Study site and population

The study was conducted in Dandé and Gourcy, two rural areas located in the west and north of the country, respectively. Gourcy serves as a sentinel site for malaria surveillance, while Dandé is an agricultural area characterized by higher water availability. The study took place between October and December 2006, which corresponds to the peak malaria transmission season.

Study Design and Participants

We conducted a randomized clinical trial comparing Artesunate-Amodiaquine (AS-AQ) with Artemether-Lumefantrine (AL). During the study period, the research team was based in health centres located in the participating localities. All patients of both sexes who were residents of these areas met the inclusion criteria and provided informed consent, which made them eligible for enrollment.

Patients presenting with clinical features suggestive of uncomplicated malaria underwent a physical examination and parasitological confirmation using thick and thin blood smears for detection of asexual forms of Plasmodium falciparum. Individuals with positive test results were further assessed for eligibility based on the World Health Organization [ 13 ] criteria for uncomplicated P. falciparum malaria and subsequently enrolled into the study.

Inclusion criteria

Subjects with a confirmed parasitological diagnosis were then selected according to the following inclusion criteria:

Data Collection and Patient Follow-up

Study Procedures

Patients were followed at the clinic on days 1, 2, 3, 7, 14, 21, and 28, as well as on any additional day if they reported illness. At each visit, a complete clinical examination was performed to assess new symptoms or the progression of existing symptoms. Thick and thin blood smears were prepared to detect asexual forms of Plasmodium falciparum and gametocytes. For molecular analysis, two to three drops of blood were collected on Whatman filter paper at each visit, except on day 1. Haemoglobin concentration was measured at inclusion and at the final follow-up visit Table 1 .

Criteria for assessing treatment efficacy

Treatment efficacy was assessed according to the criteria contained in the [ 1 ] protocol as follows:

Early Treatment Failure (ETF)

PCR = Polymerase Chain Reaction; J0 = the first visit, before the first dose is given; J1 = first day; J2 = second day; J3= third day; J7 = seventh day; J14 = fourteenth day; J21 = twenty-first day; J28 = twenty-eight days.

Late Therapeutic Failure (LTF) is divided into two categories:

Late Clinical Failure (LCF):

Late Parasitological Failure (LPF)

Presence of parasitaemia on day 28 with an axillary temperature < 37.5˚C, in the absence of any criteria for LCF or LPF.

Adequate Clinical and Parasitological Response (ACPR)

Absence of parasitaemia on day 28 regardless of temperature without meeting the criteria for ETF, LCF or LPF.

Molecular Studies

The molecular study consisted of two major analyses:

1) Genetic polymorphism of msp1 and msp2 genes in Plasmodium falciparum

2) Genotyping of the Pfcrt K76T allele

Both were performed using Polymerase Chain Reaction (PCR), which involves four steps:

Extraction of Parasitic DNA

(Method adapted from Plowe et al., 1995, using Chelex-100)

Procedure:

DNA Amplification (Nested PCR)

Nested PCR Principle:

PCR Components

The reaction mixture was incubated in a thermocycler according to the program shown in Table 3 . For the second PCR series, the mixture was prepared with the CQR-A and CQR-B primers, to which 2 µl of the product from the first amplification was added. This mixture was then incubated in a thermocycler under the program specified in Table 3 below.

Pfcrt = Plasmodium falciparum chloroquine resistance transporter.

Digestion of DNA by the restriction enzyme ApoI (NEB)

Digestion is carried out at a temperature of 50˚C for 2 hours in a thermocycler or incubator. The Apo1 enzyme (NEB) will only cut the wild-type strain (HB3), leaving the mutant strain (Dde2) intact.

In order to digest the nested PCR product, 10 µL of a reaction solution with APO1 (NEB) was prepared Table 4 . To this reaction solution, we added 10 µL of the nested PCR amplification product. The entire mixture (20 µL) was placed in an Eppendorf microtube and incubated at 50˚C in a water bath. APO1 has optimal activity at 50˚C.

Amplification product disclosure

Electrophoresis:

Migration is performed on 2.5% agarose gel:

Interpretation of the photograph:

A good reaction is indicated by the presence of bands consistent with those expected. The size of the expected product and that of the positive controls must be checked for consistency. The Apo I enzyme (NEB) only cuts wild-type strains. Bands of the same size as the wild-type control (100 bp) correspond to strains sensitive to chloroquine.

On the other hand, those of the same size as the resistant control (134 bp) correspond to strains resistant to chloroquine. Bands that appear at both controls correspond to mixed strains.

The expected size for the amplification products is 537 bp for the first amplification and 134 bp for the second.

Determination of genetic polymorphism of msp1 and msp2

Reaction mixture and amplification programme for msp1 and msp2

For msp1 and msp2, the constituents (volume and concentration of reagents) are identical except for the sequences of the primer pairs.

The components of the reaction mixtures for the first and second PCRs of msp1 and msp2 are shown in Table 5 . The product of the first amplification serves as the DNA source for the second amplification.

dNTPs = Deoxyribonucleoside triphosphates.

During the first PCR, the primer pairs used were: 01/02 for msp1 and S3/S2 for msp2; during the second PCR, primers N1/N2 and S1/S4 were used for msp1 and msp2, respectively. The 25 µl, contained in a sterile microtube, are incubated in a thermocycler (Master cycler gradient) under a program specific to each gene Table 6 .

Interpretation of results

1) Experimental Setup Recap

MSP1, 2 = Merozoite Surface Protein 1, 2.

2) Gel Visualization

3) Interpretation of Banding Patterns

This method is commonly used in malaria genotyping (e.g., to distinguish recrudescence from reinfection).

Non-exhaustive example of gel interpretation

For a given patient, the status is determined based on the polymorphism of msp1 and msp2.

Data analysis

We used EXCEL 2003 software to enter the data. Statistical analysis was performed using Epi Info 6.04 and SPSS 11.0 software.

The chi-square test and Fisher’s exact test were used to compare proportions and measure the association between certain variables. A p-value of <0.05 was considered statistically significant.

Ethical considerations

Prior to the start of the study, this protocol was submitted to and approved by the institutional ethics committee of the MURAZ/IRSS center.

Patients were informed that their participation in the study was entirely voluntary and that the information collected by the team was strictly confidential. They were also allowed to withdraw from the study at any time if they wished.

Between October and December 2006, a total of 497 patients were examined at two sites. Of these, 286 tested positive for thick blood smears under microscopic examination, corresponding to a plasmodial index of 57.54%. A total of 167 patients were enrolled in the study and treated either with Artesunate-Amodiaquine (AQ/AS, n = 67) or artemether-lumefantrine (AL, n = 100). During follow-up, 60 patients were excluded for various reasons (including repeated vomiting, withdrawal of consent, and loss to follow-up): 20 from the AQ/AS group and 40 from the AL group Figure 1 .

During the follow-up, 60 patients were excluded for various reasons (repeated vomiting, withdrawal of consent, lost to follow-up…) including 20 for AQ/AS and 40 for AL ( Figure 1 ). In the Amodiaquine-Artesunate (AQ-AS) arm, 20 students were excluded for various reasons:

In the Artemether-Lumefantrine (AL) arm, 40 students were excluded for various reasons:

Characteristics of the study population

Among the 47 patients included, the majority (78.72%, 37/47) were aged between 6 and 59 months. The overall age range was 6 months to 30 years, with a mean age of 3.85 ± 5.21 years.

Female patients represented 55.32% (26/47) of the study population.

Temperature

At inclusion, patient temperatures ranged from 36.1˚C to 40.2˚C, with an overall mean of 38.2˚C ± 1.03˚C. Among children under 5 years of age, the mean temperature was 38.4˚C, compared to 37.7˚C in those over 5 years. The average temperature showed minimal variation between genders.

Parasite Density

The geometric mean parasite density at inclusion was 12,751.12 trophozoites/µL, ranging from 2000 to 110,068 trophozoites/µL. The mean parasite density was 12,617.3 trophozoites/µL in children over 5 years of age, compared with 12,086.8 trophozoites/µL in children under 5 years of age.

Hemoglobin level

The mean hemoglobin level was 9.10 ± 2.27 g/dL, ranging from 5 to 14.8 g/dL. In patients older than 5 years, the mean value was 11.38 g/dL, whereas in those younger than 5 years, it was 8.10 g/dL. Overall, 55.3% (26/47) of patients were anemic at inclusion.

Gametocyte density

Among the 47 patients who completed follow-up, only one presented with gametocytes on the day of inclusion, corresponding to a prevalence of 2.13% (1/47).

Clinical and Parasitological Results

Table 7 below presents the prevalence rates obtained after treatment with AQ/AS. Across all study sites, the average prevalence of treatment failure was 14.89% (95% CI: 6.2 - 28.3). No significant variation in treatment failure prevalence was observed between sites (p = 0.45).

ETF = Early Treatment Failure; LCF = Late Clinical Failure; LPF = Late Parasitological Failure; GTF = General Therapeutic Failure; ACPR = Adequate Clinical and Parasitological Response.

No cases of early treatment failure were observed. Regardless of the study site, all recorded failures were classified as late parasitological failures. The overall therapeutic efficacy was 85.11%, with site-specific rates of 85.37% in Gourcy and 83.33% in Dandé.

Given the late onset of these failures, an analysis of parasite polymorphism will be necessary to differentiate between recrudescences and new infections. This will provide a more accurate estimate of the true treatment failure rate.

Clinical and parasitological results by site after PCR adjustment

The prevalence of treatment failure due to relapse was 12.8% (95% CI: 8.1 - 17.5) in the study population. Moreover, no significant influence of the study site on the prevalence of treatment failure was observed Table 8 .

The polymorphism analysis of msp-1 and msp-2 revealed that six (6) of the seven (7) PCR-positive clinical cases were due to recrudescent parasites, while only one (1) case, detected at the Gourcy site, represented a new infection. Thus, among all treatment failures, 14.3% were reinfections and 85.7% were recurrences, a statistically significant difference (p = 0.03).

When stratified by age, four (4) of the six (6) recurrences occurred in children under 5 years of age, compared to two (2) cases in children aged 5 years and above. This difference was not statistically significant (P = 0.56).

Prevalence of the Pfcrt-K76T allele

The prevalence of the Pfcrt 76T mutation prior to AS/AQ therapy was 48.9% (95% CI: 34.6 - 63.2) in the study population. By site, prevalence was 48.8% in Gourcy and 50% in Dandé, with no significant difference between the two locations (P = 0.70). Similarly, prevalence did not vary significantly according to age or sex (P = 0.94) ( Table 9 ).

***Due to resurgence.

Prevalence of mutations in subjects who failed treatment

To assess whether the presence of the Pfcrt 76T mutation prior to treatment was associated with failure of the AS/AQ combination, we compared the prevalence of the mutant allele between cases of treatment failure and RCPA cases. Among the six recrudescent isolates, four (66.7%) carried the Pfcrt 76T mutation, whereas 19 of 41 (46.3%) RCPA isolates also harbored this mutation. Statistical analysis showed no significant association between the presence of the Pfcrt 76T allele and treatment failure due to parasite recrudescence (P = 0.94).