Search

Plasmodium falciparum has evolved resistance to almost all antimalarial drugs through genomic changes –SNPs and copy number variants (CNVs) – and a nonspecific stress-response-based survival such as the ring-stage temporary growth arrest (TGA). The ring-stage can develop into asexual or sexual stages or stop growing when conditions are unfavorable. The ring-stage shows extreme plasticity under adverse conditions, such as artemisinin exposure. The ring-stage TGA stage exhibits an innate and passive resistance to many stressors, which ensures that some individuals in the population can survive in the host. P. falciparum ring-stage TGA may rely on environmental sensing and extracellular communication between the parasites. Ring-stage TGA may be an underestimated parasite reservoir, especially in asymptomatic individuals. No treatment that specifically targets growth-arrested ring stages is currently available. Malaria is a life-threatening tropical disease caused by parasites of the genus Plasmodium, of which Plasmodium falciparum is the most lethal. Malaria parasites have a complex life cycle, with stages occurring in both the Anopheles mosquito vector and human host. Ring stages are the youngest form of the parasite in the intraerythrocytic developmental cycle and are associated with evasion of spleen clearance, temporary growth arrest (TGA), and drug resistance. This formidable ability to survive and develop into mature, sexual, or growth-arrested forms demonstrates the inherent population heterogeneity. Here we highlight the role of the ring stage as a crossroads in parasite development and as a reservoir of surviving cells in the human host via TGA survival mechanisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Owing to the emergence of multiresistant Plasmodium falciparum parasites in Southeast Asia, along with the impressive decrease in the efficacy of the endoperoxide compound artemisinin and of artemisinin-based combination therapies, the development of novel antimalarial drugs or combinations is required. Although several antiplasmodial molecules, such as endoperoxide-based compounds, are in advanced research or development, we do not know whether resistance to artemisinin derivatives might impact the efficacy of these new compounds.Objectives: To address this issue, the antiplasmodial efficacy of trioxaquines, hybrid endoperoxide-based molecules, was explored, along with their ability to select in vitro resistant parasites under discontinuous and dose-escalating drug pressure.Methods: The in vitro susceptibilities of artemisinin- and trioxaquine-resistant laboratory strains and recent Cambodian field isolates were evaluated by different phenotypic and genotypic assays.Results: Trioxaquines tested presented strong cross-resistance with artemisinin both in the artemisinin-resistant laboratory F32-ART5 line and in Cambodian field isolates. Trioxaquine drug pressure over 4 years led to the in vitro selection of the F32-DU line, which is resistant to trioxaquine and artemisinin, similar to the F32-ART lineage. F32-DU whole genome sequencing (WGS) revealed that resistance to trioxaquine was associated with the same non-synonymous mutation in the propeller domain of the K13 protein (M476I) that was found in the F32-ART lineage.Conclusions: These worrisome results indicate the risk of cross-resistance between artemisinins and endoperoxide-based antiplasmodial drugs in the development of the K13 mutant parasites and question the usefulness of these molecules in the future therapeutic arsenal. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background The emergence of Plasmodium falciparum resistance to artemisinin and its derivatives, manifested as delayed parasite clearance following the treatment, has developed in Southeast Asia. The spread of resistance to artemisinin from Asia to Africa may be catastrophic for malaria control and elimination worldwide. Recently, mutations in the propeller domain of the Kelch 13 (k13) gene (PF3D71343700) were associated with in vitro resistance to artemisinin and with delayed clearance after artemisinin treatment in southern Asia. The aim of the study was to characterize the genetic variability of k13 and to evaluate the molecular resistance to artemisinin for the first time in Senegal. Methods Plasmodium falciparum isolates were collected from 138 malaria patients in Dakar and its districts during the rainy season of October 2012 to January 2013 at the Hôpital Principal de Dakar. The k13 gene was amplified using nested PCR and sequenced. Results A very limited variability within the k13 gene in Senegalese P. falciparum isolates was identified. No polymorphism was detected in the six k13-propeller blades. Only two mutations, T149S (6.3%) and K189T (42.2%), and one (N) or two (NN) asparagine insertion at the codon 142 (4.7 and 6.3%, respectively) were detected in the Plasmodium/Apicomplexa-specific domain. None of the polymorphisms associated with artemisinin resistance in Southeast Asia was detected in the 138 P. falciparum from Dakar. Discussion The present data do not suggest widespread artemisinin resistance in Dakar in 2012-2013. Notably, the C580Y, R539T or Y493H substitutions that were associated with in vitro resistance or delayed parasite clearance in Southeast Asia were not observed in Dakar, nor were any of the polymorphisms observed in parasites from Southeast Asia, nor the M476I mutation that was selected in vitro with artemisinin pressure in a African parasite line. [ABSTRACT FROM AUTHOR]

The uptake and digestion of host hemoglobin by malaria parasites during blood-stage growth leads to significant oxidative damage of membrane lipids. Repair of lipid peroxidation damage is crucial for parasite survival. Here, we demonstrate that Plasmodium falciparum imports a host antioxidant enzyme, peroxiredoxin 6 (PRDX6), during hemoglobin uptake from the red blood cell cytosol. PRDX6 is a lipid-peroxidation repair enzyme with phospholipase A 2 (PLA 2) activity. Inhibition of PRDX6 with a PLA 2 inhibitor, Darapladib, increases lipid-peroxidation damage in the parasite and disrupts transport of hemoglobin-containing vesicles to the food vacuole, causing parasite death. Furthermore, inhibition of PRDX6 synergistically reduces the survival of artemisinin-resistant parasites following co-treatment of parasite cultures with artemisinin and Darapladib. Thus, PRDX6 is a host-derived drug target for development of antimalarial drugs that could help overcome artemisinin resistance. [Display omitted] • Human peroxiredoxin 6 (PRDX6) is internalized by P. falciparum blood stages • Inhibition of host PRDX6 causes lethal lipid-peroxidation damage in the parasite • Co-treatment with artemisinin and PRDX6 inhibitors overcomes artemisinin resistance • Targeting of a host enzyme like PRDX6 may prevent development of drug resistance Wagner et al. find that Plasmodium falciparum blood stages internalize human PRDX6, which repairs lipid-peroxidation damage. PRDX6 inhibitors prevent lipid repair and kill the parasite. Co-treatment of artemisinin-resistant strains with PRDX6 inhibitors and artemisinin restores susceptibility to artemisinin. PRDX6 thus provides a promising host-based target for anti-malaria drug development. [ABSTRACT FROM AUTHOR]

We conducted a study in Binh Phuoc, Vietnam, in 2015 on the therapeutic efficacy of dihydroartemisinin/piperaquine for Plasmodium falciparum malaria. A high number of treatment failures (14/40) was found, and piperaquine resistance in Vietnam was confirmed. A change in the malaria treatment policy for Vietnam is in process. [ABSTRACT FROM AUTHOR]

Malaria remains a major health problem in Madagascar. Over past decades, the burden of malarial disease has fluctuated over time, partly in line with the successes and failures of antimalarial policy. In the 1950s and 1960s, a sharp decline in malaria transmission was observed in the central highlands due to indoor spraying with DDT and to the massive use of chloroquine by the population. Following this, the discontinuation of the ‘nivaquinization’ policy was followed by devastating outbreaks in the central highlands in the 1980s. Currently, the rate of in vitro chloroquine-resistant Plasmodium falciparum isolates does not exceed 5%. This figure appears disconnected from the high level of clinical treatment failure (near 40%). pfcrt mutant isolates are found in less than 1% of isolates on the Island. Conversely, pfmdr1 mutant isolates are found in more than 60% of isolates and may be responsible for the bulk of resistance to chloroquine in Madagascar. Other antimalarials remain generally effective in Madagascar. Recent clinical and in vitro data support the complete efficacy of the combination artesunate–amodiaquine in Madagascar. As such, this artemisinin combination therapy should play a central role in the control and possible elimination of P. falciparum malaria in Madagascar. [ABSTRACT FROM AUTHOR]

Background: Data concerning antimalarial combination treatment for uncomplicated malaria in Madagascar are largely lacking. Randomized clinical trial was designed to assess therapeutic efficacies of chloroquine (CQ), amodiaquine (AQ), sulphadoxine-pyrimethamine (SP), amodiaquine plus sulphadoxine-pyrimethamine combination (AQ+SP) and artesunate plus amodiaquine combination (AQ+AS). Methods: 287 children between 6 months and 15 years of age, with uncomplicated falciparum malaria, were enrolled in the study. Primary endpoints were the day-14 and day-28 risks of parasitological failure, either unadjusted or adjusted by genotyping. Results: All treatment regimens, except for CQ treatment, gave clinical cure rates above 97% by day-14 and 92% by day-28 (PCR-corrected). AQ+SP was as effective as AQ+AS. The risk of new infection within the month after therapy was generally higher for AQ+AS than AQ+SP. Conclusion: These findings show that the inexpensive and widely available combination AQ+SP may be valuable in for the treatment of uncomplicated malaria in Madagascar and could have an important role in this country, where much of the drugs administered go to patients who do not have malaria. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article "Declining efficacy of artemisinin combination therapy against P. falciparum malaria on the Thai-Myanmar border (2003-2013): the role of parasite genetic factors" by Phyo et al. in the 2016 issue.

Background: Given the risk of artemisinin resistance spreading from the Greater Mekong sub-region, prospective monitoring in sub-Saharan Africa should be expedited. Molecular biology techniques used for monitoring rely on the detection of k13 validated mutants by using PCR and Sanger sequencing approach, usually not available in malaria endemic areas. Methods: A semi-automated workflow based on the easyMAG® platform and the Argene Solution® (bioMérieux, Marcy l'Etoile, France) as a field-based surveillance tool operable at national level was developed in four steps. Clinical and analytical performances of this tool detecting five of the most frequent and validated k13 mutants (Y493H, I543T, R539T, F446I and C580Y) from dried blood spots (DBS) were compared to the gold standard approach (PCR and Sanger sequencing). Results: By using the ARMS (amplification-refractory mutation system) strategy, the best multiplexing options were found in 3 separate real-time PCR duplexes (IC as internal control/I543T, C580Y/Y493H and F446I/R539T) with limits of detection ranging from 50 (C580Y) to 6.25 parasites/µL (Y493H). In field conditions, using 642 clinical DBS (from symptomatic patients and asymptomatic individuals) collected from Cambodia, Myanmar and Africa (Chad), the overall sensitivity and specificity of the K13 bMx prototype assay developed by bioMérieux were ≥ 90%. Areas under the ROC curves were estimated to be > 0.90 for all k13 mutants in samples from symptomatic patients. Conclusion: The K13 ready-to-use bMx prototype assay, considered by the end-users as a user-friendly assay to perform (in shorter time than the K13 reference assay) and easy to interpret, was found to require less budget planning and had fewer logistical constraints. Its excellent performance qualifies the prototype as a reliable screening tool usable in malaria endemic countries recognized to be at risk of emergence or spread of validated k13 mutants. Additional multi-site studies are needed to evaluate the performances of the K13 bMx prototype assay in different epidemiological contexts such as Africa, India, or South America. [ABSTRACT FROM AUTHOR]

BACKGROUND Although the clinical efficacy of antimalarial artemisinin-based combination therapies in Africa remains high, the recent emergence of partial resistance to artemisinin in Plasmodium falciparum on the continent is troubling, given the lack of alternative treatments. METHODS In this study, we used data from drug-efficacy studies conducted between 2016 and 2019 that evaluated 3-day courses of artemisinin-based combination therapy (artesunate-amodiaquine or artemether-lumefantrine) for uncomplicated malaria in Eritrea to estimate the percentage of patients with day-3 positivity (i.e., persistent P. falciparum parasitemia 3 days after the initiation of therapy). We also assayed parasites for mutations in Pflcelchl3 as predictive markers of partial resistance to artemisinin and screened for deletions in hrp2 and hrp3 that result in variable performance of histidine rich protein 2 (HRP2)-based rapid diagnostic tests for malaria. RESULTS We noted an increase in the percentage of patients with day-3 positivity from 0.4% (1 of 273) in 2016 to 1.9% (4 of 209) in 2017 and 4.2% (15 of 359) in 2019. An increase was also noted in the prevalence of the Pfkelchl3 R622I mutation, which was detected in 109 of 818 isolates before treatment, from 8.6% (24 of 278) in 2016 to 21.0% (69 of 329) in 2019. The odds of day-3 positivity increased by a factor of 6.2 (95% confidence interval, 2.5 to 15.5) among the patients with Pfkelchl3 6221 variant parasites. Partial resistance to artemisinin, as defined by the World Health Organization, was observed in Eritrea. More than 5% of the patients younger than 15 years of age with day-3 positivity also had parasites that carried Pfkelchl3 R622I. In vitro, the R622I mutation conferred a low level of resistance to artemisinin when edited into NF54 and Dd2 parasite lines. Deletions in both hrp2 and hrp3 were identified in 16.9% of the parasites that carried the Pfkelchl3 R622I mutation, which made them potentially undetectable by HRP2-based rapid diagnostic tests. CONCLUSIONS The emergence and spread of P. falciparum lineages with both Pfkelchl3-mediated partial resistance to artemisinin and deletions in hrp2 and hrp3 in Eritrea threaten to compromise regional malaria control and elimination campaigns. (Funded by the Bill and Melinda Gates Foundation and others; Australian New Zealand Clinical Trials Registry numbers, ACTRN12618001223224, ACTRN12618000353291, and ACTRN12619000859189.). [ABSTRACT FROM AUTHOR]

The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the locus Plasmodium falciparum K13 using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites. [ABSTRACT FROM AUTHOR]