Chloroquine-primaquine therapeutic response and safety in patients with uncomplicated Plasmodium vivax malaria in the Colombian Amazon region

Abstract

Background

In Colombia, published studies on the treatment of uncomplicated Plasmodium vivax malaria with chloroquine-primaquine are scarce. The aim of this study was to evaluate the therapeutic response to two treatment regimens at the 28-day follow-up and the occurrence of adverse events in patients with P. vivax malaria.

Methods

A quasi-experimental clinical trial was conducted at 3 sites in the Department of Amazonas. Patients received supervised or unsupervised anti-malarial treatment (chloroquine plus primaquine), and the primary effectiveness endpoint was the clinical and parasitological response. Safety was assessed through adverse event surveillance.

Results

A total of 103 patients were included: 53 in the 7-day primaquine group (Group I) and 50 in the group receiving primaquine for 14 days (Group II). Among the patients in group I, an adequate treatment response of 100% and 89.5% was found in patients who received supervised and unsupervised treatment, respectively. In Group II, adequate responses of 100% and 95% were reported for patients who received supervised and unsupervised treatment, respectively. No adverse events were detected.

Conclusions

The response to combined treatment with chloroquine plus primaquine continues to be adequate for treating P. vivax malaria in the Colombian Amazon region; however, a response to unsupervised treatment in the region is recommended.

Background

Plasmodium vivax malaria is one of the most common febrile diseases in tropical and subtropical areas of the world [1]. In Colombia, 100,744 cases of uncomplicated malaria were reported in 2023, 64,565 (63.0%) of which were caused by P. vivax; of these, 1074 cases were reported from the Colombian Amazon [2]. Although the department contributes around 1.5 to 3.0% of the country’s total cases per year, P. vivax malaria is the most common in the region. Moreover, underreporting of information significantly hampers public health decision-making.

The Department of Amazonas is classified in risk stratum 4 for malaria according to the Methodological Guidelines for Stratification and Estimation of the Population at Risk for Malaria 2020–2023. However, some non-municipalized areas of the department, such as La Pedrera and Tarapacá and the municipality of Puerto Nariño, are classified in stratum 5 [3]. The environmental and geographic conditions and sociodemographic, cultural and economic factors of this region make it favourable for the development and propagation of the vector and the transmission of the parasite [4].

According to statistics reported by the National Administrative Department of Statistics (DANE), in the Department of Amazon, more than 50% of the population lives in populated centres and dispersed rural areas [5]. Displacement from communities (dispersed rural areas) with municipal capitals and vice versa is conditioned by economic and environmental factors, largely limiting access to health services.

The recommended treatment for treating P. vivax malaria in the country is the joint administration of chloroquine (4-aminoquinoline) at a total dose of 25 mg/kg for three days and primaquine (8-aminoquinoline) with a treatment protocol of 0.25 mg/kg/day for 14 days, for a total dose of 210 mg [6]. The Department of Amazonas adopted a treatment scheme with chloroquine at a total dose of 25 mg/kg concomitant with primaquine for 7 days at a dose of 0.5 mg/kg/day and a total dose of 210 mg [7] to increase patient adherence to treatment.

Low adherence to anti-malarial medications in indigenous populations is a complex issue influenced by cultural and structural factors. One of the main challenges is geographical isolation, as many communities are located in remote areas, making it difficult to ensure consistent supervision of medication supply and proper monitoring of disease progression [8].

Additionally, indigenous communities often have long-standing traditional medical systems, where they place greater trust in local healers and the use of medicinal plants passed down through generations, rather than modern treatments [9]. Furthermore, the rapid improvement of symptoms after three days of chloroquine treatment may lead to patients discontinuing the primaquine regimen prematurely [7].

In the Colombian Amazon, only one study has evaluated the efficacy of chloroquine-primaquine for treating P. vivax malaria with a 28-day follow-up in the Tarapacá municipality [10]. The limited data from this single study underscores the need for continuous monitoring of anti-malarial treatments in the region to detect potential resistance.

It is crucial to understand the treatment response in patients with P. vivax malaria to develop strategies that reduce the recurrence rate of this species. It remains uncertain whether the current doses of chloroquine and primaquine are sufficient for this purpose. Considering the debilitating impact of P. vivax on affected populations, the disease leads to a significant deterioration in health, making it vital to evaluate and improve treatment effectiveness [11].

Therefore, this study aimed to evaluate the response to chloroquine plus primaquine treatment and the occurrence of adverse events in patients with uncomplicated P. vivax malaria in the Colombian Amazon. The first pillar of the “Global Technical Strategy for Malaria 2016–2030” [12] and the World Health Organization (WHO) protocol “Methods for monitoring the efficacy of antimalarials” [13] were used as reference in conducting the study.

Methods

Study design and site

A quasi-experimental clinical trial was conducted with 2 treatment arms at 3 sites in the Colombian Amazon: the non-municipalized areas of La Pedrera (1°19′43ʺS 69°34′47ʺW), Tarapacá (2°52′44ʺS 69°44′38ʺW) and the indigenous communities of San Pedro de Tipisca and the 12 de Octubre of the municipality of Puerto Nariño (3°46′47ʺS 70°21′44ʺW) (Fig. 1). Patients were included and followed up between December 2020 and July 2022 on the basis of the “Methods for surveillance of antimalarial drug efficacy” protocol of the WHO of 2009 [13].

Fig. 1

Study sites

Study population and inclusion criteria

Residents in the area who visited the health centre for illness and were diagnosed with malaria via microscopy or a rapid test were invited to participate in the study. The inclusion criteria for enrolment were age between 18 and 65 years old, mono infection with P. vivax, axillary temperature greater than or equal to 37.5 °C and ability to pass oral medication, the age range was chosen to optimize follow up based on the availability to return to the controls. Were excluded: women who were pregnant or breastfeeding, patients with severe malaria or danger signs as defined by the Colombian Ministry of Health [7]; mixed infection or monoinfection with other Plasmodium species detected by microscopy or rapid tests, such as Plasmodium falciparum or Plasmodium malariae; the presence of febrile conditions due to diseases other than malaria (dengue, COVID-19, or unspecified fever); and a history of hypersensitivity reactions, contraindications or the consumption of anti-malarial drugs or infusions of plants used to treat malaria during the 4 weeks prior to probable inclusion. An informed consent was signed to participate.

Intervention protocols

Patients were nonrandomly assigned by study personnel to one of the 2 treatment schedules with chloroquine (CQ) plus primaquine (PQ) to match the treatment groups for 7 or 14 days of primaquine. All treatments were administered on the basis of the Guideline for the Comprehensive Clinical Care of the Malaria Patient 2011 [7]. Within each treatment schedule, there were both supervised and unsupervised patients, depending on how easy it was for the patients to return to the controls. Supervised patients were monitored on days 1, 2, 3, 7, 14, 21, and 28, while unsupervised patients were encouraged to visit health posts daily, with mandatory visits on days 3 and 28. During follow-up, all patients were assessed for signs, symptoms, and adverse effects, such as itching, headache, or vomiting, following the WHO protocol [13]. Axillary temperatures were also recorded throughout the study.

Procedures

A complete medical history (symptoms, current medications, and previous use of anti-malarial drugs) and demographic and contact details were noted. At each follow-up, clinical signs and symptoms were recorded, and blood smears were obtained to detect malaria parasites. Filter paper spot samples for molecular diagnosis by PCR were taken on day 0 and day 28, and haemoglobin was measured with HemoCue^® equipment on the day of inclusion and day 28.

Microscopic blood examination or rapid test

Thick and thin blood films were obtained, were stained with Giemsa and examined to confirm the diagnosis and were labelled anonymously. The parasite count was determined against 200 leukocytes based on optimal fields (10–15 leukocytes per field) with a 100X objective. When 200 leukocytes were counted and fewer than 10 parasites were observed, the count was increased to 500 leukocytes. If no parasites were observed on the slide, the result was considered negative after 200 fields were evaluated.

Blood smears of the enroled patients were examined by two different microscopists upon the conclusion of the study. If the difference in the quantification of parasitaemia between the first two readings varied by more than 20% or if the species diagnosis was controversial, an additional reading was performed by a third microscopist. In the case of Puerto Nariño, where microscopic evaluation was not possible, the Standard Diagnostics BioLine Malaria Ag P.f./P.v rapid test was used on the inclusion day; thick blood smears were obtained on days 0, 3 and 28; and filter card samples were obtained on days 0 and 28.

Molecular diagnosis by PCR

A nested PCR based on the protocol from Singh et al. [14] was performed to confirm the detection of parasites in all samples on days 0 and 28. The samples were individually stored in plastic bags and sent to the Malaria Group Laboratory at the University of Antioquia. DNA extraction was performed with 5% Chelex 100 as follows: filter paper was added to 0.5% saponin, the mixture was washed 3 times with 1 × PBS, and Chelex 100 was added; the mixture was denatured at 56 °C for 15 min and 100 °C for 10 min; and the supernatant was recovered after 15 min of centrifugation at 13,000 rpm. PCR confirmation was performed via a PCR protocol developed by Singh et al. via a first amplification reaction with the primers rPLU1 5′-TCAAAGATTAAGCCATGCAAGTGA-3′ and rPLU5 5′-CCTGTTGTTGCCTTAAACTCC-3 for the fragment of the 18 s-rRNA ribosomal subunit of the parasites of the Plasmodium genus. This PCR product was used for the second reaction (nested PCR) with the primers rVIV1 5′-CGCTTCTAGCTTAATCCACATAACTGATAC-3′ and rVIV2 5′-ACTTCCAAGCCGAAGCAAAGAAAGTCCTTA-3′ for the identification of P. vivax. For the identification of DNA fragments of both genus and species, electrophoresis was performed on a 2% agarose gel (Table 1).

Table 1 Nested PCR primers

Anti-malarial treatment

Chloroquine was administered once daily for 3 days in a total dose of 25 mg/kg (10 mg the day zero and 7.5 mg the days 1, 2). Two primaquine regimes were administered: the 7-day treatment group (Group I) received a 0.5 mg/kg dose once daily, and the 14-day treatment group (Group II) received a 0.25 mg/kg/day dose; both groups started from day zero.

Patients with supervised treatment in the communities 12 de Octubre and San Pedro de Tipisca, in Puerto Nariño, were visited in their homes by the health promoters. In Tarapacá and Pedrera, the patients were monitored for at least 20 min by a professional to ensure that no dose was missed and there were no adverse effects.

In case the patient was unable to attend at the scheduled time, direct communication was maintained with the patient via phone to coordinate their attendance and ensure that the dose was taken as soon as possible.

Follow-up loss and violation to protocol

Patients receiving supervised treatment who missed follow-up on days 14 or 21 were not classified as lost to follow-up, unless they had their final dose on day 14 or missed visits on days 1, 2, 3, 7, or 28. In the unsupervised group, no patients were considered lost to follow-up, except for those who did not return on day 28; these individuals were excluded from the study. Additionally, all patients who tested positive for a species other than P. vivax during the study were also excluded.

Outcomes

Treatment outcomes were assessed on the basis of parasitological and clinical results and were classified according to the WHO protocol as early treatment failure, late clinical failure, late parasitological failure or adequate clinical and parasitological response [13]:

• Early treatment failure (ETF): the development of danger signs or severe malaria on day 1, day 2 or day 3 in the presence of parasitaemia

  • Parasitaemia on day 2 was greater than that on day 0, irrespective of axillary temperature

  • Parasitaemia on day 3 with an axillary temperature ≥ 37.5 °C

  • Parasitaemia on day 3 ≥ 25% count on day 0

• Late Clinical Failure (LCF)

  • Development of danger signs or severe malaria on any day from day 4 to day 28 in the presence of parasitaemia, without previously meeting any of the criteria for early treatment failure

  • Presence of parasitaemia and axillary temperature ≥ 37.5 °C (or history of fever) on any day from day 4 to day 28, without previously meeting any of the criteria for early treatment failure

• Late Parasitological Failure (LPF): Presence of parasitaemia on any day from day 7 to day 28 and an axillary temperature < 37.5 °C, without previously meeting any of the criteria of early treatment failure or late clinical failure.

• Adequate Clinical and Parasitological Response (ACPR): Absence of parasitaemia on day 28 irrespective of axillary temperature without previously meeting any of the criteria of early treatment failure, late clinical failure or late parasitological failure

Statistical analysis

A descriptive analysis was performed on the different outcomes with respect to the different sociodemographic variables and the assigned treatment protocol; medians and interquartile ranges were estimated for quantitative variables without a normal distribution, and relative and absolute frequencies were estimated for qualitative variables. All the statistical procedures were performed via the R package for Windows, version 4.0.2 (The R Foundation for Statistical Computing, Vienna, Austria).

The final analysis included a comprehensive description of all patients who were screened, along with the reasons for their exclusion from the study. It also included detailed information on all patients who were included in the study; the proportion of adverse events and serious adverse events among these patients; and the rates of early treatment failure, late clinical failure, late parasitological failure, and adequate clinical and parasitological response by day 28.

Ethical considerations

The study was conducted according to good clinical practice guidelines, and the international ethical standards for biomedical research with human subjects established by the WHO and the norms of the Ministry of Social Protection of Colombia (Resolution 8430 of 1993) were followed; the Ethical Committee of Research Headquarters of the University of Antioquia approved the study protocol (Act 20-32-929, December 2020). Prior to patient recruitment, verbal and written consent was obtained from the indigenous authorities and health entities of the department for the study to be carried out. The informed consent process was designed with respect for the cultural and linguistic differences of the indigenous communities, avoiding technicalities. Most patients were recruited at the health centre or at their own homes, and when necessary, local health promoters explained the study in their language. The study procedures, follow-up, and potential risks and benefits were clearly outlined. Illiteracy and other barriers were addressed by involving witnesses or family members, ensuring patients fully understood the information before deciding to participate, thereby safeguarding the integrity of the informed consent.

Results

Between December 2020 and July 2022, 123 patients were screened for eligibility, and 103 were included in the study; 53 patients received primaquine treatment for 7 days, and 50 patients received treatment for 14 days. Among all the patients, were excluded 20 patients: 19 withdrawn for exclusion criteria and one for violation to protocol for noncompliance with the 28-day follow-up (Fig. 2). In total, there were 3 cases of recurrence among the 103 patients, which represents 97% therapeutic efficacy of the combination of chloroquine plus primaquine for P. vivax malaria.

Fig. 2

Flowchart of participants considered in each treatment arm

Baseline characteristics of the participants

The highest proportion of patients was men of indigenous ethnicity. A description of the age and parasitaemia status revealed that the supervised group of patients with 7 days of PQ treatment was the youngest and had the highest parasitaemia on the day of inclusion (Table 2).

Table 2 Baseline characteristics

Therapeutic response in the 7-day treatment group (group I)

Two (10.5%) of the patients who received unsupervised treatment had parasitaemia detected on day 28 by microscopy and PCR. One patient had a temperature above 37.5 °C was classified as late clinical failure, and the other patient with temperature below 37.5 °C was classified as late parasitological failure. The total (100%) of supervised patients exhibited adequate clinical and parasitological response and in summary in the 7 days scheme, two between 53 (3.7%) patients showed therapeutic failure. (Table 3).

Table 3 Therapeutic response in the two treatment groups

Therapeutic response in the 14-day treatment group (group II)

Among the patients who received unsupervised primaquine treatment, 5% (n = 1) had parasitaemia detected on day 28 and a temperature greater than 37.5 °C; the patient was classified as having late clinical failure. The total (100%) of supervised patients exhibited adequate clinical and parasitological response and in summary in the 14 days scheme, one between 50 (2%) patients showed therapeutic failure (Table 3).

Symptoms presented at inclusion and day 28 of follow-up

Patients with treatment for 7 days

At the baseline follow-up (day of enrolment), the median symptom onset in the supervised group was similar between supervised and unsupervised patients and all reported fever. By day 28 the patients in the supervised group reported no symptoms and the median Haemoglobin level was 13.0 mg/dL, in the unsupervised group 4 patients were symptomatic of the 4 patients who presented headache on day 28, two had parasitaemia; the median hemoglobin level was 13.2 mg/dL (Table 4).

Table 4 Symptoms on the day of inclusion, day 3 and day 28 of follow-up

Patients with treatment for 14 days

On day 28 of follow-up, patients in the supervised group did not report symptoms; however, in the unsupervised group, one of the patients who reported fever, headache, chills, sweating, musculoskeletal pain or anorexia was positive for P. vivax on day 28 of follow-up (Table 4).

Safety of anti-malarials

No serious adverse events were identified during the follow-up in relation to treatment, and no cases of complicated malaria occurred.

Discussion

The objective of this study was to assess the frequency of recrudescence in patients with uncomplicated P. vivax malaria treated with chloroquine and primaquine in the Amazonas Department. The study compared two primaquine regimens, administered over 7 or 14 days, both delivering a total dose of 210 mg.

The 2022 Colombian clinical practice guidelines for the diagnosis and treatment of malaria recommend the administration of chloroquine for 3 days concomitant with primaquine for 14 days (0.25 mg/kg/day) [6]; however, the administration of primaquine for 7 days (0.5 mg/kg/day) is allowed to improve adherence to this shorter regimen [7] and this scheme is adopted by the Health Secretariat of the Department of Amazonas.

Malaria caused by P. vivax presents significant challenges for control, particularly due to the potential failure to treat erythrocytic parasites causing recrudescence and the relapses originated from liver stages. Chloroquine is the first-line drug used to target the blood stage of the parasite; however, chloroquine resistance (CQR) has emerged as a major obstacle. This issue is especially concerning in South America, where treatment failures have been reported [15].

Recently, in Bolivia, of 96 patients with P. vivax infection who completed the 28-day follow-up, 10 (10.4%) had therapeutic failure on day 28, six had adequate blood concentrations (> 100 ng/m), and 6.5% chloroquine drug resistance was detected [16].

Given concerns about increasing resistance to chloroquine, alternative treatments have been evaluated. For instance, in Brazil, a randomized clinical trial assessed the efficacy of three treatment regimens over a 62-day period using one of two fixed-dose artemisinin-based combinations or chloroquine (600 mg on day 1 or 450 mg on days 2 and 3) combined with a short course of primaquine (7–9 days, with a total dose of 3–4.2 mg/kg). The cure rates across all three treatment arms exceeded 90% at 28 and 42 days. Specifically, for chloroquine, the cure rate was 100% on day 28 and 88% on day 63. This study demonstrated the efficacy and safety of all three regimens, with 42-day cure rates meeting the WHO criteria [17].

Another efficacy study in Brazil evaluated the effectiveness of a modified chloroquine formulation on day 28 in patients treated orally with 150 mg coated chloroquine tablets for 3 days (an initial dose of 450 mg, followed by two doses of 300 mg), alongside two 15 mg primaquine tablets administered for 7–9 days. Early parasitological clearance, assessed 72 h after treatment initiation, was evaluated in 84 patients. All patients achieved whole-blood chloroquine levels above 100 ng/mL by day 3, for a success rate of 100%. The cumulative success rate, or the probability of remaining parasite free on day 28, was 98.8% (95% CI 91.7–99.8) [18].

In Colombia, cases of chloroquine treatment failure have also been documented [19]. While CQR has been confirmed in Papua New Guinea, the Solomon Islands, Cambodia, South Sudan, Eritrea, and Vanuatu [20], concomitant treatment with chloroquine and primaquine has demonstrated lower parasitaemia rates by day 28 compared to chloroquine alone [21].

In a 2015 study conducted at the border between Brazil and French Guiana, a 99% adequate therapeutic response was observed in patients who received supervised treatment with chloroquine plus primaquine for 7 days [22]. This result is consistent with the findings of the present study. These findings contrast with those of a study conducted in the Brazilian Amazon, where a 5.2% therapeutic failure rate was recorded on day 28 of follow-up among 135 patients who were treated with chloroquine for 3 days in combination with primaquine for 7 days [23].

A study conducted in Loreto, Peru, in which chloroquine was administered for three days in combination with primaquine for either 7 or 14 days, with both groups receiving a total dose of 210 mg supervised, reported no cases of positive patients on day 28 of follow-up for any of the evaluated regimens. These findings are consistent with those reported for the two supervised treatment regimens in this study [24].

In Colombia's Amazonas Department in 2007 a study evaluated the efficacy of chloroquine plus primaquine for treating P. vivax related malaria. Twenty patients received a 3-day course of chloroquine (25 mg/kg total dose) combined with 14 days of primaquine (0.25 mg/kg/day). After 28 days of follow-up, 18 patients (90%) achieved an adequate treatment response, demonstrating the effectiveness of this combination therapy [10]. However, the lack of more recent studies in the Colombian Amazon, coupled with concerns about potential increases in treatment failures, highlighted the need for updated research. This study aimed to address this gap by evaluating treatment responses in areas with the highest number of reported malaria cases in the department.

In this study, the response to treatment was adequate in 100% of patients who received supervised treatment during 7 or 14 days; however, patients with unsupervised treatment presented therapeutic failures of 10.5% and 5.0%, respectively. Nonetheless, when supervised and unsupervised cases were combined, the therapeutic efficacy reached 97%.

A study conducted in Chocó, Colombia, showed adequate clinical and parasitological responses in 98.6% of patients who received supervised treatment with chloroquine plus primaquine for 14 days [25]. Similarly, a study conducted in Turbo, Antioquia, reported no positive cases on day 28 of follow-up with a supervised regimen of chloroquine plus primaquine for 14 days [26]. These results align with the findings of the present study.

Although this study does not allow for definitive conclusions regarding the efficacy of supervised versus unsupervised treatment, it suggests that treatment administration by a medical team and patient monitoring could reduce the likelihood of treatment failure, whether clinical or parasitological, by day 28. However, further research is necessary to accurately determine the effectiveness of different treatment regimens and administration methods.

Treatment outcomes in the unsupervised groups may have been influenced by reduced parasite susceptibility to chloroquine. The co-administration of primaquine could have had a synergistic effect on clearing asexual parasitaemia [27]. Parasitaemia detected on day 28 might suggest recrudescence, as the same species was identified on day 0, though this could not be genetically confirmed. Additionally, patients may have forgotten or stopped taking their medication, potentially affecting the treatment's efficacy.

A study in Afghanistan compared the relative therapeutic effectiveness of unsupervised chloroquine alone versus chloroquine plus primaquine in treating vivax malaria. The results showed that recurrences were less common with the combination therapy (hazard ratio 0.37, 95% CI 0.25–0.54), supporting the use of primaquine even in unsupervised settings [28]. Additionally, a review of primaquine therapy for malaria suggested that unsupervised primaquine regimens could be effective when accompanied by appropriate instructions [29].

However, the effectiveness of unsupervised primaquine treatment can be significantly lower in real-world settings compared to clinical trials. A hospital-based cohort study in southern Papua, Indonesia, found that unsupervised primaquine prescribed according to WHO guidelines was associated with only a minimal reduction in the risk of clinical recurrence within one year, highlighting the challenges of adherence to a regimen outside of controlled trial conditions [30].

This study revealed that the percentage of patients who were positive on day 3 was 20.6% in the 7-day supervised primaquine group and 10.0% in the 14-day supervised primaquine group. In the study conducted in Chocó, 5,5% of the patients were reported on day 3 of follow-up with supervised treatment with chloroquine plus primaquine for 14 days [25], a result similar to that reported in the corresponding treatment arm of the present study. However, none of the patients tested positive on day 3 were positive at 28 days, suggesting that a positive sample does not necessarily predict failure in the following weeks.

A decrease in parasitaemia occurred progressively during the first days of follow-up in the monitored groups; however, parasitaemia was still observed in 10 patients by day 3. This may suggest a reduced susceptibility to chloroquine, as a complete reduction in parasitaemia is typically expected at this point [7] but the final response could be masked by the synergistic effect of the PQ.

There was a greater frequency of infections in men than in women, which may be attributed to behaviors linked to economic or recreational activities that potentially increase men’s exposure to the bite of the vector. A study conducted in Panama reported similar results, with a higher frequency of infections in men (49/83; 59.0%) [31].

The symptoms observed in this study align closely with those noted in a study from Brazil, which found a high prevalence of symptoms like fever, headache, chills, and sweating at enrolment [23]. Likewise, in Colombia, the most commonly reported symptoms during enrolment included headache, musculoskeletal pain, fever, loss of appetite, chills, and sweating [32].

In Tarapacá and Puerto Nariño, all treatments were administered under supervision, largely because most patients reside in urban areas, which enabled recruitment, treatment administration, and follow-up at the local health centre. In Tarapacá this process was supported by institutional policies, as this health centre is one of the few that consistently offers supervised treatment to its patients. Concerning Puerto Nariño, health promoters from the Departmental Health Secretariat facilitated patient recruitment, which minimized issues related to providing supervised treatment and monitoring patients. However, the incidence of malaria during the study was low, less than 5 cases per week, affecting enrolment.

La Pedrera shows a predominance of malaria cases in remote communities, with fewer instances noted in urban centres. As a result, patient supervision and follow-up were limited due to geographical barriers. However, including this township in the study was crucial, as it has the highest number of reported cases in the department.

This study has significant limitations, notably, selection biases due to the nonrandomization of patients in the study groups, which may affect the generalizability of the findings to the broader population. Additionally, there were significant challenges in following patients due to several factors, such as residence in dispersed locations in jungle areas and long distances by rivers, as well as patient mobility and relocation to other places or communities, hindering continuous monitoring.

Glucose-6-phosphate dehydrogenase (G6PD) testing was not conducted before treatment assignment since it is not a standard procedure according to national care guidelines, and there was no officially endorsed test available at the time of the study. Furthermore, epidemiological records from the department and among the indigenous population did not indicate a prevalence of this deficiency or any adverse effects related to primaquine that would suggest a need for prioritizing this test under local conditions [33].

With respect to sample size, a convenience sampling approach was adopted. This decision was influenced by the fluctuating number of cases across different study sites, which varied depending on the time of year. In some locations, the case numbers were not particularly high, limiting enrolment. Furthermore, geographical and economic limitations significantly restricted the implementation of the project.

Despite these limitations, the results of this study provide important data for the department, warranting further investigation. These findings open the door to formulating new questions and hypotheses regarding the efficacy and effectiveness of anti-malarial treatments in the region. Future controlled studies with follow-up periods of 6 months to 1 year and search of resistance markers, such as pvmdr1, pvcrt, dhps and dhfr could be valuable for studying recurrences.

Conclusion

The current treatment protocol for P. vivax malaria in the Colombian Amazon involves a 7-day regimen of chloroquine combined with primaquine, whereas the rest of the country follows a 14-day regimen of chloroquine plus primaquine. This study assessed the therapeutic response to both treatment regimens and found that patients receiving supervised treatment with primaquine for either 7 or 14 days demonstrated an adequate therapeutic response, with no adverse or serious adverse events reported. Consequently, it was concluded that supervised treatment with either regimen is safe for addressing P. vivax malaria in the region.

However, it is essential to recognize that in the Amazonas department, treatment is generally not administered under supervision in any municipality, except for certain cases at the health centre in the non-municipalized area of Tarapacá. Thus, the results of this study highlight the importance of continuing to evaluate the effectiveness of standard unsupervised treatment in the department, particularly considering the therapeutic failure rates identified.