Study sites and study population
This study was part of a multi-country single arm clinical trial aimed at assessing the efficacy and safety of two artemisinin-based combinations (DPQ and artemether–lumefantrine) when used to treat malaria in HIV-infected adults on standard ART. One of the single arm trial assessing the efficacy and safety of AL was conducted in Zambia and findings have been reported elsewhere [10]. In this paper, findings from another single arm trial assessing the efficacy and safety of DPQ which was conducted from October 2013 to June 2015 at Queen Elizabeth Central Hospital and Chikhwawa District Hospital in Malawi as well as Manhiça District Hospital in Mozambique, are reported. These are settings of moderate-high transmission of malaria [11, 12] and high HIV prevalence [13, 14]. Blantyre is an urban district in Southern Malawi with an estimated population 1,239,647, while Chikhwawa is a rural district located 34 km south of Blantyre with an estimated population of 518,284. In 2014, the malaria parasite prevalence in under-five children in Malawi was 33% and higher in rural (37%) than urban areas (11%) [11]. HIV prevalence in Malawi was estimated at 10.6% in 2015 [13]. Manhiça is a rural district in Southern Mozambique located 80 km north of Maputo city, with an estimated population of 178,000 in 2014. Malaria parasite prevalence in under-five children was estimated at 51% in 2013 [12] while HIV prevalence in the district was estimated at 39.9% in 2010 [14].
During the study period, the criteria for initiating ART in the two countries were WHO HIV disease stages 3 or 4, CD4 cell count < 350, pregnancy or lactation [15]. In October–December 2013, > 87% of ART individuals in Malawi were on a fixed dose combination of tenofovir/lamivudine/efavirenz while 6% were on stavudine/lamivudine/nevirapine or zidovudine/lamivudine/nevirapine [16]. In Mozambique, the majority of the ART individuals were on fixed dose zidovudine/lamivudine/nevirapine but the use of fixed dose tenofovir/lamivudine/efavirenz increased steadily over the study period. In both countries, artemether–lumefantrine was the first-line treatment for uncomplicated malaria. DPQ was registered but not routinely available within the public health system in the two countries. Nevertheless, it was evaluated in this study because it is one of the WHO recommended artemisinin-based combinations and has a more convenient dosing schedule than AL (once daily for 3 days). The efficacy and safety of AL was assessed in a separate trial [10].
Study design and clinical procedures
This was a single arm clinical trial (Registration number: PACTR201311000659400). HIV-infected patients on nevirapine- or efavirenz-based ART suspected of having malaria were pre-screened through history taking and clinical examination to determine their eligibility for the study. The study inclusion criteria were as follows: age ≥ 15 to ≤ 65 years; weight ≥ 35 kg; documented fever (axillary ≥ 37.5 °C) or history of fever 24 h prior enrolment; smear positive P. falciparum malaria monoinfection with asexual malaria parasite densities < 200,000/µL; ability to swallow oral medications and willingness and ability to comply with scheduled visits, supervised treatment administration, laboratory tests, and other study procedures. The following were the exclusion criteria: severe malaria as per WHO criteria [17]; mixed infection with another Plasmodium species; haemoglobin (Hb) concentration < 7 g/dL; severe sickle cell disease or sickle-haemoglobin C anaemia; evidence of pregnancy or lactation; use of any anti-malarial treatment or drug with anti-malarial activity within the past 1 month, except cotrimoxazole; history of DPQ hypersensitivity reactions; gastrointestinal diseases that could alter gut absorption or motility; history of splenectomy; history of epilepsy or convulsions; pre-existing clinically-significant cardiac, liver, renal, neurological or psychiatric abnormalities; alternative clinical cause of fever other than malaria and participation in any investigational drug study in the past 30 days.
Finger-prick blood samples were taken from those who satisfied the preliminary eligibility criteria and tested for malaria using Rapid Diagnostic Test (RDT) (SD BIOLINE Malaria Ag P.f/Pan test produced by Alere) and for haemoglobin concentration using Hemocue Haemoglobinometer. Thick blood smear microscopy examinations were performed on patients with RDT positive malaria while clinical examinations were performed in those with confirmed malaria parasitaemia. Consenting participants were enrolled and scheduled for a 3-day hospital admission.
The participants received dihydroartemisinin–piperaquine (Eurartesim®, Sigma Tau): 3 tablets for study participants < 60 kg or 4 tablets study for participants ≥ 60 kg. Each tablet contained dihydroartemisinin/piperaquine 40 mg/320 mg, respectively, administered at 0 h, 24 (+ 4) and 48 (+ 4) h after the first dose. Participants’ vital signs were measured at 6-hourly intervals and adverse events were monitored. A 12-Lead electrocardiogram (ECG) was performed before the first dose of DPQ and within 2 h after administration of the third dose DPQ. Any patient with Fridericia-corrected QT (QTcF) interval of ≥ 450 ms or QTc increase of > 60 ms from the baseline underwent follow-up ECGs until resolution of the abnormality. Participants were discharged at least 24 h after taking the third (last) dose of DPQ (post-treatment day 3) and advised to come for follow up visits on post-treatment days 7 (± 1), 14 (± 1), 21 (± 2), 28 (± 2), 35 (± 2), 42 (± 2) and 63 (± 2). Participants were encouraged to return to the health facility any time they felt unwell (unscheduled visits). All adverse events were graded using the DAIDS criteria [18]. Adverse events with onset or increased severity after the first dose of DPQ were counted as treatment-emergent adverse events (TEAEs). During follow up visits, participant’s time and any incurred expenses when attending the study clinic were appropriately compensated, as approved by the ethics committees.
Laboratory procedures
During the admission period, thick blood slides were collected pre-dosing and at 6-hourly intervals until after obtaining two consecutive malaria negative smears. The slides were also collected at scheduled and unscheduled follow-up visits. The slides were Giemsa-stained and read by an experienced microscopist using standard protocols [19]. For quality control, all slides were re-read by a second microscopist; a third microscopist settled any discrepant readings. Dry blood spot (DBS) samples were collected on filter paper (Whatman 3MM®) at baseline and during recurrent malaria episodes. Parasite DNA was extracted from the DBS samples, amplified using polymerase chain reaction (PCR) and genotyped for merozoite specific protein (MSP) 1 and 2 to distinguish malaria recrudescence from re-infection, using methods previously described [20]. Samples that did not produce results were classified as indeterminate.
Venous blood samples were collected on days 0, 3, 28, 42 and 63 for biochemistry tests using a Beckman CX5® Chemistry analyzer, on days 0, 3, 7, 28, 42 and 63 for haematological tests using a Beckman Coulter® HMX Analyzer and on days 0, 28, and 63 for CD4 cell count measurement using a BD FACSCount™ machine. Plasma samples collected on days 0, 28, and 63 were stored for future HIV viral load assays.
Blood samples for sparse pharmacokinetic (PK) assays were collected in sampling windows of 0–6, 6–48, 48–60 h, and on days 7, 21, 28 or 35 from first dose, as previously recommended [21]. In this paper, the relationship between day-7 concentrations and ACPR was explored since day-7 concentrations of the slowly eliminated partner drug of ACT have been shown to be a better determinant of therapeutic response than the area under the concentration–time curve [22]. The PK samples were analysed using a previously described HPLC–UV assay method [8]. The lower limit of quantitation (LLOQ) of the piperaquine was 25 ng/mL, with a coefficient of variation of < 10%. The PK laboratory at the Malawi-Liverpool Wellcome Trust Clinical Research Programme in Blantyre, Malawi, participated in the World Wide Antimalarial Resistance Network’s external quality assurance programme [23].
Study endpoints
The primary study endpoint was proportion of patients with PCR-corrected day 42 ACPR, defined as patients who did not have parasitaemia on day 42 that exhibited identical P. falciparum malaria PCR markers (merozoite surface protein 1 and 2) with those at baseline, irrespective of axillary temperature, and who had not previously met any of the criteria of early treatment failure (ETF), late clinical failure (LCF) or late parasitological failure (LPF). Standard WHO definitions of ETF and LCF were used [9].
The other primary study end points were grade 3 or 4 TEAEs of special interest (Fridericia-corrected QT interval prolongation, dizziness, palpitations, urticaria or itchiness) and serious adverse events (SAEs) as per standard definitions [24]. Local study physicians determined the relationships between DPQ and the adverse events (AEs). A Data Safety and Monitoring Board reviewed serious AEs and adverse events of special interest (AESIs) and assessed the validity of study physicians’ decisions. Secondary endpoints, included day 42 PCR-uncorrected ACPR, time for parasite to decline by 50% (PC50) and 90% (PC90), fever clearance time and trends in haemoglobin concentrations and CD4 cell counts from baseline to day 28.
Samples size
Sample size calculation was based on estimates of total treatment failure rate (TTFR). The estimated day-42 PCR corrected TTFR was ≤ 10% [25]. A precision of 5%, around this point estimate, allowed the upper limit of the 95% Wald binomial confidence interval to be 15%. Using the formula for estimating a single study population sample size [26], our effective sample size was estimated at 138 for each ART type. The final sample size, for each ART group, was 163 after adjusting for an anticipated loss-to-follow-up rate of 15%. This sample size was achieved for the efavirenz-ART group but not for nevirapine-ART group, as the ART programs in both countries had successfully transitioned from nevirapine- to efavirenz-based ART first-line regimens. Sample size calculations including all subsequent statistical analysis were performed in STATA 13.1
Statistical analyses
For the primary end-point, three analysis populations were used. Firstly, the Intention-to-treat (ITT) population included patients who received at least 1 dose of study medication. Secondly, the per-protocol (PP) population included all participants who had the primary endpoint data at day 42, received a full course of DPQ and adhered to the follow-up visit schedule. Thirdly, the safety population included all patients who received any amount of study medication and had at least 1 assessment after dosing. ACPR plus 95% CI in PP and ITT populations were calculated. Sensitivity analyses were performed using the ITT and PP populations that first considered all participants with missing data as having parasitological failure and then considered the same participants as having treatment success.
Statistical analyses for secondary ACPR endpoints were similar to the primary endpoints. In addition, the Kaplan–Meier survival plots were used to summarize the time to PCR-corrected and uncorrected treatment failure. Parameters assessing post-treatment parasite clearance (PC50, PC90 and parasite clearance half-life) in the two ART groups were estimated using the WorldWide Antimalarial Resistance Network parasite clearance estimator, as described elsewhere [27].
Descriptive statistics were computed for baseline variables in the two ART groups. However, as this study was designed to estimate and not to compare efficacy and safety of DPQ between the two ART groups, no formal statistical comparisons of baseline characteristics efficacy or safety endpoints were made between the two ART groups. As part of a priori exploratory analysis, and where appropriate, Wilcoxon rank-sum/Mann–Whitney U test was used to compare distributions of the day-7 piperaquine concentrations in those who attained or did not attain ACPR by day 42. Piperaquine concentrations below the LLOQ were imputed to half the lower limit of quantification and included in the estimation of median piperaquine exposure if the imputed values were < 10% of the data. Additionally, Wilcoxon matched paired signed-rank test was used to compare baseline and day 28 CD4 cell and haemoglobin values in each ART group.