In addition, the NMCP recommended the intermittent preventive treatment IPT of pregnant women with sulfadoxine–pyrimethamine (SP) and seasonal malaria chemoprevention (SMC) for children with SP-amodiaquine (Bamba et al., 2013; Mbaye et al., 2017).

Unfortunately, the recent emergence of P. falciparum resistance to artemisinin derivatives in Southeast Asia challenges malaria control and elimination efforts. This situation is increasingly compromised  by concurrent resistance to partner drugs in combination therapies, such as the piperaquine  (Witkowski et al., 2017).

In 2014, the first report of DHA–PPQ treatment failures was published (Saunders et al., 2014). Therapeutic failures were estimated to reach 60% indicating a dramatic expansion of piperaquine resistance. Moreover, full P. falciparum resistance to DHA/PPQ treatment has been reported recently in Cambodia (Amato et al., 2017). These events were directly associated with increased copy number variations (CNVs) in the plasmepsin system, including the pfPM2 gene (PF3D7_1408000) coding for the food vacuole enzyme plasmepsin II (Sanogo et al., 2018). CNV is generally considered as emerging at relatively rapid mutation rates (Cheeseman et al., 2009).

The relevance of the Pfplasmepsin 2 (PfPM2) gene amplification is conferring any survival or fitness advantages in response to PPQ pressure. Piperaquine resistance is poorly characterized. It is currently identified by late clinical failures with amplification of the plasmepsin 2 copy number gene. Although several genetic variations have been associated with decreased piperaquine susceptibility: a single copy of the mdr1gene has been associated with dihydroartemisinin–piperaquine treatment failures in Cambodian patients (Witkowski et al., 2017).

However, PfPM2copy number was the most significant molecular signature associated with dihydroartemisinin–piperaquine treatment failure (Witkowski et al., 2017). The piperaquine resistance has not yet been observed in area where artemisinin resistance has not also been documented as Senegal. However, it is possible that plasmepsin 2 amplifications will be found in other regions where piperaquine has been used as a partner drug  (Bopp et al., 2018). Thus the assessment of PfPM2 gene copy number to areas where piperaquine is being used in artemisinin-based combination therapies is important. In line with the NMCP strategy for the surveillance of antimalarial drug efficacy, the aim of this study was to do a surveillance of multiple copies of PfPM2 gene, potentially involved in piperaquine resistance.

Study site and sample collection                          

In total, 120 patient samples from two regions of Senegal were selected: Dakar (60), Kedougou (60), which present different levels of malaria transmission intensity (Figure 1). In Dakar malaria transmission is low and parasite prevalence is estimated at 1.3%. In the southeastern Kedougou region, the level of malaria transmission is high with an incidence higher than fifteen malaria cases per 1000 habitants (PNLP-Senegal, 2018). In Dakar samples were collected between October and November 2015 while in Kedougou the samples were collected in September 2016. These Samples were collected in the some months because these months corresponding to periods of high transmission in Senegal. For routine surveilliance in a region of low transmission such as Dakar, we estimate that a maximum sample size would be required to obtain a positive case compared to Kedougou with high transmission. Venous blood samples were collected in 5 ml vacutainer tubes and filter paper was made for molecular testing. All individuals in this study presented with uncomplicated malaria and parasite presence and species was confirmed by microscopy.

Ethical approval

The study protocol was approved by the National Ethics Committee for Health Research of Senegal (CNERS). Before participant recruitment and sample collections were initiated, written and informed consent was obtained from all participants.

Copy number variation assays

Parasite DNA was extracted from 120 samples using the QIAamp DNA Blood Mini kit (Qiagen) according to manufacturer instructions (QIAamp DNA Blood Mini Kit, Qiagen, Valencia,CA). The relative PfPM2 copy number were assessed by qPCR using real-time PCR machine (ABI 7500) as described by Witkowski with minor modifications in the Sequences of fragments synthetic genes (Witkowski et al., 2017). As a single copy endogenous gene control, we used the single copy β-tubulin gene and P. falciparum genomic DNA from strain 3D7 which has one copy of PfPM2 was included in each run as controls.

Briefly, quantitative PCR (qPCR) was carried out in 20 μl volumes in a 96-well plate containing 10 μl qPCR EvaGreen dye Supermix, 1 μl of each forward and reverse primer (Table 1), 3 μl H₂O and 5 μl of template DNA. Amplifications were performed under the following conditions: 98°C for 3 min, followed by 40 cycles of 95°C for 10s and 58°C for 20 s and amplifications were run in triplicates.

Determination of the copy number

Amplification of the PfPM2 genes was measured by using five standards of mixed synthetic gene fragments (Table 2).The number of copies for each gene is determined relative to a standard curve. The standard curve is obtained from a mixture of fragments synthetic  genes  of  Pfplasmepsin2  and  Pfβtubulin  by making five molar ratio following theses dilutions: standard 1 (1:1 PfPlasmepsin2-Pfβtubulin), standard 2 (2:1 PfPlasmepsin2-Pfβtubulin), standard 3 (3:1 PfPlasmepsin2-Pfβtubulin) and standard 4 (4:1 PfPlasmepsin2-Pfβtubulin)

The standards were also quantified according to the following Protocol: Quantitative PCR (qPCR) was carried out in 20 μl volumes in a 96-well plate containing 10 μl qPCR EvaGreen dye Supermix,1 μl of each forward and reverse primer (Plasmepsin2 primer for the Pfplasmepsin2 gene and β-tubulin primer for the Pfβ-tubulin gene), 6 μl H₂O and 2 μl of template DNA. Amplifications were performed under the following conditions: 98°C for 3 min, followed by 40 cycles of 95°C for 10 s and 58°C for 20 s and amplifications were run in triplicates. The number of copies of PfPlasmepsin2 will be calculated according to the fold change (2 ^ -ΔC?) method.

ΔCt = Ct PfPlasmepsin2 - Ct Pfβ-tubulin and the Ct is deduced from the standard curve.

Once the 2 ^ -ΔC? is obtained for the five standards, we used it to make a standard curve with a line equation y = ax + b, it will be used to determine the number of copies of the PfPM2 gene present in each of our samples (Figure 2). In our study a copy number of PfPM2> 1.7 was defined as an amplification of this gene. Our CVN for the control (3D7) was 0.7.

Using a copy number threshold of 1.7 the relative PfPM2 copy number was assessed in 120 P. falciparum positive patients, 60 in Dakar and 60 in Kedougou. From the samples  with  a  valid  copy  number  estimate (n = 115),  1.73% carried a multiple copies of PfPM2, whilst one copy of the gene were  found  in  98.26%  of  the  isolates (Figure 3). A multiple copies were noted only in Kedougou with 3.33%. In Dakar all the samples presented a single copy.

DHA/PPQ has shown near-perfect efficacy levels in clinical trials conducted in Africa, the combination also has been proposed as second-line treatment of Plasmodium falciparum malaria in Senegal. Unfortunately, full P. falciparum resistance to DHA/PPQ treatment has been reported recently in Cambodia. These events were directly associated with increased copy number variations in the PfPM2 gene. Therefore, preexisting PfPM2 duplications in Cambodia might have been rapidly selected by DHA/PPQ, aided by a less effective protective action of the artemisinin deriva­tive (Hastings et al., 2016). Such a scenario suggests that this copy number variation may already be present in Africa. Accurate and timely surveillance of drug resistance markers aids in maintaining and prolonging the efficacy of the limited selection of anti-malarial drugs available. Thus, it becomes opportune to follow this emergence and it is for this reason that we have chosen to study the number of copies of the PfPM2 gene in order to determine the number of copies circulating in each individual of P. falciparum in Dakar and in Kedougou. 1.73% carried  a  multiple  copies  of  PfPM2,  whilst  one copy of the gene were found in 98.26% of the isolates from Kedougou (Figure 2).The copy number of PfPM2 genes was differ between isolates from different sites. An amplification of this gene was only reported in Kedougou. Kedougou is a particular site with presence of gold mining, more than 10 nationalities are represented in this region, with a strong representation of Malians, Burkinabés, and Guineans. This relative affluence favors the informal sale of antimalarial drugs and promotes abusive self-medication. This is one of the key factors in the emergence of drug resistance (Ministere de l’economie et des finances, 2018).This situation can explain the presence of multiple copies of PfPM2 gene in this area.

Our results show that the multiplications associated with resistance to piperaquine are probably already frequent in Senegal, which is of concern given the use of DHA / PPQ as a second line of treatment in Senegal. Recently complete resistance of P. falciparum following DHA / PPQ treatment has been reported in Cambodia and this resistance has been directly associated with an increase in the number of copies of the PfPM2 2 gene (Amato et al., 2017).

In Mali, in 65 out of the 96 samples it was confirmed the presence of 7 infections carrying 2 copies of PfPM2 (Sanogo et al., 2018). Previously a multiple copies of pfpm2 was also found in Mozambique with a frequency of 1.1% in 351 samples (Gupta et al., 2018).This same situation has already been found in a study carried out in high transmission areas of West Africa (Mali, Burkina Faso and Guinea) by the West African Network for Antimalarial Drugs (Sagara et al., 2018). Another study showed a strong presence of multiple copies of the PfPM2 gene in Uganda with a frequency of 33.9% or 38/112 and a low copy frequency in Gabon and Mozambique with respectively frequencies of 11.3% (8/71) and 12.5% ??(1/8) (Leroy et al., 2019).In Ethiopia and Cameroon only a single copy of PfPM2 was detected in two isolates after DHA-PPQ failures (Russo et al., 2018).Paradoxically, PfPM2 in multiple copies is generally found in parts of Africa where DHA-PPQ failures are rare and resistance to PPQ has not yet been described. However, there is no evidence of PPQ resistance in Senegal although in this study we found a presence of multiple copies of the PfPM2 gene.

Recently, the clinical and parasitological efficacy of DHA/PPQ has been estimated at 97.6% with only 2.5% of individuals experimenting late therapeutic failure (Diallo et al., 2020). The rare cases of failure of PPQ-based treatments described in Africa were not associated with amplification of the PfPM2 gene (Leroy et al., 2019).

However, a new study show that PfCRT Thr93Ser and Ile218Phe mutation have been shown to confer resistance to PPQ without the presence of pfpm2 duplications (Dhingra et al., 2019). Also multiple copies of PfPM2 and pfcrt-F145I were detected in 2014 (12.8%) and increased to 30.4% in 2015. Parasites containing either multiple PfPM2 copies with and without pfcrt-F145I or a single pfpm2 copy with pfcrt-F145I exhibited elevated IC90 values of piperaquine (Boonyalai et al., 2021).

In summary PfPM2 duplications correlates with PPQ resistance, but duplication is not essential for resistance. Therefore, plasmepsin copy number should not be used as a sole indicator of PPQ resistance. Additionally, some polymorphisms in pfcrt can confer resistance to PPQ in Dd2 parasites and the E415G pfexo mutation has been correlated with DHA-PPQ resistance in Cambodian isolates (Moss et al., 2022).

In other words, in Africa it is unclear whether the amplification of PfPM2 is necessary and/or sufficient for the development of resistance to PPQ. These observations suggest that, further molecular surveillance of PPQ resistance including multi-genes will be necessary.

The emergence of DHA–PPQ resistance greatly threatens the efficacy of the remaining ACTs worldwide. With the availability of PfPM2 copy number as molecular markers of piperaquine resistance, it is necessary to have robust assays that can be used to monitor the presence and frequency of these markers in contemporary parasite isolates across endemic regions. In this current study we found a presence of multiple copies of PfPM2 gene but the association between these amplifications and a clinical resistance to PPQ was not verifiable. However, an additional functional work is needed to better understand the mechanisms of PPQ resistance and to identify the association of increased piperaquine IC50 values with the copy-number variation. Also a continuous Surveillance for increased PfPM2 copy number could aid malaria control efforts by pinpointing areas where these drugs may be failing like in Senegal.

The authors have not declared any conflict of interests.

The authors appreciate the African Center of Excellence for Genomics of Infectious Disease (ACEGID), the International Centre for Excellence in Malaria Research (ICEMR) project and the Parasitology and Mycology Laboratory Le Dantec Hospital. They also appreciate Younouss Diedhiou, Lamine Ndiaye, Amadou MactarMbaye, Daba Zoumaro for their contribution to the sample collection and patients and staff for their contribution to this study.