Malaria drug resistance landscape in the Democratic Republic of the Congo: a spatial mapping systematic review of molecular surveillance surveys

doi:10.21203/rs.3.rs-3247384/v1

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Systematic Review

Malaria drug resistance landscape in the Democratic Republic of the Congo: a spatial mapping systematic review of molecular surveillance surveys

https://doi.org/10.21203/rs.3.rs-3247384/v1

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Context: The Democratic Republic of Congo (DRC), one of the most malaria-affected countries worldwide, is a potential hub for global drug-resistant malaria. This study aimed at summarizing and mapping surveillance surveys of malaria parasites carrying molecular markers of drug-resistance across the country.

Methods: A systematic mapping review was carried out before July 2023 by searching for relevant articles through seven databases (PubMed, Embase, Scopus, African Journal Online, African Index Medicus, Bioline and Web of Science).

Results: We identified 1541 primary studies of which 29 fulfilled inclusion criteria and provided information related to 6385 Plasmodium falciparumclinical isolates (collected from 2000 to 2020). We noted the PfCRT K76T mutation encoding for chloroquine-resistance in median 32.1% [interquartile interval, IQR: 45.2] of analyzed malaria parasites. The proportion of parasites carrying this mutation decreased overtime but wide geographic variations persisted. A single isolate had encoded the PfK13 R561H substitution that is invoked in artemisinin-resistance emergence in the Great Lakes region of Africa. Parasites carrying various mutations linked to resistance to the sulfadoxine-pyrimethamine combination were widespread and reflected a moderate resistance profile (PfDHPS A437G: 99.5% [IQR: 3.9]; PfDHPS K540E: 38.9% [IQR: 47.7]) with median 13.1% [IQR: 10.3] of them being quintuple IRN-GE mutants (i.e., parasites carrying the PfDHFR N51I-C59R-S108Nand PfDHPS A437G-K540E mutations). These quintuple mutants tended to prevail in eastern regions of the country. Among circulating parasites, we did not record any parasites harboring mutations related to mefloquine-resistance, but we could suspect those with decreased susceptibility to quinine, amodiaquine, and lumefantrine based on corresponding molecular surrogates.

Conclusion: Drug resistance poses a serious threat to existing malaria therapies and chemoprevention options in the DRC. This review provides a baseline for monitoring public health efforts as well as evidences for decision-making in support of national malaria policies and for implementing regionally tailored control measures across the country.

Parasitology

Democratic Republic of Congo

Malaria

Antimalarial drug resistance

Mutations

Molecular markers of drug resistance

The Democratic Republic of Congo (DRC) has always been highly endemic for Plasmodium falciparum malaria [1, 2]. Until the middle of the 20th century, quinine – i.e., the first drug used for malaria treatment and prophylaxis, was not supplied by extensive programs due to reduced availability and high cost of its importation from South-East Asia [1–3]. World War II led to a major shortage of quinine worldwide and led the colonial authorities to start producing the drug locally and to introduce into the country some synthetic antimalarial products newly developed, namely chloroquine and pyrimethamine [1, 2, 4]. Due to its low-cost and high efficacy at the time, chloroquine quickly became a leading antimalarial drug enabling large-scale distribution programs through a few urban and industrial cities that existed in the country in 1940s-1950s [1]. However, the efforts initiated by the colonial administration to fight against malaria were prematurely interrupted following the accession to independence of the country in 1960, which led to the hasty departure of the colonial health officials and the rapid dismantling of the Congolese health system hitherto under construction [3, 5]. Malaria control activities were revived later in 1971 at the scale of Kinshasa, the country's capital, owing to the "Programme de Lutte Antipaludique" (PLA) created with support from the United States Agency for International Development (USAID) [5]. Nationwide control activities could be launched in the 1980s when the "Projet de Lutte Antipaludique" (PLAP) created in 1977 was integrated into the "Programme Elargie de Vaccinationation et de Lutte contre les Maladies Transmissible de l’Enfance" (PEV/LMTE) in 1982 [6–8]. Finally, the National Malaria Control Program (NMCP) was only created in 1998 to address malaria with broad mitigation efforts and health policies [6–8]. Therefore, despite a relatively recent introduction of modern antimalarial drugs, malaria control efforts have often been carried out outside any strong policies and regulatory frameworks in the country. The history of malaria control has consequently been dominated by low adoption of official policies as well as popular practices dominated by self-medication, consumption of herbal medicines, and over-the-counter access to drugs of questionable quality [2, 6, 9, 10]. The resulting high drug abuse has potentially served as a setting for the emergence or spread of drug resistance. In this context, the historical effectiveness of chloroquine against malaria could not be sustained for long in the country. Chloroquine-resistant malaria, first suspected in early 1980s [11], evolved rapidly in a decade and was already widespread and associated with excessive malaria morbidity and mortality across the country by the time the NMCP was created [12, 13]. Since then, the NCMP has primarily focused on adjusting strategies to the drug resistance situation while scaling up its activities nationwide [7, 8, 13–15]. The national strategic plan established in 2002 to control malaria has thus been regularly updated and adapted to the evolving landscape of drug-resistant malaria [6–8, 13, 16–18]. Chloroquine was therefore briefly replaced by sulfadoxine-pyrimethamine (S-P) in 2002 for the first-line treatment of malaria, then by artemisinin-based combination therapies (ACTs) in 2005 [6, 8, 13]. The shift to ACTs had been imposed by the therapeutic failures observed with S-P due to the prior resistance of malaria to the component molecules of the drug. In nearly twenty years, artesunate-amodiaquine (since 2005) [8], artemether-lumefantrine (since 2011) [6, 15, 19], and artesunate-pyronaridine (since 2021) [16] have been adopted as alternatively recommended ACTs. Unlike chloroquine, which was completely removed from national guidelines, the S-P combination was limited to intermittent preventive treatment of malaria (IPT) in pregnancy since its withdrawal from curative use [6, 15]. Likewise, quinine has been dedicated to specific clinical forms of malaria (e.g., severe malaria, malaria in early pregnancy, malaria rescue therapy, malaria in young children) in an attempt to restrict its use and possibly prevent it from drug resistance emergence [6, 13, 16]. Further shifting of quinine away from the therapeutic frontline has been achieved by the recent introduction and nationwide scale-up of injectable artesunate for the treatment of severe malaria [7, 14, 15]. However, despite policy adjustments over time, the replaced or withdrawn drugs have often persisted in the market and in popular use against malaria outside of any NMCP control [6, 9, 18]. Furthermore, several drugs that had never been recommended by national guidelines, such as ACTs with mefloquine or piperaquine as a partner molecule of artemisinin, remained widely used [9]. Overall, a landscape conducive to the emergence and spread of resistance has taken shape along this history of national malaria policy. Chloroquine resistance remains at high levels in recent years [20]. Malaria resistance to S-P has been growing and now raises fears of a loss of the drug’s preventive effectiveness while alternative strategies for IPT remain limited [14, 21]. Artemisinin-resistant malaria, which appeared in the Greater Mekong sub-region with the first clinical failures of ACTs, has recently emerged in countries bordering the DRC (e.g. Rwanda and Uganda) and is causing serious concerns for Africa, a continent initially spared this resistance [22–26]. The country can thus potentially become a global hub for artemisinin-resistant malaria especially since the country kept reporting over 10% of the worldwide malaria burden yearly [27]. This situation is most worrying as alternative drugs to replace artemisinin derivatives (i.e., current front-line drugs against both uncomplicated and severe malaria) remain very limited or would require several years to be developed and implemented [28]. This emphasizes the critical need for a national system to monitor and track drug-resistant malaria for global health perspectives. Therefore, this article initiate a living systematic review aiming at periodically summarizing the distribution of malaria parasites carrying molecular markers of drug resistance across the RDC in order to support customized public health decision-making and surveillance efforts.

Search strategy and resource identification

We conducted this systematic review following the PRISMA (“Preferred Reporting Items for Systematic Reviews and Meta-Analyzes”) guidelines (Additional file Table S1) [29, 30]. The review steps were independently performed by two groups of investigators (i.e., NKK/ARA and ETK/NK) and their results were cross-checked to reduce possible errors during the search of information sources and the integration of evidence retrieved from primary articles. Any discrepancies likely arising from the process were resolved by consensus. We searched seven databases (i.e., PubMed, Embase, Scopus, African Journal Online, African Index Medicus, Bioline, and Web of Science) for articles published before July 2023. These articles had to report on clinical Plasmodium isolates sampled within the DRC and that had been genotyped for the detection of molecular markers of drug resistance. The search strategy pre-defined for this purpose was built using English and French versions of specific keywords including the names of genes potentially encoding known molecular markers of drug resistance (Additional file Table S2). No filter was applied to the literature search to ensure the widest inclusion of potentially informative resources. Bibliographic listings contained in previous articles were manually searched for additional articles to be eventually considered for the review.

Selection criteria

We used pre-defined inclusion and exclusion criteria following a PICOS framework – i.e., Population, Intervention, Comparator, Outcomes, and Study designs – (Additional file Table S3) to assess the eligibility of primary articles. Eligible articles were those reporting original observational data on molecular markers of drug resistance (genotype and frequency) in Plasmodium isolates collected from individuals residing in the DRC. When the data from a specific survey were used in subsequent publications, we captured only the most recent information in the inclusion process. Traveling studies were excluded along with articles reporting insufficient information (e.g., unknown isolate frequency), systematic reviews, case reports, conference presentations, conference abstracts and correspondence to editors. We applied a modified version of the Newcastle-Ottawa Scale (NOS) to assess the quality of primary articles based on three criteria: the representativeness of the study samples (rated on a maximum of one star), the sample size (rated on a maximum of one star), and the result of the study (rated on a maximum of three stars) [31]. Only articles of methodological quality rated as moderate (two to three stars on the NOS) or high (four to five stars on the NOS) were considered for inclusion in this systematic review.

Data collection and management

We carried out the data collection according to a sequential process (i.e., literature search, assessment and inclusion of resources, validation and extraction of data). We reviewed each study that met the selection criteria for extracting information related to study characteristics and to genotypes of drug resistance driving genes. Surveys from large geographic area (larger than a city, a town or a village) that could not be separated by sites were treated as of unknown location. Data that were not made available through primary articles were requested from corresponding authors.

Data synthesis and risk of bias assessment

A narrative summary of the information collected was produced referring to absolute isolate numbers as well as median values and corresponding interquartile ranges (IQRs) for relative proportions of isolates carrying specific genotypes such as copy number variations, wild-type genotypes, or single nucleotide polymorphisms (SNPs). Whenever possible, we summarized haplotype variations for alleles jointly reported on codon-positions 72 to 76 of the P. falciparum chloroquine resistance transporter (PfCRT), on codon-positions 51, 59, and 108 of the dihydrofolate reductase (PfDHFR) as well as on codon-positions 437 and 540 of the dihydropteroate synthase (PfDHPS). We linked each survey to its year (or midpoint year) of sampling and its geographic location to display spatial or temporal patterns of parasites potentially carrying different genotypes. The R software version 4.2.0 [32] was used to perform data analysis and mapping. The risk of bias was minimized by excluding traveling malaria cases as well as repeated communications on same isolates. In addition, the NOS criteria used to assess the methodological quality of the primary articles would have minimized the risks of selection bias, confounding factors, and performance bias in the studies considered for this review [33].

Basic characteristics of primary studies

We aggregated 1541 articles found in different databases through the literature search strategy with no additional studies obtained by hand search. By excluding 78 duplicated articles, we screened 1463 publications of which 1434 were found to be ineligible based on criteria defined for this review (Fig. 1). Finally, we could include 29 articles in the review. These articles reported on 6385 P. falciparum specimens sampled between 2000 and 2020 from different sites and that had been successfully genotyped to determine potential drug-resistance molecular markers encoded in the following genes: pfdhfr, pfdhps, pfcrt, pfk13, pfmdr1, and pfmdr2 (Additional file Table S4). An overview of the antimalarial drug resistance landscape in the country is presented in Table 1. The distribution of surveys varied considerably over time and geographical space with most frequent molecular surveillance covering Kinshasa, the country's capital city (16 out of 26 studies of known location). We noted that the largest gaps in geographic coverage of surveillance were in areas in the north and center of the country (Additional file Figure S1).

Table 1

Summary of the landscape of drug-resistant malaria in the DRC as of June 2023
Antimalarial drug	Malaria drug-resistance in the DRC as of June 2023
Quinine	• Quinine-resistant malaria was not confirmed since there is still no validated molecular marker; but it was only suspected given several isolates carrying PfCRT K76T and PfMDR-1 D1246Y mutations.
Lumefantrine	• Lumefantrine-resistant malaria was suspected given isolates potentially carrying PfMDR1 the NFD haplotype which consists of N86, Y184F, and D1246 (but there is still no know validated marker for this resistance).
Mefloquine	• Mefloquine-resistant malaria was not detected as no isolate was detected with amplified copy numbers of pfmdr1 and pfmdr2 genes.
Chloroquine	• Median 32.4% [IQR: 45.6] of isolates were chloroquine resistant as they carried a PfCRT K76T mutation predominately onto a background with CVIET haplotypes. • PfCRT K76T carriage by parasites substantially decreased from 2000 to 2020. • Wide geographic variations in the prevalence of PfCRT K76T parasites, however, was still persisting in 2020 (1.8 to 89.5%) with increased risks of rebound due to the massive reintroduction and misuse of chloroquine for putative treatment or prevention of COVID-19.
Amodiaquine	• Amodiaquine-resistant malaria was not confirmed as no parasite isolate carried a PfCRT SVMNT haplotype, but it was suspected since up several isolates carried PfCRT N86Y and D1246Y mutations (and therefore possibly encoded the YYY haplotype consisting of N86Y, Y184 and D1246Y).
Piperaquine	• Piperaquine-resistant malaria was not explored (i.e., corresponding PfCRT mutations and gene amplification for PfPM2 and PfPM3 were not analyzed).
Artemisinin and derivatives	• Artemisinin-resistant malaria was not established as only a single isolate (sampled in 2014) was detected with a R561H mutation that mediates for resistance. However, there is significant risk of local emergence or regional expansion of ART-resistant parasites from neighboring countries with reported emerging resistance (e.g., Uganda, Rwanda, and Tanzania) or from sites found with reduced levels of drug efficacy with ACTs. • Isolates harboring mutations that structurally mimic known molecular markers of artemisinin resistance need to be monitored and investigated.
Pyronaridine	• Pyronaridine-resistant malaria was not explored since corresponding mutations of the PfMRP1 were not analyzed.
Proguanil	• The genetic background of the parasites suggests that proguanil-resistant malaria is very common (e.g., > 70% of parasites carry PfDHFR S108N, N51I, and C59R), suggesting caution in the use of a chemoprophylaxis including PRO (e.g., PRO-AV combination) when traveling to the DRC.
Sulfadoxine-Pyriméthamine (S-P)	• S-P resistant malaria was widespread at high frequencies but with a moderate molecular profile (PfDHPS A437G: 88.0% [IQR: 33.6]; PfDHPS K540E: 38.9% [IQR: 47.7]). • Quintuple mutants (i.e., IRN-GE) were identified in 13.1% of parasites with highest prevalence in areas located in East parts of the country.

P. falciparum resistance to quinoline derivative drugs in the DRC

SNPs along two key transporter proteins, PfCRT (encoded by the PF3D7_0709000 gene on chromosome 7) and “Plasmodium falciparum multidrug resistance 1” (PfMDR1, encoded by the PF3D7_0523000 gene on chromosome 5), were first discovered to confer resistance to quinoline derivatives drugs in the 1990s [34]. Since then, the PfCRT K76T mutation has emerged as the main molecular marker mediating the P. falciparum chloroquine-resistance [34]. This mutation was thus extensively sought in this review (11 articles conducted from 2000 to 2019 and including total 3464 isolates) and resulted in an overall median 32.4% [IQR: 45.6] frequency among parasites collected across different locations with highest frequencies found in eastern parts of the country (Fig. 2 & Additional file Figure S2). We noted a decrease in the proportion of isolates carrying this point mutation from 100.0% [IQR: 0.0] in 2000 to 13.3% [IQR: 23.2] in 2019 (Additional file Table S5), despite that wide geographical variations (e.g., 1.8–89.5%) were still found in most recent surveys [35, 36]. Consistently, PfMDR1 SNPs involved in decreased parasite susceptibility to chloroquine were also frequently observed, including N86Y (52.6% [IQR: 28.7]), Y184F (43.8% [IQR: 8.8]), and D1246Y (23.3% [IQR: 36.4]) mutations (Additional file Figure S3) [37–39]. Interestingly, the PfMDR1 N86Y, Y184, and D1246Y genotypes (i.e., the PfMDR1 YYY haplotype) possibly occurring on a PfCRT K76T genetic background suggest that parasites with reduced susceptibility to amodiaquine were likely circulating in the country [40, 41]. Likewise, the PfMDR1 NFD haplotype (consisting of PfMDR1 N86, Y184F, and D1246 genotypes) potentially associated with a wild-type PfCRT 76 codon could possibly be harbored by circulating parasites with reduced susceptibility to lumefantrine [41]. Similarly, the data collected [37–39] provide insight into the possibility of parasites with reduced sensitivity to quinine due to the potential combination of PfMDR1 D1246Y and PfCRT K76T mutations [34] (Additional file Figure S3). To further explore the molecular profile of parasites carrying the PfCRT K76T allele, we focused on different PfCRT haplotypes resulting from amino acids variations on codon-positions 72 to 76 (Fig. 3). Therefore, we found that the PfCRT CVIET (i.e., C72-V73-M74I-N75E-K76T) was the most frequent mutant haplotype (25.4% [IQR: 48.8]) whereas other mutant haplotypes were detected at very low median frequency per site (< 1% parasites, each). Overall, median 51.2% [IQR: 77.6] of parasites carried the wild-type PfCRT CVMNK haplotype (i.e., C72-V73-M74-N75-K76). We spotted no parasite carrying a PfCRT SVMNT haplotype (i.e., C72S-V73-M74I-N75-K76T), a well-established marker of amodiaquine resistance [42] (Fig. 3). Finally, we did not identify any parasite isolates encoding gene copy number variations for the “Plasmodium falciparum multidrug resistance 2” (PfMDR2; n = 2 isolates) and the PfMDR1 (n = 366 isolates) that would have suggested potential resistance of P. falciparum to mefloquine (Additional file Figure S4).

P. falciparum resistance to artemisinin derivative drugs in the DRC

Several SNPs of a gene located on the chromosome 13 which encode the P. falciparum Kelch 13 protein (PfK13) have been involved in resistance to artemisinin and its derivatives [43–45]. Through this review, eleven articles analyzed the genetic polymorphism of the PfK13 in 5383 P. falciparum isolates collected from 2005 to 2019 (Additional file Table S4). Therefore, a median frequency of 98.9% [IQR: 0.84] isolates displayed a conserved PfK13 sequence (i.e., a PfK13 of wild-type or with only synonymous mutations) while 1.1% [IQR: 0.84] carried at least one non-synonymous mutation (Additional file Figure S5). Notably, out of 34 different non-synonymous mutations found in 78 isolates, 30 were located on the PfK13 Propeller domain (i.e., above the codon-position 440 [46]) (Additional file Table S6). Unlike other surveys that targeted the PfK13 Propeller domain, only Miotto et al. [47] sequenced the full-length PfK13 and could report four non-synonymous mutations located outside the Propeller domain (i.e., K92N, T149S, K189T and R225K). We highlighted a set of five mutations recorded at relative frequencies < 1% and located on codon-positions that had been linked to artemisinin resistance in Southeast Asia (Fig. 4). Interestingly, among these mutations, we noted a PfK13 R561H (encoded by one isolate from an unknown location) which is a marker validated in vivo and in vitro for driving artemisinin resistance, and whose emerging carrier parasites have been expanding recent three years in countries bordering the DRC, namely in Tanzania, Uganda, and Rwanda [22, 24, 48, 49]. The remaining four mutations also warrant interest as they structurally mimic SNPs linked in vivo or in vitro to artemisinin resistance in Southeast Asia (i.e., M476K mimicking M476I, G538S mimicking G538V, V568M mimicking V568G and D584E mimicking D584V). We finally observed that all other PfK13 mutations found in the country, except S522C [43], have not been explored clinically and experimentally in order to rule out any biological relevance.

P. falciparum resistance to antifolate drugs in the DRC

Genetic mutations in genes encoding two enzymes, the PfDHFR and the PfDHPS, are known as conferring resistance of P. falciparum to antifolate drugs, namely S-P, since in the 1990s [50]. These mutations are thus widely explored for surveillance purposes [34]. In this review, we gathered total 3537 isolates (ten articles) and 3518 P. falciparum isolates (twelve articles) that have been respectively genotyped, from 2002 to 2020, for specific mutations of PfDHFR (at any of the C50, N51, C59, S108, and I164 codon-positions) and PfDHPS (at any of the I431, S436, A437, K540, A581, and A613 codon-positions) (Table 2). We thus found that most prevalent mutations were PfDHFR S108N (99.5% [IQR: 3.9]) and N51I (97.9% [IQR: 25.0]) as well as PfDHPS A437G (88.0% [IQR: 33.6]) and K540E (38.9% [IQR: 47.7]) (Fig. 5). PfDHFR SNPs were ubiquitous across the country while PfDHPS ones predominated either in the western (for A437G) or in eastern parts (for K540E) of the country (Fig. 5). Furthermore, we assessed PfDHFR-PfDHPS haplotype combinations for 2098 isolates from five articles that jointly provided genetic polymorphism data on three PfDHFR (i.e., N51, C59, and S108) and two PfDHPS codon-positions (i.e., A437 and K540). We thus identified parasites harboring thirteen different PfDHFR-PfDHPS haplotypes (i.e., NCS-AK, ICN-AK, IRN-GE, ICN-GE, ICN-GK, IRN-AE, IRN-AK, IRN-GK, NCN-GK, NCS-GE, NCS-GK, NRN-AK, NRN-GK) (Additional file Table S7). In absolute terms, the most frequent PfDHFR-PfDHPS haplotypes were quadruple IRN-GK mutants (59.3%; n = 1018) comprising three PfDHFR mutations (N51I, C59R, and S108N) along with PfDHPS A437G, followed by quintuple IRN-GE mutants that encoded an additional PfDHPS K540E mutation (13.1%; n = 311). These mutants were most prevalent in eastern regions of the country (Fig. 6).

Table 2

Frequency of genetic alleles potentially linked to malaria resistance to anti-folate drugs in the DRC.
Alleles	No. of genotyped isolates	No. of mutants	Median % of mutants (IQR)
PfDHPS alleles
I431V	1588	14	1.4 (0.9)
S436A	2165	297	7.4 (13.9)
A437G	2103	1821	88.0 (33.6)
K540E	3518	1373	38.9 (47.7)
A581G	3076	431	10.7 (17.7)
A613S	1684	10	0.1 (0.5)
PfDHFR alleles
N51I	2324	2260	97.9 (25.0)
C59R	2324	1937	79.1 (0.0625)
S108N	2324	2291	99.5 (11.9)
I164L	2927	2	< 0.1 (3.94)

This review summarizes information from 6385 P. falciparum isolates sampled across the DRC over the past two decades and provides a baseline for enhanced country-level drug resistance surveillance efforts. Indeed, these parasites have been analyzed for genetic mutations that reflect antimalarial drug resistance with relevance for health policy [34]. Therefore, this work and subsequent updates through an intended living systematic review (LSR) process [51] have the potential to support a continuous monitoring of drug-resistant malaria through the country while supporting evidence-based public health decision making and identifying surveillance gaps to be addressed. So far, resistance surveillance activities targeted drugs historically used against malaria in the country, including quinolines (i.e., chloroquine, amodiaquine, mefloquine, and LU), artemisinin derivatives, and antifolate drugs (i.e., S-P) [7, 52]. Overall, we detected malaria parasites displaying mutations reflecting or raising suspicion of resistance to all these drugs, except for mefloquine. However, the magnitude of detected resistance mostly warranted additional explorations given limited number of surveys, gaps in geographic coverage, and asymmetrical surveillance activities prioritizing Kinshasa, the country's capital. In this context, we advocate for the democratization of monitoring efforts through different country’s areas to partially overcome existing disparities. Such efforts have become more achievable in resource-limited settings like most parts of the DRC, thanks to recent advances in portable, low-cost sequencing platforms that have gained momentum as an alternative to heavy central laboratories for the detection of antimalarial drug resistance markers [53, 54].

With respect to resistance to quinolone-containing antimalarial drugs, surveillance activities were dominated by monitoring PfCRT K76T mutations that confer resistance to chloroquine but possibly contribute to reduced susceptibility to other drugs such as amodiaquine and LU [34]. Consistently with outcomes from other Sub-Saharan regions [55], we found that the overall proportion of parasites carrying this mutation has decreased overtime in the DRC. This suggests a gradual recovery of chloroquine susceptibility among malaria parasites following the lifting of the drug selective pressure after its withdrawal from clinical use since 2002 [13]. However, the frequency of PfCRT K76T parasites which remains overall above 30% prevents any short-term reintroduction of chloroquine into clinical practice in the country. In addition, residual locations with very high proportions of PfCRT K76T parasites were still existing [35, 36] and could reflect a local fixation of the PfCRT K76T mutation prior to chloroquine withdrawal. Given evidence on the role of the PfCRT CVIET haplotype (which includes a K76T mutation within it) in resistance to amodiaquine [34], its widespread use as part of first-line treatment could contribute with some selective drug pressure that may support the persistence of K76T parasites over time. In addition, the sustained chloroquine resistance could also be in part driven by a persistent chloroquine use in the population at odds with national policies, as has been reported in other sub-Saharan African countries [56]. Further explorations and health policies accounting for within-country geographical variations are therefore needed [36]. Regulatory efforts to control the use of antimalarial drugs remain also relevant especially since the ongoing the coronavirus disease 2019 (COVID-19) pandemic brought back to the fore the widespread use of chloroquine (and its derivative, hydroxy-chloroquine) raising fears of further drug-resistance development [57, 58]. Unlike widespread chloroquine resistance, no evidence suggesting any mefloquine resistance could be obtained while resistance to quinine, amodiaquine, and lumefantrine could only be suspected. However, these outcomes raise some cautions given limited evidence gathered in this review. First, these suspicions were based on a combination of specific PfMDR1 and PfCRT genotypes which still require causality validation through experimental studies [34, 40, 41]. Then, data contrasting with any lumefantrine or amodiaquine resistance were also obtained, including the absence of parasites encoding PfCRT SVMNT haplotype [42] or PfMDR1 S1034C and N1042D mutations [34]. Finally, the magnitude of possible resistance to quinolines other than chloroquine could not be captured across the country as, so far, only limited studies tracked PfMDR1 SNPs [37, 38] and related haplotype combinations [39]. Likewise, resistance to piperaquine – encoded by additional PfCRT SNPs [59, 60] as well as Plasmepsins 2 and 3 [61] – was not covered so far by surveillance activities. Therefore, while continuously monitoring chloroquine resistance is needed, further investigations and surveillance efforts are warranted to clear suspicions upon resistance to other quinoline compounds [9].

Furthermore, this review showed that resistance to artemisinin derivatives is not yet established in the DRC as only a single isolate carrying a marker validated for this resistance had been reported so far [43]. However, a series of recent events raise fears of an imminent change in the local epidemiological landscape and lead us to call on the NMCP to launch urgent measures to proactively counter resistance to artemisinin. In fact, an emergence and expansion of artemisinin-resistant malaria in neighboring countries (e.g. Rwanda, Uganda, and Tanzania) has been reported during the last three years [43, 46, 48, 49]. In addition, Moriarty et al. [39] has just reported alarming evidence of efficacy below the 90% cutoff recommended by the World Health Organization (WHO) to consider a change in first-line treatment recommendations of two ACTs (i.e., artemether-lumefantrine and dihydroartemisinin-piperaquine) in Mikalayi – a town located in Kasai region not far from a site in Angola that has shown similar reduced efficacy for artemether-lumefantrine [62]. This notably led the NMCP to diversify its policy for the first-line ACTs by including the artesunate-pyronaridine combination alternatively to existing artesunate-amodiaquine and artemether-lumefantrine, which will come into effect from 2024 [16]. Applying a multiple first-line treatment is supposed to decrease the selective pressure that would have been maximal on a single drug and could delay the emergence of artemisinin resistance and its spread across the country, which seems only a matter of time. Adoption of artesunate-pyronaridine may be epidemiologically advantageous as pyronaridine has not been used in the past in the country [16] in addition to its known resilience to resistance development and lack of cross-resistance with other antimalarial drugs [63]. However, since resistance to pyronaridine is believed to have already existed in Africa since the 1980s [64], we draw attention to the need to assess in vitro the intrinsic sensitivity of ambient parasites to this drug before its actual implementation. In the same momentum, assessing molecular substitutes for possible resistance to pyronaridine (i.e., specific SNPs in the Plasmodium falciparum multidrug-associated resistance protein 1 - PfMRP1 [65]) could provide relevant information on the existing background of pyronaridine susceptibility. Either way, beyond changes made in the national ACTs policies, extended molecular surveillance activities are henceforth needed to guide artemisinin-related policies and sustain parasites susceptibility. These surveillance activities should be designed to cover expanded genetic loci currently linked to artemisinin resistance since the well-known mutations of the PfK13 Propeller domain that drive artemisinin resistance in Southeast Asia could not be observed locally in treatment failures with ACTs [39] and as African malaria parasites are known able to use a different genetic background to generate resistance [66]. Therefore, the molecular surveillance requires including both the PfK13 Propeller [45, 46] and the ‘Broad-Complex Tramtrack and Bric a brac’ or ‘Poxvirus and Zinc finger’ domains (BTB/POZ) [67] as well as other genomic loci such as the P. falciparum Coronin gene [68]. Previously, we also highlighted the need for monitoring African parasites carrying PfK13 SNPs that mimic known drug resistance markers, of which a few sporadic cases were observed in this review [46]. Beside diversifying first-line treatments and extending drug resistance surveillance activities, additional efforts should be undertaken to further reduce selective pressure in areas at high risk for the artemisinin resistance development, particularly in provinces bordering Uganda, Rwanda, Tanzania, and Angola. Campaigns to restrict suboptimal use of artemisinin (e.g., use of artemisinin-based monotherapies, consumption of Artemisia spp. plants) could therefore be considered alongside tracking resistant parasites in migratory populations.

Regarding malaria resistance to antifolate drugs, we found that despite widespread resistance to S-P across the DRC, the drug combination still retains some usefulness for malaria chemoprevention. Beyond the IPT currently implemented in the country during pregnancy, several WHO-recommended SP-based malaria chemoprevention strategies are therefore within reach, including perennial malaria chemoprevention for young children aged 12 at 24 months, seasonal malaria chemoprevention for children 3–59 months, and IPT in school-aged children 5–15 years [69]. Obviously, the molecular profile of this drug resistance corresponds to a moderate level of effectiveness for IPT in pregnancy, as per the van Eijk et al.’s definition criteria (i.e., PfDHPS A437G ≥ 90% or PfDHPS K540E ≥ 30% and < 90%) [70]. This implies that S-P may still be effective for preventing adverse pregnancy and birth outcomes (e.g., low birth weight, anemia) in the country more likely due to its additional non-malarial effects (e.g. antibiotic and immunomodulatory effects) [21, 70, 71]. However, the expected prophylactic effects of S-P against malarial infections may already have been lost; mother and fetus could therefore remain exposed to infection despite taking S-P [21, 72]. Moreover, considering that nearly 40% of parasites carried the PfDHPS K540E substitution, S-P-based chemoprevention in children would still be indicated with respect to the cutoff criteria recommended by WHO (< 50% of PfDHPS K540E parasites) [73]. The NMCP has thus already planned to implement S-P-based chemoprevention interventions in Congolese children [17]. Despite the perceived usefulness of these interventions, further implementation of S-P-based chemoprevention raises some concerns that should be brought to the attention of DRC health authorities. First, the risk of further selecting PfDHPS K540E parasites and quintuple IRN-GE mutants should be managed properly and closely monitored to avoid rapidly reaching higher resistance levels and complete loss of the clinical efficacy of the drug [70, 74–76]. Second, combining S-P with amodiaquine, which has shown its effectiveness in the Sahel subregion of Africa [77], should be considered instead of simply the S-P combination. Finally, given the regional genetic background, local evidence (e.g. provided by clinical trials) of the prophylactic efficacy and the sustainability of any S-P based strategy is needed [69, 74, 78]. As discussed for chloroquine resistance, within-country variations and evolution dynamics in resistance profiles should anyway be taken in account when up scaling any S-P-based strategy in either pregnant women or children [75]. In particular, the higher prevalence of RN-GE parasites found in the eastern parts of the country should be regarded as local barriers to S-P-based policies that warrant alternative strategies [70, 79–82]. Furthermore, the molecular profile of the parasites (i.e. 99.5%, 97.9% and 79.1% of the parasites encoding the PfDHFR mutations S108N, N51I and C59R respectively) is also suggestive of frequent resistance to Proguanil, a drug antifolate widely used in combination with Atavaquone for chemoprophylaxis of malaria in travellers [83]. People visiting the DRC must therefore be warned of the serious threat that circulating resistant parasite could pose to the effectiveness of this malaria prevention strategy.

Overall, this systematic review had several limitations, including a limited number of primary articles, gaps in geographic coverage of monitoring activities, and high methodological heterogeneity in primary studies. Genetic markers of drug resistance were presented unrelated to information from in vivo assays and in vitro studies which would have further enriched this review by providing the maximum information on the emergence and evolution of drug resistant malaria in the population [46, 84, 85]. The scarcity of existing in vivo and in vitro studies is probably due to high costs and technical requirements. All these limitations restricted this work to a narrative review; but with desired progress in national malaria resistance surveillance efforts, in the future we hope to be able to update and report this review as an improved meta-analysis that addresses these weaknesses.

Despite its shortcomings, this review highlights drug-resistant malaria as a major health problem and provides a basis for future surveillance efforts to guide public health efforts tailored to the country’s situation. Indeed, resistance to chloroquine remains high, resistance to sufadoxine-pyrimethamine undermines current chemoprevention strategies, while possible emergence of resistance to artemisinin threatens the country responsible for one-tenth of the world's malaria burden. Hopefully, the living systematic review launched with the current work will offer an approach to keep the high-quality evidence synthesis continuously up to date with most relevant and reliable information on drug resistance that can be used to inform policy and practice, and to ultimately improve quality of care and population health outcomes within the DRC and beyond.

ACTs

Artemisinin-based combination therapies

BTB/POZ

The ‘Broad-Complex Tramtrack and Bric a brac’ or ‘Poxvirus and Zinc finger’ domains

COVID-19

Coronavirus disease 2019

DRC

Democratic Republic of Congo

IPT

Intermittent preventive treatment of malaria

NMCP

National Malaria Control Program

NOS

Newcastle-Ottawa Scale

PEV/LMTE

"Programme Elargie de Vaccinationation et de Lutte contre les Maladies Transmissible de l’Enfance"

PICOS

The framework Population, Intervention, Comparator, Outcomes, and Study designs

PfCRT

The P. falciparum chloroquine resistance transporter

PfDHFR

The P. falciparum dihydrofolate reductase

PfDHPS

The P. falciparum dihydropteroate synthase

PfK13

The P. falciparum Kelch 13 protein

PfMRP1

The Plasmodium falciparum multidrug-associated resistance protein 1

PfMRP2

The Plasmodium falciparum multidrug-associated resistance protein 2

PLA

"Programme de Lutte Antipaludique"

PLAP

"Projet de Lutte Antipaludique"

PRISMA

Preferred Reporting Items for Systematic Reviews and Meta-Analyzes

SNPs

Single nucleotide polymorphisms

S-P

Sulfadoxine-pyrimethamine

USAID

United States Agency for International Development

WHO

World Health Organization

Ethics approval and consent to participate

This study is a synthesis of primary studies having benefited from ethical authorizations and consent to participate at the time of their conduct. As such, no additional permission was required for this review.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. The status of the subsequent living systematic review can be accessed at: https://nadinekayiba.pro/research/.

Competing interests

The authors declare that they have no competing interests.

Funding

NKK received a Ph.D. scholarship under a funding from the Belgian Cooperation Agency through the Academy of Research and Higher Education (ARES). This work also received a support from the Japan Society for the Promotion of Science (JSPS) KAKENHI under grant number JP18KK0454 and the Japan Agency for Medical Research and Development (AMED) under grant numbers JP21wm0125003 and JP19fm0208020 (all to YK).

Authors' contributions

NS and NKK designed the study. NKK, ETK, and ARA wrote the protocol, performed the literature search, aggregate data acquisition, primary article review, and data extraction. NKK and ETK analyzed the data and compiled summaries. NKK and ETK wrote the first draft of the manuscript. YK provided access to documentary resources based on the Embase database. NK, DYM, BD, PKZ, PDM, GML, MPH, DMM, YK, AK, YN, PLD, and NS reviewed the first draft of the manuscript. All authors provided conceptual input, revised, and approved the final version of the manuscript.

Acknowledgements

We thank all the authors of the primary articles who kindly shared with us the basic data of their respective studies.

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Malaria drug resistance landscape in the Democratic Republic of the Congo: a spatial mapping systematic review of molecular surveillance surveys

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Abstract

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Background

Methods

Search strategy and resource identification

Selection criteria

Data collection and management

Data synthesis and risk of bias assessment

Result

Basic characteristics of primary studies

Discussion

Conclusions

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