Efficacy of chloroquine with primaquine for uncomplicated Plasmodium vivax malaria without G6PD testing in Northwest Ethiopia: a one-arm in vivo prospective therapeutic efficacy study

Abstract

Background

The declining efficacy and widespread resistance to antimalarial drugs (AMD) pose significant challenges to global malaria control and elimination efforts. To enhance treatment efficacy, the World Health Organization (WHO) recommends the use of combination therapies. This study aimed to evaluate the therapeutic efficacy of chloroquine with primaquine (CQ–PQ) for the treatment of uncomplicated Plasmodium vivax malaria in the study area.

Methods

The study utilized a single-arm, 42-day follow-up design to assess the therapeutic efficacy and safety of a treatment regimen in Northwest Ethiopia. Participants, all diagnosed with uncomplicated vivax malaria, received a 3-day course of chloroquine (CQ) at 25 mg/kg, followed by a 14-day course of primaquine (PQ) at 0.25 mg/kg. Participants were monitored with follow-up visits on days 1, 2, 3, 7, 14, 21, 28, 35, and 42. Data were double-entered into a standard Excel sheet by the WHO and analysed using SPSS v.26. Statistical analyses included Kaplan–Meier survival analysis, t-tests, and ANOVA, with statistical significance set at p < 0.05.

Results

Of the 100 participants enrolled, 92% completed the study. The cumulative treatment success rate for CQ–PQ was 93.7% (95% CI 0.86–0.97) on day 42, with a 6.3% (95% CI 0.03–0.14) treatment failure rate. Asexual parasite clearance was rapid, with 97% achieving clearance by day 2 and full clearance by day 3 in all but one participant. Haemoglobin levels increased significantly from 12.3 g/dL at baseline to 13.5 g/dL by day 42, with 84.2% of mild anaemic patients and 85.7% of moderate anaemic patients showing recovery. Common adverse events included abdominal pain (8%) and diarrhea (5%), all of which resolved by day 7.

Conclusion

CQ–PQ therapy demonstrated high efficacy in clearing parasitaemia and improving haemoglobin levels in patients with vivax malaria. These results highlight the potential of CQ–PQ as an effective treatment option, with a favourable safety profile. Further studies are needed to explore long-term outcomes and the impact of this treatment on malaria control in different settings.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12936-025-05446-y.

Background

Malaria is a life-threatening acute febrile illness [1], caused by protozoan parasites of the genus Plasmodium [2]. Transmission to humans primarily occurs through the bite of infected female Anopheles mosquitoes [3]. The disease is characterized by symptoms that typically appear 10 to 30 days after infection, during the intra-erythrocytic cycle [4]. These symptoms are nonspecific and can range from mild or absent to severe and potentially fatal, making early diagnosis challenging [5].

The global burden of malaria remains significant, with the World Malaria Report estimating 263 million malaria cases worldwide in 2024, an increase of 11 million cases compared to 2023 [6]. Although malaria cases rose, the number of malaria-related deaths showed a slight decline, dropping from 600,000 in 2023 to 597,000 in 2024 [6, 7]. Among these, P. vivax malaria accounted for approximately 9.2 million cases globally, with an additional 2.9 million cases reported compared to the previous year. Ethiopia, in particular, reported 9.5 million total malaria cases, contributing to 3.1% of global malaria deaths. Notably, the country ranked among the top five nations with the highest increase in cases, with 4.5 million more cases than in 2023 [6].

In response to this ongoing challenge, the World Health Organization (WHO) has implemented successive strategic frameworks to advance malaria control and elimination globally, starting with the Global Malaria Eradication Programme (GMEP) [8]. The revised Global Technical Strategy (GTS) for malaria (2016–2030) aims to reduce malaria incidence and mortality by 90%, eliminate malaria in at least 35 countries by 2030, and prevent its resurgence [9]. Ethiopia’s National Malaria Elimination Programme (NMEP) aligns with these global priorities, setting a national goal for 2025 to reduce malaria morbidity and mortality by 50%, eliminate malaria in areas with a yearly parasite incidence below 10%, and prevent reintroduction of the disease in areas with zero reported cases [10].

Despite significant progress in malaria control, the emergence and spread of antimalarial drug resistance (AMDR) represent a growing threat that threatens to overshadow these achievements [11]. The first P. vivax strains non-susceptible to chloroquine (CQ) was reported in Papua New Guinea, [12], and later in 1995 in central Ethiopia [13]. Since then, indicators of CQ treatment failure have been reported in various parts of the country and around the world [14]. This rapid spread of CQ resistance poses a serious health risk to millions of people in affected regions [15].

Currently, limited options for effective antimalarial drugs (AMDs) exist due to increasing resistance, making it crucial to sustain the therapeutic efficacy of existing drugs until novel treatments are developed [14]. To optimize therapeutic outcomes, the WHO recommends combination antimalarial therapy [4]. In areas like Ethiopia, where CQ-resistance remains a concern, the combination of CQ with primaquine (PQ) is recommended as the first-line treatment for uncomplicated P. vivax malaria. The standard regimen involves a 3-day CQ course followed by a 14-day PQ course to achieve radical cure by targeting both blood-stage and hypnozoite-stage parasites [16]. However, the threat of resistance underscores the need for routine monitoring of therapeutic efficacy to enable early detection of resistance patterns and the implementation of timely containment measures [17]. This study, therefore, aims to evaluate the efficacy of the CQ–PQ regimen for treating uncomplicated P. vivax malaria cases, with a 42-day follow-up period in the study areas.

Methods

Study setting and design

This was a single-arm, 42-day follow-up, in vivo therapeutic efficacy and safety study conducted at Andasa Health Center, located in Northwest Ethiopia, from November 2022 to March 2023. The study site is situated 582 km northwest of Addis Ababa, approximately 22 km south of Bahir Dar city, in the Amhara Regional State, on the main road leading to the Blue Nile Falls (Tis Abay). The geographical coordinates of the site are 11° 30′ 19″ North Latitude and 37° 29′ 43″ East Longitude, with an elevation of 1716 m above sea level (PQ).

The study was carried out in the Bahir Dar Zuria district, which has a total population of 220,581 inhabitants, of whom 112,378 are male and 108,203 are female, with 100% of the population residing in rural areas [18]. The Nile River passes through this district, which is known for its extensive irrigation practices supporting vegetable and fruit cultivation. The district experiences a mean annual rainfall of approximately 1035 mm, with temperatures ranging from 10 to 30 °C. Malaria transmission in the region occurs year-round, with Plasmodium falciparum and P. vivax being the predominant species.

According to Ethiopia’s malaria stratification and mapping system, based on the annual parasite incidence (API), the study area is classified as having low malaria transmission, with an API between 5 and 10 [10]. The first-line treatment for uncomplicated P. vivax malaria in this region is a combination of CQ with PQ.

Sample size and sampling technique

The WHO protocol for the surveillance of antimalarial drug efficacy [17] was used to calculate the required sample size and sampling technique. Using a single population proportion formula by assuming an expected proportion of treatment failure in the study population of 5% with a confidence level of 95%, and a precision level of 5%. A minimum sample of 73 patients was estimated. Assuming a loss to follow-up and withdrawal of 20% over a 42-day follow-up, 90 patients were targeted. A convenient sampling technique was used.

Study population, inclusion, and exclusion criteria

Patients presenting with clinically suspected malaria, characterized by fever (axillary temperature ≥ 37.5 °C) or a history of fever within the last 48 h, and seeking treatment at Andasa Health Center during the study period, were enrolled. Inclusion criteria included individuals aged over 6 months, with confirmed P. vivax mono-infection, presenting with an asexual parasitaemia of at least 250/μL of blood as determined by microscopy. Only those living within a 10 km radius of the health center and willing to provide informed consent (or assent for minors) were included in the study.

Exclusion criteria included presence of mixed Plasmodium species infections, individuals weighing less than 5 kg, with a haemoglobin level ≤ 7 mg/dL, or with clinical conditions requiring hospitalization (e.g., coma, severe anaemia). Patients with febrile conditions caused by diseases other than malaria (such as measles, otitis media, acute lower respiratory tract infections, or severe diarrhea with dehydration) were also excluded. Additionally, patients with a history of hypersensitivity to CQ and/or PQ, recent use of any antimalarial medication within 2 weeks prior to enrollment, or those with a Hillmen urine colour chart result exceeding the 4-point mark were excluded. Patients with severe malnutrition (as defined by the WHO), as well as pregnant or breastfeeding women, were not eligible for inclusion.

Data collection

Socio-demographic data, clinical information, and laboratory results were collected in accordance with the WHO protocol. Data collection tools included enrollment forms, case report forms, and adverse event recording forms [17].

Clinical evaluation

A standard physical examination was performed at baseline (day 0) and on days 1, 2, 3, 7, 14, 21, 28, 35, and 42. Baseline data, including medical history, demographic information, body weight, height, axillary temperature, and parasite density, were recorded. Haemolysis was assessed on day 0 using the Hillman urine test (HUT), with follow-up assessments on days 1, 2, 3, 7, and 14.

Laboratory assessment

Parasitological assessment

Blood smears (thick and thin films) were collected on day 0 for species identification and parasite count. Subsequent samples were taken on days 1, 2, 3, 7, 14, 21, 28, 35, and 42, or on unscheduled visits. Microscopy was performed at 1000× magnification by two independent microscopists. Parasite density, expressed as the number of asexual parasites per µl of blood, was calculated by dividing the number of asexual parasites by the number of white blood cell (WBC) counted and then multiplying by an assumed WBC density (8000 per µl). Blood slides were considered negative when examination of 1000 WBC or 100 fields containing at least 10 WBC per field revealed no asexual parasites [19].

Haematological assessment

Haemoglobin (Hgb) levels were measured at baseline, day 14, 28, and 42 using the HemoCue HB 301 + analyzer. Anaemia was classified based on WHO Hgb thresholds; (< 11 g/dL for children under 5 years, < 11.5 g/dL for children aged 5–11 years, < 12 g/dL for children aged 12–14 years and non-pregnant women aged ≥ 15 years, and < 13 g/dL for men aged ≥ 15 years) [20].

Treatment and dosing procedure

Patients received CQ and PQ according to national malaria treatment guidelines [16]. CQ 250 mg tablet (Manufacture IPCA Laboratories Ltd. Indian, Batch number L8080013) was administered in weight-based doses over 3 days (10 mg base/kg on days 0 and 1, 5 mg base/kg on day 2). PQ 7.5 mg coated tablet (Manufacturer Remedica Ltd.UK, Batch number 92147) weight-based 0.25 mg base/kg daily for 14 days was administered, starting on day 0. All doses of CQ and the first four doses of PQ were supervised initially, with subsequent PQ doses taken at home. Patients were monitored for vomiting and re-treated if necessary. In cases of treatment failure, Artemether-Lumefantrine (AL) was administered (Manufacturer IPCA Laboratories Ltd. Indian, Batch number HWE111327).

Follow-up activities

Patients were scheduled for follow-up visits on days 1, 2, 3, 7, 14, 21, 28, 35, and 42. Axillary temperature and parasite density were recorded at each visit. Post-treatment adverse drug events were monitored, and haemolysis was assessed using the Hillman urine colour chart. Adverse events, defined as unintended symptoms resulting from CQ and PQ, were documented.

Treatment outcome classification

Outcomes were classified as per WHO guidelines [17]: adequate clinical and parasitological response (ACPR), early treatment failure (ETF), late clinical failure (LCF), or late parasitological failure (LPF). Secondary outcomes included gametocyte clearance, fever clearance, haemoglobin recovery, and adverse events.

Data quality control and management

Standard operating procedures (SOPs) for laboratory tests were followed, and slides were re-examined by senior laboratory technologists. All drugs were donated by WHO and quality-assured by the Ethiopian Pharmaceuticals Supply Agency. Data collection was meticulously monitored for accuracy, with double-entry into a database and subsequent verification.

Data entry and analysis

Data were entered using WHO Excel sheets and analyzed with SPSS (version 26). Kaplan–Meier survival analysis was used to assess treatment outcomes. Statistical comparisons of baseline temperature and parasitaemia across age groups were performed using t-tests and ANOVA. Mean Hgb levels before (day 0) and after treatment (day 42) were compared using a paired t-test. A p-values of < 0.05 was considered statistically significant.

Ethical clearance

Ethical approval was obtained from the Institutional Review Board at Ethiopian Public Health Institute (EPHI-IRB294-2022). Written informed consent was obtained from all adult participants and from parents or legal guardians of children under the age of 12 prior to enrolment. Assent was obtained from children between the ages of 12 and 17 years. Participants received transportation reimbursement (Fig. 1).

Fig. 1.

Map of the study site. Bahir Dar zuria district, Andassa kebele

Results

Baseline socio-demographic characteristics of study participants

Between November 2022 and March 2023, a total of 2915 patients with suspected malaria were screened. Among them, 23.4% (n = 683/2915) were confirmed as slide-positive for malaria. Of these, 39.7% (n = 271/683) were diagnosed with P. vivax. A total of 36.9% (n = 100/271) met the study inclusion criteria and were subsequently enrolled. Of the enrolled participants, 92% (n = 92/100) completed the follow-up, while 8% (n = 8/100) were censored (Fig. 2). The enrolled participants were predominantly male (66%, n = 66/100), with a male-to-female ratio of 2:1. The median age was 9.0 years (IQR: 5–17), with an age range of 1.3 to 65 years. Children aged 5–15 years comprised 53% (n = 53/100) of the study population, while those under 5 years accounted for 17% (n = 17/100). The mean weight and height were 27.5 kg and 130 cm, respectively. All study participants (n = 100) were urban residents (Table 1).

Fig. 2.

Flow chart of study participants recruited and followed for 42 days, showing the number of participants assessed at each follow-up visit and final classification outcomes

Table 1.

Baseline socio-demographic and clinical characteristics of the study participants

Variables	Sex		Age Category			Total
	Male	Female	Under 5	5 to 15	> 15
Participants, n	66	34	17	53	30	100
Previous malaria, n (%)	60 (69%)	27 (31%)	11 (12.6%)	49 (56.4%)	27 (31%)	87 (87%)
Previous CQ intake, n (%)	22 (61%)	14 (39%)	6 (17%)	22 (61%)	8 (22%)	36 (41%)
Temp (0C), mean ± SD	38.1 (0.77)	38.0 (0.6)	38.4 (0.8)	38.1 (0.6)	37.9 (0.69)	38.1 (0.72)
Temp ≥ 37.50C, n (%)	59 (66%)	31(34%)	16 (18%)	50 (55%)	24 (27%)	90 (90%)
Geometric mean para/μL (Min–Max) para/μL	5529 (872–36,040)	5203 (504–32,000)	9990 (1670–36,040)	6671 (504–28,800)	2648 (800–15,000)	5416 (504–36,040)
Gametocyte carriage, n (%)	32 (76%)	10 (24%)	9 (21%)	21 (50%)	12 (29%)	42 (42%)
Hb (g/dL), mean ± SD	12.2 (1.17)	11.6 (1.66)	11.6 (1.46)	11.8 (1.33)	12.5 (1.33)	12 (1.38)
Anemia status, n (%)	14 (54%)	12 (46%)	5 (19%)	13 (50%)	8 (31%)	26 (26%)

CQ Chloroquine, °C Degree Celsius, g/dL gram per deciliter, Hgb Hemoglobin, μL Microliter, n number, SD Standard Deviation

Baseline clinical characteristics

At baseline, 90% (n = 90) of participants had a fever, while the remaining had a history of fever within the past 48 h. The highest temperature recorded was 40.2 °C in an 8-year-old child. The average baseline temperature was 38.1 °C ± 0.72 °C SD. Among the 87 participants with a prior history of malaria, 41% (n = 36/87) had previously received CQ, while 59% (n = 51/87) had been treated with AL, at least once and more in their lives. However, only those who had taken the AMDs more than 2 weeks before enrolment were included in the study (Table 1).

The baseline geometric mean parasitaemia was 5416/μL, and as age increases the parasite load showed relatively declining numbers. The Pearson correlation analysis showed that < 5 children had a higher baseline parasitaemia compared to adults (r = 0.395, r² = 0.156, significant at p = 0.000) (Additional file 1). Gametocyte carriage was 42% (n = 42/100) at baseline. The average baseline Hgb level (±) SD was 12 ± 1.38 g/dL with anaemia status of 26% (Table 1).

Primary outcomes

The cure rate of CQ–PQ treatment

A total of 6 recurrence cases were observed during the 42-day follow-up period. The Kaplan–Meier survival analysis, the cumulative treatment success rate and cumulative treatment failure rate of CQ–PQ treatment on day 42 were 93.7% (95% CI 86.4–97.1) and 6.3% (95% CI 2.9–13.6), respectively. On day 28 the cumulative success rate and failure rate of CQ–PQ treatment were 94.7% (95% CI 87.8–97.8) and 5.3% (95% CI 2.2–12.2), respectively (Fig. 3, Table 2). Genotyping was not conducted in this study; therefore, the results represent PCR uncorrected.

Fig. 3.

Kaplan–Meier Survival curve in CQ–PQ therapeutic efficacy study outcome

Table 2.

Kaplan- Meier analysis of CQ–PQ treatment outcomes of study participants

Follow-up days	At-risk	Censored	Failure	Survived	K–M survival rate	K–M failure rate
D0	100	0	0	100	1	0
D1	100	0	0	100	1	0
D2	100	2	0	100	1	0
D3	98	0	1	99	0.990	0.01
D7	97	2	1	98	0.980	0.02
D14	94	0	0	98	0.980	0.02
D21	94	3	0	98	0.980	0.02
D28	91	0	3	95	0.947	0.053
D35	88	1	1	94	0.937	0.063
D42	86	0	0	94	0.937	0.063

Asexual parasitaemia and gametocyte clearance

About half of the participants (47%) achieved asexual parasite clearance by day 1 following the first dose of CQ–PQ, increasing to 97% by day 2, and complete clearance in all but one participant by day 3 (Fig. 4); the exception was a 10-year-old child who experienced ETF with a parasitaemia of 3879/μL. By day 42, ACPR was achieved in 86% of participants without recurrence of asexual parasitaemia. One 12-year-old patient was classified as having LCF on day 7 with a parasitaemia of 3293/μL and fever, while LPF was identified in three participants on day 28 (parasitaemia: 1720/μL, 320/μL, and 210/μL) and in one participant on day 35 (parasitaemia: 4636/μL). Only 33% (n = 2/6) of the treatment failure cases exhibited higher parasitaemia compared to baseline (Table 3, Additional file 2). Gametocyte clearance was observed in 73.8% (n = 31/42) of patients by day 1, and in 100% of patients by day 3, with no gametocyte recurrence detected during the 42-day follow-up period (Fig. 4).

Fig. 4.

A line graph of asexual parasite and gametocyte clearance following CQ–PQ treatment

Table 3.

Parasite density at baseline and on the day of TF of study participants

TF	Sex	Age	FUD	Parasite density		PRR/μL para. density at day0 to TF
				At day 0/μL	On the day of TF/μL
ETF	F	10	Day 3	14,200	3879	3.6
LCF	M	12	Day 7	9080	3293	2.7
LPF	F	14	Day 28	8840	1720	5.0
	M	18	Day 28	872	320	2.7
	M	25	Day 28	1201	2120	0.6
	F	6	Day 35	3760	4636	0.8

ETF Early treatment failure, FUD follow-up day, LCF late clinical failure, LPF late parasitological failure, μL Microliter, PRR Parasite reduction rate, TF Treatment failure

Fever clearance

Of the 90 participants with axillary temperature ≥ 37.5 °C at enrollment, only 10% (n = 9/90) remained febrile on day 1 and 2% remained on day 2. All participants achieved fever clearance by day 3. However, one of the participants developed recurrent fever (38.4 °C) on day 7 with asexual parasitaemia and was classified as LCF.

Haemoglobin recovery

On the day of recruitment, none of the patients had severe anaemia (Hgb ≤ 7 g/dL). A history of malaria was reported in 84% (n = 16/19) mild anaemic patients and 71% (5/7) moderate anaemic patients. Haemoglobin levels significantly increased between baseline (day 0) and day 42 (p < 0.0001). Mean haemoglobin concentration rose from 12.3 ± 1.11 g/dL (range: 8.4–15.5 g/dL) at baseline to 13.5 ± 1.126 g/dL (range: 9.5–16.3 g/dL) at day 42. By the end of the follow-up, 84.2% (n = 16/19) of mild anaemic patients showed Hgb level improvement, and 85.7% (n = 6/7) of moderate anaemic patients showed Hb recovery. The number of anaemic patients decreased from 26 on day 0 to 4 on day 42 following CQ with PQ treatment (Fig. 5).

Fig. 5.

Haemoglobin recovery following CQ–PQ treatment

Adverse events following CQ–PQ treatment

No severe adverse events occurred throughout the follow-up. The most common adverse events reported following the treatment were abdominal pain 8% (n = 8/100), followed by diarrhoea 5% (n = 5/100). Dark-coloured urine (Hillmen urine colour chart grading ≥ 5) was not reported during the follow-up period. After day 3 most of the clinical symptoms and adverse events were improved and all subsided by day 7 (Table 4).

Table 4.

Clinical symptoms and adverse events following CQ–PQ treatment during the follow-up period

Adverse events/clinical symptoms	Before treatment	After treatment
	Day 0	Day 1	Day 2	Day 3	Day 7	Day 14
Abdominal pain, n (%)	5 (5%)	8 (8%)	6 (6%)	3 (3%)	1 (1%)	–
Cough, n (%)	6 (6%)	5 (5%)	3 (3%)	1 (1%)	–	–
Diarrhea, n (%)	2 (2%)	5 (4%)	2 (2%)	1 (1%)	–	–
Dizziness, n (%)	9 (9%)	3 (3%)	–	–	–	–
Fever, n (%)	90 (90%)	9 (10%)	2 (2%)	1 (1%)	1 (1%)	–
Joint/muscle pain, n (%)	36 (36%)	12 (12%)	–	–	–	–
Headache, n (%)	86 (86%)	7 (7%)	1 (1%)	–	1 (1%)	–
Nausea, n (%)	27 (27%)	12 (12%)	3 (3%)	–	–	–
Vomiting, n (%)	7 (7%)	3 (3%)	–	–	–	–

Hillmen urine test

On day 0, all participants’ Hillmen urine colour chart results were below the 5-point mark. Nine percent (n = 9/100) of patients at baseline had a 4-point Hillman test result, which decreased to 0% by day 3 of follow-up. Conversely, the percentage of individuals with a 1-point mark Hillman test result at enrollment rose from 37% (n = 37/100) to 86% (n = 81/94) on day 14 after completing a full dose of the combined treatment. None of the participants developed PQ-induced dark-brown urination (Additional file 3).

Discussion

The monitoring of AMDs efficacy is crucial for tracking changes in parasite susceptibility, and therapeutic efficacy studies are regarded as the gold standard in this regard. The WHO recommends assessing the efficacy of first and second-line AMDs [14] at least every 2 years to inform malaria control strategies and contribute to developing effective national treatment policies [17]. In line with this, the Ministry of Health (MoH) and the Ethiopian Public Health Institute (EPHI) collaborated on this monitoring programme.

Previous studies across Ethiopia have highlighted the variability in the response of P. vivax to CQ monotherapy, indicating the potential spread of resistance [21–23]. In accordance with the WHO recommendations, the Ethiopian Ministry of Health adopted a combination therapy of CQ (25 mg/kg over 3 days) with PQ (0.25 mg/kg daily for 14 days) to achieve radical cure by targeting hypnozoites and thereby preventing relapses [16]. This follow-up study aimed to evaluate the therapeutic success of CQ–PQ in the region.

Our findings indicate that CQ–PQ treatment is highly efficacious, with therapeutic success rates of 94.7% (95% CI 87.8–97.8) on day 28 and 93.7% (95% CI 86.4–97.1) on day 42. These rates are consistent with studies from Northeastern Myanmar (94.8%) [24] and Northwestern Ethiopia (92.6%) [25]. The slight decline in therapeutic success from day 28 to day 42 may be attributed to relapses due to the reduction in CQ blood levels may allow relapses to emerge, it is the total dose of PQ that is most likely to impact on the emergence or not of relapses. In particular, use of a low total dose primaquine regimen in this study means that some relapses are likely to emerge [26].

Although the therapeutic success on day 28 is lower than studies in Southwestern India (100%), Central China (100%), Brazil (100%), and Colombia (98.6%) [27–30], as well as two Ethiopian studies (Arbamich 100%, Bishoftu-Adama 99.2%) [31, 32], the day 42 success rate in this study is comparable to findings from Arbamich [31] but lower than those from India (100%) and China (96.1%) [33, 34]. Variations in treatment outcomes may result from differences in study populations, drug administration practices, misdiagnosis, new infections, and hypnozoite reactivation [14].

Although this study did not include a direct comparison arm with CQ mono-therapy, evidence from studies conducted in Brazil, Ethiopia, Thailand, and Afghanistan has shown that the addition of PQ to CQ improves treatment efficacy and significantly reduces the risk of recurrence compared to CQ mono-therapy [14]. A study in Brazil reported a higher efficacy of CQ–PQ (98.8%) compared to CQ monotherapy (92.1%) over 28 days [35]. In Ethiopia, the treatment failure rate decreased from 5.6% with CQ to 0.75% with CQ–PQ [32], and studies in Thailand and Afghanistan also reported lower recurrence rates with CQ–PQ compared to CQ alone [36, 37]. The combination therapy’s efficacy is attributed to the complementary actions of CQ, which targets blood-stage parasites, and PQ, which targets both liver- and blood-stage parasites. Although CQ does not affect dormant hypnozoites, it suppresses relapse for up to 28 days [38], after which PQ helps prevent further relapses [39].

In this study, six treatment failure (TF) cases were identified: ETF, LCF, and four LPF. Most TF cases (67%, 4/6) occurred in children aged 5–15 years, aligning with studies from Ethiopia [25] but differing from a study in Myanmar, where TF cases were more common in adults [40]. Higher baseline parasitaemia was observed in children under 15, which may reflect the development of immunity with age, resulting in lower parasitaemia and reduced symptomatic disease in older individuals [41, 42]. Additionally, the earlier onset of malaria in children likely increases their exposure to both malaria and TF [14].

Parasite clearance from baseline parasitaemia serves as an important indicator of treatment success. In this study, 99% of participants achieved asexual parasitaemia clearance by day 3 (Fig. 4), which is consistent with findings from a meta-analysis suggesting that early clearance (by day 2 or 3) is a key indicator of treatment efficacy [43–45]. Furthermore, significant gametocyte clearance was achieved, with all participants clearing gametocytes by day 3, and no recurrence was observed. However, the occurrence of ETF on day 3 and LCF on day 7 raises concerns about the emergence of resistance to first-line treatment, necessitating further monitoring.

Regarding fever clearance, 90% of participants were febrile at enrollment, but 100% achieved fever clearance within 72 h. This finding is in line with studies from Myanmar [40] and demonstrates faster fever clearance compared to studies in Southwest Ethiopia, where CQ alone showed delayed fever clearance [23]. The rapid fever resolution may be attributed to the early parasitaemia clearance or the administration of antipyretics (paracetamol) in febrile patients.

Malaria-related anaemia is common in endemic areas, and effective treatment is critical to prevent progression to severe anaemia [46]. In this study, after completing CQ–PQ treatment, a significant increase in Hgb levels was observed (p < 0.001), which is consistent with findings from other Ethiopian studies [31] and contrasts with studies using CQ alone, where Hgb improvements were negligible [23]. While other factors like poor nutrition and intestinal parasitic infections may contribute to anaemia, most participants with a history of recurrent malaria showed Hgb improvement (9/12), suggesting the positive impact of CQ–PQ treatment on anaemia recovery [47].

Adverse events associated with CQ and PQ is well-documented. In this study, no severe adverse events were reported, and the treatment was safe and well-tolerated, consistent with studies conducted in Colombia [28]. Abdominal pain and diarrhea were the most common side effects, similar to studies in Brazil [48]. Importantly, no cases of haemolytic anaemia attributable to G6PD deficiency were observed, suggesting that PQ was safely administered without prior G6PD testing in this population. This finding is consistent with previous reports from Ethiopia, including a study conducted in Arba Minch [31]. The overall prevalence of G6PD deficiency in Ethiopia was relatively low, ranging from 1.4% and 3.5% with notable regional variation. The most frequently identified G6PD mutant was A376G, which is associated with moderate enzyme deficiency and a lower risk of severe haemolysis [49]. Given this epidemiological context, the likelihood of PQ-induced haemolytic events is considered low in most Ethiopian populations. This aligns with WHO recommendations that, in settings where G6PD deficiency prevalence is below 5% and testing is not readily available, PQ use may be considered under close clinical supervision [50]. In this study, participants were closely monitored using the Hillmen urine colour test, and no urine discoloration exceeded the 5-point threshold during follow-up. These findings support the safe and supervised use of PQ in low-prevalence and resource-limited settings.

Therapeutic efficacy studies (TES) are a cost-effective tool for monitoring the potential emergence of treatment failures with nationally recommended AMDs. Regular monitoring and surveillance in different geographical locations are essential for detecting early resistance trends. This study provides strong evidence that CQ–PQ remains an effective first-line treatment for uncomplicated P. vivax malaria, provided that regular monitoring is implemented to detect emerging resistance.

Limitations of this study include the absence of advanced diagnostic techniques, such as PCR, to differentiate between relapses and new infections, and the lack of data on CQ drug concentrations, which would have helped determine whether treatment failure was due to true parasite resistance or insufficient drug levels. Further studies employing advanced diagnostic techniques, such as molecular genotyping and drug concentration assays, are warranted to better understand treatment efficacy and relapse patterns in this specific endemic area.

Conclusion and recommendations

In conclusion, CQ–PQ therapy has proven to be an effective treatment for P. vivax malaria, with high cure rates, rapid parasite clearance, and significant improvements in haemoglobin levels, particularly in urban settings. The treatment demonstrated a favourable safety profile, with mild adverse events that resolved quickly. Based on these findings, it is recommended to continue the use of CQ–PQ as a primary treatment option for P. vivax malaria, while monitoring for potential resistance. Further research should explore long-term outcomes and the broader application of this therapy in diverse settings to strengthen malaria control efforts.

Abbreviations

ACPR

Adequate Clinical and Parasitological Response

AL

Artemether lumefentrine

AMDs

Anti-malaria drugs

CQ

Chloroquine

CQR

Chloroquine resistant

CTF

Chloroquine treatment failure

EPHI

Ethiopian Public Health Institution

ETF

Early treatment failure

Hgb

Haemoglobin

HUT

Hillmen urine test

LCF

Late clinical failure

LPF

Late parasitological failure

PQ

Primaquine

PRR

Parasite reduction ratio

SD

Standard deviation

TES

Therapeutic efficacy study

TF

Treatment failure

WHO

World Health Organization

Author contributions

EG designed and conceived the study, developed the tool, coordinated data collection, carried out the statistical analysis, and drafted the manuscript. TE, AA, AB, DGA, and BGB edited and critically reviewed the manuscript. HM, YW, GT, GT, MH, MK, AA, and BGB facilitate the site work and coordinate the whole activity. All authors read and approved the final manuscript.

Funding

This study was funded by Ethiopian Public Health Institution.

Availability of data and materials

Data will be available upon request from the Ethiopian Public Health Institution.

Declarations

Ethics approval and consent to participate

Ethical approval was obtained from the Ethiopian Public Health Institute (EPHI-IRB294-2022). Written informed consent was obtained from all participants, with assent from children aged ≥ 12 years. Participants received transportation reimbursement and insecticide-treated nets. Privacy and confidentiality were maintained throughout the study period by excluding personal identifiers during data collection.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.