Impact of piperaquine resistance in Plasmodium falciparum on malaria treatment effectiveness in The Guianas: a descriptive epidemiological study.

BACKGROUND: Plasmodium falciparum is an apicomplexan parasite responsible for lethal cases of malaria. According to WHO recommendations, P falciparum cases are treated with artemisinin-based combination therapy including dihydroartemisinin-piperaquine. However, the emergence of resistant parasites against dihydroartemisinin-piperaquine was reported in southeast Asia in 2008 and, a few years later, suspected in South America. METHODS: To characterise resistance emergence, a treatment efficacy study was performed on the reported patients infected with P falciparum and treated with dihydroartemisinin-piperaquine in French Guiana (n=6, 2016-18). Contemporary isolates collected in French Guiana were genotyped for P falciparum chloroquine resistance transporter (pfCRT; n=845) and pfpm2 and pfpm3 copy number (n=231), phenotyped using the in vitro piperaquine survival assay (n=86), and analysed through genomic studies (n=50). Additional samples from five Amazonian countries and one outside the region were genotyped (n=1440). FINDINGS: In field isolates, 40 (47%) of 86 (95% CI 35·9-57·1) were resistant to piperaquine in vitro; these phenotypes were more associated with pfCRTC350R (ie, Cys350Arg) and pfpm2 and pfpm3 amplifications (Dunn test, p<0·001). Those markers were also associated with dihydroartemisinin-piperaquine treatment failure (n=3 [50%] of 6). A high prevalence of piperaquine resistance markers was observed in Suriname in 19 (83%) of 35 isolates and in Guyana in 579 (73%) of 791 isolates. The pfCRTC350R mutation emerged before pfpm2 and pfpm3 amplification in a temporal sequence different from southeast Asia, and in the absence of artemisinin partial resistance, suggesting a geographically distinctive epistatic relationship between these genetic markers. INTERPRETATION: The high prevalence of piperaquine resistance markers in parasite populations of the Guianas, and the risk of associated therapeutic failures calls for caution on dihydroartemisinin-piperaquine use in the region. Furthermore, greater attention should be given to potential differences in genotype to phenotype mapping across genetically distinct parasite populations from different continents. FUNDING: Pan American Health Organization and WHO, French Ministry for Research, European Commission, Santé publique France, Agence Nationale de la Recherche, Fundação de Amparo à Pesquisa do Estado do Amazonas, Ministry of Health of Brazil, Oswaldo Cruz Foundation, and National Institutes of Health. TRANSLATIONS: For the French and Portuguese translations of the abstract see Supplementary Materials section.

Improving the diagnosis of severe malaria in African children using platelet counts and plasma PfHRP2 concentrations.

Severe malaria caused by Plasmodium falciparum is difficult to diagnose accurately in children in high-transmission settings. Using data from 2649 pediatric and adult patients enrolled in four studies of severe illness in three countries (Bangladesh, Kenya, and Uganda), we fitted Bayesian latent class models using two diagnostic markers: the platelet count and the plasma concentration of P. falciparum histidine-rich protein 2 (PfHRP2). In severely ill patients with clinical features consistent with severe malaria, the combination of a platelet count of ≤150,000/µl and a plasma PfHRP2 concentration of ≥1000 ng/ml had an estimated sensitivity of 74% and specificity of 93% in identifying severe falciparum malaria. Compared with misdiagnosed children, pediatric patients with true severe malaria had higher parasite densities, lower hematocrits, lower rates of invasive bacterial disease, and a lower prevalence of both sickle cell trait and sickle cell anemia. We estimate that one-third of the children enrolled into clinical studies of severe malaria in high-transmission settings in Africa had another cause of their severe illness.

Pharmacokinetic Study of Rectal Artesunate in Children with Severe Malaria in Africa.

When severe malaria is suspected in children, the WHO recommends pretreatment with a single rectal dose of artesunate before referral to an appropriate facility. This was an individually randomized, open-label, 2-arm, crossover clinical trial in 82 Congolese children with severe falciparum malaria to characterize the pharmacokinetics of rectal artesunate. At admission, children received a single dose of rectal artesunate (10 mg/kg of body weight) followed 12 h later by intravenous artesunate (2.4 mg/kg) or the reverse order. All children also received standard doses of intravenous quinine. Artesunate and dihydroartemisinin were measured at 11 fixed intervals, following 0- and 12-h drug administrations. Clinical, laboratory, and parasitological parameters were measured. After rectal artesunate, artesunate and dihydroartemisinin showed large interindividual variability (peak concentrations of dihydroartemisinin ranged from 5.63 to 8,090 nM). The majority of patients, however, reached previously suggested in vivo IC50 and IC90 values (98.7% and 92.5%, respectively) of combined concentrations of artesunate and dihydroartemisinin between 15 and 30 min after drug administration. The median (interquartile range [IQR]) time above IC50 and IC90 was 5.68 h (2.90 to 6.08) and 2.74 h (1.52 to 3.75), respectively. The absolute rectal bioavailability (IQR) was 25.6% (11.7 to 54.5) for artesunate and 19.8% (10.3 to 35.3) for dihydroartemisinin. The initial 12-h parasite reduction ratio was comparable between rectal and intravenous artesunate: median (IQR), 84.3% (50.0 to 95.4) versus 69.2% (45.7 to 93.6), respectively (P = 0.49). Despite large interindividual variability, rectal artesunate can initiate and sustain rapid parasiticidal activity in most children with severe falciparum malaria while they are transferred to a facility where parenteral artesunate is available. (This study has been registered at ClinicalTrials.gov under identifier NCT02492178.).

Protective effect of Mediterranean-type glucose-6-phosphate dehydrogenase deficiency against Plasmodium vivax malaria.

X-linked glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy. The severe Mediterranean variant (G6PD Med) found across Europe and Asia is thought to confer protection against malaria, but its effect is unclear. We fitted a Bayesian statistical model to observed G6PD Med allele frequencies in 999 Pashtun patients presenting with acute Plasmodium vivax malaria and 1408 population controls. G6PD Med was associated with reductions in symptomatic P. vivax malaria incidence of 76% (95% credible interval [CI], 58-88) in hemizygous males and homozygous females combined and 55% (95% CI, 38-68) in heterozygous females. Unless there is very large population stratification within the Pashtun (confounding these results), the G6PD Med genotype confers a very large and gene-dose proportional protective effect against acute vivax malaria. The proportion of patients with vivax malaria at risk of haemolysis following 8-aminoquinoline radical cure is substantially overestimated by studies measuring G6PD deficiency prevalence in healthy subjects.

Good participatory practice for coronavirus disease 2019 (COVID-19) research: the case of a COVID-19 prevention study.

Background: The COPCOV study (chloroquine/ hydroxychloroquine prevention of coronavirus disease), which started recruitment in April 2020, is a multi-country double-blind, randomised, placebo-controlled trial which is being conducted in healthcare facilities involved in coronavirus disease 2019 (COVID-19) case management. COPCOV aims to recruit healthcare workers and other staff employed in facilities managing people with proven or suspected COVID-19. Methods: We conducted a series of engagement sessions, each involving a short presentation of the study, a section where attendees were asked to express if they would be interested in participating in such a study and which information they would need to change their view and an open Q&A section. Answers were transcribed and coded into themes by two independent investigators. Themes were derived from the data. The aims were to assess the feasibility of the study at the respective sites, to identify context-specific ethical issues, to understand concerns potential participants might have, to fine tune research procedures and to refine COPCOV information materials. They complemented other site-specific engagement, communication and public relation activities such as press releases and websites. Results: From 16 th March 2020 to 20 th January 2021, 12 engagement sessions were conducted in Thailand, Laos, Vietnam, Nepal and the UK involving 213 attendees in total. The sessions were designed to encourage potential participants and research professionals not directly involved in the project to interact with those who planned the study and those conducting it. Many attendees were keen to join the study while others had concerns. Questions raised revolved around the social value and study rationale; safety of trial medications and risk-benefit balance; study design and commitments. Conclusions: These sessions helped us refine information materials, identify misunderstandings about the study as well as complement site feasibility assessments. Our experience strongly supports the use of participatory practices prior to conducting clinical trials.

Plasma Plasmodium falciparum Histidine-rich Protein 2 Concentrations in Children With Malaria Infections of Differing Severity in Kilifi, Kenya.

BACKGROUND: Most previous studies support a direct link between total parasite load and the clinical severity of Plasmodium falciparum malaria infections. METHODS: We estimated P. falciparum parasite loads in 3 groups of children with malaria infections of differing severity: (1) children with World Health Organization-defined severe malaria (n = 1544), (2) children admitted with malaria but without features of severity (n = 200), and (3) children in the community with asymptomatic parasitemia (n = 33). RESULTS: Peripheral parasitemias were highest in those with uncomplicated malaria (geometric mean [GM] parasite count, 111 064/µL; 95% confidence interval, CI, 86 798-141 819/µL), almost 3 times higher than in those with severe malaria (39 588/µL; 34 990-44 791/µL) and >100 times higher than in those with asymptomatic malaria (1092/µL; 523-2280/µL). However, the GM P. falciparum histidine-rich protein 2 (PfHRP2) values (95% CI) increased with severity, being 7 (4-12) ng/mL in asymptomatic malaria, 843 (655-1084) ng/mL in uncomplicated malaria, and 1369 (1244-1506) ng/mL in severe malaria. PfHRP2 concentrations were markedly lower in the subgroup of patients with severe malaria and concomitant invasive bacterial infections of blood or cerebrospinal fluid (GM concentration, 312 ng/mL; 95% CI, 175-557 ng/mL; P < .001) than in those without such infections (1439 ng/mL; 1307-1584; P < .001). CONCLUSIONS: The clinical severity of malaria infections related strongly to the total burden of P. falciparum parasites. A quantitative test for plasma concentrations of PfHRP2 could be useful in identifying children at the greatest clinical risk and identifying critically ill children in whom malaria is not the primary cause.

K13-Mediated Reduced Susceptibility to Artemisinin in Plasmodium falciparum Is Overlaid on a Trait of Enhanced DNA Damage Repair.

Southeast Asia has been the hotbed for the development of drug-resistant malaria parasites, including those with resistance to artemisinin combination therapy. While mutations in the kelch propeller domain (K13 mutations) are associated with artemisinin resistance, a range of evidence suggests that other factors are critical for the establishment and subsequent transmission of resistance in the field. Here, we perform a quantitative analysis of DNA damage and repair in the malaria parasite Plasmodium falciparum and find a strong link between enhanced DNA damage repair and artemisinin resistance. This experimental observation is further supported when variations in seven known DNA repair genes are found in resistant parasites, with six of these mutations being associated with K13 mutations. Our data provide important insights on confounding factors that are important for the establishment and spread of artemisinin resistance and may explain why resistance has not yet arisen in Africa.

Triple artemisinin-based combination therapies versus artemisinin-based combination therapies for uncomplicated Plasmodium falciparum malaria: a multicentre, open-label, randomised clinical trial.

BACKGROUND: Artemisinin and partner-drug resistance in Plasmodium falciparum are major threats to malaria control and elimination. Triple artemisinin-based combination therapies (TACTs), which combine existing co-formulated ACTs with a second partner drug that is slowly eliminated, might provide effective treatment and delay emergence of antimalarial drug resistance. METHODS: In this multicentre, open-label, randomised trial, we recruited patients with uncomplicated P falciparum malaria at 18 hospitals and health clinics in eight countries. Eligible patients were aged 2-65 years, with acute, uncomplicated P falciparum malaria alone or mixed with non-falciparum species, and a temperature of 37·5°C or higher, or a history of fever in the past 24 h. Patients were randomly assigned (1:1) to one of two treatments using block randomisation, depending on their location: in Thailand, Cambodia, Vietnam, and Myanmar patients were assigned to either dihydroartemisinin-piperaquine or dihydroartemisinin-piperaquine plus mefloquine; at three sites in Cambodia they were assigned to either artesunate-mefloquine or dihydroartemisinin-piperaquine plus mefloquine; and in Laos, Myanmar, Bangladesh, India, and the Democratic Republic of the Congo they were assigned to either artemether-lumefantrine or artemether-lumefantrine plus amodiaquine. All drugs were administered orally and doses varied by drug combination and site. Patients were followed-up weekly for 42 days. The primary endpoint was efficacy, defined by 42-day PCR-corrected adequate clinical and parasitological response. Primary analysis was by intention to treat. A detailed assessment of safety and tolerability of the study drugs was done in all patients randomly assigned to treatment. This study is registered at ClinicalTrials.gov, NCT02453308, and is complete. FINDINGS: Between Aug 7, 2015, and Feb 8, 2018, 1100 patients were given either dihydroartemisinin-piperaquine (183 [17%]), dihydroartemisinin-piperaquine plus mefloquine (269 [24%]), artesunate-mefloquine (73 [7%]), artemether-lumefantrine (289 [26%]), or artemether-lumefantrine plus amodiaquine (286 [26%]). The median age was 23 years (IQR 13 to 34) and 854 (78%) of 1100 patients were male. In Cambodia, Thailand, and Vietnam the 42-day PCR-corrected efficacy after dihydroartemisinin-piperaquine plus mefloquine was 98% (149 of 152; 95% CI 94 to 100) and after dihydroartemisinin-piperaquine was 48% (67 of 141; 95% CI 39 to 56; risk difference 51%, 95% CI 42 to 59; p<0·0001). Efficacy of dihydroartemisinin-piperaquine plus mefloquine in the three sites in Myanmar was 91% (42 of 46; 95% CI 79 to 98) versus 100% (42 of 42; 95% CI 92 to 100) after dihydroartemisinin-piperaquine (risk difference 9%, 95% CI 1 to 17; p=0·12). The 42-day PCR corrected efficacy of dihydroartemisinin-piperaquine plus mefloquine (96% [68 of 71; 95% CI 88 to 99]) was non-inferior to that of artesunate-mefloquine (95% [69 of 73; 95% CI 87 to 99]) in three sites in Cambodia (risk difference 1%; 95% CI -6 to 8; p=1·00). The overall 42-day PCR-corrected efficacy of artemether-lumefantrine plus amodiaquine (98% [281 of 286; 95% CI 97 to 99]) was similar to that of artemether-lumefantrine (97% [279 of 289; 95% CI 94 to 98]; risk difference 2%, 95% CI -1 to 4; p=0·30). Both TACTs were well tolerated, although early vomiting (within 1 h) was more frequent after dihydroartemisinin-piperaquine plus mefloquine (30 [3·8%] of 794) than after dihydroartemisinin-piperaquine (eight [1·5%] of 543; p=0·012). Vomiting after artemether-lumefantrine plus amodiaquine (22 [1·3%] of 1703) and artemether-lumefantrine (11 [0·6%] of 1721) was infrequent. Adding amodiaquine to artemether-lumefantrine extended the electrocardiogram corrected QT interval (mean increase at 52 h compared with baseline of 8·8 ms [SD 18·6] vs 0·9 ms [16·1]; p<0·01) but adding mefloquine to dihydroartemisinin-piperaquine did not (mean increase of 22·1 ms [SD 19·2] for dihydroartemisinin-piperaquine vs 20·8 ms [SD 17·8] for dihydroartemisinin-piperaquine plus mefloquine; p=0·50). INTERPRETATION: Dihydroartemisinin-piperaquine plus mefloquine and artemether-lumefantrine plus amodiaquine TACTs are efficacious, well tolerated, and safe treatments of uncomplicated P falciparum malaria, including in areas with artemisinin and ACT partner-drug resistance. FUNDING: UK Department for International Development, Wellcome Trust, Bill & Melinda Gates Foundation, UK Medical Research Council, and US National Institutes of Health.

Delayed diagnosis of T3 supplementation in a bodybuilder presenting with tachycardia and features of sepsis.

A 25-year-old man presented generally unwell to the emergency department. Initial assessment identified systemic inflammatory response syndrome markers with an insect bite as a potential source of infection and he was treated for presumed sepsis. Tachycardia persisted and baseline thyroid function testing showed undetectable free thyroxine and thyroid-stimulating hormone (TSH), prompting further endocrine investigation. Triiodothyronine (T3) was markedly raised with normal TSH receptor antibodies, and the patient later confessed to supplementary testosterone and T3 use as part of bodybuilding activities. Following counselling, thyroid function normalised and the patient returned to his usual health. This case describes the diagnostic work up in a case of persistent tachycardia caused by T3 supplementation, demonstrating the potential for endocrine supplementation by bodybuilders which may be poorly understood and recognised by clinicians. T3 supplementation should be considered and a thorough drug history obtained in bodybuilders presenting with symptoms of thyrotoxicosis and deranged thyroid function tests.

Case Report: Application of hepatitis B virus (HBV) deep sequencing to distinguish between acute and chronic infection.

Deep sequencing of the full-length hepatitis B virus (HBV) genome provides the opportunity to determine the extent to which viral diversity, genotype, polymorphisms, insertions and deletions may influence presentation and outcomes of disease. Increasing experience with analysis of HBV genomic data opens up the potential for using these data to inform insights into pathophysiology of infection and to underpin decision making in clinical practice. We here set out to undertake whole genome HBV sequencing from an adult who presented acutely unwell with a new diagnosis of HBV infection, and tested positive for both HBV anti-core IgM and IgG, possibly representing either acute hepatitis B infection (AHB) or chronic hepatitis B with an acute reactivation (CHB-AR). The distinction between these two scenarios may be important in predicting prognosis and underpinning treatment decisions, but can be challenging based on routine laboratory tests. Through application of deep whole-genome sequencing we typed the isolate as genotype-D1, and identified several minority variants including G1764A and G1986A substitutions in the pre-core promoter and pre-core regions, which support CHB-AR rather than AHB. In the longer term, enhanced deep sequencing data for HBV may provide improved evidence to distinguish between acute and chronic infection, to predict outcomes and to stratify treatment.

HRP2: Transforming Malaria Diagnosis, but with Caveats.

The major growth in point-of-care malaria diagnosis over the past decade has been based on immunochromatographic malaria rapid diagnostic tests (mRDTs), which generally detect Plasmodium falciparum via its abundant histidine-rich protein 2 (HRP2). Here, we review the discovery and biology of HRP2, as well as the strengths and weaknesses of HRP2-based diagnosis compared with alternative antigens. We highlight recent studies describing HRP2 deletion in Latin America, Eritrea, and possibly other regions, and the methodological challenges of confirming deletion of the pfhrp2 gene. We also discuss the mechanism of persistent HRP2 positivity after effective antimalarial treatment, along with other emerging HRP2-based applications, including detection of submicroscopic malaria and diagnosis of severe malaria.

Isolated leptomeningeal carcinomatosis and possible fungal meningitis as late sequelae of oesophageal adenocarcinoma.

We describe a case of a 67-year-old man with known chronic obstructive pulmonary disease, type 2 diabetes mellitus, hypertension, osteoarthritis, previous history of excess alcohol intake, and oesophagectomy 3 years earlier for T3N0 adenocarcinoma, referred by his general practitioner with confusion, weight loss and several recent falls. CT of the chest, abdomen and pelvis revealed a right middle-lobe pulmonary embolism, while CT of the head revealed a communicating hydrocephalus. Lumbar puncture was performed, and empirical treatment for tuberculous and fungal meningitis was commenced. Unfortunately, he suffered a rapid neurological deterioration with markedly elevated cerebrospinal fluid (CSF) pressures, leading to an external ventricular drain. Cytological analysis of a CSF sample revealed a cellular infiltrate consistent with leptomeningeal carcinomatosis (adenocarcinoma), with the previous oesophageal malignancy the likely primary. He passed away 17 days after hospital admission. Prolonged culture of CSF later produced evidence of two distinct phaeomycotic moulds (Cladosporium sp and Exophiala sp), suggesting that fungal meningitis may also have contributed to the clinical picture.

The efficacy of dihydroartemisinin-piperaquine and artemether-lumefantrine with and without primaquine on Plasmodium vivax recurrence: A systematic review and individual patient data meta-analysis.

BACKGROUND: Artemisinin-based combination therapy (ACT) is recommended for uncomplicated Plasmodium vivax malaria in areas of emerging chloroquine resistance. We undertook a systematic review and individual patient data meta-analysis to compare the efficacies of dihydroartemisinin-piperaquine (DP) and artemether-lumefantrine (AL) with or without primaquine (PQ) on the risk of recurrent P. vivax. METHODS AND FINDINGS: Clinical efficacy studies of uncomplicated P. vivax treated with DP or AL and published between January 1, 2000, and January 31, 2018, were identified by conducting a systematic review registered with the International Prospective Register of Systematic Reviews (PROSPERO): CRD42016053310. Investigators of eligible studies were invited to contribute individual patient data that were pooled using standardised methodology. The effect of mg/kg dose of piperaquine/lumefantrine, ACT administered, and PQ on the rate of P. vivax recurrence between days 7 and 42 after starting treatment were investigated by Cox regression analyses according to an a priori analysis plan. Secondary outcomes were the risk of recurrence assessed on days 28 and 63. Nineteen studies enrolling 2,017 patients were included in the analysis. The risk of recurrent P. vivax at day 42 was significantly higher in the 384 patients treated with AL alone (44.0%, 95% confidence interval [CI] 38.7-49.8) compared with the 812 patients treated with DP alone (9.3%, 95% CI 7.1-12.2): adjusted hazard ratio (AHR) 12.63 (95% CI 6.40-24.92), p < 0.001. The rates of recurrence assessed at days 42 and 63 were associated inversely with the dose of piperaquine: AHRs (95% CI) for every 5-mg/kg increase 0.63 (0.48-0.84), p = 0.0013 and 0.83 (0.73-0.94), p = 0.0033, respectively. The dose of lumefantrine was not significantly associated with the rate of recurrence (1.07 for every 5-mg/kg increase, 95% CI 0.99-1.16, p = 0.0869). In a post hoc analysis, in patients with symptomatic recurrence after AL, the mean haemoglobin increased 0.13 g/dL (95% CI 0.01-0.26) for every 5 days that recurrence was delayed, p = 0.0407. Coadministration of PQ reduced substantially the rate of recurrence assessed at day 42 after AL (AHR = 0.20, 95% CI 0.10-0.41, p < 0.001) and at day 63 after DP (AHR = 0.08, 95% CI 0.01-0.70, p = 0.0233). Results were limited by follow-up of patients to 63 days or less and nonrandomised treatment groups. CONCLUSIONS: In this study, we observed the risk of P. vivax recurrence at day 42 to be significantly lower following treatment with DP compared with AL, reflecting the longer period of post-treatment prophylaxis; this risk was reduced substantially by coadministration with PQ. We found that delaying P. vivax recurrence was associated with a small but significant improvement in haemoglobin. These results highlight the benefits of PQ radical cure and also the provision of blood-stage antimalarial agents with prolonged post-treatment prophylaxis.

The haematological consequences of Plasmodium vivax malaria after chloroquine treatment with and without primaquine: a WorldWide Antimalarial Resistance Network systematic review and individual patient data meta-analysis.

BACKGROUND: Malaria causes a reduction in haemoglobin that is compounded by primaquine, particularly in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. The aim of this study was to determine the relative contributions to red cell loss of malaria and primaquine in patients with uncomplicated Plasmodium vivax. METHODS: A systematic review identified P. vivax efficacy studies of chloroquine with or without primaquine published between January 2000 and March 2017. Individual patient data were pooled using standardised methodology, and the haematological response versus time was quantified using a multivariable linear mixed effects model with non-linear terms for time. Mean differences in haemoglobin between treatment groups at day of nadir and day 42 were estimated from this model. RESULTS: In total, 3421 patients from 29 studies were included: 1692 (49.5%) with normal G6PD status, 1701 (49.7%) with unknown status and 28 (0.8%) deficient or borderline individuals. Of 1975 patients treated with chloroquine alone, the mean haemoglobin fell from 12.22 g/dL [95% CI 11.93, 12.50] on day 0 to a nadir of 11.64 g/dL [11.36, 11.93] on day 2, before rising to 12.88 g/dL [12.60, 13.17] on day 42. In comparison to chloroquine alone, the mean haemoglobin in 1446 patients treated with chloroquine plus primaquine was - 0.13 g/dL [- 0.27, 0.01] lower at day of nadir (p = 0.072), but 0.49 g/dL [0.28, 0.69] higher by day 42 (p < 0.001). On day 42, patients with recurrent parasitaemia had a mean haemoglobin concentration - 0.72 g/dL [- 0.90, - 0.54] lower than patients without recurrence (p < 0.001). Seven days after starting primaquine, G6PD normal patients had a 0.3% (1/389) risk of clinically significant haemolysis (fall in haemoglobin > 25% to < 7 g/dL) and a 1% (4/389) risk of a fall in haemoglobin > 5 g/dL. CONCLUSIONS: Primaquine has the potential to reduce malaria-related anaemia at day 42 and beyond by preventing recurrent parasitaemia. Its widespread implementation will require accurate diagnosis of G6PD deficiency to reduce the risk of drug-induced haemolysis in vulnerable individuals. TRIAL REGISTRATION: This trial was registered with PROSPERO: CRD42016053312. The date of the first registration was 23 December 2016.

Artemisinin Resistance and Stage Dependency of Parasite Clearance in Falciparum Malaria.

BACKGROUND: Artemisinin resistance in falciparum malaria is associated with kelch13 propeller mutations, reduced ring stage parasite killing, and, consequently, slow parasite clearance. We assessed how parasite age affects parasite clearance in artemisinin resistance. METHODS: Developmental stages of Plasmodium falciparum parasites on blood films performed at hospital admission and their kelch13 genotypes were assessed for 816 patients enrolled in a multinational clinical trial of artemisinin combination therapy. RESULTS: Early changes in parasitemia level (ie, 0-6 hours after admission) were determined mainly by modal stage of asexual parasite development, whereas the subsequent log-linear decline was determined mainly by kelch13 propeller mutations. Older circulating parasites on admission were associated with more-rapid parasite clearance, particularly in kelch13 mutant infections. The geometric mean parasite clearance half-life decreased by 11.6% (95% CI 3.4%-19.1%) in kelch13 wild-type infections and by 30% (95% CI 17.8%-40.4%) in kelch13 mutant infections as the mean age of circulating parasites rose from 3 to 21 hours. CONCLUSION: Following the start of antimalarial treatment, ongoing parasite sequestration and schizogony both affect initial changes in parasitemia. The greater dependency of parasite clearance half-life on parasite age in artemisinin resistant infections is consistent with ring stage resistance and consequent parasite clearance by sequestration. The stage of parasite development should be incorporated in individual assessments of artemisinin resistance.

Correction to: Analysis of anti-malarial resistance markers in pfmdr1 and pfcrt across Southeast Asia in the Tracking Resistance to Artemisinin Collaboration.

Following publication of the original article [1], one of the authors has highlighted an xml-related discrepancy concerning the author group titled 'Additional Tracking Resistance to Artemisinin Collaboration authors (TRAC Group Authorship)', listed under the Acknowledgements section.

The effect of chloroquine dose and primaquine on Plasmodium vivax recurrence: a WorldWide Antimalarial Resistance Network systematic review and individual patient pooled meta-analysis.

BACKGROUND: Chloroquine remains the mainstay of treatment for Plasmodium vivax malaria despite increasing reports of treatment failure. We did a systematic review and meta-analysis to investigate the effect of chloroquine dose and the addition of primaquine on the risk of recurrent vivax malaria across different settings. METHODS: A systematic review done in MEDLINE, Web of Science, Embase, and Cochrane Database of Systematic Reviews identified P vivax clinical trials published between Jan 1, 2000, and March 22, 2017. Principal investigators were invited to share individual patient data, which were pooled using standardised methods. Cox regression analyses with random effects for study site were used to investigate the roles of chloroquine dose and primaquine use on rate of recurrence between day 7 and day 42 (primary outcome). The review protocol is registered in PROSPERO, number CRD42016053310. FINDINGS: Of 134 identified chloroquine studies, 37 studies (from 17 countries) and 5240 patients were included. 2990 patients were treated with chloroquine alone, of whom 1041 (34·8%) received a dose below the target 25 mg/kg. The risk of recurrence was 32·4% (95% CI 29·8-35·1) by day 42. After controlling for confounders, a 5 mg/kg higher chloroquine dose reduced the rate of recurrence overall (adjusted hazard ratio [AHR] 0·82, 95% CI 0·69-0·97; p=0·021) and in children younger than 5 years (0·59, 0·41-0·86; p=0·0058). Adding primaquine reduced the risk of recurrence to 4·9% (95% CI 3·1-7·7) by day 42, which is lower than with chloroquine alone (AHR 0·10, 0·05-0·17; p<0·0001). INTERPRETATION: Chloroquine is commonly under-dosed in the treatment of vivax malaria. Increasing the recommended dose to 30 mg/kg in children younger than 5 years could reduce substantially the risk of early recurrence when primaquine is not given. Radical cure with primaquine was highly effective in preventing early recurrence and may also improve blood schizontocidal efficacy against chloroquine-resistant P vivax. FUNDING: Wellcome Trust, Australian National Health and Medical Research Council, and Bill & Melinda Gates Foundation.

Plasmepsin II-III copy number accounts for bimodal piperaquine resistance among Cambodian Plasmodium falciparum.

Multidrug resistant Plasmodium falciparum in Southeast Asia endangers regional malaria elimination and threatens to spread to other malaria endemic areas. Understanding mechanisms of piperaquine (PPQ) resistance is crucial for tracking its emergence and spread, and to develop effective strategies for overcoming it. Here we analyze a mechanism of PPQ resistance in Cambodian parasites. Isolates exhibit a bimodal dose-response curve when exposed to PPQ, with the area under the curve quantifying their survival in vitro. Increased copy number for plasmepsin II and plasmepsin III appears to explain enhanced survival when exposed to PPQ in most, but not all cases. A panel of isogenic subclones reinforces the importance of plasmepsin II-III copy number to enhanced PPQ survival. We conjecture that factors producing increased parasite survival under PPQ exposure in vitro may drive clinical PPQ failures in the field.

Malaria.

Following unsuccessful eradication attempts there was a resurgence of malaria towards the end of the 20th century. Renewed control efforts using a range of improved tools, such as long-lasting insecticide-treated bednets and artemisinin-based combination therapies, have more than halved the global burden of disease, but it remains high with 445 000 deaths and more than 200 million cases in 2016. Pitfalls in individual patient management are delayed diagnosis and overzealous fluid resuscitation in severe malaria. Even in the absence of drug resistance, parasite recurrence can occur, owing to high parasite densities, low host immunity, or suboptimal drug concentrations. Malaria elimination is firmly back as a mainstream policy but resistance to the artemisinin derivatives, their partner drugs, and insecticides present major challenges. Vaccine development continues on several fronts but none of the candidates developed to date have been shown to provide long-lasting benefits at a population level. Increased resources and unprecedented levels of regional cooperation and societal commitment will be needed if further substantial inroads into the malaria burden are to be made.