Study design

This single-arm, open-label cohort event monitoring study was conducted at 6 health centers in Cameroon, Democratic Republic of Congo, Gabon, Ivory Coast, and Republic of Congo between 22 June 2017 and 10 April 2019. The protocol adhered to Good Clinical Practice, the Declaration of Helsinki, and relevant regulations in the respective countries and was reviewed and accepted as a valid postmarketing safety assessment by the EMA. The authors vouch for the accuracy and completeness of the data and the fidelity of the trial conduct, analysis, and reporting to the protocol. A copy of the protocol and statistical analysis plan is provided in the Supporting information (S1 Protocol and SAP). This study is reported as per the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline (S1 STROBE Checklist).

Ethics statement

All patients, or their parents/guardians, provided written informed consent, and children able to understand the study gave signed assent. The protocol was conducted in compliance with the Declaration of Helsinki (amended Tokyo 2004) and the directives in the respective countries, in particular concerning the submission to Ethics Committees and the protection of personal data. Additionally, the study was conducted in accordance with the principles of the Good Clinical Practice according to the International Council for Harmonization (ICH) Harmonized Tripartite Guidance (CPMP/ICH/135/95). The ethics committee at each study site granted ethical approval as follows. Cameroon: Comité National d’Ethique de la Recherche pour la Santé Humaine; Democratic Republic of Congo: Comité d’Ethique de l’Université Protestante au Congo; Gabon: Comité Nationale d’Ethique et de la Recherche Scientifique and Comité d’Ethique Institutionnel Albert Schweitzer Hospital, Lambaréné; Ivory Coast: Comité Nationale d’Ethique et de la Recherche Scientifique and Comité National d’Ethique des Sciences de la Vie et de Santé; and Republic of Congo: Institutional Ethics Committee of the Fondation Congolaise pour la Recherche Médicale.

Patients

Eligible patients were male or female, of any age, with body weight of at least 5 kg. All patients had acute uncomplicated malaria and a fever or history of fever within the previous 24 hours and/or anemia. Exclusion criteria were severe malaria, clinical signs or symptoms of hepatic injury (nausea and abdominal pain associated with jaundice), known severe liver disease (i.e., decompensated cirrhosis, Child–Pugh stage C or D), known allergy to artemisinin and/or pyronaridine, known pregnancy, lactation, treatment with pyronaridine–artesunate in the previous 28 days, or if the patient was considered by the investigator to be at particular risk.

Treatment

Fixed-dose pyronaridine–artesunate (Shin Poong Pharmaceutical, Seoul, South Korea) was administered once daily for 3 days with 2 formulations provided according to body weight and without regard to food intake. Daily doses for pyronaridine–artesunate tablets (180:60 mg) were 1 (≥20 to <24 kg), 2 (≥24 to <45 kg), 3 (≥45 to <65 kg), or 4 (≥65 kg); and for granule sachets (60:20 mg) were 1 (≥5 to <8 kg), 2 (≥8 to <15 kg), or 3 (≥15 to <20 kg). To reflect real-world practice, patients could be included more than once in the study, following a 28-day washout period between consecutive pyronaridine–artesunate treatments. Patients received the first pyronaridine–artesunate dose under supervision and were provided with the remaining doses to be taken unsupervised on the subsequent 2 consecutive days.

Procedures

At enrollment on day 0, eligible patients underwent physical examination, with demographic data and medical history noted.

Blood samples (5 mL) were collected for hematology, clinical chemistry, and confirmation of malaria diagnosis. Blood samples were stored for retrospective analysis of baseline clinical chemistry and viral hepatitis assessment: hepatitis A, B, C, and E (anti-HAV IgM, anti-HBc IgM, HBsAg, hepatitis C RNA, and hepatitis E IgM antibody) and hepatitis delta antibody if positive for hepatitis B. All laboratory analysis was performed at the site where the patient was recruited by technically certified staff, and results were validated by external independent laboratories. Standard quality control procedures were in place, including daily internal controls and the quality control process assessed by the clinical monitors.

Malaria was diagnosed using a rapid diagnostic test (RDT) or Giemsa-stained thick blood smear. The RDT was consistent with local treatment guidelines and used according to the manufacturer’s instructions by staff trained according to the local standard operating procedure (S1 Methods). Microscopic diagnosis was performed in accordance with standard protocols by qualified microscopists [22], with 10% of slides undergoing quality control checks by an independent microscopist. Blood spots were obtained at screening and stored on filter paper for polymerase chain reaction (PCR) genotyping. Patients were visited by a community health worker (CHW) on days 7 and 28 who inquired regarding any signs or symptoms of malaria. In addition, patients were requested to present to the health center should they have malaria symptoms at any time during follow-up. Any patient who presented with suspected malaria was clinically examined and malaria confirmed by microscopy. At follow-up day 28, blood samples were collected from all patients to confirm parasite clearance by microscopy. In the case of P. falciparum recurrence, PCR genotyping was used to differentiate between recrudescence and reinfection by comparing blood spot samples taken at baseline versus recurrence, using published criteria [23].

Adverse events were assessed without knowledge of baseline ALT/AST. Patients were to present at the clinic should any adverse event occur within 28 days of pyronaridine–artesunate dosing. Home follow-up visits were conducted by CHWs on days 7 and 28 to assess treatment adherence, clinical condition, adverse events, and concomitant medications. CHWs were trained before the study and retrained during the study by clinical investigators in the assessment of safety and tolerability using questionnaire forms. In addition, investigators accompanied CHWs to field visits to assure quality and consistency in adverse event assessment. All clinical information provided by CHWs was assessed by clinical investigators to ensure accurate medical recording of all safety and tolerability signs. CHWs were provided with pictorial information specifically identifying the signs and symptoms of liver injury. Furthermore, any potential hepatotoxic signs or symptoms noted by CHWs were specifically discussed with the study doctor, with patients referred to the clinic if necessary.

Hepatic events prompted blood sampling for clinical chemistry and hematology and a full hepatitis panel. Assessment at the health facility was triggered by serious or severe adverse events or adverse events of special interest, i.e., hepatotoxicity (jaundice, dark urine, putty/mastic stool, worsening of fatigue, nausea, vomiting, anorexia, abdominal pain, itching, rash, spontaneous bruising, or appearance of red spots), serious liver reactions, and hypersensitivity (flushing, wheals/urticaria, breathlessness, faintness, or a fall in blood pressure). Female participants had a urinary pregnancy test at day 28, with provision in the case of pregnancy for extended follow-up until the birth or other outcome.

Outcomes and trial populations

The safety population included all enrolled patients who received at least 1 dose of study medication for any malaria episode. The primary outcome was the incidence of hepatic events in the safety population following pyronaridine–artesunate treatment of malaria patients with baseline ALT or AST >2×ULN, defined as clinical signs and symptoms of possible hepatotoxicity, i.e., fatigue, nausea, abdominal pain, itching, or signs of jaundice (dark urine, putty or mastic colored stools, and yellowing of the whites of the eyes or skin), associated with a rise in ALT/AST >2× the baseline value.

Safety outcomes were the incidence of adverse events in the safety population overall and categorized by normal/abnormal/unknown baseline ALT/AST, any underlying hepatic disease identified on the hepatitis panel, HIV status (where known), age, sex, weight, and nutritional status. Malnutrition was defined for patients <5 years old as a mid-upper arm circumference <115 mm, in patients aged ≥5 years to ≤19 years using the World Health Organization age- and sex-specific body mass index (BMI)-for-age z-scores [24], and for adults >19 years old as a BMI <18.5 kg/m².

Treatment adherence was self-assessed by patients and also estimated by CHWs based on empty packaging provided by patients versus the number of tablets/sachets dispensed.

Effectiveness was evaluated on day 28 in the intention-to-treat and per-protocol populations, with cure defined as the absence of microbiologically confirmed malaria without previous failure. Cure rates were reported unadjusted and PCR adjusted for P. falciparum reinfection. Day 28 effectiveness subgroup analysis was planned for by country, dosing (granules versus tablets), malnutrition, and age. Additional outcomes were time between malaria episodes and the frequency of repeat episodes. The intention-to-treat population included all malaria episodes treated with at least 1 dose of study medication with confirmed positive parasitemia at baseline. The per-protocol population included all malaria episodes in the intention-to-treat population in patients who were treated with a full course of study medication (adherence per patient self-assessment), had a day 28 effectiveness endpoint, did not vomit after study drug administration (except where vomiting occurred after the first dose and dosing was successfully repeated), and who had no prior or concomitant medication, which had an antimalarial effect and so could interfere with the treatment outcome. All outcomes and trial populations were prespecified (S1 Protocol and SAP).

Statistical analysis

Sample size was calculated based on the primary objective, i.e., the incidence of protocol-defined hepatic events in malaria patients with baseline ALT/AST >2×ULN. The screening rate in previous studies for malaria patients with baseline ALT/AST >2×ULN was 1.4% [25]. To detect 1 protocol-defined hepatic event in patients with baseline ALT/AST >2×ULN with a probability of at least 80% (81.6%), 120 malaria episodes occurring in patients with a baseline ALT/AST value >2×ULN were required. Thus, to observe 1 severe hepatic event, at least 8,572 malaria episodes were required (i.e., 1.4% of 8,572 = 120). There was no country-specific recruitment target, and the trial was to continue until observation of at least 120 malaria episodes in patients with elevated baseline ALT/AST. As a secondary endpoint, a multivariate logistic regression model was planned in order to compare the incidence of hepatic events in patients with elevated baseline ALT/AST values versus those with normal values. However, as the primary endpoint did not occur in any patient during the trial, this analysis was not conducted.

Descriptive statistics were used for analysis of all other outcomes, with 95% CIs calculated (Clopper–Pearson). To better mimic the clinical reality, patients could be included more than once in the study. For the outcome of clinical effectiveness, each malaria episode was treated as an independent event, with no adjustment for repeated measures. This was to better reflect the endpoint of operational effectiveness, i.e., to include patient-related as well as drug-related contributors and to be consistent with previous analyses of randomized controlled trials of repeated treatment with pyronaridine–artesunate [11,25]. For the determination of cure rates, patients with a missing parasitological assessment at day 28 were considered as failures in the intention-to-treat population and excluded from the per-protocol population. For further details, please see S1 Protocol and SAP. Statistical analysis used SAS (version 8.2) and GraphPad Prism (version 8.4.3). Adverse events were coded using MedDRA (version 22).

Patients

Of the 8,609 malaria episodes that met study eligibility criteria, 8,560 were treated with at least 1 dose of study medication, comprising the safety population (Fig 1). Of the 1.9% (158/8,560) of malaria episodes occurring in patients with baseline ALT/AST elevations >2×ULN, 19.0% (30/158) were positive for viral hepatitis. The safety population included similar proportions of males and females; most patients had 1 malaria episode, but the maximum was 9 episodes (Table 1). Repeated malaria episodes accounted for 16.7% (1,414/8,480) of all episodes in the intention-to-treat population (Table 1). Time between repeated malaria episodes tended to decrease for each subsequent episode (S1 Fig). Based on patient self-assessment, treatment adherence was achieved in 94.5% (8,085/8,560) of malaria episodes versus 81.5% (6,974/8,560) based on empty medication packaging presented to CHWs (Table 1).

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Fig 1. Screening and analysis populations.

There may have been more than 1 reason for exclusion from enrollment into the study or exclusion from a study population. Normal LFTs were ALT or AST ≤2×ULN and abnormal values were ALT or AST >2×ULN at baseline. “Patient considered to be at risk from participation” included patients that investigators considered inappropriate for trial inclusion, mainly because of low hemoglobin or an inability to draw blood for assessments. Note that no patient with clinically evident preexisting liver injury was excluded from the screening population. ALT, alanine aminotransferase; AST, aspartate aminotransferase; LFT, liver function test; ULN, upper limit of normal.

https://doi.org/10.1371/journal.pmed.1003669.g001

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Table 1. Baseline demographic and clinical characteristics in the safety population.

https://doi.org/10.1371/journal.pmed.1003669.t001

Hepatic safety

There were no malaria episodes with protocol-defined hepatic events in any patient in the safety population. No further planned secondary analyses of the primary endpoint could, therefore, be conducted.

Safety

Adverse events of any cause occurred in 20.8% (1,490/7,154) of patients, most frequently pyrexia (5.1% [366/7,154]) and vomiting (4.2% [303/7,154]) (Table 2, S1 Table). There was no difference in adverse event frequency between patients with normal versus abnormal baseline ALT/AST values (Fig 2), although those with unknown ALT/AST values experienced more adverse events (40.0% [14/35] 95% CI, 25.6 to 56.4).

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Fig 2. Frequency of adverse events of any cause subgroup analysis in the safety population.

Data are shown for the number of patients. Patients may have been categorized differently for different malaria episodes. Categorical data were not available for all patients. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CI, confidence interval; LFT, liver function test.

https://doi.org/10.1371/journal.pmed.1003669.g002

Adverse event frequency was not affected by sex, weight, nutritional status, HIV status, or first versus repeated malaria episode (Fig 2). The nature of adverse events and their incidence was comparable for first versus repeated episodes (S2 Table). Four of the 30 patients positive for viral hepatitis had adverse events (13.3%).

Higher adverse event rates were noted for patients aged under 1 year (29.1% [39/134] 95% CI, 21.6 to 37.6) and those ≥18 years of age (27.0% [459/1,700] 95% CI, 24.9 to 29.2) versus those aged 5 to <18 years (16.9% [611/3,609] 95% CI, 15.7 to 18.2) (Fig 2, S3 and S4 Tables). In particular, vomiting was associated with age, occurring in 11.2% [15/134] of children under 1 year old, 7.7% (131/1,711) of those aged 1 to <5 years, 3.1% (112/3,609) of children and adolescents 5 years to 18 years, and in just 2.8% (47/1,700) of adults (S3 Table). The frequency of pyrexia and cough also tended to decrease with increasing age (S3 Table).

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Table 2. Most common adverse events of any cause in patients treated with pyronaridine–artesunate for acute uncomplicated malaria (safety population).

https://doi.org/10.1371/journal.pmed.1003669.t002

There were 9.6% (685/7,154) of patients with adverse events considered related to pyronaridine–artesunate treatment, most commonly vomiting (3.2% [228/7,154]) and headache (1.0% [72/7,154]) (S5 Table). Treatment was discontinued early owing to adverse events in 0.8% (54/7,154) of patients, mostly because of vomiting (0.5% [37/7,154]) (S6 Table). Severe adverse events occurred in 0.4% (32/7,154) of patients and life-threatening events in 0.1% (10/7,154) (S7 Table).

Serious adverse events were reported in 0.4% (29/7,154) of patients, with the most frequent being malaria (0.1% [9/7,154]) and anemia (0.1% [7/7,154]) (S8 Table). Four patients had serious adverse events considered possibly related to pyronaridine–artesunate treatment (epistaxis, gastroenteritis, and 2 cases of anemia).

There were 3 deaths during the study (diarrhea, drowning, and severe malaria), none of which were considered related to pyronaridine–artesunate treatment. The case of severe malaria occurred in a 6-year-old girl from the Democratic Republic of Congo who was enrolled with acute uncomplicated malaria, presenting with asthenia and vomiting. She was diagnosed with severe malaria 1.5 hours following the first dose of pyronaridine–artesunate and admitted to hospital after symptoms of asthenia worsened. Pyronaridine–artesunate was discontinued, and the patient treated with intravenous quinine. She also received intravenous phenobarbital and diazepam for convulsions and cefotaxime and gentamycin for suspected meningitis, but her condition rapidly worsened, and she unfortunately died later the same day. As the patient died of severe malaria within 24 hours of her first dose of pyronaridine–artesunate, it is usual to consider the cause as disease progression, and so the death was not attributed to drug treatment. The death from diarrhea occurred in a 66-year-old man who received 6 pyronaridine–artesunate tablets over 3 days, who died 29 days after the first dose of study medication from severe diarrhea, with no further information available. The other death was that of a 9-year-old boy who drowned in a river 12 days after the first administration of pyronaridine–artesunate.

An adverse event of special interest occurred in 9 patients (0.1%, N = 7,154); in all cases, this was hypersensitivity occurring only in 1 episode: 6 occurred in the first episode (6/7,085; 0.09% 95% CI, 0.03 to 0.18), and the other 3 occurred for the first time in episodes 2, 3, and 4, respectively (3/1,051; 0.3%; 95% CI, 0.06 to 0.8). All patients were aged ≥5 years with normal baseline ALT/AST, 4 had urticaria, and 1 each had maculo-papular rash, papular rash, hypersensitivity (symptomatology of flushing and wheals), lip swelling, and eye pruritis; all were of mild-to-moderate intensity.

CHWs identified 595 cases that required discussion of adverse events with the investigational site, with 165 referrals. An additional 223 cases self-referred. Of these, further blood testing was clinically indicated in 7 patients, with no concerning results (S9 Table). Eleven pregnancies were confirmed during the study. Three were terminated for social reasons. The remaining 8 (6 of which were exposed to study treatment during the first trimester) progressed normally with spontaneous deliveries, except for 1 case of uterine hypertonus at full term, requiring cesarean section.

Effectiveness

P. falciparum was the predominant pathogen, present in 8,478 of 8,480 malaria episodes (intention-to-treat population). Plasmodium ovale (n = 70) and Plasmodium malariae (n = 55) occurred as mixed Plasmodium infections; 1 P. ovale/P. malariae coinfection and 1 Plasmodium vivax mono-infection were identified (S10 Table). Adjusting for P. falciparum reinfection, treatment effectiveness at day 28 was 98.6% ([7,369/7,746] 95% CI 98.3 to 98.9) in the per-protocol population and 90.9% ([7,705/8,478] 95% CI 90.2 to 91.5) in the intention-to-treat population. The unadjusted cure rate in the per-protocol population was 93.2% ([7,221/7,746] 95% CI 92.6 to 93.8) and 85.9% ([7,285/8,480] 95% CI 85.1 to 86.6) in the intention-to-treat population (S11 Table).

Subgroup analyses of the day 28 PCR-adjusted cure rate in the per-protocol population are shown in Fig 3 and S11 Table. Cure rates across the 5 participating countries exceeded 98%. The cure rate was lower for the granule formulation (97.4% [2,849/2,925] 95% CI 96.8 to 97.9) versus tablets (99.4% [4,790/4,821] 95% CI 99.1 to 99.6) and lower in infants (<1 year old; 96.9% [126/130] 95% CI, 92.3 to 99.2) versus older children and adults (98.6% [7,513/7,616] 95% CI, 98.4 to 98.9), though CIs overlapped. In malnourished patients, the cure rate was 100% (370/370).

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Fig 3. Day 28 PCR-adjusted treatment effectiveness in the per-protocol population subgroup analysis.

Data are shown for the number of malaria episodes. CI, confidence interval; PCR, polymerase chain reaction.

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