Ethics and regulatory statement
The clinical study protocol and informed consent were reviewed and approved by the Ethics Committee at the Tropical Medicine Research Center of Rondônia (Nº 01/11 CEP/CEPEM e Nº 0001.0.046.000-11 CAAE-SISNEP). The Brazilian National Council on Ethics in Research (CONEP), Ministry of Health, accredits this committee. The clinical study was conducted in accordance with the Helsinki Declaration, Good Clinical Practice [
21] and the Brazilian National Health Council (CNS) resolutions 196/1996 and 466/2011. The study is registered with the Brazilian Register of Clinical Trials (RBR-77q7t3-UTN: U1111-1121-2982). The protocol was also approved by the National Regulatory Agency, ANVISA (CE No. 188/2012-0896111125), and this study was conducted according the Good Clinical Practice (GCP) [
22]. During the study, visits were conducted to ensure GCP adherence. Written informed consent was obtained for every subject prior to enrolment. If the study subject was illiterate, an impartial third party witnessed the informed consent process. All subjects were informed of the nature of the trial and the possible risks bound to it and that they were free to withdraw their consent to participate at any time. The investigators and study staff ensured confidentiality of all records.
Study design and drug administration procedures
This single arm open-label non-comparative trial was conducted according the WHO recommendations [
23] at the Tropical Medicine Research Centre of Rondônia (CEPEM). This centre is located in the Amazon Region of Brazil. The demonstration of at least a 90 % cure rate is considered by the WHO to be satisfactory evidence of efficacy to support the National Malaria Control Programme treatment guidelines [
23]. The sample size was calculated with an expected failure rate of 5 %. To achieve a precision level of 5 % and a possible loss of 20 % of participants by the follow-up, 88 patients were included.
Eligible patients were treated in accordance with the Brazilian Ministry of Health recommendation [
24]. All patients who were included were orally treated with 150 mg coated chloroquine tablets (Farmanguinhos—Fiocruz, batch number 11090655) for 3 days. On the first day, a higher dose of chloroquine was administered (450 mg) followed by 2 days of 300 mg doses per day. Patients were also concomitantly treated with two tablets of primaquine 15 mg (Farmanguinhos—Fiocruz, batch numbers 12010038; 13030282) for 7, 8 or 9 days according three weight ranges (≥50–69; 70–79; 80–90 kg), respectively. The total primaquine dose was never lower than 3 mg/kg or higher than 4.2 mg/kg. An illustrative table is provided as an additional file (Additional file
2).
Chloroquine and primaquine administrations were supervised during the first 3 days. The first dose was taken after diagnosis, and the following doses were administered between 8 and 10 a.m. If the patient vomited within 30 min after a dose, the same dose was administered again. Treatment adherence to primaquine between days 4 and 7 was assessed by inquiring with the patients at the day 7 follow-up visit. The patients were also asked about the use of concomitant therapies at every visit and recorded.
Safety and efficacy evaluations
Patients were evaluated on the day of enrolment and on days 1, 2, 3, 7, 14, 21 and 28 after the study period. The scheduled study procedures included a complete clinical anamnesis, a physical exam and a urinalysis at enrolment and an assessment of the most frequent clinical signs of malaria and adverse events (AEs) at all the follow-up visits. Additionally, blood samples were collected for parasite counts (0, 2, 3, 7, 14, 21 and 28 days), haemoglobin and haematocrit evaluations (0, 14 and 28 days), genotyping (0, 7, 14, 21 and 28 days) and chloroquine pharmacokinetic analyses (0, 3, 7, 14, 21 and 28 days).
The primary efficacy endpoint was the proportion of the population with an adequate clinical and parasitological response; otherwise, it was classified either as early treatment failure or late treatment failure.
Both per protocol (PP) and intention-to-treat (ITT) analyses were conducted, for which the PP was the primary analysis, and the ITT analysis was performed to confirm the results. The PP population excluded any participant associated with a protocol violation or who was lost by the follow-up visit at the primary endpoint on day 28, whereas the ITT population included any violation or loss by the follow-up as parasitological and clinical failure. The cumulative success rate by day 28, corresponding to the probability of remaining parasite-free at day 28, was calculated using a Kaplan Meyer survival curve. The absolute number of patients who presented symptoms and parasitaemia 72 h after treatment were defined as early treatment failures. Demographic data were presented for both populations. All the data were summarized as frequencies and percentages for categorical variables and as the means ± standard deviations (SD) for quantitative variables.
The safety analysis was conducted in the ITT population describing the frequency of AEs and stratifying the AEs among the follow categories: serious AEs, AEs leading to treatment suspension, and AEs corresponding to causalities described as doubtful, unlikely, possible or probable. The mean haemoglobin and haematocrit results at baseline and at the follow-up visits are also presented.
The patients were encouraged to seek unscheduled assessments if any AE was suspected. All clinical or laboratory abnormalities were categorized as grade I to IV according to the common terminology criteria for adverse events (CTCAE) of the National Cancer Institute [
25]. Any suspected serious AE as defined by good clinical practice was reported to the sponsor and the Ethical Review Committee. Any disorder recorded at the inclusion anamnesis or the recurrence of the disorder were not recorded as an AE. However, any exacerbation of a previous disorder, either in frequency or intensity, was described as an AE. Known drug-related events were recorded as an AE, even if it could be classified as a malaria symptom.
The parasitological densities were estimated using Giemsa-stained blood slides at a magnification of 1000× as the WHO recommended methodology for the surveillance of anti-malarial drug efficacy [
23]. Two trained microscopists provided independent results, and the final densities corresponded to the average of the two independent assessments. A third microscopist evaluated discordant results pertaining to the presence or absence of parasites, the diagnosis of different species, or the report of a parasite density greater than 50 % in the same slide. In this case, the final densities corresponded to the average of the two closest counts. Negative results were issued only after the evaluation of 1000 leucocytes in the microscopic fields. Gametocyte presence was also recorded. Genotyping samples were not collected to correct results by PCR, as a high frequency of heterologous hypnozoite activation during vivax relapse is known [
26].
To access chloroquine pharmacokinetics, samples of the peripheral blood were collected either at domiciliary visits or at the day of a clinic visit and transferred to Whatman (USA) ET 31 CHR E 3MM filter papers. The whole blood concentrations of chloroquine were measured using validated high performance liquid chromatography tandem to a mass spectrometry (HPLC–MS/MS) method. This method was in accordance with Brazilian [
27] and international [
28] regulatory requirements for bioanalytical methods. The pharmacokinetics laboratorial analysis was conducted at the Sefar/Oswaldo Cruz Foundation, which is accredited by the Brazilian regulatory agency, ANVISA. The main pharmacokinetic parameters of C
max, T
max, AUC
0–t and AUC
0–inf were calculated using the software WinNonlinTM version 6.3 Pharsight and Microsoft Excel version 14.4.0. The presence of parasites with chloroquine blood levels above the 100 ng/mL threshold was considered to characterize resistance according the protocol and the WHO.