Investigating selected host and parasite factors potentially impacting upon seasonal malaria chemoprevention in Bama, Burkina Faso

In Burkina Faso, malaria is still the leading cause of morbidity and mortality with 7875,575 cases and 12,725 deaths in 2018 [1]. With these statistics, Burkina Faso has one of the highest malaria incidence rates in the world, and is one ‘high burden to high impact’ countries of the World Health Organization (WHO). In Burkina, malaria is highly seasonal with peak transmission occurring during the rainy season (June-October). Since 2005, the country has adopted several malaria control strategies to reduce the burden of the disease, including the provision of artemisinin-based combination therapy (ACT) and distribution of insecticide-treated mosquito nets (ITN) and long-lasting insecticidal nets (LLINs). In 2014, the Burkinabe National Malaria Control Programme introduced seasonal malaria chemoprevention (SMC) with amodiaquine–sulfadoxine–pyrimethamine (AQ–SP). This strategy targets children aged 3–59 months, excluding those with known allergies to sulfonamides or AQ, those who received a dose of AQ or SP within the past month, those with known HIV-positive status and under cotrimoxazole treatment, and those severely ill or experiencing a presumptive malaria episode [2].

A concern with the implementation of SMC is the absence of malaria infection screening using available diagnostic methods before the treatment of eligible children. Studies have demonstrated that in malaria endemic countries, a large proportion of malaria infections are asymptomatic [3, 4]. While guidelines do not currently recommend the treatment of asymptomatic parasitaemia with artemisinin-based combinations as is recommended for symptomatic cases, the lack of screening for malaria infection during SMC results in some number of individuals receiving AQ–SP as “treatment” for asymptomatic malaria [1]. Moreover, while the efficacy of AQ–SP as a chemoprevention has been well studied, the efficacy of AQ–SP as treatment in most of these settings has not been well characterized since the roll-out of SMC [5‐7].

Therapeutic drug concentrations are influenced by pharmacokinetics, host immunity, and parasite susceptibility to the drug. Amodiaquine is metabolized to its primary active metabolite N-desethylamodiaquine (DEAQ) by the cytochrome P450 enzyme (CYP) 2C8. Two of the more common CYP2C8 genetic variants have been described to have altered drug metabolism, including the impaired conversion of amodiaquine to DEAQ; CYP2C8*2, which is more prevalent in people of African descent, and CYP2C8*3, which is more prevalent in Caucasians [8]. The role of genetic variation on the metabolism of anti-malarial drugs—known as pharmacogenomics—is not well understood, but CYP2C8 variations may be partly responsible for the wide interindividual variability in plasma levels of DEAQ, possibly contributing to inconsistencies in the efficacy and safety of AQ in the region.

Drug resistance to amodiaquine is primarily mediated by a single nucleotide polymorphism (SNP) that results in a K76T substitution in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene. Resistance is augmented by polymorphisms in another transporter gene, pfmdr1. Before SMC implementation in Burkina Faso, AQ resistance mediating polymorphisms have been reported in several studies [5, 9, 10]. Selection of these drug resistance-associated SNPs has been reported after several months of SMC administration in Burkina Faso [11]. Intriguingly, artemether–lumefantrine—the most widely used first-line ACT in Burkina Faso—is known to exert an opposing selective pressure in both pfcrt and pfmdr1 towards wild-type SNPs [12]. Currently, it is unknown which selective pressure predominates, but transmission of these drug resistance polymorphisms is likely to be impacted by the prevalence of asymptomatic infections and treatment with AQ–SP.

The aim of this study is to report the prevalence of microscopic and submicroscopic malaria infections before and after the first round of SMC in children under five in Bama, Burkina Faso. This study also provides a more comprehensive view of the host and parasite factors that may influence SMC, specifically day 7 plasma concentrations of DEAQ, the presence of CYP2C8 genetic variants, and the prevalence of parasite drug resistance-mediating polymorphisms.

Blood samples were collected from 106 children at the start of SMC by randomly selecting one child under 5 years in each enrolled household. In the second survey, the same households were revisited and blood samples were collected from 93 children; 31 were absent from their households for three or more visits and thereby excluded. The ages of children ranged between 6 and 48 months old, with an average of 29 months. The ratio of male to female children were comparable at both time points (0.93 to 1.1).

The present study assessed the prevalence of malaria infection by microscopy and PCR in a cohort of children under 5 years old after the first round of SMC with AQ–SP in Bama, Burkina Faso. The results of this study demonstrated a relatively low but non-negligible parasitaemia in SMC eligible children after drug administration. These results further support that the majority of malaria infections are sub-microscopic [17‐21]. All microscopy-positive samples were also PCR-positive and there was no discrepancy between both techniques in identifying parasite species. These results further demonstrate that a significant proportion of SMC eligible children were infected with malaria at the time they received SMC for prevention, but more notably, nearly 15% of the children were re-infected following insufficient protection from SMC, with a single case positive for malaria at both time points. This proportion of infected children following SMC represents a failure of chemoprevention and demonstrates the limitation of SMC. These failures may be explained by the high transmission intensity, drug factors that limit the duration of prophylaxis following administration. A limitation to this analysis is that approximately 30% of the samples were collected within 2 days after administration of the first dose of SMC. Although infected children are not expected to have cleared all parasites within 48 h of drug administration, this factor may have reduced the prevalence by microscopy and possibly PCR.

To probe the potential drug-related impacts, day 7 DEAQ levels were further assessed in children after SMC administration. The median day 7 DEAQ concentration in the study cohort is consistent with previous PK studies of amodiaquine and DEAQ [22, 23]. Though the influence of CYP2C8 polymorphisms on amodiaquine metabolism has been described in vitro, data on the impact of these polymorphisms in vivo is lacking. Of interest in the context of SMC, is the potential for increased toxicity associated with impaired amodiaquine metabolism; however, there has been no clear association between the CYP2C8*2 allele and amodiaquine toxicity or efficacy. In this study population CYP2C8*2 variant was found to be relatively common with a prevalence of 17.1%, comparable to the published allelic frequencies of other studies from West Africa [8, 15, 24]. To explore the potential impact of the CYP2C8*2 variant on amodiaquine metabolism, day 7 DEAQ concentrations were compared between children with homozygous wild-type, heterozygous, and homozygous CYP2C8*2 mutant alleles. While a trend towards a decrease in DEAQ concentrations could be observed from wild-type to heterozygous alleles, this decrease was not statistically significant. Children homozygous for the CYP2C8*2 allele had the lowest concentrations of DEAQ in this study but the small number of homozygous CYP2C8*2 children (n = 3) precluded any meaningful analysis. It is possible that a single wild-type and mutant allele provides enough metabolic turnover of amodiaquine to maintain therapeutic ranges of DEAQ. Caution should be used to interpret these results; given the sample size in this study of 110 children and a predicted 3:1 ratio of wild-type to CYP2C8*2 heterozygous children, the study had 80% power to detect a 33.3% decrease in DEAQ levels. It is important to note that this was an “effectiveness” study, and consistent with national SMC guidelines, only the first dose of amodiaquine was directly observed. There are supervisors in each village that perform household visits to improve programme implementation and treatment adherence, but this cannot guarantee compliance. Caregiver delay or omission of subsequent doses would impact drug concentration–time profiles, which would complicate interpretation of day 7 DEAQ levels. Whether or not homozygous/heterozygous CYP2C8*2 children are at increased risk of amodiaquine toxicity or reduced efficacy warrants consideration for further study.

As reported in other studies [11, 25, 26], a major concern with the use of SMC is an increase in the proportion of parasites carrying AQ and SP resistance-mediating polymorphisms, jeopardizing the future of malaria prevention and control. However, it is not yet well established whether the prophylactic failure of SMC is associated with emergence of resistance. Genotyping for putative AQ resistance-associated markers revealed that there was a large proportion of mixed infections during the first survey, present in more than half of genotyped samples. While the second survey showed a much higher proportion of pfcrt 76T mutations, associated with reduced amodiaquine sensitivity, less than half of the PCR-positive samples could be genotyped at this time point by our methods. Genotyping failure is likely attributed to the low-density of these infections, reflected in the limited number of these detected by microscopy, and also in the lack of successful SP-associated mutation typing. In contrast to pfcrt polymorphisms, there was limited genetic diversity at the pfmdr1 N86Y loci, with all but one sample positive for the N86 wild-type SNP. As noted earlier, lumefantrine selects toward the wild-type N86, in opposition to the direction of selection for amodiaquine. Careful monitoring of both AQ and SP-resistance associated polymorphisms and of possible prophylactic failures of SMC should be ensured in order to understand the potential population-level impacts of the opposing selection pressure by the first-line treatment, artemether–lumefantrine, and the widespread SMC regimen, AQ–SP. If possible, future studies should encompass multiple rounds of SMC, with testing at baseline, throughout, and after SMC to assess for the selection of resistance. Finally, more in-depth pharmacokinetic studies with detailed drug resistance typing may be required to understand the relationship between AQ–SP exposure and the development of resistance.

This study estimated the prevalence of malaria infection in children at two time points after treatment with the first round of seasonal chemoprophylactic AQ–SP, demonstrating a notable prevalence of low-level malaria parasitaemia in children 3 weeks after the first round of SMC. Day 7 concentrations of the active DEAQ metabolite varied widely, likely reflecting variability in adherence and possibly metabolism. The findings of this study highlight factors that may contribute to the effectiveness of SMC in children in a high transmission season.