Investigating host and parasite factors surrounding seasonal malaria chemoprevention in Bama, Burkina Faso

Background: Since 2014, seasonal malaria chemoprevention (SMC) with amodiaquine-sulfadoxine-pyrimethamine (AQ-SP) has been implemented on a large scale during the high malaria transmission season in Burkina Faso. We report in this paper the prevalence of microscopic and submicroscopic malaria infection at the outset and after the first round of SMC in children under five years old in Bama, Burkina Faso, as well as host and parasite factors involved in mediating the efficacy and tolerability of SMC. Methods: Two sequential cross-sectional surveys were carried out in the first month of SMC in a rural area in southwest Burkina Faso. Blood smears and dried blood spots were collected from 106 and 93 children under five, respectively, at the start of SMC and again three weeks later. Malaria infection was detected by microscopy and by PCR from dried blood spots. For all children, day 7 plasma concentrations of desethyl-amodiaquine (DEAQ) were measured and CYP2C8 genetic variants influencing AQ metabolism were genotyped. Samples were additionally genotyped for pfcrt K76T and pfmdr1 N86Y, molecular markers associated with reduced amodiaquine susceptibility. Results: 2.8% (3/106) of children were positive for Plasmodium falciparum infection by microscopy and 13.2% (14/106) by nested PCR within 2 days of SMC administration. Three weeks after SMC administration, in the same households, 4.3% (4/93) of samples were positive by microscopy and 14.0% (13/93) by PCR (p=0.0007) . CYP2C8*2, associated with impaired amodiaquine metabolism, was common with an allelic frequency of 17.1% (95%CI=10.0-24.2). Day 7 concentration of DEAQ ranged from 0.48 to 362.80 ng/mL with a median concentration of 56.34 ng/mL. Pfmdr1 N86 predominated at both time points, whilst a non-significant trend towards a higher prevalence of pfcrt 76T was seen at week 3. Conclusion: This study showed a moderate prevalence of low-level malaria parasitemia in children 3 weeks following SMC during the first month of administration. Day 7 concentrations of the active DEAQ metabolite varied widely, likely reflecting variability in adherence and possibly metabolism. Our findings highlight factors that may contribute to the effectiveness of SMC in children in a high transmission setting. opposing selection pressure by the first-line treatment, artemether-lumefantrine, widespread regimen, 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 parasitemia in children three 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. Our findings highlight factors that may contribute to the effectiveness of SMC in children in a high transmission season.


Background
In Burkina Faso, malaria is still the leading cause of morbidity and mortality with 7,875,575 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 of the World Health Organization's "high burden to high impact" countries. 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 combinations treatments (ACTs) 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 parasitemia with artemisinin-based combination therapies (ACTs), 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][6][7].
Therapeutic drug concentrations are influenced by pharmacokinetics, host immunity, and parasite susceptibility to the drug. Amodiaquine is metabolized to its primary active metabolite Ndesethylamodiaquine (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 antimalarial 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. Selection of drug resistance-associated SNPs has been reported after several months of SMC administration in Burkina Faso. [9] 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 [10]. 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. We also provide 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.

Study Site And Population
Samples for the present study were collected in Bama, Burkina Faso, a rural area located in the district of Dandé in the Kou Valley. Bama is a rice-growing area of 1200 hectares located 30 km from Bobo-Dioulasso in the southwest of Burkina Faso. The rainy season in this area extends from June to October and the dry season from November to May. The Kou Valley is a permanent source of irrigation water with two rice crops per year from July to November and from January to May. Malaria transmission is perennial with a peak during the rainy season when the density of An. gambiae is very high with an annual entomological inoculation rate of up to 200 infective bites/person/night [11]. The

Participant Selection And Inclusion
Two weeks' prior the delivery of the first round of SMC, 124 randomly selected households were visited by the study team. EPI-style random walk method was utilized to randomly select households with children under 5 years of age in a two-step process which involved first selecting a starting point and secondly selecting households from that point onward. The EPI method was appropriate due to lack of local census data and boundary maps. Only one child was selected per household. Households with children aged 3 to 59 months were selected to participate in the study after the parents/guardians gave informed consent. Each house was visited three times thereafter. For the first malaria prevalence survey, conducted in late July 2017, within the period of distribution of the first round of SMC. CHWs were accompanied and the study team collected blood smears and dried blood spots (DBS) by finger-prick. Houses were visited +/-2 days of the 1st day of SMC administration. The team returned to collect blood samples for pharmacokinetic analyses (see Quantification of DEAQ below) 7-8 days post-SMC administration. For the second malaria prevalence survey in late August 2017, one week before the second round of SMC, the study team visited the same households and collected smears and DBS from the same children, if available for resampling.

Preparation Of Blood Smears And Microscopy Examination
Thick and thin blood films obtained from children by finger prick were air dried, stained in Giemsa 2% for 30 min, and examined by light microscopy fitted with 100x oil immersion lens in the laboratory of IRSS. A smear was considered negative after examination of at least 100 fields.

Dna Extraction And Polymerase Chain Reaction (pcr)
Parasite DNA was extracted from DBS using chelex-100. Plasmodium species were subsequently detected by nested PCR as previously described [12]. Species were determined by amplification of 18 s RNA using nested PCR with secondary primers specific to the species Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax. For quality control, a template free control was used in all reactions and genomic DNA from laboratory strains (www.beiressource.com) were used as positive control for respective species. All positive samples by microscopy and negative by PCR were reanalyzed by PCR before confirmation of the microscopy results.

Quantification Of Desethylamodiaquine (deaq)
A total of 200 µL of capillary whole blood was collected in EDTA-containing microtubes on day 7.
Samples were centrifuged at 2000 × g for 10 minutes and the plasma was then transferred to cryovials and stored at -80 °C. DEAQ plasma concentrations were quantified by reverse phase liquid chromatography and detection with an AB Sciex API 5000 tandem mass spectrometer (LC-MS-MS) in turbo-ion spray-positive mode by NorthEast BioLab (Hamden, CT). 2 H 5 N-Desethylamodiaquine was used as an internal standard and the total assay coefficient of variation were < 7%. The lower limit of 8 quantification (LLOQ) of DEAQ was 0.34 ng/mL.

Cyp2c8 Genotyping
Genotyping for two nonsynonymous CYP2C8 variants was performed on DNA extracted from DBS using the QIAamp DNA Mini Kit (Qiagen). CYP2C8*2 (805A > T) and CYP2C8*3 (416G > A and 1196A > G) alleles were determined using a TaqMan Drug Metabolism Genotyping Assay (assay ID C_30634034_10 and C_25625794_10, respectively). PCR was performed in a 25 µL reaction with 12.5 µL of TaqMan Universal PCR Master Mix, 1.25 µL of the drug Metabolism Genotyping Assay Mix, and at least 3 ng of DNA template, per the manufacturer's instructions. Quality control was maintained with no template controls with every reaction, along with previously determined wild type, heterozygous, or homozygous CYP2C8*2 or *3 allele positive controls (from K. Kidd Lab, Yale University) [13].

Genotyping Of Drug Resistance Markers
Genotyping was performed on parasite DNA extracted from DBS using the QIAamp DNA Mini Kit (Qiagen). Nested PCR was performed to amplify amodiaquine resistance-associated alleles for pfcrt and pfmdr1. Amplicons were used for a ligase detection reaction (LDR) which contained an allelespecific and conserved sequence primer [14]. Allele-specific primers contained a 5' nucleotide sequence unique to a MagPlex bead tagged with a complementary sequence. The 3' end of the allelespecific primer corresponded to a particular drug resistance polymorphism: pfcrt K76T or pfmdr1 N86Y. Conserved sequence primers were modified by 5' phosphorylation and 3' biotinylation. After primer ligation, LDR reaction products were hybridized to their respective MagPlex beads in 1.5 TMAC buffer (3 M tetramethylammonium chloride, 50 mM Tris-HCl pH 8, 3 mM EDTA, and 0.1% Nlauroylsarcosine sodium). Fluorescent labeling was performed with a 1:50 dilution of streptavidin-Rphycoerythrin. Drug resistance polymorphisms were determined by measuring fluorescent intensity using xPonent software (Luminex) on a Bio-Plex 200 instrument (Bio-Rad). Genotyping was attempted for SP-associated mutations, but due to a low success rate, data is not included.

Statistical analysis
Data were collected with Excel and analyzed by R version 3.4.0 (2017-04-21). Chi-square was used to compare proportions and a p value of < 0.05 was considered statistically significant.

Results
We collected samples from 106 children at the start of SMC by randomly selecting one child under five years in each enrolled household. During the second survey, 31 of the original households were unoccupied on the day of visit, leaving 93 children for sampling. 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).

Malaria Prevalence In First Survey
DBS were collected one day before (16.7%) or on the day of (54.6%) SMC, while 28.7% were collected within two days after SMC due to field logistics. Results of Plasmodium infection detected by both microscopy and PCR are summarized in Table 1 (Fig. 1).
With respect to genotype, 28.7% (31/108) were heterozygous and only 2.8% (3/108) were homozygous for the CYP2C8*2 allele. Genotyping for CYP2C8*3 variants was successful in 97.3% (107/110) of the samples; however, the CYP2C8*3 allele was not detected in any of the children genotyped. Results of the genotyping are summarized in Table 3.
We further assessed any relationship between DEAQ drug levels and CYP2C8*2 genotype, however there was no statistically significant difference in DEAQ concentration between those with homozygous wild-type alleles (median 65.93 ng/mL, IQR 30.31-101.22 ng/mL) or heterozygous CYP2C8*2 alleles (median 53.64 ng/mL, IQR 42.54-72.82 ng/mL) (P = 0.5216, Mann-Whitney U test).  To probe the potential drug-related impacts, we further determined day 7 DEAQ levels in children after SMC administration. The median day 7 DEAQ concentration in our cohort is consistent with previous PK studies of amodiaquine and DEAQ [20,21]. 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 our study population we found that the CYP2C8*2 variant was relatively common with a prevalence of 17.1%, comparable to the published allelic frequencies of other studies from West Africa [8,13,22]. To explore the potential impact of the CYP2C8*2 variant on amodiaquine metabolism, we compared day 7 DEAQ concentrations in children with homozygous wild-type, heterozygous, and homozygous CYP2C8*2 mutant alleles. While we observed a trend towards a decrease in DEAQ concentrations 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 our 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. It is important to note that this was an "effectiveness" study, and only the first dose of amodiaquine was directly observed. 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 [9,23,24], 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 also 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

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Not applicable Figure 1 Scatterplot of Day 7 DEAQ results.