Study design, setting and patient population
A single center, randomized, open-label primaquine efficacy trial enrolled Indonesian soldiers with P. vivax malaria after returning from malarious Papua (Fig. 1) [24]. The study took place at an army base at Sragen, Central Java, between March 2013 and July 2014. The trial team established permanent residence, offices, clinic, pharmacy, and laboratory on that base.
The battalion identified for study returned to Java by ship, disembarking at Tanjung Mas at Semarang on 28 February 2013. Routine microscopic screening for malaria at portside revealed 67 of 536 soldiers had malaria (30 P. falciparum, 33 P. vivax, and 4 mixed infections). These soldiers received treatment according to national guidelines [12], but not primaquine because screening for glucose-6-phosphate dehydrogenase (G6PD) deficiency was not available. The soldiers departed on liberty to visit with families prior to returning to base. They had done so by 20 March 2013, when study familiarization began, including emphasis on the importance of voluntary consent. Enrollments commenced on 29 March 2013.
Soldiers on base faced no known risk of acquiring malaria locally. Sragen District (population 896,201 residents) occupies 942 km2 with an average density of 947 residents/km2 with 25 primary health care centers and 10 hospitals. During 2012–2013, authorities reported eight malaria cases, all imported. No known cases of locally acquired malaria were reported during 2013–2014. Reinfection at Sragen was highly improbable.
Eligibility and randomization
Consenting soldiers with confirmed P. vivax were screened for eligibility by obtaining a medical history, and conducting physical and laboratory examinations that included electro-cardiogram (ECG; BTL-08, BTL Industries, Framingham, MA, USA; 12-lead, 50 mm/s, 10 mm/mV), complete blood counts (Coulter Ac-T; Beckman Coulter, Fullerton, CA, USA), full blood chemistry panel (Cobas-111, Roche, Rotkreuz, Switzerland), and G6PD deficiency screening (NADPH kit 203A, Trinity Biotech, Wicklow, Ireland). Eligible subjects had uncomplicated vivax malaria, were not under treatment for another illness, had not recently consumed antimalarials, and showed normal ECG, blood laboratory values, and G6PD activity by fluorescent spot testing (Trinity Biologics, Ireland).
The trial statistician block-allocated treatment assignments by varying blocking number at random [26]. A plain, consecutively numbered and sealed envelope prepared by the trial administrator contained therapy assignment and was opened in sequence as each eligible and consenting soldier enrolled. The study pharmacist supervised therapy and adherence to assigned treatment, including direct observation of each of the daily 14 doses of PQ.
Study treatments
Groups received one of the following treatments under directly observed, signature-affirmed supervision by a member of the research team:
AS + PQ – 200 mg AS (Arsuamoon®; a 50-mg artesunate tablet co-packaged with an amodiaquine hydrochloride tablet [discarded]; Guilin Pharmaceuticals Co. Ltd., Shanghai, China) was administered, followed by a daily dose of 100 mg daily for six days. After a 48-hour pause for AS washout, PQ was administered with a daily dose of two tablets, each containing 15 mg PQ base for 14 days. As in all other groups, Sanofi Pharmaceuticals (PRIMAQUINE®, Primaquine phosphate tablets USP 26.3 mg, 15 mg equivalent base, Sanofi-Aventis Canada Inc. Laval, Quebec) provided the PQ administered.
AS-PYR + PQ – single daily dose of three tablets, each containing 60 mg AS and 180 mg of PYR provided as Pyramax® from Shin Poong Pharmaceuticals, Seoul, South Korea, for three days given concurrently with a daily dose of 30 mg PQ base for 14 days.
DHA-PP + PQ – a single daily dose of three tablets, each containing 40 mg DHA and 320 mg of PP base provided as Eurartesim® from Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Pomezia, Rome, Italy, for three days given concurrently with a daily dose of 30 mg PQ base for 14 days.
Subjects weighing >70 kg were treated as above except instead receiving 45-mg daily dose PQ (three tablets) for 14 days, five daily AS tablets instead of four, and four rather than three tablets of either AS, AS-PYR or DHA-PP daily. All subjects were offered a non-fatty carbohydrate snack prior to dosing to mitigate stomach upset caused by PQ [27]. Subjects were monitored for an hour and dosing repeated if vomiting occurred.
Follow-up
Physical complaints and methemoglobin (Masimo Blood Oximeter, Masimo Corp., Irvine, CA, USA) were recorded daily during therapy. Routine blood film examinations occurred on days 3, 7, 14, 21, 28, 35, 42, 56, 63, 70, 84, 126, 140, 180, and 365 post-enrollment. Subjects reporting to the clinic with illness were examined the same day. Subjects positive for P. vivax received DHA-PP + PQ (0.5 mg/kg/day × 14 days) and were released from study after 28 days. Subjects not relapsing were followed for one year.
Endpoints and definitions
Primary endpoint was incidence density (events/person-year) of first relapse by P. vivax in the year following radical cure. Patients reaching that endpoint contributed time at risk up to that event or withdrawal. Microscopic diagnoses of P. vivax were confirmed by another microscopist on site, and later by a blinded third microscopic read in Jakarta. Blood blots were collected onto Whatman FTA Classic™ filter paper for PCR confirmation of the diagnosis, and DNA extracted (QIAmp DNA Blood Mini™) before nested PCR Plasmodium genus-specific primers (Nest 1) and then with species-specific primers for Nest 2 as detailed elsewhere [28]. Secondary endpoints included frequencies of physical complaints, laboratory abnormalities, reported adverse events of grade 3 or higher during treatment, and serious adverse events possibly, probably, or definitely related to study treatment at any time during treatment or follow-up.
Statistical considerations
We powered the trial for precision in independent estimates of PQ efficacy when combined with AS, AS-PYR, or DHA-PP. The precision estimate was derived iteratively through analysis of binomial distribution of variance around a predicted 98 % efficacy [7]. A sample size of 60 individuals per arm delivered the desired 3.5 % precision around the estimate of efficacy.
The primary endpoint of incidence density informed the estimate of efficacy derived from a relapse control. That control adjusts for geographic variability in relapse attack rates [4, 29]. We employed a relapse control cohort from a previous study [7] as relapse control in the current study. The cohort came from a prior battalion of study from Lumajang, East Java. They had been deployed to precisely the same locale in Papua as the current battalion of study at Sragen, Central Java. We reasoned the relapse attack rate would be similar. Therapeutic efficacy among treatment groups at Sragen was thus calculated using incidence density of relapse control (IDAS-Lumajang) against incidence density (ID) of recurrent P. vivax post-therapy among groups as follows:
$$ \left[\left(\mathrm{I}{\mathrm{D}}_{\mathrm{AS}\hbox{-} \mathrm{Lumajang}}\hbox{-} \mathrm{I}{\mathrm{D}}_{\mathrm{Sragen}\kern0.5em \mathrm{treatment}\kern0.5em \mathrm{groups}}\right)/\mathrm{I}{\mathrm{D}}_{\mathrm{AS}\hbox{-} \mathrm{Lumajang}}\right]\times 100 $$
The 95 % confidence interval (CI) for each of the three estimates of efficacy was calculated from the binomial distribution.
Incidence density of relapse represents the preferred mathematical treatment for events occurring over prolonged periods [30, 31]. Like efficacy of chemoprophylaxis against malaria [32], incidence density within groups was the number of P. vivax attacks divided by the sum of person-years at risk at end of study. Therapeutic efficacy expressed as proportion of subjects remaining free of recurrence after six months was included in this trial analysis for sake of reference to other hypnozoitocidal trials where reinfection possibly confounded recurrence rates [10].
Ethics
Institutional ethics review boards of the Faculty of Medicine, University of Indonesia (ref.no. 13/H2.F1/ethic/2013) and the Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford (ref.no.179-12) reviewed and approved a protocol detailing this trial. Indonesian food and drug regulatory authorities (Badan Pengawasan Obat dan Makanan, Jakarta; ref.no. PN.01.06.313.03.13.998) approved and monitored this trial.