Measuring ex vivo drug susceptibility in Plasmodium vivax isolates from Cambodia

Cambodia continues to be at the epicenter of globally emerging multidrug resistant malaria. This has prompted intensive elimination efforts accompanied by surveillance to characterize Plasmodium falciparum resistance. Over the past several decades, P. falciparum has developed resistance to numerous drugs, particularly in Southeast Asia where high grade treatment failures have been documented [1‐4]. Since 2000, the use of artemisinin (ART)-based combination therapy (ACT) as first-line treatment has been implemented in nearly all malaria endemic areas to overcome resistance developing as a result of monotherapy treatments. However, P. falciparum resistance to ART emerged in 2006, just a few years after introduction of artesunate–mefloquine (AS–MQ) in Cambodia [5, 6], and was later confirmed on a large scale by intensive multidisciplinary surveillance studies [7]. In 2013, the first high grade clinical failures of dihydroartemisinin–piperaquine (DHA–PPQ) were reported [8]. DHA–PPQ had only recently been introduced as first-line therapy in Cambodia. Reduced in vitro P. falciparum susceptibility to PPQ developed on a background of artemisinin resistance here [9, 10], with specific molecular mechanisms elucidated soon thereafter [11, 12].

While intensive surveillance for P. falciparum continues in Cambodia, little is known about Plasmodium vivax drug resistance. Clinical P. vivax resistance is far more difficult to characterize due to difficulties distinguishing true recrudescence of resistant parasites, reinfections with new blood-stage P. vivax infections, and relapsing infections by liver stage hypnozoites. Moreover, inability to maintain long-term culture of P. vivax parasites prevents reproducible assessments of parasite drug susceptibilities. Plasmodium vivax resistance is generally assumed to be less pronounced than P. falciparum, as blood-stage P. vivax infection remains clinically susceptible to most available anti-malarials. CQ resistant P. vivax was first reported in 1989 from Papua New Guinea (PNG) and Papua Indonesia [13‐16], where CQ monotherapy remains ineffective. Chloroquine-resistant vivax isolates in this region have been found to harbour polymorphisms in the pvmdr1 (P. vivax multidrug resistance 1) gene, whereas amplification of the gene has been associated with reduced susceptibility to MQ and other drugs in vitro [17‐20]. Sporadic cases of CQ failure have since been reported in parts of Southeast Asia and South America [21‐24], but there is no clear evidence that these are associated with pvmdr1 mutations in these regions. Thus, the usefulness of this putative drug resistance marker for detecting emerging resistance remains uncertain.

Artemisinin-based combination therapy is now recommended for use as first-line agent for all malaria in areas of multidrug resistant P. falciparum, particularly where clinical failures have been documented [25, 26]. In Cambodia, DHA–PPQ was used as first-line therapy for both vivax and falciparum malaria since 2011 to respond to declining efficacy of CQ for P. vivax treatment in specific northern and western provinces where the clinical cure rate had dropped to 80–90%, though cure rates have remained near 100% elsewhere [27‐29]. The approach was also implemented to simplify drug administration and overcome diagnostic difficulties. Limited diagnostic capacity makes distinguishing P. vivax from P. falciparum microscopically challenging, despite overall increases in diagnostic capacity after years of effort. Frequent relapse of latent P. vivax following treatment for blood stage P. falciparum infections, and high prevalence of difficult to distinguish mixed species infections pose further challenges. While the use of DHA–PPQ to treat CQ-sensitive P. vivax is thought to be effective and convenient [26], it may in fact exacerbate resistance to both P. vivax and P. falciparum [30].

To investigate P. vivax anti-malarial susceptibility in Cambodia, fresh P. vivax isolates collected from Northern provinces from 2013 to 2015 were assessed for sensitivity to commonly used drugs in short-term culture. Growth inhibition was measured by both the microscopy-based schizont maturation test (SMT) and Plasmodium pan-species lactate dehydrogenase (pLDH) ELISA. SMT had been considered the conventional method for P. vivax drug testing, but not sensitive for low parasitaemia samples. It is also labour-intensive and interpretation of results is subjective. pLDH ELISA has emerged, offering notable advantages. pLDH has demonstrated higher sensitivity for detection in settings of low P. vivax growth rate and very low parasitaemia, improving yields for IC₅₀ determination [31]. Results from these assays were then compared with those from concurrently collected P. falciparum isolates analysed in both the novel pLDH assay and the previously established HRP-2 assay [32]. Plasmodium vivax multidrug resistance gene 1 (pvmdr1) mutations and copy number, which have been proposed as candidate markers for drug resistance, were analyzed in a subset of isolates. This is the first report that we are aware of that documents ex vivo P. vivax drug susceptibility in Cambodia, and provides baseline data for future surveillance and elimination efforts.

This is the first report of ex vivo P. vivax drug susceptibility testing of field isolates in Cambodia, providing important baseline data for ongoing resistance surveillance. The focus of containment and elimination efforts in Cambodia to date has been multidrug resistant P. falciparum malaria. Relatively little is known about P. vivax drug sensitivity due to the inability to culture P. vivax long term. The pLDH method bypasses this critical limitation by using fresh isolates, making it potentially practical and informative for measuring ex vivo P. vivax resistance. While further validation of the assay is needed, the correlations observed with the IC₅₀ results with P. falciparum, in both the pLDH and previously established HRP-2 assays, lend support to its utility as a surveillance tool [32]. The P. falciparum HRP-2 assay has been carefully standardized in an effort to reliably produce interpretable IC₅₀ results in P. falciparum field isolates over time [32]. However, it cannot be used in P. vivax which does not produce HRP-2. Unfortunately, we were unable to establish the P. vivax SMT using previously described methods. Potential explanations for the poor success rate of SMT could include low parasitaemia and drug residue in samples. Nearly 40% of P. vivax samples has parasitaemia < 0.1%, and issues of self-medication and unregulated anti-malarial distribution in Cambodia are well documented [42]. Further, WBC depletion process seems to retard P. vivax growth leading to failure for SMT. The loss of P. vivax and time spent during the WBC filtration process could be possible reasons. However, less effect was observed in pLDH ELISA in which IC₅₀ from whole blood, and WBC depleted samples testing were well correlated. This corresponded with the previous finding on P. falciparum HRP-2 method, suggesting the reliability of assay on whole blood sample without WBC removing [43]. Overall, in addition to being a field expedient method, the pLDH method was able to reveal some important information about P. vivax susceptibility.

With the possible exception of chloroquine, blood stage P. vivax is generally thought to have remained susceptible to a wide variety of anti-malarials, though data is limited. Using the pLDH-ELISA, P. vivax appeared significantly more susceptible to dihydroartemisinin (DHA), artesunate (AS), and chloroquine (CQ) than P. falciparum,but less susceptible to mefloquine (MQ), and artemisone (ATM), and similarly susceptible to lumefantrine (LUM), piperaquine (PPQ) and doxycycline (DOX). It should be noted that although DOX IC₅₀ values were in the micromolar range, they were still below previously proposed values for resistance [44]. The present study reveals Cambodian P. vivax isolates appear to remain sensitive to CQ while resistance to other anti-malarials may be worse than previously assumed, though the absence of baseline values precludes definitive conclusions. Although previously established, well-controlled pLDH methods were used here to test both P. vivax and P. falciparum, inter-species difference and assay bias between 2 species cannot be ruled out. Inter-species comparisons require careful interpretation, especially in the absence of baseline data. In addition, mixed parasite stages found in P. vivax samples may confound pLDH results for drugs with stage-specific activity. It is possible that the pLDH results of P. vivax here represent an average overall susceptibility of mixed parasite stages. As an example, our findings indicated that mixed stage P. vivax remained susceptible to CQ, though CQ has specific ring stage activity [37].

The present study brings to light important methodologic considerations for assessing P. vivax resistance in vitro. The effect of parasite growth efficiency on P. vivax pLDH assay was minimal. There were not significant differences in pLDH-ELISA IC₅₀ for most drugs tested between isolates reaching ≥ 40% schizonts and those with less growth. Thus, the pLDH assay had utility even in P. vivax isolates failing to reach the 40% schizont target required for the SMT. pLDH-ELISA validity was also confirmed by successful discrimination of known susceptibility profiles for 3D7 (CQ sensitive, MQ-resistant) and W2 (CQ-resistant, MQ-sensitive) P. falciparum laboratory strains. When testing P. falciparum clinical isolates, pLDH-ELISA was able to detect PPQ resistance at several 100-fold higher IC₅₀, corresponding with results from the 72 h HRP-2 ELISA. Comparison with these previously well benchmarked assays further supports use of pLDH-ELISA for P. vivax isolate drug susceptibility testing.

Based on our pLDH-ELISA results, stage-specific drug activity on P. vivax growth was apparent for DOX and AS, but less pronounced for DHA, LUM and MQ (Table 1). Isolates initially at the trophozoite stage had significantly higher IC₅₀s to these drugs than those initially at the ring stage. Specific activity of chloroquine on ring stages, previously described for the schizont maturation test (SMT), was not detected here using the pLDH-ELISA. Comparative P. vivax testing at ring and trophozoite stages for the same isolates may confirm stage-specific activity of these drugs. Duration of drug incubation in the SMT is another factor previously reported to influence in vitro drug responses for P. vivax [37]. A prior statistical modelling study of SMT dose–response data indicated that only assays with initial ring stage parasitaemia ≥ 65% and a duration ≥ 35 h produce robust IC₅₀ values [45]. More data is required to identify the threshold where the association between IC₅₀ assay duration and parasite stage composition disappears in the pLDH ELISA.

Comparing findings of the present study to those reported previously for P. falciparum and P. vivax isolates from the Brazilian Amazon [46, 47] and Indonesia [36, 48, 49] using pLDH ELISA and SMT assays, Cambodian isolates were found to be less susceptible to MQ and PPQ. However, Cambodian and Brazillian P. vivax isolates were more sensitive to CQ than in Papua Indonesia where P. vivax CQ resistance has emerged. Another ex vivo study reported P. vivax CQ resistance in 60% of isolates collected from the Thai-Myanmar border, and higher median IC₅₀ than in Cambodia [50]. Although, comparability of the SMT and pLDH ELISA have yet to be formally established, these regional differences are not surprising. Reduced MQ and PPQ susceptibility of Cambodian isolates reflects higher drug pressure in the region from long term use. High grade PPQ resistance recently emerged in P. falciparum with resultant effects on sympatric P. vivax infection [10, 30]. Chloroquine resistant P. vivax infections have been detected in Indonesia since the 1990s and, in 2008, the national treatment guidelines for P. vivax were changed to ACT [51]. Approximately 60% of P. vivax patients treated with chloroquine experienced a recurrence within 28 days in studies from Malaysia and Vietnam [24, 52]. Yet chloroquine sensitivity was better preserved in studies conducted in Cambodia [29, 44], the Brazilian Amazon [53], Myanmar [54], India [55] and Ethiopia [56].

Piperaquine phosphate IC₅₀s of Cambodian P. vivax isolates were higher than Indonesia, but the median was similar to those of P. falciparum from the same region. However, some Cambodian P. falciparum isolates were able to grow in much higher PPQ concentrations with up to 100-fold greater IC₅₀s than P. vivax, suggesting greater overall susceptibility in P. vivax compared to P. falciparum. In 2012, AS–MQ was replaced with DHA-PPQ as national first-line treatment for both P. falciparum and P. vivax [57]. Since reduced ex vivo P. falciparum PPQ sensitivity of Cambodian isolates has corresponded with high grade failure of DHA–PPQ treatment in this region [10, 35], it raises a concern that DHA–PPQ may also become less effective for P. vivax. Unlike P. falciparum, P. vivax does not exhibit concomitant artemisinin resistance. In the absence of an established baseline value, it is possible that P. vivax has higher intrinsic PPQ IC₅₀ in the assay than P. falciparum. Further, the decline observed in P. vivax IC₅₀ after PPQ was introduced in 2013 may have been the result of a decline in overall parasite fitness in response to developing mutations, similar to previous observations in P. falciparum [58]. Regardless, clinical correlations are necessary to define the impact of increased PPQ IC₅₀s for P. vivax. Also to be explored is whether the change in treatment policy for P. vivax may have relieved chloroquine pressure. However, ongoing P. falciparum chloroquine resistance comparable to previously observed levels [9, 34] argues against this. Resistance in both parasite species would have been expected to subside if exposure had truly been reduced [59]. Definitive demonstration of assay utility will require comparison of ex vivo pLDH-ELISA results with clinical response in well-controlled P. vivax field studies. Limited ability to distinguish recrudescence, relapse, and reinfection clinically may confound interpretation.

While molecular data in the present study was limited by convenience sampling, and available resources, it does offer a few useful observations. The mechanism of CQ resistance in P. vivax remains unclear although a few studies have suggested associations with P. vivax multi-drug resistance gene (pvmdr1) Y976F mutation and pvcrt-o expression [17, 60]. In northern Cambodian isolates, pvmdr1 Y976F mutants were observed at extremely high frequency yet when tested in the ex vivo assay, the isolates are CQ-sensitive. Accordingly, CQ has retained clinical efficacy as a P. vivax rescue agent in trials conducted there over the past several years by the USAMD-AFRIMS [29]. This argues against the usefulness of the Y976F mutation as a CQ resistance marker [17]. No pvmdr1 amplification was detected in Cambodian isolates, precluding correlation with ex vivo drug susceptibility to chloroquine or other anti-malarials. No attempt to measure stage-specific pvcrt-o expression in these clinical isolates was made here. Comparing our findings to those reported previously from other regions revealed geographical differences in pvmdr1 amplification and mutation prevalence. The prevalence of Y976F and F1076L mutations were high in Cambodia, Indonesia, and Papua New Guinea (70–100%) [36, 61, 62], but no mutant was detected in Brazil [47, 60]. Moderate rates of mutation were previously found in Thailand (18–23% for Y976F and 53–61% for F1076L) [50, 62]. Corresponding to previous findings [40], most Cambodian isolates were double mutants (74%), with single F1076L mutants found in < 10%. The prevalence here differed compared to Thailand where a single F1076L mutation was seen in > 60%. No pvmdr1 amplification was detected in northern Cambodian isolates tested in the present study, despite reduced susceptibility to MQ. It is possible that if more sensitive assays targeting the pvmdr1 breakpoint were used [63], minority clones with pvmdr1 amplification may have been detected, given the polyclonal nature of vivax infections in this region [64‐66]. Pvmdr1 amplification rates of 4–37% have been observed in other regions of the country [30, 40]. Similarly, a 7–39% amplification rate was reported in Thailand [40, 62]. There was no pvmdr1 amplification in Indonesia [36, 67] and low prevalence in Brazil (0.9–4%) [67, 68]. The lack or low rate of pvmdr1 amplification in some areas of Thailand and Cambodia with intense MQ pressure does not support previous evidence associating pvmdr1 amplification with MQ pressure [67].

Although the pLDH-ELISA IC₅₀ assay as described here represents an important first step to assess P. vivax drug susceptibility, the following caveats must be considered. The confounding factors of mechanism and speed of drug action must be taken into account when interpreting results obtained using different in vitro methods. High optical density (OD) background interference in wells containing maximal concentrations of some drugs tested caused low growth ratios and failure to achieve sigmoidal IC₅₀ curves. This phenomenon was reported previously to depend on the nature of the drug being tested and detection methodology. Relatively higher OD background tends to be observed in non-artemisinin drugs with a more pronounced effect in ELISA-based assays [69, 70]. This may relate to the later onset of action of non-artemisinins in the parasite life cycle, allowing ring stage parasites to continue to produce the proteins being assayed despite high drug concentrations. This may explain paradoxical growth seen at high concentrations of non-artemisinins common in the pLDH-ELISA, but less frequently in the HRP2-ELISA, and not at all in the microscopy-based SMT [69, 70]. This corresponds with the observation of parasite growth in high concentrations of atovaquone, mefloquine, and quinine in the pLDH-ELISA assay for both P. falciparum and P. vivax, but not in the HRP2 ELISA or SMT. The SMT’s relatively low throughput, and challenges interpreting concentration–response curves for parasite cultures unable to develop to the threshold of ≥ 40% schizonts may limit its usefulness. The assay requires extensive experience on stage differentiation by operators to be effective and replicable. However, as the malaria map continues to shrink in Cambodia, it may be possible to better concentrate the expert microscopy skills needed to adequately perform the SMT in areas of greatest need.

Overall, the pLDH-ELISA has the potential to be a useful and replicable method to assess ex vivo P. vivax resistance in field isolates in Cambodia with reasonable throughput. Results correlated well with those observed for P. falciparum in both the pLDH and better established HRP-2 assays. While current malaria elimination efforts are focused on resistant P. falciparum malaria, P. vivax malaria may ultimately prove more difficult to eliminate due to its ease of transmission, dormancy, and only primaquine being effective at preventing P. vivax relapse. Current tools are also inadequate to differentiate relapse and recrudescence from re-infection. Evaluation of ex vivo P. vivax susceptibility using field isolates will likely prove a useful part of the armamentarium to monitor for growing resistance and predict risk of treatment failures. Clinical P. vivax resistance can be difficult to detect due to difficulty distinguishing relapsing disease from recrudescence and/or reinfection, and the pLDH assay may prove a useful adjunct in this regard. Incorporating ex vivo pLDH monitoring with therapeutic efficacy studies for individuals with P. vivax is advised to further validate this field-expedient method.