Chloroquine has shown high therapeutic efficacy against uncomplicated Plasmodium vivax malaria in southern Ethiopia: seven decades after its introduction.

BACKGROUND: Plasmodium vivax malaria is a leading cause of morbidity in Ethiopia. The first-line treatment for P. vivax is chloroquine (CQ) and primaquine (PQ), but there have been local reports of CQ resistance. A clinical study was conducted to determine the efficacy of CQ for the treatment of P. vivax malaria in southern Ethiopia. METHODS: In 2021, patients with P. vivax mono-infection and uncomplicated malaria were enrolled and treated with 25 mg/kg CQ for 3 consecutive days. Patients were followed for 28 days according to WHO guidelines. The data were analysed using per-protocol (PP) and Kaplan‒Meier (K‒M) analyses to estimate the risk of recurrent P. vivax parasitaemia on day 28. RESULTS: A total of 88 patients were enrolled, 78 (88.6%) of whom completed the 28 days of follow-up. Overall, 76 (97.4%) patients had adequate clinical and parasitological responses, and two patients had late parasitological failures. The initial therapeutic response was rapid, with 100% clearance of asexual parasitaemia within 48 h. CONCLUSION: Despite previous reports of declining chloroquine efficacy against P. vivax, CQ retains high therapeutic efficacy in southern Ethiopia, supporting the current national treatment guidelines. Ongoing clinical monitoring of CQ efficacy supported by advanced molecular methods is warranted to inform national surveillance and ensure optimal treatment guidelines.

No Association between the Plasmodium vivax crt-o MS334 or In9pvcrt Polymorphisms and Chloroquine Failure in a Pre-Elimination Clinical Cohort from Malaysia with a Large Clonal Expansion.

Increasing reports of resistance to a frontline malaria blood-stage treatment, chloroquine (CQ), raises concerns for the elimination of Plasmodium vivax. The absence of an effective molecular marker of CQ resistance in P. vivax greatly constrains surveillance of this emerging threat. A recent genetic cross between CQ sensitive (CQS) and CQ resistant (CQR) NIH-1993 strains of P. vivax linked a moderate CQR phenotype with two candidate markers in P. vivax CQ resistance transporter gene (pvcrt-o): MS334 and In9pvcrt. Longer TGAAGH motif lengths at MS334 were associated with CQ resistance, as were shorter motifs at the In9pvcrt locus. In this study, high-grade CQR clinical isolates of P. vivax from a low endemic setting in Malaysia were used to investigate the association between the MS334 and In9pvcrt variants and treatment efficacy. Among a total of 49 independent monoclonal P. vivax isolates assessed, high-quality MS334 and In9pvcrt sequences could be derived from 30 (61%) and 23 (47%), respectively. Five MS334 and six In9pvcrt alleles were observed, with allele frequencies ranging from 2 to 76% and 3 to 71%, respectively. None of the clinical isolates had the same variant as the NIH-1993 CQR strain, and none of the variants were associated with CQ treatment failure (all P > 0.05). Multi-locus genotypes (MLGs) at 9 neutral microsatellites revealed a predominant P. vivax strain (MLG6) accounting for 52% of Day 0 infections. The MLG6 strain comprised equal proportions of CQS and CQR infections. Our study reveals complexity in the genetic basis of CQ resistance in the Malaysian P. vivax pre-elimination setting and suggests that the proposed pvcrt-o MS334 and In9pvcrt markers are not reliable markers of CQ treatment efficacy in this setting. Further studies are needed in other endemic settings, applying hypothesis-free genome-wide approaches, and functional approaches to understand the biological impact of the TGAAGH repeats linked to CQ response in a cross are warranted to comprehend and track CQR P. vivax.

Analysis of the relapse of imported Plasmodium vivax and Plasmodium ovale in five provinces of China.

BACKGROUND: The global battle against malaria is facing formidable challenges, particularly in controlling Plasmodium vivax and Plasmodium ovale, whose cases have not been reduced as effectively as Plasmodium falciparum because of their relapse. This study investigates the current situation and underlying factors contributing to relapse or recrudescence of imported cases of P. vivax and P. ovale, and seeks to provide a reference for reducing relapse or recrudescence in malaria-free areas and offers a scientific basis for designing strategies to prevent imported re-transmission. METHODS: This study analysed imported P. vivax and P. ovale in Anhui, Zhejiang, Henan, Hubei, and Guangxi provinces during 2014-2021 by retrospective analysis. A case-control study was conducted on patients who experienced relapse or recrudescence. RESULTS: From 2014 to 2021, 306 cases of P.vivax and 896 cases of P.ovale were included in the study, while 75 cases had relapse or recrudescence, including 49 cases of P. ovale (65.33%) and 26 cases of P. vivax (34.67%). Within less than 5 weeks after returning to the country, 122 cases of P. vivax (39.87%, 122/306) and 265 cases of P. ovale (29.58%, 265/896) occurred. Within less than 53 weeks, the ratio of P. vivax was 94.77% (290/306), and that of P. ovale was 89.96% (806/896). Among the cases experiencing relapse or recrudescence, only 1 case of P. vivax (1/26 3.85%) and 3 cases of P. ovale (3/49 6.12%) occurred within less than 5 weeks after the first onset, whereas 21 cases of P. vivax (21/26 80.77%) and 42 cases of P. ovale (42/49 85.71%) occurred within less than 53 weeks after the first onset. The difference in relapse or recrudescence due to different drugs and medication regimens and medical activities at various levels of medical institutions was statistically significant. CONCLUSION: In areas where malaria has been eliminated, routine health screening in a scientific time frame for people returning from at-risk areas can effectively improve the efficiency of preventing re-transmission, thereby reducing prevention costs and disease burden. Preventing patients from self-treating and strengthening medication regulations in health facilities are key measures to reduce relapse or recrudescence.

Plasmodium malariae and P. ovale genomes provide insights into malaria parasite evolution.

Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri). These species are prevalent across most regions in which malaria is endemic and are often undetectable by light microscopy, rendering their study in human populations difficult. The exact evolutionary relationship of these species to the other human-infective species has been contested. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole.

Emergence of artemisinin-resistant Plasmodium falciparum with kelch13 C580Y mutations on the island of New Guinea.

The rapid and aggressive spread of artemisinin-resistant Plasmodium falciparum carrying the C580Y mutation in the kelch13 gene is a growing threat to malaria elimination in Southeast Asia, but there is no evidence of their spread to other regions. We conducted cross-sectional surveys in 2016 and 2017 at two clinics in Wewak, Papua New Guinea (PNG) where we identified three infections caused by C580Y mutants among 239 genotyped clinical samples. One of these mutants exhibited the highest survival rate (6.8%) among all parasites surveyed in ring-stage survival assays (RSA) for artemisinin. Analyses of kelch13 flanking regions, and comparisons of deep sequencing data from 389 clinical samples from PNG, Indonesian Papua and Western Cambodia, suggested an independent origin of the Wewak C580Y mutation, showing that the mutants possess several distinctive genetic features. Identity by descent (IBD) showed that multiple portions of the mutants' genomes share a common origin with parasites found in Indonesian Papua, comprising several mutations within genes previously associated with drug resistance, such as mdr1, ferredoxin, atg18 and pnp. These findings suggest that a P. falciparum lineage circulating on the island of New Guinea has gradually acquired a complex ensemble of variants, including kelch13 C580Y, which have affected the parasites' drug sensitivity. This worrying development reinforces the need for increased surveillance of the evolving parasite populations on the island, to contain the spread of resistance.

The changing epidemiology of Plasmodium vivax: Insights from conventional and novel surveillance tools.

Sarah Auburn and co-authors discuss the unique biology and epidemiology of P. vivax and current evidence on conventional and new approaches to surveillance.

Glucose-6-phosphate dehydrogenase activity in individuals with and without malaria: Analysis of clinical trial, cross-sectional and case-control data from Bangladesh.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) activity is dependent upon G6PD genotype and age of the red blood cell (RBC) population, with younger RBCs having higher activity. Peripheral parasitemia with Plasmodium spp. induces hemolysis, replacing older RBCs with younger cells with higher G6PD activity. This study aimed to assess whether G6PD activity varies between individuals with and without malaria or a history of malaria. METHODS AND FINDINGS: Individuals living in the Chittagong Hill Tracts of Bangladesh were enrolled into 3 complementary studies: (i) a prospective, single-arm clinical efficacy trial of patients (n = 175) with uncomplicated malaria done between 2014 and 2015, (ii) a cross-sectional survey done between 2015 and 2016 (n = 999), and (iii) a matched case-control study of aparasitemic individuals with and without a history of malaria done in 2020 (n = 506). G6PD activity was compared between individuals with and without malaria diagnosed by microscopy, rapid diagnostic test (RDT), or polymerase chain reaction (PCR), and in aparasitemic participants with and without a history of malaria. In the cross-sectional survey and clinical trial, 15.5% (182/1,174) of participants had peripheral parasitemia detected by microscopy or RDT, 3.1% (36/1,174) were positive by PCR only, and 81.4% (956/1,174) were aparasitemic. Aparasitemic individuals had significantly lower G6PD activity (median 6.9 U/g Hb, IQR 5.2-8.6) than those with peripheral parasitemia detected by microscopy or RDT (7.9 U/g Hb, IQR 6.6-9.8, p < 0.001), but G6PD activity similar to those with parasitemia detected by PCR alone (submicroscopic parasitemia) (6.1 U/g Hb, IQR 4.8-8.6, p = 0.312). In total, 7.7% (14/182) of patients with malaria had G6PD activity < 70% compared to 25.0% (248/992) of participants with submicroscopic or no parasitemia (odds ratio [OR] 0.25, 95% CI 0.14-0.44, p < 0.001). In the case-control study, the median G6PD activity was 10.3 U/g Hb (IQR 8.8-12.2) in 253 patients with a history of malaria and 10.2 U/g Hb (IQR 8.7-11.8) in 253 individuals without a history of malaria (p = 0.323). The proportion of individuals with G6PD activity < 70% was 11.5% (29/253) in the cases and 15.4% (39/253) in the controls (OR 0.7, 95% CI 0.41-1.23, p = 0.192). Limitations of the study included the non-contemporaneous nature of the clinical trial and cross-sectional survey. CONCLUSIONS: Patients with acute malaria had significantly higher G6PD activity than individuals without malaria, and this could not be accounted for by a protective effect of G6PD deficiency. G6PD-deficient patients with malaria may have higher than expected G6PD enzyme activity and an attenuated risk of primaquine-induced hemolysis compared to the risk when not infected.

Epidemiological profile of Plasmodium ovale spp. imported from Africa to Anhui Province, China, 2012-2019.

BACKGROUND: Although autochthonous malaria cases are no longer reported in Anhui Province, China, imported malaria has become a major health concern. The proportion of reported malaria cases caused by Plasmodium ovale spp. increased to levels higher than expected during 2012 to 2019, and showed two peaks, 19.69% in 2015 and 19.35% in 2018. METHODS: A case-based retrospective study was performed using data collected from the China Information System for Disease Control and Prevention (CISDCP) and Information System for Parasitic Disease Control and Prevention (ISPDCP) from 2012 to 2019 to assess the trends and differences between Plasmodium ovale curtisi (P. o. curtisi) and Plasmodium ovale wallikeri (P. o. wallikeri). Epidemiological characteristics were analyzed using descriptive statistics. RESULTS: Plasmodium o. curtisi and P. o. wallikeri were found to simultaneously circulate in 14 African countries. Among 128 patients infected with P. ovale spp., the proportion of co-infection cases was 10.16%. Six cases of co-infection with P. ovale spp. and P. falciparum were noted, each presenting with two clinical attacks (the first attack was due to P. falciparum and the second was due to P. ovale spp.) at different intervals. Accurate identification of the infecting species was achieved among only 20.00% of cases of P. ovale spp. infection. At the reporting units, 32.17% and 6.96% of cases of P. ovale spp. infection were misdiagnosed as P. vivax and P. falciparum infections, respectively. CONCLUSION: The present results indicate that the potential of P. ovale spp. to co-infect with other Plasmodium species has been previously underestimated, as is the incidence of P. ovale spp. in countries where malaria is endemic. P. o. curtisi may have a long latency period of > 3 years and potentially cause residual foci, thus posing challenges to the elimination of malaria in P. ovale spp.-endemic areas. Considering the low rate of species identification, more sensitive point-of-care detection methods need to be developed for P. ovale spp. and introduced in non-endemic areas.

Short-course primaquine for the radical cure of Plasmodium vivax malaria: a multicentre, randomised, placebo-controlled non-inferiority trial.

BACKGROUND: Primaquine is the only widely used drug that prevents Plasmodium vivax malaria relapses, but adherence to the standard 14-day regimen is poor. We aimed to assess the efficacy of a shorter course (7 days) of primaquine for radical cure of vivax malaria. METHODS: We did a randomised, double-blind, placebo-controlled, non-inferiority trial in eight health-care clinics (two each in Afghanistan, Ethiopia, Indonesia, and Vietnam). Patients (aged ≥6 months) with normal glucose-6-phosphate dehydrogenase (G6PD) and presenting with uncomplicated vivax malaria were enrolled. Patients were given standard blood schizontocidal treatment and randomly assigned (2:2:1) to receive 7 days of supervised primaquine (1·0 mg/kg per day), 14 days of supervised primaquine (0·5 mg/kg per day), or placebo. The primary endpoint was the incidence rate of symptomatic P vivax parasitaemia during the 12-month follow-up period, assessed in the intention-to-treat population. A margin of 0·07 recurrences per person-year was used to establish non-inferiority of the 7-day regimen compared with the 14-day regimen. This trial is registered at ClinicalTrials.gov (NCT01814683). FINDINGS: Between July 20, 2014, and Nov 25, 2017, 2336 patients were enrolled. The incidence rate of symptomatic recurrent P vivax malaria was 0·18 (95% CI 0·15 to 0·21) recurrences per person-year for 935 patients in the 7-day primaquine group and 0·16 (0·13 to 0·18) for 937 patients in the 14-day primaquine group, a difference of 0·02 (-0·02 to 0·05, p=0·3405). The incidence rate for 464 patients in the placebo group was 0·96 (95% CI 0·83 to 1·08) recurrences per person-year. Potentially drug-related serious adverse events within 42 days of starting treatment were reported in nine (1·0%) of 935 patients in the 7-day group, one (0·1%) of 937 in the 14-day group and none of 464 in the control arm. Four of the serious adverse events were significant haemolysis (three in the 7-day group and one in the 14-day group). INTERPRETATION: In patients with normal G6PD, 7-day primaquine was well tolerated and non-inferior to 14-day primaquine. The short-course regimen might improve adherence and therefore the effectiveness of primaquine for radical cure of P vivax malaria. FUNDING: UK Department for International Development, UK Medical Research Council, UK National Institute for Health Research, and the Wellcome Trust through the Joint Global Health Trials Scheme (MR/K007424/1) and the Bill & Melinda Gates Foundation (OPP1054404).

Implementing parasite genotyping into national surveillance frameworks: feedback from control programmes and researchers in the Asia-Pacific region.

The Asia-Pacific region faces formidable challenges in achieving malaria elimination by the proposed target in 2030. Molecular surveillance of Plasmodium parasites can provide important information on malaria transmission and adaptation, which can inform national malaria control programmes (NMCPs) in decision-making processes. In November 2019 a parasite genotyping workshop was held in Jakarta, Indonesia, to review molecular approaches for parasite surveillance and explore ways in which these tools can be integrated into public health systems and inform policy. The meeting was attended by 70 participants from 8 malaria-endemic countries and partners of the Asia Pacific Malaria Elimination Network. The participants acknowledged the utility of multiple use cases for parasite genotyping including: quantifying the prevalence of drug resistant parasites, predicting risks of treatment failure, identifying major routes and reservoirs of infection, monitoring imported malaria and its contribution to local transmission, characterizing the origins and dynamics of malaria outbreaks, and estimating the frequency of Plasmodium vivax relapses. However, the priority of each use case varies with different endemic settings. Although a one-size-fits-all approach to molecular surveillance is unlikely to be applicable across the Asia-Pacific region, consensus on the spectrum of added-value activities will help support data sharing across national boundaries. Knowledge exchange is needed to establish local expertise in different laboratory-based methodologies and bioinformatics processes. Collaborative research involving local and international teams will help maximize the impact of analytical outputs on the operational needs of NMCPs. Research is also needed to explore the cost-effectiveness of genetic epidemiology for different use cases to help to leverage funding for wide-scale implementation. Engagement between NMCPs and local researchers will be critical throughout this process.