Background: Drugs that kill or inhibit sexual stages of Plasmodium such as Primaqiune (PQ) could potentially amplify or synergize the impact of first line antimalarials by blocking transmission to mosquitoes. This study examined the effect of Primaquine on gametocyte carriage in the case management of uncomplicated falciparum malaria with artemisinin-based combination therapy (ACT) with the overall purpose of possibly recommending it as an adjunct drug for malaria control. Methods: A total of 181 patients with uncomplicated falciparum malaria, normal glucose-6-phosphate dehydrogenase (G6PD) enzyme levels, and haemoglobin levels ≥ 8 g/dL completed this two-arm randomized blinded clinical trial to test the efficacy of a single dose PQ (0.75 mg/kg) on falciparum gametocytaemia. 88 subjects were assigned to a standard 3-day course of Dihydroartemisinin-Piperaquine (DHP) alone (n = 88) while 93 others had DHP combined with a single dose of PQ on day 3 (n = 93). A 28-day follow-up schedule carried out in the outpatient clinic of a Primary health facility in Vom, Plateau State Nigeria where study participants were seen on days 1, 3, 7 and then weekly to assess the presence of asexual parasites and gametocytes by microscopy. A Kaplan-Meier analysis was employed to determine the survival function of gametocytes on day 3. The data was analyzed using Epi info version 7.1.5. Results: With a gametocyte prevalence of 27.1%, gametocyte carriage rate was lower in the PQ group due to higher probability of clearing gametocytes (Breslow test χ2 = 8.306, df = 1, p = 0.004) and significantly less likely to harbor gametocytes by day 7 when compared to the DHP-alone group (χ2 = 6.218, df = 1, p = 0.013). Conclusion: Addition of single-dose 0.75 mg/kg PQ was associated with reduced gametocyte carriage as a result of faster gametocyte clearance and lower incidence of gametocyte development in DHP-treated patients. PQ as gametocytocidal drug may be useful in combination with artemisinin-based combination therapy (ACT) regimen to clear gametocytes and thereby interrupt malaria transmission to mosquito vector more effectively than ACT alone.
Malaria has remained a global public health challenge for decades [
The efforts to reduce the impact of malaria on the population at risk have compelled several malaria endemic nations to implement a number of prevention and control strategies [
Interestingly, antimalarial treatment failure has been associated with increased gametocyte carriage after treatment implicating gametocytes as early indicators of parasite resistance [
Clinical reports and mathematical models have shown that reduction in the incidence of disease and elimination goals can be achieved with combination therapies that incorporate drugs that not only cure patients but also decrease transmission of Plasmodium falciparum sexual stage parasites to the mosquito vector [
The implementation of artemisinin-based combination therapy (ACT) may have beneficial influence on malaria transmission in the general population [
This inadequacy of ACTs prompts the use of Primaquine (PQ) [
There is paucity of data on efficacy of PQ in post-treatment gametocytaemia in ACT treated patients especially in malaria endemic regions where ACTs are recommended first line drugs for malaria treatment [
The aim of this study was to assess the effect of Primaquine on gametocyte carriage in the treatment of uncomplicated falciparum malaria with Dihyroartemisinin-Piperaquine (DHP) and the gametocyte carriage rate as well as incidence of gametocyte development/clearance time with the overall purpose of possibly recommending it as an adjunct drug for artemisinin-based combination therapy for malaria control. Findings from this study could help inform formulation of national policy where Primaquine, in line with WHO recommendation, may be proffered as an adjunct drug for Artemisinin-based combination therapy for malaria control.
A blinded randomized controlled clinical conducted between July 2015 and September 2015 was carried out in the General Outpatient Department of Vom Christian Hospital, Plateau State, Nigeria.
This comprised all patients presenting with fever or history of fever within the past 24 hours and microscopically confirmed P. falciparum infection regardless of the presence of gametocyte stages attending the general outpatient clinic of the hospital.
The study was devised to test the hypothesis that the addition of a single dose of Primaquine (0.75 mg/kg) to ACTs for the treatment of uncomplicated falciparum malaria shortens gametocyte clearance time and reduces gametocyte prevalence.
1) Adults and children aged one year and above or weight ≥ 10 kg, 2) Plasmodium parasite density in the range of 1000 asexual parasites per µl and above, 3) Presence of fever or axillary temperature ≥ 37.5˚C, or positive history of fever in the preceding 24 hrs, 4) Normal glucose-6-phosphate dehydrogenase (G6PD) enzyme levels based on a qualitative test, 5) Haemoglobin level of 8 g/dL and above, 6) Residence in study area and or ability to come for the stipulated follow-up visits, 7) Ability to take oral medications, 8) Informed consent of participant or parent/guardian of participant.
1) General danger symptoms and signs of severe malaria; as oral therapy is not indicated in the treatment of severe malaria, 2) Febrile conditions caused by diseases other than malaria; will not respond to anti-malaria, 3) Allergy to any of the drug components, or had used any of the component drugs within 28 days of enrolment, to avoid complications to therapy and masking effects of previous drug use, 4) Serious concomitant medical illness; or any medical illness not compatible with the study objectives, 5) Pregnancy detected clinically or by urine β-subunit hCG or lactating mother, drug safety in pregnancy and breastfeeding not ascertained.
All eligible subjects were recruited using a structured questionnaire after training of research assistants. Relevant socio-demographic characteristics (bio-data); medical history, physical examination findings and laboratory investigation were conducted for each patient by the study physician. Physical examination including body temperature was measured using a digital thermometer (˚C). The patients were weighed using a reliable standardized weighing scale (Health scale, Techmel & Techmel, Texas, USA) and recorded to the nearest kilogram, with minimal clothing. The weighing scale was calibrated daily using a known weight. The patients were also examined for pallor, jaundice, splenomegaly and hepatomegaly. The diagnosis of malaria was confirmed by collecting finger prick blood to make both thick and thin film on a slide with 3% Giemsa stain for 30 minutes [
Parasite Density = ( Number of asexual parasite counted × WBCcount ) Number of leucocytes counted [
Gametocyte Density = ( Number of gametocytes × WBCcount ) Number of leucocytes counted [
Quality control was done by masked re-reading of 10% of all slides selected randomly by an independent scientist and was considered negative if no parasite were detected in 200 oil immersion fields. G6PD deficiency was defined using qualitative assays based on the fluorescent spot test (Trinity Biotech, cat no. 203-A) with results classified into three groups normal, intermediate and deficient [
All consecutively enrolled patients were given an inclusion number and then randomly assigned (in blocks of 12 patients weekly) to receive either a standard 3-day course of DHP (fixed-dose tablets of 40 mg Dihydroartemisinin and 320 mg Piperaquine) with Primaquine (DHP + PQ arm) or without it (DHP-only arm) using computer-generated random number list concealed in sequentially numbered, sealed opaque envelopes and administered (by daily balloting) by the study pharmacist. The study pharmacist was the only member of the team not blinded to the treatment arm and was not involved in assessing patient or assigning outcomes. The daily DHP regimen was based on weight (≥41 kg: 3 tablets; 31 - 40 kg: 2 tablets; 18 - 30 kg: 1 tablet; 10 - 17 kg: 1/2 tablet). The total dose of Dihydroartemisinin ranged 4 - 10.9 mg/kg, and Piperaquine ranged 32 - 87.3 mg/kg. In the DHP + PQ arm, a single dose of PQ was given on day 3 to achieve a dose of 0.75 mg/kg, rounded to the nearest half tablet. The mean dose was 0.74 mg/kg (range, 0.5 - 0.94 mg/kg). The first daily dose for both study groups was administered at the study center supervised by the study physician or an assistant, the second dose the following day by the patient at home and the third (last) dose at the study center. A full drug dose was re-administered if patient spits out or vomits within 30 minutes or half drug dose administered if between 30 mins and one hour. If the patient rejects the drug again, he/she received another anti-malaria drug in accordance with the national guide line but excluded from the study. Patients were instructed on how to administer the doses which fell outside the study time.
Follow up clinical assessment with examination of body temperatures was done on days 0, 3, 7, 14, 21 and 28 or at any time if the patient felt unwell. Subjects with fever (≥38.5˚C using axillary temperature measurement) received Acetaminophen (Paracetamol). Blood sample from finger prick was obtained to make thick and thin smear during each of these follow up days for parasitological examination. Individuals, for whom the treatment failed as defined, were given rescue treatment according to the national guideline and withdrawn from the study [
The primary outcome measure was the effect of the standard single dose of Primaquine in addition to the completed 3-day treatment with DHP on gametocyte carriage. This was done by comparing the overall risk of gametocyte carriages on day 7 and then weekly till day 28, to measure the combined effect on gametocyte development and clearance (primary endpoint).
This two-arm trial required 84 patients per arm to detect a 7% absolute reduction in the prevalence of gametocytes following the week after treatment with PQ from 9% in the DHP-only arm to 2% in the DHP + PQ arm with 80% power and a 2-sided α of 0.05. The anticipated gametocyte prevalence of 9% was based on the day 7 prevalence in earlier studies with DHP [
The impact of PQ on gametocytes was assessed using the following secondary endpoints: 1) The gametocyte clearance rates by day 28 in patients with gametocytes on day 3, 2) The incidence of gametocyte development by day 28 in patients who were gametocyte-free on day 3, 3) Gametocyte densities between days 3 and 28 inclusive.
Data generated were analyzed using Epi-info 7.1.5 version, Centre for Disease Control and Prevention Atlanta Georgia software for computer analysis [
A total of 181 patients (88 DHP-only arm and 93 DHP + PQ arm) were studied (
The mean and standard deviation of the parasite count at baseline between the treatment arms revealed that there was no significant difference (p = 0.402). There was significant difference in mean parasite count between the two treatment arms at day 3 (t = 2.912, p = 0.004), and there was a significant mean difference in parasite count on day 7 (t = 5.380, p = 0.0005); day 14 (t = 6.690, p = 0.0005).
The gametocyte count was found in the peripheral blood of a total of 49 patients (27.1%); and results revealed that there was no significant mean difference in gametocyte count at baseline between treatment arms (p = 0.658); and also, there was no significant mean difference in gametocyte count after 72 hours (p = 0.407) and day 7 (p = 0.120).
In the DHP treatment arm, 9 study subjects completely cleared gametocytes within 72 hours, while 6 subjects cleared in 168 hours. Therefore,
Gametocyte clearance time ( GCT ) DHP = ( 9 × 72 ) + ( 11 × 168 ) + ( 3 × 336 ) 23 = 1656 23 = 152.3 hours
Drug Treatment Arm | Chi-square/ t-test value | P-value | ||
---|---|---|---|---|
DHP (n = 87) | DHP + PQ (n = 93) | |||
Mean ± SD age (Years) | 32.48 ± 14.49 | 35.77 ± 14.50 | 1.530 | 0.128ǂ |
Adults Children (age > I year) | 69 8 | 80 6 | 10.211 | 0.511ǂǂ |
Male Female | 26 30 | 62 63 | 0.156 | 0.693ǂǂ |
Mean ± SD weight (kg) | 59.91 ± 18.35 | 61.60 ± 16.36 | 0.656 | 0.513ǂ |
Mean ± SD temperature(˚C) | 37.54 ± 0.89 | 37.60 ± 0.65 | 0.591 | 0.555ǂ |
Range of parasitemia (count/L) | 1000 - 1980 | 1170 - 1996 | ||
Mean ± SD (parasite count) (u/l) | 1437.31 ± 190.50 | 1460.62 ± 183.10 | 0.840 | 0.402ǂ |
Mean ± SD (gametocyte count) (u/l) | 16.02 ± 30.50 | 20.32 ± 28 | 0.983 | 0.327ǂ |
Haemoglobin Estimation (%) | 16.02 ± 30.50 | 14.19 ± 24.86 | 0.548 | 0.584ǂ |
ǂIndependent t-test; ǂǂChi-square.
Day | Treatment Arm | Mean | t-test | p-value |
---|---|---|---|---|
1 | DHP DHP + PQ | 1437.31 ± 190.50 1460.62 ± 183.10 | 0.840 | 0.402 |
3 | DHP DHP + PQ | 713.57 ± 168.57 634.04 ± 196.87 | 2.912 | 0.004 |
7 | DHP DHP + PQ | 298.58 ± 135.66 186.29 ± 144.66 | 5.380 | 0.0005 |
14 | DHP DHP + PQ | 26.44 ± 29.18 3.95 ± 13.77 | 6.690 | 0.0005 |
Day | Treatment Arm | N | Mean | t-test | p-value |
---|---|---|---|---|---|
1 | DHP DHP + PQ | 23 26 | 16.02 ± 30.50 14.19 ± 24.86 | 0.443 | 0.658 |
3 | DHP DHP + PQ | 14 12 | 5.28 ± 12.67 3.87 ± 10.11 | 0.832 | 0.407 |
7 | DHP DHP + PQ | 3 0 | 0.28 ± 1.76 0.00 ± 0.00 | 1.562 | 0.120 |
In the DHP + PQ treatment arm, 14 study subject completely cleared gametocytes within 72 hours, while 12 subjects cleared in 168 hours. Therefore,
Gametocyte clearance time ( GCT ) DHP + PQ = ( 14 × 72 ) + ( 12 × 168 ) 26 = 648 26 = 116.3 hours
The gametocyte clearance time in the DHP + PQ treatment arm (116.3 hours) was faster than that of the DHP treatment arm (152.3 hours) p = 0.589.
The Kaplan-Meier test was used to determine the rate of gametocyte clearance. The time of visit was measured in time intervals of 28 days in between visits. The Kaplan-Meier chart (
Results revealed that there was no significant mean haemoglobin difference between the DHP and DHP + PQ treatment arms respectively (p = 0.584); there was no significant mean haemoglobin difference between the treatment arms in day 3 (p = 0.783); also, there was no significant mean difference in day 7 (p = 0.639); day 14 (p = 0.724). Furthermore, there was no significant mean difference in haemoglobin between the treatment arms in day 21 (p = 0.842), and in day 28 (p = 0.318).
This study showed that gametocyte prevalence at enrolment was 27.1%. This is
Day | Treatment Arm | Mean ± SD Haemoglobin (%) | t-test | p-value |
---|---|---|---|---|
1 | DHP DHP + PQ | 13.92 ± 1.78 14.07 ± 1.89 | 0.644 | 0.584 |
3 | DHP DHP + PQ | 13.87 ± 1.71 13.94 ± 1.70 | 0.276 | 0.783 |
7 | DHP DHP + PQ | 13.89 ± 1.60 14.01 ± 1.68 | 0.470 | 0.639 |
14 | DHP DHP + PQ | 14.02 ± 1.58 13.93 ± 1.67 | 0.353 | 0.724 |
21 | DHP DHP + PQ | 13.96 ± 1.62 14.01 ± 1.63 | 0.200 | 0.842 |
28 | DHP DHP + PQ | 14.23 ± 1.71 14.47 ± 1.51 | 1.001 | 0.318 |
higher than the 25.5% prevalence documented by Oguche and colleagues in a study in Nigerian children published in 2014 [
The mean duration of gametocyte carriage in the study participants was seven days. A similar result was reported by Eziefula et al. where a mean duration of 6.6 days in the group with 0.75 mg/kg PQ was observed [
Gametocyte clearance time (GCT) was the time from the initiation of treatment until the first time a slide was gametocyte negative, and remained so for the next 24 hours. The mean gametocyte clearance time was 163 hours without intervention (e.g. PQ) [
The survival analysis also showed that participants in the DHP + PQ arm had significantly lower probability of having/clearing gametocytes than the DHP treatment group during the study period (p = 0.004). In addition, between the day 3 and day 28, study results revealed that participants in the DHP study arm had a significantly higher probability of having/clearing gametocytes than the DHP + PQ arm (p = 0.007). This finding agrees with that observed by Sutanto and colleagues [
ACTs have been shown to have benefits beyond parasitologic and clinical cure but also proved from studies to reduce malaria transmission to the mosquito vector [
This result is consistent with previous reports by Tangpukdee et al. in symptomatic malaria patients treated with Artemisinins or ACTs showing persistent mature blood-stage gametocytemia that were not affected by the Artemisinins/ACTs [
While PQ has no effects on asexual parasites, it has synergistic activity with ACTs [
A likely reason for this similarity may be that they also used microcopy for assessment of gametocyte in their study. PQ on the other hand, has gametocytocidal effects on the mature gametocytes and accelerates their clearance from the circulation [
On the contrary, studies from Sudan and India showed reduced or no effect of PQ [
The short duration of the study could not allow conclusive assessment of the effect of gametocytes on recrudescent infections as well as reinfections. Additionally, most studies attest to the considerable contribution of sub-microscopic gametocytaemia to mosquito infection [
The addition of a single dose of PQ could thus have a major effect on malaria transmission from treated patients by shortening the period of infectivity to mosquito vector without compromising efficacy and safety as demonstrated in the study. PQ added to the current first-line ACTs rapidly reduced microscopically patent gametocytaemia within a week of administration with minimum reportage of adverse events as well as insignificant changes in haemoglobin concentration between the study groups.
This study contributes to the fight against malaria in a two-pronged approach: by providing research on the effect of antimalarials in disrupting malaria transmission from vectors to humans as well as prevention of the development of antimalarial drug resistance partly attributed to persistent gametocyte carriage.
Further studies of longer duration using real-time PCR assays for gametocyte counts including mosquito feeding studies needs to be conducted to fully elucidate the transmission blocking activity of PQ. In addition, larger studies involving asymptomatic patients should be done to evaluate the efficacy, tolerability and appropriate dose of PQ especially in areas where G6PD testing is not widely available. These will add to the existing evidence for advocacy of policy recommendation of PQ as an adjunct to existing ACT regimen for a durable malaria control in Nigeria.
Ethical approval for the study was obtained from the Jos University Teaching Hospital ethical committee and permission from the management of the Vom Christian Hospital. Funding for the research was provided by the authors. There is no conflict of interest as the authors have no financial or personal relationships that may have influenced this study.
The authors thank Professor S. Manu for editorial assistance with this article. We remain grateful to Chundusu Daniel, Yusuf Bulus and Noro Samson for their selfless laboratory support.
Tsuung, A.B., Pitmang, S.L. and Dassak, F.L. (2018) Effect of Primaquine on Gametocyte Carriage in the Case-Management of Uncomplicated Falciparum Malaria with Acts: Nigerian Perspective. Advances in Infectious Diseases, 8, 39-53. https://doi.org/10.4236/aid.2018.82006