Background
Artemisinin combination therapy (ACT) remains the most effective currently available regimen for multi-drug-resistant falciparum malaria, and as such is key to reducing the global malaria burden [
1,
2]. While artemisinin-resistant
Plasmodium falciparum has emerged in the western provinces of Cambodia, and some neighboring countries in Southeast Asia [
3‐
6], ACT appears to remain clinically effective where partner drug efficacy is preserved. Dihydroartemisinin-piperaquine (DHA-PPQ) has been widely adopted in the region as a first-line agent to treat multi-drug resistant falciparum malaria. In 2008, it replaced artesunate-mefloquine (AS-MQ) in select areas of western Cambodia due to AS-MQ treatment failures [
7]. In early studies, various PPQ-containing ACT showed excellent safety and tolerability with efficacy of 96–98 % in Cambodia [
8‐
12], and DHA-PPQ was implemented as the drug of choice countrywide a few years later [
13].
Treatment failures were detected soon after, as early as 2010, with PCR-corrected day-42 failure rate of 25 % in Pailin and 11 % in Pursat Provinces in Cambodia [
14]. In 2013, high grade DHA-PPQ failure of 53 % was first reported in Oddar MeanChey province with corresponding increases in PPQ in vitro IC
50 higher than patient plasma PPQ levels during the terminal elimination phase [
15‐
17]. Subsequent reports confirmed rapidly increasing DHA-PPQ failure rates in areas with previously documented artemisinin (ART) resistance [
18,
19]. Despite the rapid loss of PPQ sensitivity, multiple studies detected a simultaneous decline in
pfmdr1 copy number amplification and increased in vitro MQ sensitivity among PPQ-resistant isolates [
15,
18,
20]. The resurgence in MQ sensitivity suggested that short-term re-introduction MQ-containing therapy or perhaps triple artemisinin combination therapy (TACT) containing both PPQ and MQ might be effective. While approaches are currently being pursued to various degrees by public health authorities and research organizations [
18], there is no consensus regarding safety and resistance concerns related to the use of TACT [
21].
To address this growing public health crisis, intensive monitoring of drug resistance profiles remains crucial information to determine appropriate alternatives in settings of rapidly emerging multi-drug anti-malarial resistance. In the absence of a known molecular PPQ resistance marker, field-based ex vivo parasite drug susceptibility testing using fresh
P. falciparum isolates remains a cost-effective, rapid, surveillance tool to track resistance [
22]. Here, parasite drug susceptibilities to ART, PPQ and MQ between 2010 and 2015 on both sides of the Thai–Cambodian border, and in southwestern Thailand along the border with Myanmar were reported.
Discussion
Monitoring ex vivo drug susceptibility of
P. falciparum revealed rapid progression of PPQ resistance in northern Cambodia between 2013 and 2015, following reports of severe clinically significant resistance in 2013 [
15,
18], while western Thai isolates collected from the same time period remained sensitive to PPQ corresponding with a recent report of 94 % efficacy of DHA-PPQ in this region [
5]. The northeastern Thai isolates showed modest worsening of PPQ resistance given their close proximity to the Cambodian parasite populations studied (within 100 km). Increased PPQ IC
50 and IC
90 associated with recrudescence in DHA-PPQ clinical studies from 2010 to 2013 corresponded with the first reports of DHA-PPQ clinical treatment failures in Cambodia [
18,
19,
26]. The findings enforce those of recent studies, indicating the rapid expansion of PPQ-resistant parasites in Cambodia, as well as early signs that piperaquine resistance is emerging in Thailand. Given rapid emergence in Cambodia, and the fact that DHA-piperaquine has only recently been introduced on a large scale, the possibility for rapid worsening of resistance in Thailand is high. Limited treatment to choose from there is an urgent need for alternative therapies in the region. Whether the rapid expansion of PPQ resistance is due to increased transmission potential of PPQ-resistant isolates as observed for ART-resistant parasites [
6] or slow-clearing parasites following DHA-PPQ treatment [
27], requires further investigation.
The majority of both Cambodian and Thai isolates were resistant to ARTs in a RSA, consistent with a high prevalence of mutations in the propeller domain of the
P. falciparum kelch13 associated with ART resistance in nearly 100 % of isolates [
15]. This supports emerging PPQ resistance on an ART resistance background given the high rate of clinical DHA-PPQ failures [
18,
26], although there was no clear cross-resistance between PPQ and the ARTs, in part given the very high rate of ex vivo ART resistance. Similar to previous reports [
15,
18,
26], the opposite trend was observed for MQ with increased ex vivo sensitivity noted in Cambodian isolates from 2013-2015. Studies also revealed the association of DHA-PPQ treatment failure with single copy of
P. falciparum multi-drug resistance gene
(pfmdr1) and decreased MQ IC
50. The concomitant increase in MQ sensitivity coupled with worsening PPQ resistance could be explained by declining PPQ susceptibility within parasite population, being genetically distinct from those of MQ resistance. A recent population genetics study revealed emergence of PPQ resistance related to clonal expansion of MQ-sensitive parasites with a single
pfmdr1 copy (Parobek et al. pers. comm.)
Ex vivo parasite susceptibility to PPQ, particularly the IC
90, appears to be a sensitive, field-expedient and relatively cost-effective marker to detect emerging PPQ resistance at sentinel sites in high-risk areas. Surveillance data first revealed the occurrence of PPQ-resistant parasites in Cambodia in 2010, a year before the clinical failure rate of DHA-PPQ began to increase. Although isolates from 2010 largely remained in the IC
50 susceptible range, elevated IC
90 was detected in three isolates, two of which were in patients who failed DHA-PPQ treatment [
28]. In 2013 when clinical failure for DHA-PPQ was prevalent, IC
90 was elevated in 40 % of isolates though more than half still had IC
50 in the sensitive range. A rapid progression of resistance was detected in years following with rapid rises in both IC
90 and IC
50 for most isolates. Decreased parasite susceptibility to PPQ was associated with DHA-PPQ treatment failure, notably for IC
90, which was three-fold higher in subjects failing treatment. IC
90 appeared to better elucidate shifts in resistance patterns over time and their association with treatment outcomes compared to IC
50. Anomalous curves observed with PPQ-resistant isolates in the HRP-2 assay required PPQ concentrations up to 53,905 nM (>25 times higher than the maximum concentration used by Duru et al. [
26] before 100 % growth inhibition producing an interpretable, sigmoidal, dose–response curve could be achieved (Fig.
2). Similar to findings with HRP-2, a recent study reported poor performance of the standard hypoxanthine uptake assay due to the high frequency of non-interpretable curves observed with PPQ-resistant isolates. The anomalous curves produced suggested a paradoxical increase in parasite growth at PPQ concentrations above 100–200 nM, reflecting a resistance mechanism induced at physiological concentrations (~200 nM) observed in the blood of patients treated with DHA-PPQ [
26,
29]. Evidence of paradoxical growth at high concentrations of drugs was previously described in non-ART drug assays [
30]. This was suggested to reflect biological properties of drugs, including mechanism of action. Amendment of the lower constraint of the sigmoid model was suggested to provide a more accurate measurement. However, this is not the case for the paradoxical growth seen among resistant isolates in the PPQ assay demonstrated here, or in another recent report [
26], and may represent a phenomenon of PPQ-resistant isolates.
To compensate for these challenges for interpretation, a novel in vitro assay for PPQ resistance, the PPQ survival assay (PSA), was designed to mimic in vivo exposure of parasites to a pharmacologically relevant dose of PPQ (200 nM) for 48 h with the % parasite survival rate measured at 24 h after drug exposure [
18,
21,
26]. A 48-h drug incubation period allows adequate PPQ exposure for all parasite stages from 0-h rings to 48-h schizonts, given PPQs having long clinical half-life (~9 days) [
26,
29]. As a result, the assay does not assess parasite susceptibility to PPQ at a specific parasite stage, but rather the entire life cycle. PSA was demonstrated to be a useful tool to differentiate PPQ resistance among individual fresh isolates, and the % survival rate was found to correlate with DHA-PPQ treatment outcome. Future investigations employing this method are likely to further elucidate emerging piperaquine resistance, although the requirement for multiple individual microscopy readings on each sample limits assay throughput to some degree. The choice of method (PSA, HRP-2, etc.) should be based on available resources and experience. In our view, consistency of application, reproducibility and interpretability of results across time and place are far more important than the technique selected.
The limited sample size for Thai isolates, as well as lack of more recently collected samples are important limitations of the current study. This was mitigated by the sequential approach employed at the sentinel sites, increasing the degree of confidence that median values presented here were reflective of relative drug sensitivities at the community level. Isolates from the Thai–Myanmar border remained highly sensitive to PPQ despite ART resistance comparable to the levels seen in Cambodia, as well as modest MQ resistance. Samples were collected before the widespread use of PPQ in Thailand when AS-MQ remained the first-line drug. While there has clearly been rapid expansion of resistance in northern Cambodia, there appears to be only limited spread across the Thai border, with isolates from nearby PL, Thailand having only moderate declines in PPQ susceptibility. This may be due in part to a lower burden of disease in northeastern Thailand permitting more stringent control measures, and anti-malarial drug administration limited to well-resourced government public health clinics. Regardless, ongoing monitoring is imperative in this region.
Several reports have suggested that ART resistance could promote development of resistance to PPQ, as ART-resistant isolates surviving in the presence of ACT are more likely to spontaneously develop resistance to partner drugs [
18,
21]. The association between PPQ resistance and the K13 mutation likely reflects selection of PPQ-resistant parasites as those initially resistant to ARTs remain viable well after ACT treatment has been completed. This has been supported by apparent correlations between increasing DHA-PPQ failure rates on a background of ART-resistant isolates with K13 mutations [
17‐
19]. A recent study reported that all PPQ-resistant isolates carried the K13 mutation, although direct correlation between PPQ and ART susceptibility was not clear as PPQ-sensitive isolates also carried K13 mutation [
26]. This may be due to the high prevalence of K13 in the parasite populations studied, and limits potential analysis within the present dataset. Cutoff IC
50 or IC
90 values for PPQ resistance have yet to be defined, we have not made an effort here to compare % survival rate to ART between PPQ ‘resistant’ and ‘sensitive’ isolates. We performed a Spearman correlation analysis between % survival rate and PPQ IC
50 or IC
90 and also compared PPQ IC
50/IC
90 between ART-sensitive (RSA ≤ 1 %) and resistant isolates (RSA > 1 %). A significant correlation between ART and PPQ resistance was not found (see Additional file
1).
Making use of these data for public health purposes presents important challenges, although there does appear to be a key opportunity in the inverse resistance pattern between PPQ and MQ. PPQ resistance appears to be developing in MQ-sensitive parasites with single-copy
Pfmdr-
1 and low MQ IC
50, as PPQ-resistant isolates in a recent study (PSA survival rates ≥10 %) all had a single
Pfmdr-
1 copy and were largely MQ sensitive [
26]. Correspondingly, a parasite population genetics study identified the development of resistance to PPQ and MQ in genetically distinct parasite populations (Parobek et al. pers. comm.). In the interim, the national control programme in Cambodia has reverted to the re-introduction of AS-MQ ACT in selected areas of PPQ resistance, and previous studies have observed excellent cure rates using AS-MQ as rescue therapy for DHA-PPQ treatment failures [
17,
28]. Recent investigations have proposed triple-drug therapy such as simultaneous AS, MQ and PPQ [
18], although the safety of such combinations has not been studied. Further, the possibility of inducing simultaneous resistance to all three drugs amidst rising resistance to the individual components would argue for preserving the advantages afforded by inverse MQ and PPQ resistance patterns by rotating two drug ACT combinations sequentially, combined with inpatient follow-up of all cases to ensure compliance [
21]. Doing so in a carefully coordinated fashion on a biannual basis would permit adequate time to restore at least partial partner drug sensitivity, while holding the current second-line therapy in reserve for clinical treatment failures. Atovaquone-proguanil used as part of public health containment activities in Cambodia may prove to be a useful albeit costly third-line agent, as isolates from the same areas of PPQ resistance in northern Cambodia reported here were recently shown to remain highly susceptible to atovaquone (ATQ) in vitro without evidence of pre-treatment mutations in the cytochrome b gene codon 268 marker for ATQ resistance [
24]. However, this liability is well known, with several studies reporting rapid development of single point mutations in cytochrome b conferring ATQ resistance during treatment [
31‐
33]. Neither approach will adequately address the current high-grade ART resistance in the region, although there is hope they may extend the life of current ACT until novel compounds in development, such as the spiroindolones [
34] and synthetic ozonides [
35], become available.
Authors’ contributions
Study design Cambodia: SC, CL, DS, SP, and CL; study design Thailand: KJ, NU, MMF, JG, NS, DS, DB, and DSW; data collection: all; data analysis and interpretation: SC, DS, SSundrakes, KJ, and CLon; wrote manuscript: SC, DS, KJ, and CL; All authors read and approved the final manuscript.