Therapeutic Efficacy of Pyronaridine-artesunate (Pyramax ® ) Against Uncomplicated Plasmodium Falciparum Infection at Hamusit Health Center, Northwest Ethiopia

doi:10.21203/rs.3.rs-2430925/v1

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Research Article

Therapeutic Efficacy of Pyronaridine-artesunate (Pyramax ® ) Against Uncomplicated Plasmodium Falciparum Infection at Hamusit Health Center, Northwest Ethiopia

https://doi.org/10.21203/rs.3.rs-2430925/v1

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Background

Malaria remains a major public health problem in the world. Early detection of cases and prompt treatment are the major malaria control strategies implemented in many endemic countries, including Ethiopia. Artemisinin-based combination therapy is currently recommended by the World Health Organization (WHO) for the management of uncomplicated P. falciparum and P. vivax malaria cases. However, resistance to antimalarial drugs is a major challenge to the emergence and rapid spread of drug-resistant Plasmodium strains. Pyronaridine-artesunate (Pyramax) is an artemisinin combination therapy that has shown good efficacy for uncomplicated malaria in large-scale clinical trials conducted in Asia and Africa. This study reports the first therapeutic efficacy profile of Pyronaridine-artesunate against uncomplicated P. falciparum in Ethiopia.

Methods

A single-arm, prospective study with a 42-day follow-up period was conducted from March to May 2021 at Hamusit health center, according to the WHO protocol. A total of 90 adult P. falciparum mono-infection malaria cases (age ≥ 18 years) consented and were enrolled in the study. A standard single-dose regimen of Pyronaridine-artesunate was administered daily for 3 days, and clinical and parasitological outcomes were assessed at 42 days follow-up. Thick and thin blood films were prepared from capillary blood and examined by microscopy. Hemoglobin was measured using HemoCue® and dried blood spots were collected on day 0 and on the day of failure.

Results

Out of 90 patients, 86 (95.6%) had completed the 42-day follow-up study period. The overall PCR-corrected cure rate (adequate clinical and parasitological response) was very high at 98.9% (95% CI: 92.2–99.8%) with no serious adverse events. The parasite clearance rate was high with fast resolution of clinical symptoms; 95.6% and 100% of the study participants cleared parasitemia and fever on day 3, respectively. The mean hemoglobin concentration was significantly increased (p < 0.001) on day 14 compared to that on day 0.

Conclusion

Pyronaridine-artesunate was highly efficacious and safe against uncomplicated P. falciparum in the study population.

Efficacy

Ethiopia

Hamusit

P. falciparum

Pyronaridine-artesunate

Malaria continues to be a public health problem in the world. It is one of the major tropical diseases adversely affecting population health and economic growth of many developing countries, particularly in sub-Saharan Africa (1). The World Health Organization (WHO) African Region accounted for an estimated 228 million cases and 602,000 deaths in 2020 (1). Ethiopia is one of the African countries where both Plasmodium falciparum and Plasmodium vivax malaria species co-exist. Malaria affects more than 60% of the population and covers 75% of the country’s land mass. Malaria is endemic and unstable, with seasonal transmission and variability across elevations, where areas with altitudes less than 2000 meters above sea level are generally considered malarious (2).

Artemisinin-based combination therapy (ACT) for the management of uncomplicated P. falciparum and P. vivax malaria cases is recommended worldwide. ACT reduces both malaria-related morbidity and mortality and also hampers transmission by acting on gametocytes and reducing the likelihood that drug resistance develops (3). Pyronaridine-artesunate employs the use of a potent, short-acting artemisinin and a less-potent, long-acting partner drug, and is important to ensure effective malaria case management (4). Resistance to artemisinin has been reported in the global drug resistance epicenter (Thai-Cambodian border) and its spread to the Great Mekong region poses yet another threat to malaria control efforts (5).

Several clinical and molecular studies show that artemisinin resistance is widespread in Cambodia, Thailand, Myanmar, and Vietnam, with delayed clearance and Kelch protein (K13) mutations being common (6). Recently, K13 gene polymorphism mutation-related resistance markers have been reported in Africa (7).

The development of alternative combination therapies and artemisinin derivatives is essential to overcome the spread of drug-resistant malaria (8). The combination of pyronaridine with other antimalarial agents, including artesunate, has an additive effect (9, 10). Artesunate is the most important semisynthetic derivative due to its rapid antimalarial action, lack of considerable clinical resistance, and significantly greater solubility in water than other artemisinin derivatives (7, 8). Pyronaridine-artesunate (Pyramax) is the first ACT for which the European Medicines Agency (EMA) has adopted a positive scientific opinion, and it is also the first Korean product included in the WHO list of prequalified medicines for malaria (13, 14). The ACT, pyronaridine–artesunate, has shown good efficacy for uncomplicated P. falciparum and P. vivax malaria in large-scale clinical trials conducted in Asia and Africa (15, 16). This study reports the first safety and efficacy profile of pyronaridine-artesunate for treatment of adult patients with uncomplicated P. falciparum malaria attending at Hamusit Health Center, North west Ethiopia.

Study design, period and area

A single-arm, in vivo, prospective therapeutic efficacy study of pyronaridine-artesunate treatment against uncomplicated P. falciparum malaria was conducted, and clinical, parasitological, and hematological assessments were performed. The study was conducted from March to May 2021 in Hamusit Health Center, Dera Woreda, South Gonder, North West Ethiopia: 11° 43' 0'' North and 37° 38' 0'' East. The study area is located 2077 meters above sea level, and receives 1300 mm of annual rainfall on average, with a 26°C mean annual temperature. The area is characterized by unstable seasonal malaria that peaks following the major rainy season (June to September). The health center serves about 54,940 people, and encompasses eight primary health units (health posts).

Source Population

The study participants were recruited from laboratory-confirmed, P. falciparum-infected patients attending Hamusit health centre during the study period. Consented adults (above 18 years) with confirmed uncomplicated P. falciparum mono-infection who attended the outpatient department (OPD) of the health centre and fulfilled the study inclusion criteria were recruited. As the study drug is new to Ethiopia, the study was limited to adults (18 years of age) by institutional ethics committees.

Sample Size Determination

The sample size was determined according to the WHO (2009) protocol, using the single population proportion formula and calculated assuming a 5% margin of error, a 95% confidence interval (CI), 5% treatment failure and 20% adjustment for loss to follow up and withdrawal compensation. Accordingly, 88 study participants were required.

Baseline Evaluation

Physical and clinical examinations were assessed by a clinical study team, with particular attention to any danger signs or symptoms associated with severe malaria. Clinical history and demographic data were collected, axillary temperature and body weight were measured. Patients who met the study inclusion criteria at this stage were assigned a patient identification number and referred for further clinical, laboratory investigation, sample collection and enrolment.

Clinical Evaluations

A standard physical examination, body weight, axillary temperature, and clinical conditions were performed at baseline (day 0 before dosing) and on days 1, 2, 3, 7, 14, 21, 28, 35, and 42. A complete medical history, including prior and concomitant medications, demographic information, and contact details, were recorded at baseline.

Treatment, Dosing, and follow up

Drug dosage was determined according to the revised WHO protocol. Accordingly, enrolled patients were treated with the standard three-dose regimen of 180mg/60mg film-coated tablet Pyronaridine tetra phosphate/Artesunate, given once daily for three consecutive days. All doses of the medication were administered in the health center under the direct supervision of the study clinical team. The doses on Day 0, Day 1, and Day 2 were administered consecutively in 24 hours interval.

The patients were observed for 30 minutes after drug administration. If vomiting occurred before 30 minutes, the dose was repeated and observed for an additional 30 minutes. A patient vomiting more than once was withdrawn from the study and was referred immediately to the OPD for rescue treatment with intramuscular or intravenous quinine.

On Day 0 (enrollment day), each patient who was successfully treated with the first dose was given an appointment card with their name, PIN, and next scheduled visit date written on the front of it. Then patients were asked and advised to come back for treatment the following two days for a total of 42 follow-up days according to scheduled visits on Day 1, Day 2, Day 3, Day 7, Day 14, Day 21, Day 28, Day 35, and Day 42.

Laboratory Procedures

Microscopic Investigation

Thick and thin blood smears were prepared on the same slide for detection, identification, and quantification of parasites at all scheduled and unscheduled visits during the 42-day follow-up period. Two smears were prepared: the first was stained rapidly with 10% Giemsa for 10–15 minutes for initial screening and examined by light microscopy immediately, and the second one was stained slowly with 3% Giemsa for 45–60 minutes and examined later to provide a definitive parasite count.

Slides were examined by the principal investigator and other lab personnel, and parasite density was calculated according to the WHO revised protocol (WHO 2009). Accordingly, asexual parasite density was estimated from thick blood smears by counting the number of asexual parasites against 200 WBC or against 500 WBC (if the count was below 10 parasites/200 WBC) assuming an average WBC count of 8,000/l of blood.

Parasite density (µl) = Number of parasite count × 8000
----------------------------------
Number of WBC counted

Measurement of Blood Hemoglobin Level

Hemoglobin levels were measured from a finger-prick blood sample. Finger-prick blood samples were collected on Day 0, Day 14, Day 28 and Day 42 using HemoCue microcuvets and were measured with a portable spectrophotometer (HemoCue, Ängelhom, Sweden). Anemia was defined according to the WHO classification to mild, moderate and severe (mild: Hb = 10.0-11.9 and 11.0-12.9 g/dl for women and men, respectively; moderate: Hb = 7.0-9.9 g/dl and 8-10.9 g/dl for women and adult men, respectively; Hb 5.0 g/dl was considered severe anemia and an exclusion criteria) (17).

Genotyping

Dried blood spots were obtained for PCR analysis on Day 0 and on days of treatment failure to differentiate a recrudescence (same parasite strain) from a newly acquired infection (different parasite strain). Genotype analysis was conducted on paired dried blood spots (Day 0 and day of treatment failure) for treatment outcomes classified as late treatment failures (LCF or LPF). This was based on the extensive genetic diversity among the malaria parasite genes msp2 and Glurp. Polymerase chain reaction (PCR) genotyping of the polymorphic genes was done at the Ethiopian Public Health Institute (EPHI) and confirmed at the University of North Carolina, Chapel Hill, according to the recommendation of the WHO.

Study Endpoints

The WHO treatment outcome definition was used; valid study endpoints included treatment failure during the study period (early treatment failure [ETF], late clinical failure [LCF], and late parasitological failure [LPF)), completion of the follow-up period without treatment failure (adequate clinical and parasitological response [ACPR]), and loss to follow-up (LFU).

Safety and Quality Assessment

Safety and adverse events were assessed by recording the nature and incidence of any events following treatment. Adverse events were classified and reported to the Ethiopian Public Health Institute, as required. Data was collected by trained senior clinicians in the study health centre and standard operating procedures were followed for each laboratory activity. The quality of the reagent and equipment was maintained as per the standard operating procedures.

Statistical Analysis

Data were double-entered into the WHO Excel sheet, which is designed for therapeutic efficacy study data analysis. SPSS (version 20) software was used to calculate descriptive statistics (mean, median, standard deviation, and range). Independent sample t-test was used to compare baseline temperature, parasitemia, and mean blood Hb level at Day 0, Day 14, Day 28 and Day 42 between patients with parasitemia ≥ 10,000 and < 10,000/µl blood. Paired sample t-test was used to compare the mean Hb level between Day 0 and Day 14, Day 0 and Day 28, Day 14 and Day 28, Day 0 and Day 42, Day 14 and Day 42, Day 28 and Day 42. All comparisons were performed at 95% CI and a significance level of 0.05.

Kaplan Meier (K-M) survival analysis and per protocol (PP) analysis were used for the estimation of primary outcomes; PP analysis method was used to analyze secondary outcomes.

Ethical considerations

The study protocol was approved by the Ethiopian Public Health Institute (EPHI) and by the Institutional Review Boards (IRBs) of Bahir Dar University. In addition, permission was obtained from the Amhara regional health office and Hamusit health center. Written consent was obtained from study participants.

Characteristics of the Study Participants

A total of 4372 malaria-suspected symptomatic patients visiting Hamusit health center were screened for malaria during the study period. Of these, 427 (9.8%) were malaria slide positive; 345 (81%) were P. falciparum, 82 (19%) were P. vivax. Most of those patients were aged less than 18 years and consequently excluded from the study. A total of 109 patients were eligible to be included in the study. Five patients refused to give consent, seven were with parasitemia below 1000/µL, seven were lactating mothers, and were consequently excluded.

Ninety patients were qualified and enrolled. The majority (75.6%) of the participants were males (Table 1). The mean body temperature was 38.3 ± 0.94°C and the mean body weight was 52.69 ± 6.25, while 78 patients (86.7%) were febrile.

The mean Hb level was 14.2 ± 2.02 and 19 patients (21.1%) were anemic (5.5% moderate and 15.6% mild anemia). Overall, 17 (89.5%) of the 19 anemic patients were males. The baseline-mean parasitemia was 7562 per µl of blood. Nearly 67% of the study participants/parents had previous malaria history and 31.1% had bed nets at home.

Among the 90 participants, 4 exclusions were recorded on different follow-up days. Two were excluded on day 28 due to LFU, two had P. falciparum infection on day 35 and on day 40. All 90 enrolled cases were presented up to day 28th scheduled day, 88 cases were presented on day 35, and 86 cases were presented on day 42 (Fig. 1).

Table 1

Base line characteristics of the study participants at Hamusit health center, northwest Ethiopia, 2021
Variables		Frequency (%)
Mean age (range) in Years		33.47 (18–76)
Sex (n) (%)	Male	68 (75.6)
Sex (n) (%)	Female	22 (24.4)
Mean body temperature (°C) (M ± SD)		38.3 ± 0.94
Average body weight (Kg) (M ± SD)		52.69 ± 6.25
Hemoglobin (g/dL) level (M ± SD)		14.2 ± 2.02
Mean paras. (Per µl)		7562
Bed net availability at home (%)	yes	28 (31.1%)
Bed net availability at home (%)	No	62 (68.9%)
Previous malaria attack (%)	Yes	60 (66.7%)
Previous malaria attack (%)	No	30 (33.3%)
M ± SD: Mean ± Standard deviation, Kg: in Kilo gram, Paras.: Parasitemia

Cure Rate

The PCR-corrected cure rate of Pyronaridine-artesunate was 98.9% based on the PP analysis method with one LPF (parasitemia 4520/µl on Day 35) and no ETF or LCF (Table 2). The overall PCR-corrected cure rate by K-M survival estimate was 98.9% (95% CI: 92.2– 99.8%) (Table 3).

Table 2

Summary of treatment outcomes based on PP analysis among patients who treated Pyramax at Hamusit health center, northwest Ethiopia, 2021
Treatment outcomes	Frequency (%)
ETF	0(0)
LCF	0(0)
LPF	2(2.3%)
TTF	2(2.3%)
ACPR	86(97.7%)
LFU	2(2.2%)
Withdrawal	0(0)

ACPR: Adequate clinical and parasitological response, ETF: Early treatment failure, LCF: Late clinical failure, LFU: Lost follow up, LPF: Late parasitological failure, N: number, TTF: Total treatment failure.

Table 3

Summary of PCR-corrected cure rate based on K-M survival analysis at Hamusit health center, northwest Ethiopia, 2021
Follow up N days		Censored	Failure	Survived	K-M success	K-M failure
0	90	0	0	90	1	0
1	90	0	0	90	1	0
2	90	0	0	90	1	0
3	90	0	0	90	1	0
7	90	0	0	90	1	0
14	90	0	0	90	1	0
21	90	0	0	90	1	0
28	90	2	0	90	1	0
35	88	0	1	87	0.988636	0.011364
42	87	0	1	86	0.988506	0.011494

Clearance of Parasitemia following Treatment

Parasite clearance rate was high ,86.7% of the patients cleared parasitemia on Day 2, 95.6% on Day 3 and 100% on Day 7. The mean parasitemia declined from 5923 on Day 0 (baseline) to 127 on Day 2, 90 on Day 3 and 0 on Day 7 (Fig. 2).

Progress of Clinical Signs and Symptoms

At baseline, 78 (86.7%) had a fever during enrollment. All the participants had self-reported fever within 24 hours before enrollment. The mean body temperature declined from 38.3 0C on Day 0 to 36.90C on Day 1, 36.7 0C on Day 2 and to 36.60C on Day 3 (Fig. 3), with 80% of the participants clearing fever on Day1, and 100% on Day 3.

At baseline, a number of signs and symptoms were reported. Headaches were reported by all patients. The following signs and symptoms were reported by the participants: anorexia (29%), nausea (21.1%), dizziness (19%), abdominal pain (14.4%), diarrhea (13.3%), vomiting (16%), and cough (2.2%). During follow-up, some of these events persisted among some participants and new ones also emerged in total of 62 (68.3%) participants (Table 4). Most of the AEs disappeared with the clearance of parasitemia except headache, cough, abdominal pain, and mouth ulcer.

Table 4

Probable drug-related AEs observed during study at Hamusit health center, northwest Ethiopia, 2021
Adverse events	Follow-up days and Number (frequency) of Adverse events (N = 90)
	Day-0	Day-1	Day-2	Day-3	Day-7	Day-14	Day-21	Day-28		Day-35	Day-42
Headache	5(5.5)	3(3.3)	3(3.3)	-	-	-	-	-		-	-
Anorexia	2(2.2)	4(4.4)	3(3.3)	-	-	-	-	-		-	-
Mouth ulcer	2(2.2)	4(4.4)	2(2.2)	1(1.1)	-	-	-	-		-	-
Vomiting	2(2.2)	1(1.1)	-	-	-	-	-	-		-	-
Abdominal pain	-	3(3.3)	3(3.3)	2(2.2)	-	-	-	-		-	-
Diarrhea	-	1(1.1)	2(2.2)	2(2.2)	-	-	-	-		-	-
Cough	2(2.2)	2(2.2)	2(2.2)	2(2.2)	1(1.1)	-	-	-		-	-
Dizziness	3(3.3)	2(2.2)	2(2.2)	1(1.1)	-	-	-	-		-	-
Total n (%)	16(17.6)	20(22.0)	17(18.7)	8(8.8)	1(1.1)	-	-	-	-	-

The Prevalence of Anemia

The number of moderately- and mildly- anemic patients was 19 at baseline and had risen to 44, 30 and 25 on Days 14, 28, and 42, respectively. Accordingly, the mean Hb level showed a slight decline from Day 0 (14.2 ± 2.03) to Day 14 (12.7 ± 1.75) and a recovery on day 28 (13.4 ± 1.52) and 42 (13.5 ± 1.79) (Table 5). The mean baseline hemoglobin was significantly higher than on Day 14 p = 0.0001, Day 28 p = 0.002 and Day 42 p = 0.007. Comparison of Day 0, Day 14, Day 28 and Day 42 mean Hb level between patients with parasitemia ≥ 10,000 and < 10,000/µl showed no significant difference (14.4 ± 1.96, 14.1 ± 2.05, p = 0.452; 12.5 ± 1.49, 12.8 ± 1.87, p = 0.534; 13.6 ± 1.68, 13.5 ± 1.47, p = 0.436, 13.9 ± 2.03, 13.3 ± 1.68, p = 0.201 for ≥ 10,000 and < 10,000/µl; and for Day 0, Day 14 Day 28 and Day 42 respectively.

Table 5

Mean hemoglobin concentration (g/dl) of study population during follow-up days at Hamusit health center, northwest Ethiopia, 2021
Variables			Follow up days
			Day 0	Day 14	Day 28	Day 42
Mean Hb ± SD (g/dl)			14.2 ± 2.03	12.7 ± 1.75	13.4 ± 1.52	13.5 ± 1.79
Anemia status	Mild n (%)	Male	14 (21.2)	27 (40.9)	20 (30.3)	18 (27.3)
		Female	0 (0)	3 (15)	4 (20)	2 (10)
		Total	14 (21.2)	30 (55.9)	24 (50.3)	20 (37.3)
	Moderate (%)	Male	3 (4.5)	12 (18.2)	6 (9.1)	5 (7.5)
		Female	2 (10)	2 (10)	0 (0)	0 (0)
		Total	5 (15.5)	14 (18.2)	6 (9.1)	5 (7.5)
	Total (%)		19 (22.1)	44 (51.2)	30 (34.9)	25 (29.1)

The ACT, Pyronaridine-artesunate, has shown high therapeutic efficacy for uncomplicated P. falciparum and P. vivax malaria in large-scale clinical trials conducted in Asia and Africa (15, 16). In this study (the first study in Ethiopia), Pyronaridine–artesunate showed high therapeutic efficacy at a 98.9% PCR-corrected cure rate. It is in line with studies done in five African countries (98.6%), Koh Gnek (98.3%), central and southern Vietnam (96.1%), and Veun Sai of eastern Cambodia (96.7%) (18–21). A study on the efficacy of pyronaridine–artesunate and artemether-lumefantrine showed 98.9% efficacy for pyronaridine-artesunate and 96.4% efficacy for artemether-lumefantrine. Pyronaridine-artesunate was found to be non-inferior to artemether-lumefantrine (21). The efficacy report is in line with our study. However, the cure rate of the Pyronaridine-artesunate in the present study was higher than in the study in western Cambodia (87.9%) (22). It is possible that either the parasites are resistant to pyronaridine-artesunate or that recrudescence could have been overestimated by using PCR methods because of a clonal parasite population. Reinfection with the clonal parasite population could not be easily distinguishable from recrudescence (22).

The absence of ETF and low parasitological failure (only single LPF) in this study indicates the high therapeutic efficacy of both components of Pyronaridine-artesunate in the study setting. Studies from Vietnam and western Cambodia reported six and fifteen late treatment failures after 21 follow-up days of Pyronaridine-artesunate treatment (24, 26). This might be due to insufficient drug levels or parasite resistance to this drug. The short half-life of artemisinin results in plasma drug concentrations not being above the minimum inhibitory concentration (MIC) for periods long enough to kill all parasites (24). Moreover, the asexual blood stage P. falciparum parasites may become temporarily dormant and survive the therapeutic concentration of artemisinin derivatives (25). However, the PCR-confirmed recrudescence reported in this study warrants follow-up study and advanced molecular screening for resistance markers.

In the current study, 95.6% of the patients cleared parasitemia on Day 3, which is consistent with a study done in Myanmar (19). Other previous studies reported lower rates of parasite clearance: 74% in Vietnam, 56.4% in Pailin, and 86.7% in Pursat on the third day of drug administration (23, 26). The parasite clearance rate can be affected by drug blood concentration profiles, host-defense mechanisms, the initial parasitemia, concomitant infection, as well as pharmacodynamic properties (26). It is therefore essential to control for such potential confounding factors in order to identify temporal changes in parasite clearance resulting from reduced anti-malarial drug susceptibility. Artesunate's inherent ability to rapidly metabolize to its active ingredient, dihydroartemisinin, and rapidly absorb into the bloodstream may result in a rapid parasite clearance. The rate of elimination of artesunate is also rapid, with a half-life ranging from 2–3 hours, and the partner drug pyronaridine is slow-acting drug with a longer half-life (13–17 days) (27). Thus, the presence of parasites on Day 3 in this study might be due to the immune status of the patients, parasite susceptibility to anti-malarial drugs and partner drug efficacy, which lags parasite clearance.

The baseline mean body temperature was 38.3 ± 0.94⁰C and 86.7% had fever during enrollment. Fever clearance was rapid and a 100% clearance rate was observed on Day 3. A rapid fever clearance rate for Pyronaridine artesunate was reported from an efficacy study conducted in central and southern Vietnam (23). In addition to the delay in parasite clearance, the dalliance in the clearance of fever might be suggestive of artemisinin resistance in Southeast Asia.

The mean hemoglobin level at baseline (14.2 ± 2.03) was significantly higher than on Day 14 (12.7 ± 1.75), Day 28 (13.4 ± 1.52) and Day 42 (13.5 ± 1.79). This is not in line with previous studies on ACTs where, following parasite clearance, Hb levels had recovered. Reports from other studies conducted in Ethiopia and elsewhere on other ACTS (artemisinin lumefantrine) showed a slight to significant increase following treatment (28–30). However, anemia was reported as a severe adverse event in another study (31). Decreased hemoglobin level after the drug administration was found from other studies (32–34). This might be partially explained by the fact that a transient and clinically moderate but significant decrease in hemoglobin after the treatment initiation with artemisinin derivative occurs due to hemolysis of parasitized and non-parasitized red cells (35).

The slight increase in mean hemoglobin from day 14 to day 42 in this study is also consistent with another study(36). It suggests that oral artemisinin derivative therapy may not have a late clinically relevant deleterious effect on hemoglobin. According to a preclinical study, artemisinin derivatives have been shown to induce reticulocytopaenia due to the suppression of erythroblasts (36), although the reticulocytes have not been described in this study. Post-treatment hemolysis is commonly associated with the treatment of severe malaria and hyperparasitemic patients. Furthermore, this study was conducted in patients with uncomplicated falciparum malaria and the comparison of Day 14, Day 28 and Day 42 mean blood Hb levels between patients with parasitemia ≥ 10,000/µl and < 10,000/µl showed no significant difference.

Rehman and colleague (37) reported delayed hemolysis 1–3 weeks after treatment with artemisinin derivatives. Artesunate-containing drugs are effective in their rapid killing of ring stage parasites, and most of the killed ring stage parasites are cleared rapidly by the spleen, whereby the dead parasite is removed from within the erythrocytes (38). These infected erythrocytes are returned to the circulation, but they have a reduced lifespan of about 7–15 days. The delayed destruction of the infected erythrocytes corresponds with the time course of post- treatment delayed anemia. Rehman and colleagues reviewed the evidence of delayed anemia occurring in patients from malaria endemic regions of Southeast Asia and Africa. This review indicated that late hemolysis leading to a significant decrease in hemoglobin was associated with all artemisinin derivatives and all routes of administration (37).

Several adverse events were reported in this study. These events were similar to the symptoms of malaria, and there were no serious adverse events observed. The most common adverse events observed were consistent with previous studies These studies have also reported that the adverse event profile for pyronaridine artesunate was similar to that of artemether-lumefantrine and mefloquine artesunate in falciparum malaria.(39–41). Although headache, cough and mouth ulcer are common symptoms of malaria, their persistence after the recovery of other malaria symptoms observed in this study makes them potential drug-related adverse events. But further study may be required to classify the events as drug-related or due to other factors.

LIMITATION OF THE STUDY

This study has the following limitations: blood drug level was not measured as well as other malaria drug resistance detecting methods (prevalence of resistance molecular markers, in-vitro susceptibility tests) that complement therapeutic efficacy studies were not performed. The potential occurrence of post-treatment delayed hemolytic anemia in patients treated with Pyronaridine artesunate was not investigated. The study was conducted only in adults (18 and above).

The current study reports the first therapeutic efficacy study for Pyronaridine artesunate in Ethiopia. In addition, pyronaridine artesunate demonstrated effective therapeutic efficacy against uncomplicated P. falciparum malaria in adults in the current study setting, with rapid parasite clearance and fever resolution. There was no serious drug adverse event observed, implying that the drug is safe.

ACT Artemisinin combination therapy

APCR Adequate parasitological and clinical response

DBS Dry blood spot

DHA Dihydroartemisinin

EPHI Ethiopian Public Health Institute

ETF Early treatment failure

FMoH Federal ministry of health

Hb Hemoglobin

K-M Kaplan Meier

PCR Polymerase chain reaction

LFU Lose of follow up

LTF Late treatment failure

OPD Outpatient department

PP Per protocol

RBC Red blood cell

TF Treatment failure

WBC White blood cell

WHO World Health Organization

Acknowledgments

We would like to thank the Ethiopian Public Health Institute (EPHI), Amhara Regional Health Bureau, Hamusit Health Center, Bahir Dar University and University of North Carolina for their unlimited support and expertise input for this project. We would also like to acknowledge the ministry of health through EPHI for financial and technical support. WHO and Shin Poong Pharm. are acknowledged for donating the study drug. Rachel Sendor is acknowledged for proofreading the draft manuscript. We also acknowledge the study team members and study participants.

Availability of data and material

The datasets used and/or analyzed during the current study are available from the corresponding author and EPHI on a reasonable request.

Funding

The study was funded by the Global Fund to Fight AIDS, Tuberculosis and Malaria via the Federal Ministry of Health in Ethiopia and World health organization. The opinions expressed herein are those of the author(s) and do not necessarily reflect the views of any collaborating institutes.

Ethical approval and consent to participate

The study received ethical approval from the scientific and ethical review board of Ethiopian Public Health Institute (EPHI) (protocol number 294/2022) and the Institutional Review Boards (IRBs) of Bahir Dar University (protocol number 155/2021). In addition, permission was obtained from the Amhara regional health office and Hamusit health center. Written informed consent was obtained from study participants

Consent for publication

All authors have given their consent for publication.

Author contributions

MA, WG, MAA and AA conceptualize and wrote the main manuscript and analysis. MA, HM, HHG and AA carried out the data collection. H.S., H.H., B.G., M.H., G.A., W.B., H.B., J.B.P., G.T. and H.M. have involved in reviewing the paper. All authors reviewed the manuscript.

Competing interests

The authors declare that they have no competing interests

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No competing interests reported.

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Therapeutic Efficacy of Pyronaridine-artesunate (Pyramax ® ) Against Uncomplicated Plasmodium Falciparum Infection at Hamusit Health Center, Northwest Ethiopia

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Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Material And Methods

Study design, period and area

Source Population

Sample Size Determination

Baseline Evaluation

Clinical Evaluations

Treatment, Dosing, and follow up

Laboratory Procedures

Microscopic Investigation

Measurement of Blood Hemoglobin Level

Genotyping

Study Endpoints

Safety and Quality Assessment

Statistical Analysis

Ethical considerations

Results

Characteristics of the Study Participants

Cure Rate

Clearance of Parasitemia following Treatment

Progress of Clinical Signs and Symptoms

The Prevalence of Anemia

Discussion

LIMITATION OF THE STUDY

Conclusions

Abbreviations

Declarations

References

Additional Declarations

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