Prevalence of Plasmodium falciparum plasmepsin2/3 gene duplication in Africa and Asia: a systematic review and meta-analysis

Abstract

Background

The Plasmodium falciparum delayed clearance phenotype due to the emergence of partial artemisinin resistance has been documented in Asia and Africa, where it is associated with treatment failure of artemisinin-based combination therapy (ACT). The amplification of the Plasmodium falciparum plasmepsin2/3 gene (pfpm2/3) has been shown to decrease the susceptibility of P. falciparum to piperaquine, leading to treatment failure among patients on dihydroartemisinin-piperaquine. The present systematic meta-analysis summarises the evidence of pfpm2/3 gene amplification in Asia and Africa.

Methods

The protocol for the review was registered at the PROSPERO (Reference number: CRD42024599774). Thirty-four studies conducted in Africa and Asia, reporting pfpm2/3 gene amplification among P. falciparum isolates, were identified through the Medline, Google Scholar, Cochrane Central Register of Controlled Trials (CENTRAL), LILACS, and EMBASE online databases. The potential for publication bias was evaluated by examining asymmetry in funnel plots and using Egger’s test. Pooled proportions estimates were calculated using the random effects model, while heterogeneity was assessed through I² statistics. Sub-group analysis was performed based on the year of sample collection and continent.

Results

The heterogeneity among the studies included in the meta-analysis was high (I² > 95%, p < 0.01). The funnel plot was asymmetrical, suggesting that publication bias affected the meta-analysis. However, Egger’s test and Begg’s (adjusted to Kendall’s) scores for the pooled proportions of the pfpm2/3 gene confirmed no potential publication bias (p = 0.083 and 0.163, respectively). A total of 34 studies involving 4,005 P. falciparum isolates were included in this review. Of the 34 studies, 18 (53%) were conducted in Asia, and 16 (47%) were conducted in Africa. The samples for these studies were collected from 2009 to 2019. Among these studies, 15 (44%) were performed before 2016. The estimated pooled proportions of pfpm 2/3 gene amplification via the random effects model were 16.0% (95% CI 8.0–26.0%). Subgroup analysis (per continent and year of sample collection) revealed that the pooled proportions estimates of pfpm2/3 gene amplification were greater in Asia (25.0%, 95% CI 9.0–45.0%) than in Africa (8.0%, 95% CI 2.0–15.0%) and lower before 2016 than 2016 to 2020 (11%, 95% CI 3.0–23% and 19%, 95% CI 7.0–36%, respectively).

Conclusion

The present review provides up-to-date evidence on the pfpm2/3 gene amplification. A substantial pooled proportion of pfpm2/3 gene amplification was reported, and many of the amplifications were observed in isolates from Asia rather than Africa. This calls for further efforts to monitor/control the emergence and spread of partner drug resistance in the regions to avoid the emergence of total ACT resistance, which will compromise global efforts toward eliminating malaria.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12936-025-05423-5.

Background

Malaria treatment over the past two decades has primarily relied on artemisinin-based combination therapy (ACT), which remains effective even in regions with multidrug-resistant parasites. Resistance has been a driving force behind the shift in falciparum malaria treatment from chloroquine (CQ) to sulfadoxine-pyrimethamine (SP) and then to artemisinin monotherapy. The World Health Organization (WHO) recommended using ACT, citing its advantages of high efficacy even in areas with CQ resistance and fewer side effects compared to SP and CQ [1, 2].

In most endemic countries in sub-Saharan Africa (sSA), artemether-lumefantrine (AL), artesunate-amodiaquine (AS-AQ), and dihydroartemisinin-piperaquine (DHA-PPQ) are the most widely used artemisinin-based combinations [3]. However, the emergence and spread of resistance to artemisinin derivatives, as well as their partner drugs in the greater Mekong subregion of Southeast Asia (SEA), pose significant challenges to achieving the targets of the Global Technical Strategy for Malaria 2016–2030 established by the WHO [4].

The partial resistance of Plasmodium falciparum to artemisinin, defined as delayed parasite clearance (half-life ≥ 5 h) following treatment with ACT, was first reported in SEA approximately 14 years ago [5, 6]. Recently, the de novo emergence and spread of partial artemisinin resistance have been confirmed in East African countries (Rwanda, Uganda, and Tanzania) [7–9]. The reported partial artemisinin resistance could compromise the sensitivity of partner drugs, which have relatively longer half-lives than artemisinin derivatives, resulting in increased clinical treatment failure.

Since the discovery of artemisinin resistance markers that decrease artemisinin susceptibility in SEA, much of the focus has been on tracking the emergence of these markers through surveillance studies. Various systematic reviews have been conducted to establish the status of the gene encoding the Kelch protein (kelch13; PF3D7_1343700) [10–15] that mediates artemisinin resistance. However, the efficacy of ACT is a synergistic function of both artemisinin and partner drug components [16]. In instances of decreased parasite susceptibility to artemisinin derivatives, ACT can still be effective if the efficacy of the partner drug has not been compromised; thus, resistance to the partner drug is critical [16]. This scenario is only feasible if the parasite biomass exposed to the partner drug is not high. In cases with high parasite biomass exposed to a partner drug after artemisinin action (short half-life), inadequate clearance of parasites due to artemisinin derivatives may subject the partner drug (with a relatively longer half-life) to increased selection pressure [17]. Resistance to partner drugs results in treatment failure and total ACT resistance [18–21]. This emphasises the need to focus on detecting partner drug resistance markers in efforts to control and eliminate malaria. Piperaquine (PPQ) is a partner drug included in DHA-PPQ, one of the six forms of ACT prequalified by the WHO and used in many sSA and SEA malaria-endemic countries for the treatment of uncomplicated P. falciparum malaria, as well as in intermittent preventive therapy in pregnancy (IPTp) and mass drug administration (MDA) in areas approaching malaria elimination [22, 23].

Piperaquine was discovered and used as a monotherapy in SEA in the 1960s; however, due to the risk of resistance, it was later combined as a partner drug in DHA-PPQ [24]. PPQ, a synthetic 4-aminoquinoline, is thought to act by accumulating in high concentrations in the parasite’s digestive vacuole, thereby inhibiting the conversion of toxic haem to nontoxic haemozoin crystals [25]. Resistance to PPQ is linked to the duplication of the pfpm2/3 gene encoding proteases in the parasite digestive vacuole. In SEA, pfpm2/3 gene duplication was shown to decrease parasite susceptibility, leading to treatment failure among patients on DHA-PPQ treatment [26–29]. The pfpm2/3 gene modulates the metabolism of haemoglobin in the digestive vacuole, which provides essential amino acids to the parasite [30]. Thus, amplification of the pfpm2/3 gene antagonises the effect of PPQ [22] and may confer a survival advantage to the parasite [28, 31, 32]. To the best of our knowledge, several individual studies have been conducted in malaria-endemic regions to determine the amplification of the pfpm2/3 gene; however, systematic reviews and meta-analyses to guide future efforts are lacking. The rise in pfpm2/3 gene amplification would lead to DHP-PPQ failure, thus compromising the progress made in the control and elimination of malaria.

The objective of this review was to determine the pooled estimates of pfpm2/3 gene amplification in P. falciparum isolates from Asia and Africa, the two regions most impacted by malaria.

Methods

Study protocol registration

The protocol for this systematic review and meta-analysis was developed and registered in the International Prospective Register of Systematic Reviews- PROSPERO (Reference number: CRD42024599774) using the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) for protocol development [33].

Search strategy

A literature search for published studies assessing the amplification of plasmepsin2/3 in Asia and Africa was performed six times (from April to August 2024) through the Cochrane Central Register of Controlled Trials (CENTRAL), EMBASE, Google Scholar, Medline, and LILACS online databases. The search terms were used separately and in combination with Boolean operators such as “OR” or “AND.” The keywords used in the Medline, Embase, Cochrane, and the other databases search include the following: (“amplificate” OR “amplificates” OR “amplification” OR “amplifications”) AND (“plasmodium falciparum” [MeSH Terms] OR (“plasmodium”) AND “falciparum” OR “Plasmodium falciparum”) AND (“plasmepsin” OR “plasmepsins”). Since word duplication is used synonymously with word amplification, the following search terms were also used: (“duplicate” OR “duplicated” OR “duplicates” OR “duplication” OR “duplications” OR “duplicative”) AND (“plasmodium falciparum” [MeSH Terms] OR (“plasmodium”) AND “falciparum” OR “Plasmodium falciparum”) AND (“plasmepsin” OR “plasmepsins”). References were cited and managed via EndNote software version X8. Duplicate articles were detected through the EndNote software and removed.

Inclusion criteria

All published studies on plasmepsin2/3 anti-malarial resistance markers were screened. Studies describing the amplification or duplication of the pfpm2/3 gene among P. falciparum isolates in Asia and Africa were included. These studies were original research articles published in peer-reviewed journals.

Exclusion criteria

This review excluded studies describing other markers of antimalarial drug resistance (pfcrt, pfmdr1, pf coronin, pfATPase6, other plasmepsin genes (1, 3, 4, 5, 9 and 10), cysteine desulfurase, pf Kelch12, pf kelch13 and other P. falciparum resistance genes since meta-analyses for these genes have been published elsewhere or were out of the scope of this review. Studies describing pfpm2/3 gene amplification in P. vivax were also excluded. Literature reviews, non-primary research studies, and conference abstracts were deemed not to have sufficient information or reliable sources and hence were not included in the present review. These ineligible articles were removed manually.

Data extraction

Two independent reviewers (KJM and AK) screened the literature search results and selected studies based on the inclusion criteria. When a full article was unavailable online, a complete version was requested from the corresponding author. There were no differences of opinion between the reviewers concerning the inclusion of the studies. Abstracted information and data were entered into the extraction sheet. The basic information extracted included general details (author’s names, the country where the study was conducted, sample collection year, publication year, and year of ACT introduction), study characteristics (study design, sample size, study participants, genotyping methods, and transmission intensity), the total number of samples genotyped, and the number of samples with pfpm2/3 amplification. For studies conducted in multiple countries, data for each country were extracted.

Methodological and data quality assessment

The National Institutes of Health (NIH) study quality assessment tools for controlled intervention studies, observational cohort studies, and cross-sectional studies were employed in the methodological quality assessment [34]. The score range for the NIH tool scale was from 0 to 14. Each criterion was given one point, resulting in a total of 14 points. The conversion of scores into percentages was performed. The score ranges were classified as 0–60% (low quality), 61–80% (good quality), and 81–100% (excellent quality). There were no disagreements on the extracted data or methodological quality assessment between the two independent reviewers. All included studies were of good to excellent quality according to the NIH scale, as shown in Table 1.

Table 1.

Characteristics of the studies included in the systematic review and meta-analysis

SN	Country	Continent	Study design	Authors	Publication year	Sample collection	ACT introduction	Transmission	Methods	Score (%)	References
1	Nigeria, Ivory Coast, and other African Countries	Africa	Retrospective	L'Episcopia M et al	2021	2014–2015	2005 for Nigeria & Ivory Coast	Not stated	Positron emission tomography-PCR-based assay	93	[57]
2	Mali, Burkina Faso and Guinea	Africa	Randomised, double-blind trial	Inoue et al	2018	2011–2016	2005	Not stated	qPCR	86	[41]
3	Cambodia	Asia	Clinical trial	Witkowski et al	2017	2009–2015	2000	Not stated	qPCR	93	[26]
4	Equatorial Guinea	Africa	Therapeutic efficacy	Liu et al	2022	2017–2019	2008	High	Real-time PCR	100	[58]
5	Tanzania	Africa	Open-label single-arm prospective	Kakolwa et al	2018	2011–2015	2006	Not stated	qPCR	86	[59]
6	Burkina Faso, Uganda, and other African Countries	Africa	Randomised, double blind trial	Leroy et al	2019	2014–2015	2005 for Burkina Faso &Uganda	Not stated	qPCR	93	[60]
7	Vietnam	Asia	Randomised, double blind trial	Leroy et al	2019	2014–2015	2005	Not stated	qPCR	86	[60]
8	Cambodia, Thailand & Vietnam	Asia	Clinical trial	Pluijm et al	2019	2015–2018	2000,1995 &2005 respectively	Not stated	Sequencing	93	[29]
9	Thailand	Asia	Retrospective analysis	Win et al	2022	2013–2019	1995	Not stated	qPCR	93	[61]
10	China-Myanmar border	Asia	Therapeutic efficacy	Huang et al	2020	2010–2014	2005 and 2002, respectively	Not stated	qPCR	86	[45]
11	Kenya	Africa	Not stated	Diarra et al	2022	2016–2018	2006	High	qPCR	100	[62]
12	Liberia	Africa	One-arm efficacy cohort	Koko et al	2022	2017–2018	2003	Not stated	qPCR	93	[63]
13	Uganda	Africa	Cross-sectional	Asua et al	2019	2016–2017	2005	Not stated	qPCR	86	[64]
14	Congo DRC, Burkina Faso, and other African Countries	Africa	Not stated	Gendrot et al	2021	2015–2019	2005 for Congo DRC & Burkina Faso	endemic	Sanger & qPCR	86	[44]
15	Mozambique	Africa	Cross-sectional survey	Gupta et al	2020	2015–2017	2009	Not stated	qPCR	86	[54]
16	China-Myanmar border	Asia	study (TES)	Li et al	2020	2012–2016	2005 and 2002, respectively	Not stated	qPCR	93	[43]
17	Cambodia	Asia	Prospective, open-label, single-arm observational trial	Mairet-Khedim et al	2021	2016–2017	2000	Not stated	qPCR	86	[46]
18	Vietnam	Asia	Prospective, open-label, single-arm observational trial	Bui Quang et al	2020	2017–2018	2005	Not stated	qPCR	100	[65]
19	Myanmar	Asia	Observational	Landier et al	2018	2014–2017	2002	Not stated	qPCR	100	[66]
20	Vietnam	Asia	Prospective,open-label, observational clinical trial	Manh et al	2021	2018–2019	2005	Not stated	qPCR	100	[67]
21	Burkina Faso	Africa	Two-arm randomized control trial	Gansane et al	2021	2017–2018	2005	Not stated	qPCR	93	[68]
22	Cambodia &Thailand	Asia	Clinical trial	Agrawal et al	2017	2006–2014	2000&1995 respectively	Not stated	Sequencing	93	[69]
23	Ghana	Africa	Not stated	Duah-Quashie et al	2022	2015–2016	2004	Not stated	Real-time PCR	86	[70]
24	Vietnam	Asia	Randomized open-label	Rovira-Vallbona et al	2020	2015–2017	2005	Not stated	qPCR	93	[71]
25	Cameroon	Africa	Nonrandomized open-label	Mairet-Khedim et al	2021	2018	2004	Not stated	qPCR	93	[47]
26	Vietnam	Asia	Double-blind clinical trial	Son et al	2017	2016	2005	Not stated	qPCR	93	[72]
27	Cambodia	Asia	Open-label, prospective	Leang et al	2019	2017	2000	Not stated	qPCR	86	[73]
28	Uganda	Africa	double-blind randomized trial	Conrad et al	2017		2005		qPCR	93	[74]
29	Vietnam	Asia	Randomized Open-label	Phong et al	2019	2015–2016	2005	Low	Sequencing	93	[75]
30	Uganda	Africa	open-label, phase IV clinical trial	Ebong et al	2021	2018–2019	2005	High	qPCR	86	[76]
31	Vietnam	Asia	Longitudinal surveillance	Thanh et al	2017	2011–2015	2005	Not stated	Real-time qPCR	100	[21]
32	Kenya	Africa	Therapeutic efficacy	Chebore et al	2020	2016–2017	2006	High	Real-time PCR	93	[77]
33	Thailand-Myanmar border	Asia	Not stated	Ye et al	2022	2009–2013	1995&2002 respectively	Not stated	qPCR	100	[78]
34	Thailand-Cambodia border	Asia	Not stated	Ye et al	2022	2012–2014	1995& 2002, respectively	Not stated	qPCR	100	[78]

Data collection and analysis

A number of samples with pfpm2/3 gene duplication and the total number of samples analysed were entered into an Excel spreadsheet. Meta-analyses were performed via STATA 17 (Statistical Corporation, College Station, TX, US). Sub-group analyses of pfpm2/3 gene duplication were performed according to (a) continent and (b) duration of sample collection (before 2016 and post-2016) to compare recent data and previous data, whereby 2016 represents ten years post-adoption of ACT.

A random effects model was used to combine information from comparable studies. The possibility of publication bias was assessed by examining asymmetry on funnel plots and Egger’s test, where a p-value of < 0.05 was regarded as indicative of statistically significant publication bias [35]. The bias-adjusted effect estimates were obtained using the trim-and-fill method. The heterogeneity across studies was evaluated through Cochran’s Q test and I² [36]. Heterogeneity was considered substantial when the p-value of Q was < 0.10 and/or I² was > 50% [36]. Sensitivity analysis was carried out to assess the influence of each study on the overall proportion estimation. The results of the review were reported by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses ‘PRISMA 2020’ guidelines [37] PRISMA statement (prisma-statement.org).

Results

Study characteristics

A total of 598 records were identified through the electronic database search, as shown in the PRISMA flow chart (Fig. 1). Fifty-one articles were included for full-text review. A total of 34 studies were eligible for data extraction and were therefore included in the meta-analysis for the estimation of the pooled proportions of pfpm2/3 gene amplification. These studies were conducted in African (Uganda, Burkina Faso, Democratic Republic of Congo, Tanzania, Guinea, Mali, Equatorial Guinea, Nigeria, Ivory Coast, Ghana, Cameroon, Mozambique, Liberia, and Kenya) and Asian countries (Cambodia, Vietnam, Thailand, Myanmar, and China) from a total of 4,005 P. falciparum isolates. Of 34 studies, 18 (53%) were conducted in Asia, and 16 (47%) were conducted in Africa. Studies included were published between 2016 and 2022, while samples for these studies were collected from 2009 to 2020. Among these studies, 15 (44%) were conducted before 2016.

Fig. 1.

PRISMA flow chart illustrating the article selection steps [37]

Heterogeneity, publication bias, and sensitivity analysis

The heterogeneity among the studies included in the present meta-analysis was substantial (I² > 95% and p < 0.01). The funnel plot was asymmetrical, suggesting that the meta-analysis conducted was affected by publication bias, as shown in Fig. 2. However, Egger’s test and Begg’s test (adjusted to Kendall’s) scores for the pooled proportions of the pfpm2/3 gene confirmed that there was no potential publication bias (p = 0.083 and 0.163, respectively). Furthermore, the trim and fill analyses for the bias-adjusted effect for pooled proportions estimates of pfpm2/3 gene amplification confirmed that the estimates were not impacted by publication bias (p = 0.354). The sensitivity analysis used to evaluate the influence of a single study in the meta-analysis estimation (pooled estimates) indicated the robustness of the aggregated estimates because of the insignificance of the analysis (Fig. 3).

Fig. 2.

Funnel plot for determining the pooled prevalence of pfmp2/3 gene amplification. The 95% confidence interval (CI) is represented by the dashed lines. The plot shows an uneven distribution of effect size estimates (represented by blue dots) in relation to the middle line, which suggests potential publication bias or heterogeneity among the included studies

Fig. 3.

Sensitivity analysis for the prevalence of pfmp2/3 gene amplification

Pooled proportions of pfpm2/3 gene amplification

The estimated pooled proportions of pfpm2/3 gene amplification via the random effects model were 16.0% (95% CI 8.0%-26.0%). The highest pooled estimate proportions values for pfpm2/3 gene amplification were recorded in Cambodia (79%) and Vietnam (76%) (Fig. 4). Subgroup analysis revealed that the pooled proportions of pfpm2/3 gene amplification were greater in Asia (25.0%, 95% CI 9.0–45.0%) than in Africa (8.0%, 95% CI 2.0–15.0%). For the Asian continent, the highest and lowest pooled estimate proportions values for pfpm2/3 gene amplification were recorded in Cambodia (79%) and the China-Myanmar border (0%), respectively (Fig. 4). Concerning the African continent, the highest pooled estimate proportions values for pfpm2/3 gene amplification were recorded in Equatorial Guinea (68%), whereas the lowest estimates were observed in Uganda, Cameroon, Congo, and Burkina Faso (≤ 1%) (Fig. 4). Subgroup analysis per year of sample collection indicated that the pooled proportions of pfpm2/3 gene amplification were lower before 2016 than after 2016 (11%, 95% CI 3.0–23% and 19%, 95% CI 7.0–36%, respectively) (Fig. 5).

Fig. 4.

Forest plot representing pooled estimates of pfpm2/3/gene amplification across studies from Africa and Asia. **OAC Other African Countries

Fig. 5.

Subgroup analysis of the pooled estimates of pfpm2/3/ gene amplification by sample collection year. **OAC Other African Countries

Discussion

Surveillance of artemisinin and its partner drug resistance markers is crucial for preventing the spread of parasite resistance, as recommended by the WHO, serving as a key pillar in recent resistance mitigation strategies in Africa [38]. PPQ is a partner drug in DHA-PPQ, which is utilised as an alternative ACT for managing malaria clinical cases. DHA-PPQ is also widely implemented for mass drug administration (MDA) in SEA and sSA due to its safety, tolerability, and extended post-treatment prophylactic effects [39, 40]. However, pfpm2/3 multiple copies were first detected in Cambodia in 2013 and have been linked to an increase in vitro PPQ resistance [41, 42]. This is concerning, as SEA has consistently been the epicenter for the emergence and spread of antimalarial drug resistance [17]. This review provides pooled estimates of pfpm2/3 amplification in P. falciparum isolates from the two regions most affected by malaria (Asia and Africa).

The pooled proportions of pfpm2/3 gene amplification in Asia and Africa were lowest (0%) in the Democratic Republic of Congo, Burkina Faso, and the China–Myanmar border [43–45] and highest (79%) in Cambodia [46]. The overall pooled proportions of pfpm2/3 gene amplification recorded in this study were substantial (16%). This prevalence provides unequivocal evidence of the growing risk of PPQ resistance globally, considering that DHA-PPQ is widely used in chemoprophylaxis through MDA and is a potential replacement of SP as part of intermittent preventive therapy in pregnancy ‘IPTp’ [22, 23].

Due to high heterogeneity among the studies included in this review, a continent-wide subgroup analysis was conducted. The pooled proportions of pfpm2/3 gene amplification were higher in Asia than in Africa. DHA-PPQ was adopted as a first-line treatment in most Asian countries, such as Vietnam, and PPQ resistance was first reported in SEA more than 10 years ago, which may explain why Asia has the highest proportion of pfpm2/3 gene amplification. Studies suggest that PPQ resistance in SEA has emerged in the context of artemisinin resistance.

The recorded pooled proportions of pfpm2/3 gene amplification in Africa, although lower than those in Asia, are still substantial and may suggest that PPQ resistance exists in Africa, even though more than 50% of African countries reported an absence or prevalence of less than 1%. Despite the established evidence of significant parasite pfpm2/3 gene amplification in Africa, clinical resistance to DHA-PPQ has not been confirmed in the region, unlike in SEA [27, 47, 48].

Interestingly, it remains unconfirmed whether pfpm2/3 gene amplification is directly involved in mediating PPQ resistance or if it is the parasite mechanism that compensates for the fitness cost of resistance development [49]. Additionally, since pfpm2/3 serves as a surrogate marker of PPQ, the interpretation of results may need to consider evidence from other genetic predictors of resistance. Pfcrt mutations have been shown to play a role in modulating PPQ resistance by facilitating the efflux of PPQ away from its haem target in the parasite’s digestive vacuole [50]. Studies have indicated that the co-occurrence of novel Pfcrt mutations with the exonuclease (Pfexo-E415G mutation) is associated with PPQ resistance phenotypes [51].

The recorded noteworthy prevalence of pfpm2/3 gene amplification from African P. falciparum isolates is alarming since DHA-PPQ is used as an alternative first-line drug to AL in most African countries [52, 53]; thus, drug pressure is expected to be lower. The substantial pooled proportions of pfpm2/3 gene amplification in Africa suggest that pfpm2/3 amplification exists in P. falciparum isolates from Africa despite the low or absence of drug pressure, as reported in Mozambique [54].

A previous meta-analysis reported high DHA-PPQ efficacy in sSA compared to AL and AS-AQ [55], which are the most used forms of ACT [3]. However, the notable pfpm2/3 gene amplification highlighted in this review calls for strict measures to mitigate resistance to DHA-PPQ, taking into account what happened in Asia as a learning platform. Additionally, pfpm2 gene amplification has been recorded to be higher from 2016 to 2020 compared to before 2016.

The emergence and spread of partial artemisinin resistance and partner drug resistance could undermine the gains achieved through decades of investment in malaria control and impact the clinical management of both uncomplicated and severe malaria. It is worth noting that research efforts to develop triple artemisinin-based combination therapies and new non-artemisinin-based antimalarials, such as KAF156-lumefantrine, are ongoing; however, new treatment options are not yet available to provide alternatives for treatment [56].

As DHA-PPQ is increasingly deployed for malaria control through MDA strategies and the future use in ACT diversification as an approach to mitigate artemisinin resistance, increased efforts to implement genomic surveillance to characterise the evolution of parasite genetic signatures due to selection pressure need to be prioritised to inform appropriate resistance mitigation strategies.

Like any other study, this review is not without limitations. First, there may be unpublished data that could not be assessed during the online search. Second, pfpm2/3 gene amplification has been documented in only a few countries, and the review search was restricted to English, excluding data from other languages; thus, the picture presented in this study may not accurately reflect the extent of pfpm2/3 gene amplification across the two continents. Lastly, the influence of transmission on the magnitude of pfpm2/3 gene amplification was not studied due to limited information from the authors. Despite these limitations, this review is unique in its own right; it is the first study to establish pooled estimates of pfpm2/3 gene amplification in P. falciparum isolates, providing a snapshot of PPQ resistance.

Conclusion

The substantial amplification of the pfpm2/3 gene in both Asia and Africa is a public health concern regarding PPQ resistance; therefore, further molecular surveillance studies and interventions are necessary to mitigate P. falciparum resistance to piperaquine as part of ACT. This is essential to prevent total resistance to ACT, which could hinder global efforts to eliminate malaria.

Abbreviations

ACT

Artemisinin-based combination therapy

AL

Artemether-lumefantrine

AS-AQ

Artesunate-amodiaquine

CQ

Chloroquine

DHA-PPQ

Dihydroartemisinin-piperaquine

IPTp

Intermittent preventive therapy in pregnancy

MDA

Mass Drug Administration

PPQ

Piperaquine

pfcrt

Plasmodium falciparum chloroquine transporter

pfK13

Plasmodium falciparum Kelch 13

pfpm2/3

Plasmodium falciparum plasmepsin2/3

SEA

South East Asia

sSA

Sub-Saharan Africa

SP

Sulfadoxine-Pyrimethamine

WHO

World Health Organization

Author contributions

KJM participated in concept development, screening of studies, data analysis, drafting and submission of the manuscript and correspondence. AK and MZ participated in concept development, screening of studies, and data analysis. RHM, RM, VB and MK participated in manuscript drafting, revision, and submission. BPM, EK and JPAL participated in critically reviewing the manuscript. All the authors read and approved the final manuscript for submission and publication.

Funding

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable because it is a systematic review and metanalysis.

Consent for publication

Not applicable for a systematic review and meta-analysis.

Competing interests

The authors declare that they have no competing interests.