Malaria accounted for an estimated 246 million cases and 569,000 deaths globally in 2023. ¹ The African region contributed 95% of malaria cases and 96% of malaria-related deaths, with children under five years of age (U5) being disproportionately affected. ¹ Effective treatment and prevention of Plasmodium falciparum infections with antimalarial drugs are critical for malaria control and elimination. To treat P. falciparum malaria, the WHO recommends six artemisinin-based combination therapies (ACTs): artemether-lumefantrine (AL), artesunate-amodiaquine (ASAQ), artesunate-mefloquine (ASMQ), dihydroartemisinin-piperaquine (DP), artesunate-sulfadoxine/pyrimethamine (ASSP), and artesunate-pyronaridine (AP). ²

Chemoprevention is a key strategy for malaria control in areas with stable transmission. The WHO recommends three chemoprevention approaches involving sulfadoxine-pyrimethamine (SP) administered at curative doses, irrespective of infection status: intermittent preventive treatment in pregnancy (IPTp), seasonal malaria chemoprevention (SMC), and intermittent preventive treatment in infants (IPTi), now referred to as Perennial Malaria Chemoprevention (PMC). ² SP remains the preferred drug for IPTp, PMC, and SMC (the latter co-packaged with amodiaquine) due to its safety, tolerability, and cost-effectiveness in reducing malaria-related morbidity and neonatal mortality. ^{3, 4} Experiences of SP resistance in Southeast Asia regularly raised concerns about its use for malaria prevention in Africa. ⁵

Pyrimethamine and other antifolates target P. falciparum dihydrofolate reductase (DHFR), while sulfadoxine and other sulfonamides act on dihydropteroate synthase (DHPS). Resistance arises from single nucleotide polymorphisms (SNPs) in the dhfr and dhps genes. Mutations in dhfr at codons 51, 59, 108, and 164 confer resistance to pyrimethamine, while SNPs in dhps at codons 437, 540, 581, and 613 drive resistance to sulfadoxine. ^{6– 8} These mutations typically accumulate stepwise, leading to stronger drug resistance and the dhfr/dhps quintuple mutant (51I, 59R, 108N + 437G, 540E) has been associated with SP treatment failure. ^{9, 10} On the other hand, a pooled study suggested that in areas where the sextuple mutant (quintuple + dhps 581G) is present, the effectiveness of IPTp with SP may decline when the prevalence of this mutation exceeds 10%. ¹¹

Currently, validated resistance markers for other antimalarial drugs include some single nucleotide polymorphisms (SNPs) in the kelch 13 gene, providing partial resistance to artemisinin, ¹² and the K76T in the pfcrt gene ( P. falciparum chloroquine-CQ- resistance transporter) regarding chloroquine. ¹³

In Sierra Leone, malaria is endemic with perennial transmission and seasonal peaks occurring from May to October. Over 90% of cases are caused by P. falciparum, with pregnant women and U5 being the most vulnerable to the infection. ¹⁴ Malaria prevalence in U5 measured by rapid diagnostic tests (RDTs), declined from 40% in 2016 to 22% in 2021. ^{15, 16} Malaria-attributed mortality has declined significantly since 2000 in Sierra Leone, driven by free diagnostic testing, widespread deployment of insecticide-treated bed nets, and malaria chemoprevention programs. Nationwide IPTp has been implemented in antenatal clinics since 2014 and PMC has been integrated into the Expanded Program on Immunization (EPI) alongside immunization contacts at 10 weeks, 14 weeks, and 9 months, since 2018. Chloroquine, introduced as the first-line treatment for uncomplicated malaria in the 1940s, was replaced in 2004 with artemether-lumefantrine. ¹⁷

The main objective of this study is to report on the prevalence of molecular markers associated with SP resistance in Sierra Leone seven and three years after the nationwide implementation of IPTp and PMC, respectively. As a secondary objective, we also assessed the prevalence of molecular markers associated with resistance to chloroquine and artemisinin. ¹⁷

The study was conducted in Sierra Leone seven and three years after the nationwide implementation of IPTp and PMC, respectively. Since 2015, artemisinin-lumefantrine (AL) is the first-line treatment for uncomplicated malaria in the country. The latest national Malaria Indicator Survey and a household survey from another study, both conducted in 2021, reported that more than half of pregnant women received at least three doses of IPTp, and nearly 60% of infants had received three PMC doses. ^{16, 30} Despite the wide implementation of these SP-based strategies the findings from this study show a low prevalence (4.6%) of the pfdhfr/pfdhps quintuple mutant and no presence of the sextuple mutant.

The prevalence of pfdhfr triple mutations was nearly fixed (99.5%), consistent with country reports from 2016 and 2018. ^{31, 32} Nevertheless, the prevalence of the pfdhps/pfdhfr quintuple mutant was found at a lower prevalence (4.6%) in the current study as compared to the prevalence observed (10%) in the previous survey in 2016, with no data on quintuple mutant reported in 2018. ³² The low prevalence of the quintuple mutant in Sierra Leone aligns with rates reported in West Africa, like 15.4% in Guinea (2013-2016), ³³ 0% in Senegal (2010) ³⁴ and 1.6% in Mali (2012). ³⁵

The low prevalence of the quintuple mutant and the absence of the sextuple mutant in this study support the continued use of SP for chemoprevention in young children and pregnant women in Sierra Leone. Although the presence of SP resistance markers alone does not determine drug efficacy, especially when used for prevention, sustained drug pressure may drive these mutations toward saturation. ³⁶ Therefore, regular monitoring of molecular resistance markers is important to understand how their prevalence affects drug effectiveness and to tailor malaria prevention programs.

In this study, we also assessed the prevalence of molecular markers associated with chloroquine and artemisinin resistance in the country. Mutations in the pfcrt gene at codon 72-76 have been associated with P. falciparum tolerance to chloroquine and other 4-aminoquinolines such as amodiaquine and piperaquine. The pfcrt prevalence reported in this study is higher than the 22% observed in 2018 in a Southern district in the country, though the latter was based on a sample size of only 95 participants. ³⁷ The pfcrt prevalence varies across West Africa, ranging from the highest in Liberia (87.9%, 2018), ³⁸ a moderate level in the Democratic Republic of Congo (22.7%, 2019), ³⁹ to low in Equatorial Guinea (2.8%, 2019) ⁴⁰ and Togo (0.6%, 2021). ⁴¹ Mutations in the pfmdr1 gene also modulate parasite susceptibility to several ACT partner drugs (e.g. lumefantrine, mefloquine, and piperaquine). The predominant pfmdr1 NFSND haplotype detected in Sierra Leone is consistent with trends observed across West African countries that adopted artemether-lumefantrine (AL) and discontinued chloroquine as first-line treatment. ^{41– 45} pfmdr1 mutations exhibit pleiotropic effects, with N86Y driving resistance to chloroquine and amodiaquine while sensitizing parasites to lumefantrine, mefloquine, and artemisinin derivatives. ^{46– 49} These findings underscore the importance of molecular surveillance to optimize ACT use in different regions.

Several mutations in the pfK13 gene are associated with artemisinin resistance. ¹² However, none were detected in this study, indicating no molecular evidence of compromised efficacy of artemisinin and its derivatives in Sierra Leone. Nonetheless, the emergence of such mutations has been recently reported in Rwanda, ⁵⁰ Uganda, ⁵¹ and the Democratic Republic of Congo, ⁵² mirroring the early stages of resistance observed in Southeast Asia. In that region, P. falciparum developed partial resistance to artemisinin-based combination therapies (ACT), leading to treatment failures and the spread of resistance strains. ⁵ To prevent a similar trend in Africa, regular surveillance and monitoring of artemisinin resistance are crucial.

Nonetheless, the presence of resistance markers alone does not explain the reduced efficacy of antimalarial drugs. A meta-analysis of seven clinical trials concluded that IPTp with SP reduces placental malaria, low birthweight, and anemia in pregnant women, even in areas with SP treatment failure in children. ¹¹ Factors such as patient adherence, metabolism, host immunity, and nutritional status also influence drug efficacy. These factors vary by individual, population, and region, requiring careful interpretation of molecular resistance markers and their impact on chemoprevention. ⁵

This study has some strengths and limitations. A key strength is its geographical coverage, spanning three districts in Sierra Leone’s Northern Province, making it more representative than previous surveys in 2016 and 2018, which were limited to the Kambia or Bo districts. ^{31, 32, 37}

The study was limited by the fact that 20.5% (61/298) of blood samples from RDT-confirmed participants tested negative for P. falciparum by qPCR. These potential false positives may be due to the residual presence of HRP2 antigens in the bloodstream, which can persist in the blood for up to 28 days after infection clearance. ⁵³ In addition, of the 61 participants with negative qPCR results, 28 reported having taken an antimalarial drug within the past month, which would be in line with the false-positive RDT results and better agrees with previously observed false positive rates of 10% with this RDT test. ⁵⁴ Another explanation is a potential DNA degradation during sample preservation.

In 2022, the WHO recommended additional PMC-SP doses to extend protection beyond the first year of life. ⁵⁵ The current findings showing a low prevalence of the pfdhfr/pfdhps quintuple mutant and the absence of the sextuple mutant support the continued use of SP for IPTp and PMC, as well as the expansion of PMC with additional SP doses administered in the second year of life in Sierra Leone.