Prevalence and dynamics of antimalarial drug resistance mutations among the Plasmodium falciparum isolates in TAK Province, Thailand, during the period of 1998-2001.

Despite the overall decline in malaria cases in Thailand, continuous surveillance in endemic areas remains crucial. This retrospective analysis examined Plasmodium falciparum samples from Tak province, Thailand, collected in 1998, 1999, and 2001, to investigate the prevalence and evolution of antimalarial genotypic drug resistance. The study revealed a high prevalence of drug-resistant P. falciparum, particularly to mefloquine and sulfadoxine/pyrimethamine, with significant mutations in genes associated with resistance. Notably, mutations indicative of artemisinin resistance, such as those in the kelch13 gene, were detected at low frequencies, suggesting an evolving resistance pattern. The underlying cause of these resistance mutations appears to be the historical and widespread use of these antimalarial drugs, which exerted selective pressure on the parasite population. These findings underscore the necessity of ongoing surveillance and adaptive control strategies to manage drug resistance, guide treatment policies, and prevent potential outbreaks, even as malaria cases decrease. Continuous monitoring and research are imperative to sustain malaria elimination efforts and address the dynamic challenges posed by evolving drug-resistant strains.

Plasmodium falciparum kelch13 polymorphisms identified after treatment failure with artemisinin-based combination therapy in Niger.

BACKGROUND: Artemisinin-based combination therapy (ACT) is the most effective treatment for malaria, and has significantly reduced morbimortality. Polymorphisms associated with the Plasmodium falciparum Kelch gene (Pfkelch13) have been associated with delayed parasite clearance even with ACT treatment. METHODS: The Pfkelch13 gene was sequenced from P. falciparum infected patients (n = 159) with uncomplicated malaria in Niger. An adequate clinical and parasitological response (ACPR) was reported in 155 patients. Four (n = 4) patients had treatment failure (TF) that were not reinfections-two of which had late parasitological failures (LPF) and two had late clinical failures (LCF). RESULTS: Thirteen single nucleotide polymorphisms (SNPs) were identified of which seven were non-synonymous (C469R, T508S, R515T, A578S, I465V, I437V, F506L,), and three were synonymous (P443P, P715P, L514L). Three SNP (C469R, F506L, P715P) were present before ACT treatment, while seven mutations (C469R, T508S, R515T, L514L, P443P, I437V, I465V) were selected by artemether/lumefantrine (AL)-five of which were non-synonymous (C469R, T508S, R515T, I437V, I465V). Artesunate/amodiaquine (ASAQ) has selected any mutation. One sample presented three cumulatively non-synonymous SNPs-C469R, T508S, R515T. CONCLUSIONS: This study demonstrates intra-host selection of Pfkelch13 gene by AL. The study highlights the importance of LCF and LPF parasites in the selection of resistance to ACT. Further studies using gene editing are required to confirm the potential implication of resistance to ACT with the most common R515T and T508S mutations. It would also be important to elucidate the role of cumulative mutations.

Ex vivo RSA and pfkelch13 targeted-amplicon deep sequencing reveal parasites susceptibility to artemisinin in Senegal, 2017.

BACKGROUND: Malaria control is highly dependent on the effectiveness of artemisinin-based combination therapy (ACT), the current frontline malaria curative treatment. Unfortunately, the emergence and spread of parasites resistant to artemisinin (ART) derivatives in Southeast Asia and South America, and more recently in Rwanda and Uganda (East Africa), compromise their long-term use in sub-Saharan Africa, where most malaria deaths occur. METHODS: Here, ex vivo susceptibility to dihydroartemisinin (DHA) was evaluated from 38 Plasmodium falciparum isolates collected in 2017 in Thiès (Senegal) expressed in the Ring-stage Survival Assay (RSA). Both major and minor variants were explored in the three conserved-encoding domains of the pfkelch13 gene, the main determinant of ART resistance using a targeted-amplicon deep sequencing (TADS) approach. RESULTS: All samples tested in the ex vivo RSA were found to be susceptible to DHA (parasite survival rate < 1%). The non-synonymous mutations K189T and K248R in pfkelch13 were observed each in one isolate, as major (99%) or minor (5%) variants, respectively. CONCLUSION: The results suggest that ART is still fully effective in the Thiès region of Senegal in 2017. Investigations combining ex vivo RSA and TADS are a useful approach for monitoring ART resistance in Africa.

Therapeutic efficacy of artesunate-amodiaquine and artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in Chad: clinical and genetic surveillance.

BACKGROUND: Artesunate-amodiaquine (AS-AQ) and artemether-lumefantrine (AL) are the currently recommended first-and second-line therapies for uncomplicated Plasmodium falciparum infections in Chad. This study assessed the efficacy of these artemisinin-based combinations, proportion of day 3 positive patients, proportions of molecular markers associated with P. falciparum resistance to anti-malarial drugs and variable performance of HRP2-based malaria rapid diagnostic tests (RDTs). METHODS: A single-arm prospective study assessing the efficacy of AS-AQ and AL at three sites (Doba, Kelo and Koyom) was conducted between November 2020 to January 2021. Febrile children aged 6 to 59 months with confirmed uncomplicated P. falciparum infection were enrolled sequentially first to AS-AQ and then AL at each site and followed up for 28 days. The primary endpoint was PCR-adjusted adequate clinical and parasitological response (ACPR). Samples collected on day 0 were analysed for mutations in pfkelch13, pfcrt, pfmdr-1, pfdhfr, pfdhps genes and deletions in pfhrp2/pfhrp3 genes. RESULTS: By the end of 28-day follow-up, per-protocol PCR corrected ACPR of 97.8% (CI 95% 88.2-100) in Kelo and 100% in Doba and Kayoma were observed among AL treated patients. For ASAQ, 100% ACPR was found in all sites. All, but one patient, did not have parasites detected on day 3. Out of the 215 day 0 samples, 96.7% showed pfkelch13 wild type allele. Seven isolates carried nonsynonymous mutations not known to be associated artemisinin partial resistance (ART-R). Most of samples had a pfcrt wild type allele (79% to 89%). The most prevalent pfmdr-1 allele detected was the single mutant 184F (51.2%). For pfdhfr and pfdhps mutations, the quintuple mutant allele N51I/C59R/S108N + G437A/540E responsible for SP treatment failures in adults and children was not detected. Single deletion in the pfhrp2 and pfhrp3 gene were detected in 10/215 (4.7%) and 2/215 (0.9%), respectively. Dual pfhrp2/pfhrp3 deletions, potentially threatening the efficacy of HRP2-based RDTs, were observed in 5/215 (2.3%) isolates. CONCLUSION: The results of this study confirm that AS-AQ and AL treatments are highly efficacious in study areas in Chad. The absence of known pfkelch13 mutations in the study sites and the high parasite clearance rate at day 3 suggest the absence of ART-R. The absence of pfdhfr/pfdhps quintuple or sextuple (quintuple + 581G) mutant supports the continued use of SP for IPTp during pregnancy. The presence of parasites with dual pfhrp2/pfhrp3 deletions, potentially threatening the efficacy of HRP2-based RDTs, warrants the continued surveillance. Trial registration ACTRN12622001476729.

Multidrug-Resistant Plasmodium falciparum Parasites in the Central Highlands of Vietnam Jeopardize Malaria Control and Elimination Strategies.

Plasmodium falciparum resistance to dihydroartemisinin-piperaquine has spread through the Greater Mekong Subregion to southwestern Vietnam. In 2018 to 2019, we collected 127 P. falciparum isolates from Dak Nong (36), Dak Lak (55), Gia Lai (13), and Kon Tum (23) provinces in Vietnam's Central Highlands and found parasites bearing the Pfkelch13 C580Y mutation and multiple plasmepsin 2/3 genes (mean prevalence, 17.9%; range, 4.3% to 27.8%), conferring resistance to dihydroartemisinin-piperaquine. This information is important for drug policy decisions in Vietnam.

Pyronaridine-Artesunate (Pyramax) for Treatment of Artemisinin- and Piperaquine-Resistant Plasmodium falciparum in the Central Highlands of Vietnam.

The rise in Plasmodium falciparum resistance to dihydroartemisinin-piperaquine in Vietnam justifies the need to evaluate alternative artemisinin-based combination therapies. Between July 2018 and October 2019, a single-arm trial of pyronaridine-artesunate (Pyramax, PA) was conducted in Dak Nong province, Vietnam. PA (3-day course) was administered to adults and children infected with P. falciparum. PA was well tolerated by the participants. The proportion of patients with Day 42 PCR-corrected adequate clinical and parasitological response was 95.2% (95% confidence interval [CI], 82.3 to 98.8, n = 40/42) for treating falciparum malaria. The median parasite clearance half-life was 6.7 h (range, 2.6 to 11.9) and the median parasite clearance time was 72 h (range, 12 to 132) with 44.9% (22/49) of patients having positive blood films at 72 h. The two patients that recrudesced had comparable Day 7 blood pyronaridine concentrations (39.5 and 39.0 ng/ml) to the 40 patients who did not recrudesce (median 43.4 ng/ml, 95% CI, 35.1 to 54.9). Ring-stage and piperaquine survival assays revealed that of the 29 P. falciparum isolates collected from the patients before PA treatment, 22 (75.9%) had reduced susceptibility to artemisinins and 17 (58.6%) were resistant to piperaquine. Genotyping confirmed that 92.0% (46/50) of falciparum patients were infected with parasites bearing the Pfkelch13 C580Y mutation associated with artemisinin resistance. Of these, 56.0% (28/50) of the isolates also had multiple copies of the plasmepsin 2/3 genes responsible for piperaquine resistance. Overall, PA was effective in treating P. falciparum in the Central Highlands of Vietnam. (This study has been registered at AustralianClinicalTrials.gov.au under trial ID ACTRN12618001429246.).

A randomized controlled trial of dihydroartemisinin-piperaquine, artesunate-mefloquine and extended artemether-lumefantrine treatments for malaria in pregnancy on the Thailand-Myanmar border.

BACKGROUND: Artemisinin and artemisinin-based combination therapy (ACT) partner drug resistance in Plasmodium falciparum have spread across the Greater Mekong Subregion compromising antimalarial treatment. The current 3-day artemether-lumefantrine regimen has been associated with high treatment failure rates in pregnant women. Although ACTs are recommended for treating Plasmodium vivax malaria, no clinical trials in pregnancy have been reported. METHODS: Pregnant women with uncomplicated malaria on the Thailand-Myanmar border participated in an open-label randomized controlled trial comparing dihydroartemisinin-piperaquine (DP), artesunate-mefloquine (ASMQ) and a 4-day artemether-lumefantrine regimen (AL+). The primary endpoint for P. falciparum infections was the PCR-corrected cure rate and for P. vivax infections was recurrent parasitaemia, before delivery or day 63, whichever was longer, assessed by Kaplan-Meier estimate. RESULTS: Between February 2010 and August 2016, 511 pregnant women with malaria (353 P. vivax, 142 P. falciparum, 15 co-infections, 1 Plasmodium malariae) were randomized to either DP (n=170), ASMQ (n=169) or AL+ (n=172) treatments. Successful malaria elimination efforts in the region resulted in premature termination of the trial. The majority of women had recurrent malaria (mainly P. vivax relapses, which are not prevented by these treatments). Recurrence-free proportions (95% confidence interval [95% CI]) for vivax malaria were 20.6% (5.1-43.4) for DP (n=125), 46.0% (30.9-60.0) for ASMQ (n=117) and 28.7% (10.0-50.8) for AL+ (n=126). DP and ASMQ provided longer recurrence-free intervals. PCR-corrected cure rates (95% CI) for falciparum malaria were 93.7% (81.6-97.9) for DP (n=49), 79.6% (66.1-88.1) for AMSQ (n=55) and 87.5% (74.3-94.2) for AL+ (n=50). Overall 65% (85/130) of P. falciparum infections had Pfkelch13 propeller mutations which increased over time and recrudescence occurred almost exclusively in them; risk ratio 9.42 (95% CI 1.30-68.29). Among the women with falciparum malaria, 24.0% (95% CI 16.8-33.6) had P. vivax parasitaemia within 4 months. Nausea, vomiting, dizziness and sleep disturbance were more frequent with ASMQ. Miscarriage, small-for-gestational-age and preterm birth did not differ significantly among the treatment groups, including first trimester exposures (n=46). CONCLUSIONS: DP was well tolerated and safe, and was the only drug providing satisfactory efficacy for P. falciparum-infected pregnant woman in this area of widespread artemisinin resistance. Vivax malaria recurrences are very common and warrant chloroquine prophylaxis after antimalarial treatment in this area. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT01054248 , registered on 22 January 2010.

Absence of Plasmodium falciparum artemisinin resistance gene mutations eleven years after the adoption of artemisinin-based combination therapy in Nigeria.

BACKGROUND: The occurrence of artemisinin resistance (ART)-associated polymorphism of Plasmodium falciparum K13-propeller (pfk13) gene before and after the introduction of artemisinin-based combination therapy (ACT) in two regions of Nigeria was investigated in this study. Regular surveillance is necessary to make a definite conclusion on the emergence and pattern of possible resistance to ART. METHODS: This cross-sectional study was carried out in the Southwestern and Southeastern geopolitical zones of Nigeria. A total of 150, 217, and 475 participants were enrolled for the study in the Southwest (2004_Group A), Southwest (2015_Group B), and southeast (2015_Group C), respectively. Blood samples were collected from the study participants for DNA extraction and a nested PCR for P. falciparum identification. Samples that were positive for P. falciparum were genotyped for the pfk13 gene using the Sanger sequencing method. The single nucleotide polymorphisms were analysed using the Bioedit software. RESULTS: A total of 116, 125, and 83 samples were positive for P. falciparum, respectively for the samples collected from the Southwest (2004 and 2015) and southeast (2015). Parasite DNA samples collected from febrile children in 2004 (Group A; n = 71) and 2015 (Group B; n = 73) in Osogbo Western Nigeria and 2015_Group C (n = 36) in southeast Nigeria were sequenced successfully. This study did not observe mutations associated with the in vitro resistance in southeast Asia, such as Y493H, R539T, I543T, and C580Y. Two new polymorphisms V520A and V581I were observed in two samples collected in Osogbo, Southwest Nigeria. These two mutations occurred in the year 2004 (Group A) before the introduction of ACT. Six mutations were identified in 17% of the samples collected in southeast Nigeria. One of these mutations (D547G) was non-synonymous, while the remaining (V510V, R515R, Q613Q, E688E, and N458N) were synonymous. Also, one (2%) heterozygote allele was identified at codon 458 in the 2015 (Group C) samples. CONCLUSIONS: None of the mutations observed in this study were previously validated to be associated with ART resistance. These results, therefore, suggest that artemisinin is likely to remain highly effective in treating malaria in the study areas that are malarious zone.

A LAMP-SNP Assay Detecting C580Y Mutation in Pfkelch13 Gene from Clinically Dried Blood Spot Samples.

Artemisinin resistance (ART) has been confirmed in Greater Mekong Sub-region countries. Currently, C580Y mutation on Pfkelch13 gene is known as the molecular marker for the detection of ART. Rapid and accurate detection of ART in field study is essential to guide malaria containment and elimination interventions. A simple method for collection of malaria-infected blood is to spot the blood on filter paper and is fast and easy for transportation and storage in the field study. This study aims to evaluate LAMP-SNP assay for C580Y mutation detection by introducing an extra mismatched nucleotide at the 3' end of the FIP primer. The LAMP-SNP assay was performed in a water bath held at a temperature of 56°C for 45 min. LAMP-SNP products were interpreted by both gel-electrophoresis and HNB-visualized changes in color. The method was then tested with 120 P. falciparum DNA from dried blood spot samples. In comparing the LAMP-SNP assay results with those from DNA sequencing of the clinical samples, the 2 results fully agreed to detect C580Y. The sensitivity and specificity of the LAMP-SNP assay showed 100%. There were no cross-reactions with other Plasmodium species and other Pfkelch13 mutations. The LAMP-SNP assay performed in this study was rapid, reliable, and useful in detecting artemisinin resistance in the field study.

Molecular assessment of kelch13 non-synonymous mutations in Plasmodium falciparum isolates from Central African Republic (2017-2019).

BACKGROUND: Over the last decade, artemisinin-based combination therapy (ACT) has contributed substantially to the decrease in malaria-related morbidity and mortality. The emergence of Plasmodium falciparum parasites resistant to artemisinin derivatives in Southeast Asia and the risk of their spread or of local emergence in sub-Saharan Africa are a major threat to public health. This study thus set out to estimate the proportion of P. falciparum isolates, with Pfkelch13 gene mutations associated with artemisinin resistance previously detected in Southeast Asia. METHODS: Blood samples were collected in two sites of Bangui, the capital of the Central African Republic (CAR) from 2017 to 2019. DNA was extracted and nested PCR were carried out to detect Plasmodium species and mutations in the propeller domain of the Pfkelch13 gene for P. falciparum samples. RESULTS: A total of 255 P. falciparum samples were analysed. Plasmodium ovale DNA was found in four samples (1.57%, 4/255). Among the 187 samples with interpretable Pfkelch13 sequences, four samples presented a mutation (2.1%, 4/187), including one non-synonymous mutation (Y653N) (0.5%, 1/187). This mutation has never been described as associated with artemisinin resistance in Southeast Asia and its in vitro phenotype is unknown. CONCLUSION: This preliminary study indicates the absence of Pfkelch13 mutant associated with artemisinin resistance in Bangui. However, this limited study needs to be extended by collecting samples across the whole country along with the evaluation of in vitro and in vivo phenotype profiles of Pfkelch13 mutant parasites to estimate the risk of artemisinin resistance in the CAR.

Susceptibility of Plasmodium falciparum to artemisinins and Plasmodium vivax to chloroquine in Phuoc Chien Commune, Ninh Thuan Province, south-central Vietnam.

BACKGROUND: Reduced artemisinin susceptibility and artemisinin-based combination therapy (ACT)-resistance against Plasmodium falciparum and chloroquine (CQ)-resistant P. vivax malaria has been reported in Vietnam. Two therapeutic efficacy studies were conducted in Thuan Bac District (Ninh Thuan Province, Vietnam) in 2015 and 2016 to determine the extent of reduced artemisinin susceptibility and ACT resistant falciparum malaria, and CQ-resistant vivax malaria were present. METHODS: Twenty-seven patients with falciparum malaria were randomized to receive artesunate alone (AS ~ 4 mg/kg/day) for 4 days followed by dihydroartemisinin (DHA) (2.2 mg/kg)-piperaquine (PPQ) (18 mg/kg) daily for 3 days or artemether (AM) (1.7 mg/kg)-lumefantrine (LUM) (12 mg/kg) twice daily for 3 days. Sixteen subjects with vivax malaria received CQ (total 25 mg/kg over 3 days). The therapeutic efficacy study for treating falciparum malaria was complemented with molecular analysis for artemisinin and piperaquine resistance, and in vitro drug susceptibility testing. Patient's drug exposure following both falciparum and vivax treatment studies was determined. RESULTS: Twenty-five of 27 patients treated with the artemisinin regimens completed the 42-day follow-up period. None had parasites present on day 3 after commencing treatment with no incidence of recrudescence (100% curative rate). One patient on AS + DHA-PPQ was lost to follow-up and one patient had Plasmodium falciparum and Plasmodium vivax infection on day 0 by PCR. Of the vivax patients, 15 of 16 completed CQ treatment and two had a recurrence of vivax malaria on day 28, a failure rate of 13.3% (2/15). No mutations in the Pfkelch-13 gene for artemisinin resistance or exo-E415G gene polymorphism and amplification in plasmepsins 2 and 3 for piperaquine resistance were observed. In vitro testing of patient's falciparum parasites indicated susceptibility (low IC50 nM values) to dihydroartemisinin, lumefantrine, piperaquine and pyronaridine. Patient's drug exposure to artesunate and lumefantrine was comparable to published data, however, blood CQ concentrations were lower. CONCLUSIONS: Clinical findings, molecular analysis and in vitro testing revealed that the falciparum parasites at Phuoc Chien Commune were artemisinin susceptible. The clinical failure rate of the 15 vivax patients who completed CQ treatment was 13%. Further studies are required to determine whether CQ-resistant vivax malaria is present at the commune.

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

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

Multi-omics Based Identification of Specific Biochemical Changes Associated With PfKelch13-Mutant Artemisinin-Resistant Plasmodium falciparum.

Background: The emergence of artemisinin resistance in the malaria parasite Plasmodium falciparum poses a major threat to the control and elimination of malaria. Certain point mutations in the propeller domain of PfKelch13 are associated with resistance, but PfKelch13 mutations do not always result in clinical resistance. The underlying mechanisms associated with artemisinin resistance are poorly understood, and the impact of PfKelch13 mutations on cellular biochemistry is not defined. Methods: This study aimed to identify global biochemical differences between PfKelch13-mutant artemisinin-resistant and -sensitive strains of P. falciparum by combining liquid chromatography-mass spectrometry (LC-MS)-based proteomics, peptidomics, and metabolomics. Results: Proteomics analysis found both PfKelch13 mutations examined to be specifically associated with decreased abundance of PfKelch13 protein. Metabolomics analysis demonstrated accumulation of glutathione and its precursor, gamma-glutamylcysteine, and significant depletion of 1 other putative metabolite in resistant strains. Peptidomics analysis revealed lower abundance of several endogenous peptides derived from hemoglobin (HBα and HBß) in the artemisinin-resistant strains. Conclusion: PfKelch13 mutations associated with artemisinin resistance lead to decreased abundance of PfKelch13 protein, decreased hemoglobin digestion, and enhanced glutathione production.

Artemisinin resistance without pfkelch13 mutations in Plasmodium falciparum isolates from Cambodia.

BACKGROUND: Artemisinin resistance is associated with delayed parasite clearance half-life in vivo and correlates with ring-stage survival under dihydroartemisinin in vitro. Both phenotypes are associated with mutations in the PF3D7_1343700 pfkelch13 gene. Recent spread of artemisinin resistance and emerging piperaquine resistance in Southeast Asia show that artemisinin combination therapy, such as dihydroartemisinin-piperaquine, are losing clinical effectiveness, prompting investigation of drug resistance mechanisms and development of strategies to surmount emerging anti-malarial resistance. METHODS: Sixty-eight parasites isolates with in vivo clearance data were obtained from two Tracking Resistance to Artemisinin Collaboration study sites in Cambodia, culture-adapted, and genotyped for pfkelch13 and other mutations including pfmdr1 copy number; and the RSA0-3h survival rates and response to antimalarial drugs in vitro were measured for 36 of these isolates. RESULTS: Among these 36 parasites one isolate demonstrated increased ring-stage survival for a PfKelch13 mutation (D584V, RSA0-3h = 8%), previously associated with slow clearance but not yet tested in vitro. Several parasites exhibited increased ring-stage survival, yet lack pfkelch13 mutations, and one isolate showed evidence for piperaquine resistance. CONCLUSIONS: This study of 68 culture-adapted Plasmodium falciparum clinical isolates from Cambodia with known clearance values, associated the D584V PfKelch13 mutation with increased ring-stage survival and identified parasites that lack pfkelch13 mutations yet exhibit increased ring-stage survival. These data suggest mutations other than those found in pfkelch13 may be involved in conferring artemisinin resistance in P. falciparum. Piperaquine resistance was also detected among the same Cambodian samples, consistent with reports of emerging piperaquine resistance in the field. These culture-adapted parasites permit further investigation of mechanisms of both artemisinin and piperaquine resistance and development of strategies to prevent or overcome anti-malarial resistance.

Absence of in vivo selection for K13 mutations after artemether-lumefantrine treatment in Uganda.

BACKGROUND: Individual drug treatment may select resistant parasites in the human body, a process termed in vivo selection. Some single nucleotide polymorphisms in Plasmodium falciparum chloroquine-resistance transporter (pfcrt) and multidrug resistance gene 1 (pfmdr1) genes have been reportedly selected after artemether-lumefantrine treatment. However, there is a paucity of data regarding in vivo selection of P. falciparum Kelch propeller domain (pfkelch13) polymorphisms, responsible for artemisinin-resistance in Asia, and six putative background mutations for artemisinin resistance; D193Y in ferredoxin, T484I in multiple resistance protein 2, V127M in apicoplast ribosomal protein S10, I356T in pfcrt, V1157L in protein phosphatase and C1484F in phosphoinositide-binding protein. METHODS: Artemether-lumefantrine efficacy study with a follow-up period of 28 days was conducted in northern Uganda in 2014. The above-mentioned genotypes were comparatively analysed before drug administration and on days; 3, 7, and 28 days after treatment. RESULTS: In 61 individuals with successful follow-up, artemether-lumefantrine treatment regimen was very effective with PCR adjusted efficacy of 95.2%. Among 146 isolates obtained before treatment, wild-type alleles were observed in 98.6% of isolates in pfkelch13 and in all isolates in the six putative background genes except I356T in pfcrt, which had 2.4% of isolates as mixed infections. In vivo selection study revealed that all isolates detected in the follow-up period harboured wild type alleles in pfkelch13 and the six background genes. CONCLUSION: Mutations in pfkelch13 and the six background genes may not play an important role in the in vivo selection after artemether-lumefantrine treatment in Uganda. Different mechanisms might rather be associated with the existence of parasites after treatment.

How do the mutations in PfK13 protein promote anti-malarial drug resistance?

Plasmodium falciparum develops resistance to artemisinin upon exposure to the anti-malarial drug. Various mutations in the Plasmodium falciparum Kelch13 (PfK13) protein such as Y493H, R539T, I543T and C580Y have been associated with anti-malarial drug resistance. These mutations impede the regular ubiquitination process that eventually invokes drug resistance. However, the relationship between the mutation and the mechanism of drug resistance has not yet been fully elucidated. The comparative protein dynamics are studied by performing the classical molecular dynamics (MD) simulations and subsequent analysis of the trajectories adopting root-mean-square fluctuations, the secondary-structure predictions and the dynamical cross-correlation matrix analysis tools. Here, we observed that the mutations in the Kelch-domain do not have any structural impact on the mutated site; however, it significantly alters the overall dynamics of the protein. The loop-region of the BTB-domain especially for Y493H and C580Y mutants is found to have the enhanced dynamical fluctuations. The enhanced fluctuations in the BTB-domain could affect the protein-protein (PfK13-Cullin) binding interactions in the ubiquitination process and eventually lead to anti-malarial drug resistance.Communicated by Ramaswamy H. Sarma.

The many paths to artemisinin resistance in Plasmodium falciparum.

Emerging resistance against artemisinin (ART) poses a major challenge in controlling malaria. Parasites with mutations in PfKelch13, the major marker for ART resistance, are known to reduce hemoglobin endocytosis, induce unfolded protein response (UPR), elevate phosphatidylinositol-3-phosphate (PI3P) levels, and stimulate autophagy. Nonetheless, PfKelch13-independent resistance is also reported, indicating extensive complementation by reconfiguration in the parasite metabolome and transcriptome. These findings implicate that there may not be a single 'universal identifier' of ART resistance. This review sheds light on the molecular, transcriptional, and metabolic pathways associated with ART resistance, while also highlighting the interplay between cellular heterogeneity, environmental stress, and ART sensitivity.

Emergence of artemisinin-based combination treatment failure in patients returning from sub-Saharan Africa with P. falciparum malaria.

BACKGROUND: Artemisinin-based combination therapies (ACTs) are recommended as first-line treatment against uncomplicated Plasmodium falciparum infection. Mutations in the PfKelch13 (PF3D7_1343700) gene led to resistance to artemisinin in Southeast Asia. Mutations in the Pfcoronin (PF3D7_1251200) gene confer reduced artemisinin susceptibility in vitro to an African Plasmodium strain, but their role in clinical resistance has not been established. METHODS: We conducted a retrospective observational study of Israeli travellers returning from sub-Saharan Africa with P. falciparum malaria, including patients with artemether-lumefantrine (AL) failure. Blood samples from all malaria-positive patients are delivered to the national Parasitology Reference Laboratory along with personal information. Confirmation of malaria, species identification and comparative parasite load analysis were performed using real-time PCR. DNA extractions from stored leftover samples were analysed for the presence of mutations in Pfkelch13 and Pfcoronin. Age, weight, initial parasitaemia level and Pfcoronin status were compared in patients who failed treatment vs responders. RESULTS: During 2009-2020, 338 patients had P. falciparum malaria acquired in Africa. Of those, 15 (24-69 years old, 14 males) failed treatment with AL. Four were still parasitemic at the end of treatment, and 11 had malaria recrudescence. Treatment failure rates were 0% during 2009-2012, 9.1% during 2013-2016 and 17.4% during 2017-2020. In all patients, the Pfkelch13 propeller domain had a wild-type sequence. We did find the P76S mutation in the propeller domain of Pfcoronin in 4/15 (28.6%) of the treatment-failure cases compared to only 3/56 (5.5%) in the successfully treated patients (P = 0.027). CONCLUSION: AL treatment failure emergence was not associated with mutations in Pfkelch13. However, P76S mutation in the Pfcoronin gene was more frequently present in the treatment-failure group and merits further investigation. The increase of malaria incidence in sub-Saharan-Africa partly attributed to the COVID-19 pandemic might also reflect a wider spread of ACT resistance.

Genomic analysis of Indian isolates of Plasmodium falciparum: Implications for drug resistance and virulence factors.

The emergence of drug resistance to frontline treatments such as Artemisinin-based combination therapy (ACT) is a major obstacle to the control and eradication of malaria. This problem is compounded by the inherent genetic variability of the parasites, as many established markers of resistance do not accurately predict the drug-resistant status. There have been reports of declining effectiveness of ACT in the West Bengal and Northeast regions of India, which have traditionally been areas of drug resistance emergence in the country. Monitoring the genetic makeup of a population can help to identify the potential for drug resistance markers associated with it and evaluate the effectiveness of interventions aimed at reducing the spread of malaria. In this study, we performed whole genome sequencing of 53 isolates of Plasmodium falciparum from West Bengal and compared their genetic makeup to isolates from Southeast Asia (SEA) and Africa. We found that the Indian isolates had a distinct genetic makeup compared to those from SEA and Africa, and were more similar to African isolates, with a high prevalence of mutations associated with antigenic variation genes. The Indian isolates also showed a high prevalence of markers of chloroquine resistance (mutations in Pfcrt) and multidrug resistance (mutations in Pfmdr1), but no known mutations associated with artemisinin resistance in the PfKelch13 gene. Interestingly, we observed a novel L152V mutation in PfKelch13 gene and other novel mutations in genes involved in ubiquitination and vesicular transport that have been reported to support artemisinin resistance in the early stages of ACT resistance in the absence of PfKelch13 polymorphisms. Thus, our study highlights the importance of region-specific genomic surveillance for artemisinin resistance and the need for continued monitoring of resistance to artemisinin and its partner drugs.

Ring stage dormancy of Plasmodium falciparum tolerant to artemisinin and its analogues - A genetically regulated "Sleeping Beauty".

The appearance in 2008 in western Cambodia of Plasmodium falciparum tolerant to artemisinin, defined by longer parasite clearance time following drug administration and in vitro by a slightly higher survival rate of the ring stage after a 3-h treatment with 700 nM artemisinin (or analogues, collectively termed ART), has raised concerns of the possible loss of this frontline antimalarial [used in the form of an artemisinin combination therapy (ACT)], with its low IC50 value against the ring stage and pleiotropic pro-drug/poison property. The key genetic marker of ART tolerance phenotype is a number of non-synonymous mutations in Pfkelch13 propeller domain. This results in defective assembly at the ring stage of a cytostome structure located at cytoplasmic side of the parasite membrane required for invagination of a double-membrane endosome carrying host cytosol haemoglobin to the digestive vacuole. The consequential deprivation of amino acids initiates ring stage parasites bearing the causal mutations in PfK13 (or other key cytostome components) entry into a dormant state ("Sleeping Beauty"), which, after a duration longer than that the short-lived ART, "Sleeping Beauty" ring parasite resumes its normal, but accelerated, development to maintain the 48-h intra-erythrocytic life-cycle. We posit that when ART-tolerant P. falciparum has acquired under ART stress the causative PfK13 mutation (not obligatory if mutations occur in other critical cytostome components), together with other necessary mutations to adjust to the new normalcy and to provide survival competitiveness, ART-tolerant parasite has now evolved into a genetically programmed "Sleeping Beauty". The onus of preventing the spread of ART-tolerant P. falciparum lies with the efficacy of ACT partner drug, hence the recommendation of a triple ACT (TACT). Nevertheless, attention should also be focussed on understanding the mechanisms of dormancy, such as induction, maintenance and recovery, to enable discovery and development of novel antimalarials targeting this unique parasite stage.