugs should be intensified to reduce chances of artemisinin resistance in endemic areas.

Plasmodium falciparum Kelch 13 mutations and treatment response in patients in Hpa-Pun District, Northern Kayin State, Myanmar

BackgroundArtemisinin resistance, linked to polymorphisms in the Kelch gene on chromosome 13 of Plasmodium falciparum (k13), has outpaced containment efforts in South East Asia. For national malaria control programmes in the region, it is important to establish a surveillance system which includes monitoring for k13 polymorphisms associated with the clinical phenotype.MethodsBetween February and December 2013, parasite clearance was assessed in 35 patients with uncomplicated P. falciparum treated with artesunate monotherapy followed by 3-day ACT in an isolated area on the Myanmar–Thai border with relatively low artemisinin drug pressure. Molecular testing for k13 mutations was performed on dry blood spots collected on admission.ResultsThe proportion of k13 mutations in these patients was 41.7%, and only 5 alleles were detected: C580Y, I205T, M476I, R561H, and F446I. Of these, F446I was the most common, and was associated with a longer parasite clearance half-life (median) 4.1 (min–max 2.3–6.7) hours compared to 2.5 (min–max 1.6–8.7) in wildtype (p = 0·01). The prevalence of k13 mutant parasites was much lower than the proportion of k13 mutants detected 200 km south in a much less remote setting where the prevalence of k13 mutants was 84% with 15 distinct alleles in 2013 of which C580Y predominated.ConclusionsThis study provides evidence of artemisinin resistance in a remote part of eastern Myanmar. The prevalence of k13 mutations as well as allele diversity varies considerably across short distances, presumably because of historical patterns of artemisinin use and population movements.

Detection of the Plasmodium falciparum Kelch-13 gene P553L mutation in sporozoites isolated from mosquito salivary glands in South-Central Vietnam

BackgroundPlasmodium falciparum has developed resistance against artemisinin in Southeast Asia. Mutations in the P. falciparum Kelch-13 (Pfk13) gene are associated with artemisinin resistance in vitro and in vivo. We investigated the prevalence of mutations in PfK13 from sporozoite-stage parasites isolated from the salivary glands of Anopheles dirus mosquitoes.MethodsMosquitoes were caught by human-landing catches at two locations within the Khanh Phu commune, South-Central Vietnam. Identification of Anopheles species was performed based on morphological features and nucleotide sequence analysis. Sporozoite-infected salivary glands were stored on filter paper and at 4–6 °C. A nested-PCR targeting the small subunit ribosomal RNA gene was used for Plasmodium species identification. Pfk13 was amplified by nested PCR, and subjected to nucleotide sequencing.ResultsFive of 33 P. falciparum sporozoite samples carried the P553L mutation at the PfK13 locus. This mutation has been recorded previously in Vietnam, but not in Khanh Hoa province, were surveys of K13 polymorphism have not previously been carried out.ConclusionThese results demonstrate the utility of mosquito-stage malaria parasite samples for studies on the molecular epidemiology of drug resistance.

K13 Kelch Propeller Domain and mdr1 Sequence Polymorphism in Field Isolates From Northeast Region, India

Emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia has made an intense warning to global malaria control programme since Southeast Asia is playing a key role in the evolution of drug resistant malaria parasites. Several polymorphisms in K13 gene have been designated with developing resistance either in vitro or in vivo . In this study, polymorphisms within K13 kelch propeller domain and Pf mdr1 gene were investigated in a total of 134 P. falciparum field isolates circulating in 3 states of Northeast region of India, namely, Assam, Arunachal Pradesh, and Tripura because information regarding this is limited from this region. Six polymorphisms were detected, out of which 2 were new. A481 V mutation was found in 4.5% of the total field isolates. A675 V and D702 N new mutations were present in 3.7% and 1.5% isolates, respectively. Synonymous polymorphism at codon 703 was highly observed (6.7%) than other polymorphisms. N86 Y allele in Pf mdr1 gene was also noticed in isolates that contained mutation in K13 propeller domain, but N86 wild-type allele was found comparatively higher in frequency within overall isolates. Moreover, both nucleotide and haplotype diversities in K13 gene were high in Arunachal Pradesh isolates than other 2 states. This is the first report from Northeast region of India with evidence of A675 V candidate mutation along with 2 other new mutations which pays attention for further molecular surveillance in this region to document detailed scenario of K13 polymorphism pattern corresponding to artemisinin resistance.

Drug-Resistant Polymorphisms and Copy Numbers in Plasmodium falciparum, Mozambique, 2015.

One of the fundamental steps toward malaria control is the use of antimalarial drugs. The success of antimalarial treatment can be affected by the presence of drug-resistant populations of Plasmodium falciparum. To assess resistance, we used molecular methods to examine 351 P. falciparum isolates collected from 4 sentinel sites in Mozambique for K13, pfmdr1, pfcrt, and pfdhps polymorphisms and for plasmepsin2 (pfpm2) and pfmdr1 copy numbers. We found multiple copies of pfpm2 in 1.1% of isolates. All isolates carried K13 wild-type alleles (3D7-like), except 4 novel polymorphisms (Leu619Leu, Phe656Ile, Val666Val, Gly690Gly). Prevalence of isolates with pfcrt mutant (K76T) allele was low (2.3%). Prevalence of isolates with pfdhps mutant alleles (A437G and K540E) was >80%, indicating persistence of sulfadoxine/pyrimethamine resistance; however, markers of artemisinin were absent, and markers of piperaquine resistance were low. Piperaquine resistance isolates may spread in Mozambique as dihydroartemisinin/piperaquine drug pressure increases.

Artemisinin Resistance-Associated K13 Polymorphisms of Plasmodium falciparum in Southern Rwanda, 2010-2015.

Emerging artemisinin resistance is a threat to global malaria control. Mutations in the Plasmodium falciparum Kelch 13 (K13) propeller domain confer artemisinin resistance and constitute molecular markers for its detection and monitoring. We sequenced 222 P. falciparum isolates obtained from community children in the Huye District of southern Rwanda in 2010, 2014, and 2015 to investigate the presence of K13 polymorphisms. No polymorphisms were observed in 2010 but they were present in 2.5% and 4.5% in 2014 and 2015, respectively. In 2015, two isolates showed candidate K13 resistance mutations (P574L and A675V), which are common in southeast Asia and associated with delayed parasite clearance. K13 polymorphisms in southern Rwanda are infrequent but include variants associated with artemisinin resistance. Establishing correlations with local treatment response and in vitro resistance assays are needed in addition to further monitoring K13 polymorphisms in the study area.

Polymorphisms in K13, pfcrt, pfmdr1, pfdhfr, and pfdhps in parasites isolated from symptomatic malaria patients in Burkina Faso.

Background: The emergence of resistance to artemisinin derivatives in western Cambodia is threatening to revert the recent advances made toward global malaria control and elimination. Known resistance-mediating polymorphisms in the K13, pfcrt, pfmdr1, pfdhfr, and pfdhps genes are of greatest importance for monitoring the spread of antimalarial drug resistance. Methods: Samples for the present study were collected from 244 patients with uncomplicated malaria in health centers of Bobo-Dioulasso, Burkina Faso. Blood sample was collected on filter paper before the subject received any treatment. The parasite DNA was then extracted and amplified by Polymerase Chain Reaction (PCR) to evaluate the prevalence of polymorphism of pfcrtK76T, pfmdr1 (N86Y, Y184F), and pfdhps (A437G, K540E). The K13 gene polymorphism was analyzed by nested PCR followed by sequencing. Results: The overall results showed 2.26% (5/221) of K13 synonymous mutant alleles (two C469C, one Y493Y, one G496G, and one V589V), 24.78%, 19.58%, 68.75%, 60.9%, 53.7%, 63.8%, and 64.28%, respectively, for mutant pfcrt 76T, pfmdr1-86Y, pfmdr1-184F, pfdhfr51I, pfdhfr59R, pfdhfr108N, and pfdhps 437G. We did not report any mutation at codon 540 of pfdhps. Conclusion: These results provide baseline prevalence of known drug resistance polymorphisms and suggest that artemisinin combination therapies may retain good efficacy in the treatment of uncomplicated malaria in Burkina Faso.