In vivo efficacy of chloroquine plus primaquine combination therapy against uncomplicated Plasmodium vivax malaria in Limu Kossa District, Jimma Zone, Southwest Ethiopia

Abstract

Background

Plasmodium vivax is the second most common malaria parasite in Ethiopia. It has been treated with chloroquine (CQ) for the past seven decades. However, the emergence of CQ-resistant strains in the nation urged the Federal Ministry of Health of Ethiopia to review its national malaria treatment guideline in 2018. In the revised guideline, the first-line treatment for uncomplicated P. vivax infection is a combination of CQ and primaquine (PQ). Thus, the present study was designed to evaluate the in vivo efficacy of CQ and PQ combination therapy against clinical P. vivax mono-infection in one of the malaria-endemic areas of Ethiopia.

Methods

An open-label prospective clinical trial was conducted in the Limmu Kossa District, Jimma zone, Southwest Ethiopia, from September 2023 to March 2024. A total of 108 patients were recruited for the study. All participants received treatment with CQ at a dosage of 25 mg/kg over three days, followed by PQ at 0.25 mg/kg for 14 consecutive days. Patients were monitored for 42 days for any signs of treatment failure and malaria clinical symptoms, as per the World Health Organization (WHO) guidelines for anti-malarial drug evaluation. Additionally, haemoglobin (Hb) levels, body temperature, any adverse events, and signs of haemolysis were assessed. Data was analysed using R-software (version 4.0.0) and a significant level was considered at p < 0.05.

Results

The median age of the patients was 23 years, ranging from 2.5 to 62 years. Of the 108 patients initially recruited, 100 completed the 42-day follow-up period. The combination therapy of CQ and PQ for uncomplicated clinical P. vivax malaria demonstrated excellent therapeutic efficacy, with a 100% cure rate observed at both day 28 and day 42. Additionally, the recommended low dose of PQ (0.25 mg/kg) was well-tolerated, with no signs of. Additionally, most common malaria symptoms were disappeared early in the follow-up period.

Conclusion

The combination of CQ plus PQ has exhibited excellent efficacy against uncomplicated P. vivax malaria mono-infections. To preserve this efficacy, it is critical to ensure patients adhere to the full course of PQ treatment, despite its extended duration. Therefore, health authorities should put emphasis on the boosting of the public on the importance of finishing the prescribed medication regimen.

Background

Plasmodium vivax is the most widely distributed malaria parasite globally, with approximately 7 million clinical cases [1]. While it is commonly known that P. vivax has African origins [2], its existence on the continent is rare. For many decades, it was considered a benign malaria parasite [3]. However, the parasite is predominantly prevalent and steadily causing significant clinical disease in countries in the Horn of Africa including Ethiopia, Eritrea, Sudan, Djibouti, and Madagascar [4, 5]. Plasmodium vivax accounts for 3% of malaria cases worldwide, with about 12% of the global cases and deaths attributed to this parasite [1]. According to the Federal Ministry of Health of Ethiopia’s Malaria Strategic Plan and a comprehensive systematic review and meta-analysis by Ketema et al. [6, 7], P. vivax accounts for nearly 31% of malaria infections in Ethiopia. In recent years, the prevalence of P. vivax has been steadily increasing, particularly in the central west region, and gradually spreading to the western part of the country [7, 8]. Despite significant efforts over many years to eradicate malaria in endemic areas, the disease remains the most pressing health concern in Ethiopia [9, 10].

In Ethiopia, chloroquine (CQ) was the first-line treatment for uncomplicated Plasmodium falciparum and P. vivax malaria. In 1998, following the widespread of chloroquine (CQ) resistant P. falciparum, CQ was replaced by sulfadoxine-pyrimethamine (SP) and subsequently, in 2004, SP was replaced artemether-lumefantrine (AL) for the treatment of uncomplicated falciparum malaria in Ethiopia [11, 12]. However, uncomplicated P. vivax infection was treated with CQ alone until recently. Then, the emergence of chloroquine-resistant P. vivax strains was reported in most vivax-endemic areas, as reviewed by Ketema et al. [13], the national malaria treatment guideline was revised in 2018 [14]. The updated treatment regimen recommends the use of a combination of CQ (25 mg/kg for three consecutive days) and primaquine (PQ, 0.25 mg/kg for 14 consecutive days) for uncomplicated P. vivax infections [14]. This combination therapy is advised for patients in endemic areas, as well as for those in endemic regions in specific districts targeted for malaria elimination and patients outside malaria-endemic areas. In the same guideline the second line drug in case of treatment failure or patients failed to respond to CQ and PQ, a 7-day quinine or artesunate was recommended [14]. The Ethiopian National Malaria Control Programme (NMCP) has also developed a strategic plan for malaria elimination [15].

An imperative challenge with this combination treatment is poor patient adherence to the 14-day PQ regimen, which may lead to the emergence of resistance due to subtherapeutic dosing [16]. Although the use of CQ plus PQ in combination for uncomplicated vivax malaria is relatively recent, studies from high-transmission settings in Northern and Southern Ethiopia have reported significant late treatment failures, with rates of 7.4% and 10.9% [17, 18]. Therefore, this study aimed to evaluate the in vivo efficacy of CQ plus PQ against uncomplicated clinical P. vivax infections in low vivax malaria transmission settings in Ethiopia.

Methods

Description of study area

The study was conducted at Laku Chime Health Centre located in Limu Kossa district, Jimma Zone, Oromia Regional State, Southwest Ethiopia (Fig. 1). The District is located 420 km southwest of Addis Ababa, the capital city of Ethiopia and lies between latitude of 7°50′ and 8°36′ North and longitude of 36°44′ and 37° 29′ East. The district’s elevation was 1761 m. The area is characterized by several perennial rivers, intermittent streams, springs, and notable landmarks including Cheleleki Lake and Bolo Caves (data from the Limu Kosa District Agricultural and Rural Development Office). According to the 2007 national census [19], the District has a population of 161,338 with 81,462 men and 79,876 women. The majority of the population (72.6%) are Muslims, and 14,842 people, or 9.2%, are urban dwellers. The district is served by one hospital, seven health centres, 14 clinics of various levels, and 36 health posts. Laku Chime Health Centre is one of the district’s health centres. Malaria is a significant health issue in the zone and district. Although there was no comprehensive studies on the malaria prevalence and effect of seasonal variability on the disease burden, the Serological study on the aetiologies of human malaria exposure showed a frequent response was against P. falciparum and P. vivax [20]

Fig. 1

The map of study site

Study design and source population

From September 2023 and March 2024, an open-label prospective clinical trial was carried out. It involved patients with uncomplicated P. vivax mono-infection. Inclusion criteria: patients with P. vivax mono-infection, over six months of age, experiencing symptoms of uncomplicated infection with a parasite load of greater than 250 parasite/µl, no known allergies to CQ and PQ, not-pregnant or breastfeeding, residing near the health facility, informed consent from the patient or from a parent or guardian in the case of children, no severe malnutrition as per the World Health Organization (WHO) child growth standards guideline, absence of regular medication, which might interfere with anti-malarial pharmacokinetics, and required commitment to attending all scheduled visits.

Exclusion criteria: severe malaria symptoms or complications, known contraindications to CQ plus PQ, comorbidities or chronic infections, inability to participate due to physical or mental health limitations, infections other plasmodium species, severe malnutrition as defined by WHO, haemoglobin level below 5 mg/dl in children and below 7 mg/dl in adults, inability to take oral PQ medication, and receipt of anti-malarial drugs within two weeks before the enrolment date [1].

Sample size determinations

The sample size for this study was determined based on an anticipated 5% rate of treatment failure with CQ plus PQ in a low transmission setting. The calculation aimed for a ± 5% precision and a 5% significance level. Additionally, a 20% loss to follow-up rate over the 42-day study period was expected. As a result, 108 study participants were included. The participants were purposely recruited according to the study’s inclusion criteria.

Data collection procedures

The socio-demographics, and clinical data for patients recruited for efficacy testing (n = 108) were collected by health professionals at the health facility using a pre-designed data collection format. Blood samples were taken from each patient via a lancet-pricked finger. The sample was used to prepare blood films and measure haemoglobin (Hb) levels. Thin and thick smears were prepared on a clean glass slide, air air-dried, and the thin film was fixed with 70% methanol. One of the smears was stained with 10% Giemsa and examined under a 100X oil immersion objective. The other blood film used for the parasite load counting was stained with 3% Giemsa. Slides were deemed positive for specific Plasmodium species or mixed infection if any parasite stage was observed, and negative if no malaria parasites were seen under all fields of view. Parasite density (asexual stage) per microlitre (µl) of the blood was determined by counting the number of parasites per 200 white blood cells, assuming a standard human leukocyte count of 8000/µl). Gametocyte density was quantified against 1000 leukocytes and converted to the number of gametocytes/µl) of blood, assuming a mean leukocyte count of 8000/µl). As additional information to demonstrate pieces of evidence on the malaria positivity rate in the study area, retrospective data was collected from patient’s medical records and the district health bureau from 2019 to 2023.

Haemoglobin (Hb) measurement

A lancet was used to prick the patient’s finger and collect a single drop of blood in a cuvette. This blood sample was then used to determine the patients blood Hb level at enrollment (Day 0), Day 14, and Day 42 using a Hemocue machine [HemoCue^® Hb 301 System (haemoglobinometer, Angelholm, Sweden)] following the manufacturer’s instructions.

Patient treatment and follow-up

A 42-day in vivo drug efficacy test was conducted following WHO recommended methods [18]. This test involved treating symptomatic and parasitaemic P. vivax patients with a known dose of CQ and PQ. The CQ (chloroquine phosphate 250 mg, batch number 33342, Addis Pharmaceutical Factory PLC) was administered for 3 days, with a total dose of 25 mg per kg under professional supervision. Following this, PQ (Primaquine 7.5 mg film-coated tablets, batch number: 110701, Remedica Ltd., Aharnon Str., Limassol Industrial Estate, 3056 Limassol, Cyprus) was given for 14 days at a dose of 0.25 mg/kg per day. To ensure the correct administration of each dose, the patients were instructed to take all their medication under the supervision of a health professional only in the health centre. Those patients who missed any of their doses were excluded from the study and were not considered in the analysis.

Patients were monitored for parasitological and clinical responses over 42 days. All drug doses were administered under the supervision of professionals. Any physiological complaints were recorded during each visit. Patients were observed vomiting for 30 min after drug ingestion; those who vomited the first dose and expelled the entire drug were re-treated with an identical dose.

The axillary temperature and other clinical symptoms of the vivax malaria patients were assessed by an experienced nurse at the health centre and recorded in a specific format. Subjects who vomited the medication twice were excluded from the study. The participants were instructed not to take any other drugs except paracetamol if their axillary temperature was > 37.5 °C. Patients were scheduled for a follow-up examination on Days 1, 2, 3, 7, 14, 21, 28, 35, and 42 including temperature monitoring, and were advised to return at any time if they experienced malaria-like symptoms [21]. Blood smears were taken at all follow-up visits except on day 1. Patients who missed their follow-up schedules were visited at home. Additionally, haemolysis was assessed vissualy using urine colour estimation. A small portion of the patients urine was placed in a clear test tube and held up against a white piece of paper. Then colour was compared against the Hillmen Colour Chart for any evidence of haemoglobinuria on each patient from Day 1 to 14.

Treatment outcomes and endpoint classification

Treatment outcomes were assessed based on the parasitological and clinical results and classified according to the WHO [21] protocol as follows: clinical deterioration due to P. vivax illness requiring hospitalization in the presence of parasitaemia, and axillary temperature ≥ 37.5 °C on any day between days 3 and 28, and presence of parasitaemia on any day between days 7 and 28, irrespective of clinical condition was considered as treatment failure. Treatment success or adequate clinical and parasitological response (ACPR): was defined as the absence of parasitaemia on day 28 and beyond, regardless of axillary temperature., in patients who did not previously meet any of the criteria of treatment failure. In case of treatment failure detected, recrudescence from reinfections will be distinguished using molecular technique.

Quality management

Every data collector received training on adherence to the study protocol, and every day, all of the recorded data was verified. Before collecting data, all equipment and devices utilized in the process, including weight balances, haemoglobin meters, and thermometers, were calibrated. To guarantee precision, a certified medical laboratory technologist from Jimma University blindly reexamined 10% of all positive and negative blood smears. The Ethiopian Pharmaceuticals Supply Service provided all of the drugs necessary for the patient’s treatment, and their quality was guaranteed.

Data analysis

The data was entered into a Microsoft Excel sheet and checked for completeness. It was then exported to R software (version 4.0) for analysis. Descriptive and inferential statistics were used to compute some baseline data such as age, sex, temperature, parasite load and others. The tools employed included the Pearson correlation, which was to examine associations between variables, between variables, and the Mann–Whitney U test, which analysed Hb recovery in patients with adequate clinical and parasitological responses. Kaplan–Meier survival probability analysis was conducted to evaluate treatment outcomes during the follow-up period. A significance level of 95% confidence interval was used for all statistical tests.

Results

Characteristics of the study participants

During the study period, 1426 individuals suspected of having malaria were tested microscopically. Of these, 19.8% (n = 282/1426) tested positive for malaria, with 51.4% (n = 145/282) attributed to P. vivax. A total of 108 individuals (74.5%) infected solely with P. vivax from the rural areas met the inclusion criteria and were recruited for the study. In the initial follow-up phase (days 1 to 14), eight participants were excluded: one due to an adverse drug reaction, and seven due to loss to follow-up (Fig. 2).

Fig. 2

The study flow diagram

At baseline, 55.6% (n = 60/108) of participants were male, with a median age of 23 years (± 15.2 years, range 2.5 to 62 years). Children under five accounted 8.3% (n = 9/108) of the total participants. Approximately, 62.04% (n = 67/108) of the patients had access (availability of at least one bed net) to abed net, and 36.11% (n = 39/108) use it regularly (every night). Among the 108 participants, 72.2% (n = 78) had experienced previous malaria episodes, all of which were treated with CQ plus PQ combination therapy (Table 1). The mean body temperature was 38.6 °C (range 37.4–39.5 C). The geometric mean parasite count was 5,135/parasites/µl (range 550–69,817 parasites/µl), with significant variation in baseline mean parasitaemia (p < 0.001). The overall gametocyte carriage rate was 93.5% (n = 100/108). The mean Hb level at enrollment was 12 g/dL. A total of 40.7% (n = 44/108) of the participants had varying levels of anaemia, though none of them had severe anaemia (Table 1).

Table 1 Baseline characteristics of the study participants in Laku Chime health center, Southwest Ethiopia

There was a significant negative corelation between age and parasitaemia, with older individuals showing a notable reduction in parasite load (r = − 0.304, p = 0.0021). Children exhibited a higher baseline parasite burden compared to adults. The geometric mean parasite load for nearly all patients was below 35,000 parasites/µl, except for with the exception of one outlier, which had a a parasite load of 69,800 parasites/µl (Fig. 3).

Fig. 3

Comparion of age of the patients and parasite load at enrollment in Laku Chime health center, Southwest Ethiopia

Efficacy of CQ plus PQ against uncomplicated P. vivax infection

The treatment of the P. vivax mono-infected case with CQ plus PQ demonstrated exceptional efficacy. None of the patients who completed their follow-up period experienced malaria symptoms or parasitaemia between days 7 and 42, resulting in a 100% cure rate and an adequate clinical and parasitological response rate (ACPR) (Table 2).

Table 2 Cure rate of CQ plus PQ against clinical uncomplicated P.vivax malaria infection after 42 days of follow-up period in Laku Chime health center, Southwest Ethiopia

Rate of parasite and fever clearance post-treatment of CQ plus PQ

During the follow-up period, gametocytes and all asexual stages began to clear by day two. Initially, most of participants presented with fever, which resolved within the first few days. At the baseline, the average body temperature was 38.58 °C. Following treatment with CQ plus PQ, the average body temperature decreased to 37.69 °C on day two, 37.33 °C on day three, 37.22 °C on day four, and remained below 37 °C for the rest of the follow-up period. By the end of the follow-up period, 69% of the patients had recovered from fever by day one, 87% by day two, and 98% by day three. Nearly all patients had recovered from fever by day seven of the follow-up period (Fig. 4).

Fig. 4

Mean axillary temperature clearance during the follow-up periods in Laku Chime health center, Southwest Ethiopia

Hb levels and recovery

At enrollment (day 0), approximately 22% of participants had moderate anaemia and 19% had mild anaemia. Of the 41 anaemic individuals on day 0, only 84.5% had a history of malaria episodes. By the end of the follow-up period, these patients had not fully recovered their Hb levels, 7% still had moderate anaemia and 11% had mild anaemia. Only a small subset of the anaemic patients showed partial Hb recovery at the end of the follow-up. Among those with a history of malaria, 89% were able to normalize their Hb levels, while, the remaining 11% did not show improvement by day 42. Overall, the number of anaemic patients decreased from 41% at enrollment to 21% on day 14, and further 11% on day 28 following CQ plus PQ treatment (Table 3).

Table 3 Anaemia condition and Hb recovery among patients treated with CQ plus PQ in Laku Chime health centre, Southwest Ethiopia

During the follow-up days, there was a progressive increment on the mean Hb level; on days 0, 14, and 42. It was 11.4, 12.1, and 12.9 g/dl, respectively. The individuals’ Hb levels were positively impacted by the CQ plus PQ medication (Fig. 5).

Fig. 5

Mean hemoglobin level on different follow-up days post CQ and PQ treatment in Laku Chime health center, Southwest Ethiopia

Clinical signs observed during the follow-up period

At enrollment, the mean baseline body temperature was 38 ± 1 °C, with males at 38 ± 0.3 °C and females at 38 ± 0.4 °C. In the early days following treatment, many patients experienced various side effects from the medication or the disease symptoms, but most of these symptoms resolved within the first week of follow-up. The most common early symptoms were fever (91%) and headache (82%), followed by weakness (41%), and nausea (39%). Less common but notable symptoms such as anorexia, cough, and abdominal pain also resolved within the first week. Throughout the 42-day follow-up period, the majority of patients demonstrated good tolerability to the medication, with only a few exhibiting residual symptoms by Day 7 (Table 4).

Table 4 Malaria clinical symptoms and their prognosis after CQ-PQ treatment in Laku Chime health centre, Southwest Ethiopia

Discussion

In this open-labeled prospective clinical trial, the combination therapy of CQ and PQ proven outstanding efficacy (100%) against clinical P. vivax malaria patients in a 42-day observation period. The study confirmed that CQ plus PQ treatment was safe, effective, and potent in easing common malaria symptoms within the first few days of the treatment start. One of the key challenges in managing, controlling, and preventing malaria is the emergence of drug-resistant strains of the Plasmodium parasites. In Ethiopia, CQ was the first-line treatment for uncomplicated P. vivax malaria for many decades. However, due to the rise of CQ-resistant strains of P. vivax [22,23,24,25], the Federal Ministry of Health of Ethiopia revised its malaria treatment guideline in 2018 [14]. The updated guidelines incorporated CQ plus PQ as the first line of treatment for uncomplicated vivax malaria to ensure radical parasite clearance and accelerate the achievement of the malaria elimination target [6, 14, 26].

Despite the importance of this decision, patients’ compliance with the prolonged 14-day PQ treatment is a concern since poor adherence may result in the emergence of PQ-resistant strains and recurrence [27]. Plasmodium vivax infections accounted for 4.08% of the 14% five-year malaria-positivity rate reported in the study area (Supplementary Table 1 and Supplementary Fig. 1). The vivax malaria-positivity rate at this study site was relatively low (< 5%) given the current state of the nation [10, 28]. It indicates a low-vivax malaria transmission scenario according to the WHO classifications for transmission conditions [29]. In such instances, anti-malarial therapy seems appropriate and a wider reach of interventional measures has the potential to halt the spread of malaria and lower malaria-related mortality [29]. Contrary to the high transmission settings, there is little chance that patients would re-infect with the same or a different species of Plasmodium since PQ eliminates the latent stage that causes relapse. Therefore, the reappearance of the parasitaemia could be the cause of any actual therapeutic failure found in these conditions. Nevertheless, none of the study’s subjects who finished their 42-day follow-up exhibited any signs of treatment failure. Every patient responded to the combined therapy with a 100% adequate clinical and parasitological response (ACPR). This result is consistent with reports from various regions of the country, such as research done in Debre Zeit and Nazarate in East Shoa 14 years ago, as well as a study from Arba Minch in 2023, which found that a low dose of PQ (0.25 mg/kg per day for 14 days) did not result in any significant early or late treatment failures until day 28 [17, 30]. Furthermore, our results align with the outstanding effectiveness (100%) seen in Colombia and Pakistan some years ago [31, 32]. It conflicts with a recent Ethiopian study reported from Hamusit, which showed early treatment failures although did not provide genomic proof [18]. Additionally, despite knowing the exact G6PD enzyme levels before treatment, the lack of side effects, such as haemolysis, linked to PQ treatment in this trial indicates good tolerance to the low dose of PQ [33]. Thus, this study offers compelling evidence that treating vivax malaria in low-transmission conditions with the CQ plus PQ regimen now in use in Ethiopia is successful.

Higher treatment failure rates for PQ have been reported worldwide, especially in Western Thailand and the Northwest Frontier Province of Pakistan, where failure rates have reached as high as 7.7% [34, 35], this is in contrast to the current findings. PQ’s failure to offer an effective treatment for P. vivax malaria has impeded efforts to prevent and control the disease in other areas [34, 35]. The explanations for these differences might be due to factors such as the duration of PQ that has been used to treat vivax or other illnesses (whether long or short), the level of drug supervision (whether supervised or unsupervised), the timing of the primary outcomes assessments (14, 28 or 42), and the patient adherence levels (ranging from poor to good) [17, 18, 30, 35].

Three patients quit their treatment during the follow-up days, which resulted in their exclusion from the study. However, because of a recurrence of malaria symptoms, these patients were brought back to the health centre. In the health facility, they received a full dose of both PQ and CQ for treatment. This incident raises a concern about the need for patients to strictly adhere to their treatment plans and for healthcare workers at the health posts to provide appropriate guidance [16]. Such actions could be a contributing factor in the future establishment of drug-resistant strains of the parasite, since the sub-therapeutic dose may allow residual parasites to survive in deep tissues or blood [36, 37].

After a few days of CQ plus PQ administration, the patient’s parasite density rapidly decreased. On day three, all patients cleared their parasitaemia, and there was no variation in the average time required for parasite clearance between the regimens. It is almost identical to the study conducted in Debre Zeit and in East Shoa of Ethiopia, where all the study participants cleared their parasites on day 3 [30]. This could be due to the drug’s quick and full absorption in the digestive system after oral treatment and its quick metabolism [37]. PQ reaches its peaks in plasma levels within two to three hours and then quickly decreases, with a terminal phase of elimination half-life of 7.1 ± 1.6 h [38]. As one of the criteria for tracking and categorizing responses to PQ treatment, fever clearance time is critical for assessing the therapeutic efficacy of PQ in the treatment of P. vivax. On CQ plus PQ treatment, 98% of the participants cleared fever within three days of the follow-up period, which further supports the fact that PQ acts fast and quickly clears such symptoms. This finding supports similar reports from Ethiopia by Mekonned et al. [28], where fast fever clearance was reported.

In this study, one of the typical clinical manifestations (50%) of the patients was different levels of anaemia (mild and moderate). Treatment with CQ plus PQ brought a significant Hb recovery between the baseline and day 42, which aligns with a similar report from Hosanna, Ethiopia [25]. However, a small number of anaemic patients who had a previous malaria episode were unable to exhibit improvement in their Hb level, even on day 42. Since repeated malaria episodes and frequent relapse could cause impairment of Hb levels [39], poor malaria control is often related to weak interventional activities and a high prevalence of other infectious diseases and nutritional deficiencies, all of which contribute to the incidence of anaemia [40].

Limitation of the study

In this study the representation of infants < 2.5 years were limited as there was not patients encountered during recruitment. Thus, this findings might not be generalized to the whole population in the study site. In addition, as this study was conducted in low malaria transmission settings of Ethiopia, it might not be generalized to other settings in the Ethiopia such as low land rift vally areas commonly identified as vivax endmic areas in Ethiopia.

Conclusion

The study demonstrated a 100% cure rate for CQ (25 mg/kg) combined with PQ (0.25 mg/kg) treating uncomplicated clinical vivax malaria. The treatment resulted in rapid and effective clearance of parasites and fever and significant improvement in common malaria symptoms during the early follow-up period. Furthermore, the low dose of PQ (0.25 mg/kg for 14 days) was safe, with no patients showing signs of haemolysis. To preserve the effectiveness of this combination therapy, ensuring complete patient adherence to the full PQ regimen is crucial. Future studies should focus on evaluating the efficacy of CQ plus PQ in vivax malaria-endemic areas. Moreover, local, and national health authorities should implement community sensitization efforts to promote adherence to PQ and maintain the drug’s effectiveness.