Molecular genotypes of culture-adapted clinical isolates for ART- and PPQ drug resistance characterization
Of 221 isolates available for molecular marker testing, all samples held a single copy number of pfmdr1, 211 samples (95.5%) contained C580Y K13 propeller artemisinin resistance mutations, and 184 samples (83.3%) and 37 samples (16.7%) harbored multiple copies and single copy of PM2, respectively . Table 1 illustrates the molecular genotypes of 17 culture-adapted isolates. All isolates in our study carried a single copy of pfmdr1, fifteen isolates harbored the C580Y-K13 mutation, a marker for ART resistance, and two parasite isolates (14 and 17) contained K13 wild-type. Twelve isolates had multiple copies of PM2 and five isolates (4, 9, 13, 14, and 17) had a single copy of PM2. Fourteen isolates carried the exo-E415G mutations, while the remaining three isolates (13, 14, and 17) were wild type. Thirteen isolates contained novel PfCRT mutations (1 isolate with T93S, 1 isolate with H97Y, 6 isolates with F145I, and 5 samples with I218F mutation). Two parasite isolates (14 and 17) were classified as ART-sensitive (ART-S) and PPQ-sensitive (PPQ-S) since they did not have the SNPs of K13, Exo, and PfCRT as well as the amplification of PM2 copy. One isolate (13) was classified as ART-resistant (ART-R) but PPQ-S since it had C580Y-K13 mutation. Fourteen samples were classified as ART-R and PPQ-resistant (PPQ-R). Of the fourteen samples, isolates 4 and 9 contained both exo-E415G and novel PfCRT mutations, and a single copy of PM2, while the rest harbored both exo-E415G, novel PfCRT mutations, and multiple copies of PM2.
In vitro RSA0-3h and PSA0-3h correlate with ART- and PPQ-resistant phenotypes.
The in vitro RSA0-3h and PSA0-3h were carried out in numeric order by the study team that was blinded to the results of molecular markers. Fig. 1A illustrates % survival rate from RSA. P. falciparum W2 was used as ART-sensitive control line while and IPC-4884 and IPC-5202 are ART-resistant control lines, respectively. IPC-4884 and IPC-5202 have a reported %RSA survival value of 6.2 % and 88.2 %, respectively . Parasites without C580Y-K13 mutation (isolates 14 and 17) exhibited % RSA survival rate of less than 1 (a cut-off for ART resistance), while parasites with C580Y-K13 mutation all had % survival rate of greater than 1, clearly demonstrating the correlation between the C580Y-K13 marker and ART resistance.
PSA0-3h testing was used to determine the relationship between PM2 copy number, the exo-E415G mutations and novel PfCRT mutations. Fig. 1B shows the % PSA survival of the parasites used in this study and P. falciparum W2, IPC-4884, and IPC-5202 represented PPQ-sensitive parasite lines. Three isolates 13, 14, and 17 showed % PSA survival of less than 10, indicative of PPQ-sensitivity. All three isolates lacked PPQ-resistance markers investigated in this study, with only parasite isolate 13 harboring the C580Y-K13 mutation. These results correlated well with the genotyping results in that the parasite isolate 13 was as ART-R and PPQ-S while the parasite isolates 14 and 17 were both sensitive to ART and PPQ. Most of our isolates harbored multiple copies of PM2, except for isolates 4, 9, 13, 14, and 17; the latter three were classified as PPQ sensitive. Our PSA results then confirmed that parasite isolates 4 and 9 were PPQ-R and this phenotype stemmed from the presence of exo-E415G and PfCRT-mutations but a single copy of PM2.
In vitro drug susceptibility testing demonstrates correlation between PPQ-resistance and sensitivity to quinine, chloroquine, mefloquine and lumefantrine.
We next examined the drug sensitivity to other antimalarials. Fig. 2 and Table S3 shows in vitro drug susceptibility. The samples were categorized into two groups including PPQ-R (red bars) and PPQ-S (blue bars). Compared to PPQ-S, PPQ-R parasites exhibited similar IC50 toward AS, DHA, DOX, and ATQ but higher IC50 for CYC. Interestingly, IC50 values for LUM, QN, CQ, and MQ were significantly lower for PPQ-R parasites than those demonstrating PPQ-S phenotype.
PPQ-resistant parasites exhibit an unusual bimodal dose-response curve.
Bopp et. al.  reported that by increasing the starting PPQ concentration (from 0.5 to 50 mM) and extending the dilution series (from 12 to 24 points), PPQ resistant parasites exhibited the second peak of survival around 0.1-10 mM. Fig. 3 illustrates 24 point-PPQ dose-response curves of selected culture-adapted clinical isolates from each group. It can be seen that PPQ-R parasites (isolates 3, 4, 6, 9, 11, 12, and 15) exhibited the second peak of survival around 78 -20,000 ng/ml (or 0.08-21 µM), indicating a bimodal dose-response curve. Unlike PPQ-R parasites, PPQ-S parasites (isolates 14 and 17) did not show the second peak, and their dose-response curves were similar to that of the reference clone W2.
Parasites with PfCRT-F145I mutation but a single copy of PM2 showed a distended digestive vacuole (DV).
Ross et. al.  has shown previously that pfcrt-edited Dd2 parasites developed a distended and translucent DV phenotype during the development from mid-trophozoites to mid-schizonts. This trait was specific to the pfcrt-edited Dd2 with PfCRT-F145I, PfCRT-M343L and PfCRT-G353V mutations and a single copy of PM2 but not observed in the PPQ-resistant Cambodian lines PH1008-C or PH1263-C. According to this observation, we then looked at the schizont morphology of our selected parasites as shown in Fig. 3. Only parasite isolate 9 exhibited the swollen and translucent DV, corresponding to parasite that contained PfCRT-F145I and exo-E415G mutations but a single copy of PM2. We also further examined parasite isolate 4, containing a single copy of PM2, exo-E415G mutation, and PfCRT-I218F mutation. However, even though this parasite isolate contain the novel PfCRT mutations (I218F) with a normal PM2 CN, no distended DV from this sample was observed. On the contrary, parasites carrying both PfCRT-F145I mutation and multiple copies of PM2 (such as parasite isolates 3 and 6) did not show the swollen DV.
The cloning of Cambodian P. falciparum isolate 14 resulted in four clones exhibiting ART- and PPQ – sensitive phenotypes.
Clinical isolates of P. falciparum are a genetically heterogeneous population of parasites. To obtain stable strains of the parasites for long term experiments, a rapid method of cloning was developed using a combination of limiting dilution and plaque assay . We attempted to clone 8 Cambodian P. falciparum isolates (isolates 3, 4, 6, 9, 12, 14, 15, and 17) by the combination of limiting dilution and plaque assay. All of the selected samples generated a single plaque after 7 days, but only clones from isolate 14, an ART-S and PPQ-S isolate, could be expanded. This reflected the intrinsic difference in their respective growth rate. After 1 month, four clones from isolate 14 were established including 14-B5, 14-C6, 14-C7, and 14-F5. Subsequently, homogeneous parasite clones prepared by this technique were genotypically and phenotypically characterized and compared with the parent isolate as well as the standard lab clone P. falciparum 3D7 and W2 (Fig 4). Fig. 4A shows the example of DNA sequencing chromatogram for E415 SNP of exo gene. Clear single peaks are seen from the cloned parasites, all of which contain the codon GAG, coded for glutamic acid at position 415. Other molecular genotype profiles of the clones and parent isolate shown in Fig. 4B confirmed that all 4 clones possessed the identical genotypes to the parent isolate. RSA and PSA survival assay also supported the genotypes in that all clones and parent isolate 14 are sensitive to ART and PPQ.
Drug susceptibility profiles of the clones and parent isolate are illustrated in Fig. 4C and Table S4. All clones obtained from this method resembled the parent isolate 14. When comparing to 3D7 (CQ-sensitive), all clones exhibited high IC50s toward CQ similar to that of W2 (CQ-resistance), indicating that all clones are CQ-resistant. MQ IC50s for the clones were much higher than for W2 isolate (MQ-sensitive), and comparable to IC50 of D6 (MQ-resistance, IC50-MQ = 130.8 ± 15.96 nM). This suggests that all four clones had reduced MQ sensitivity. Collectively, based on the drug susceptibility profile and survival assay, all four clones (14-B5, 14-C6, 14-C7, and 14-F5) were classified as ART- and PPQ- sensitive, but CQ-resistant.
P. falciparum 14-B5 exhibited antagonistic interactions in several drug combinations
To assess how different drug combinations affect P. falciparum growth, we used the HRPII-ELISA assay to establish synergistic, additive, and antagonistic in vitro antimalarial drug interactions. We first tested fixed ratio combinations of DHA-PPQ, CQ-CQ, and ATQ-PG against P. falciparum 3D7, W2, D6, C2B, and IPC-5202 strains as well as our 14-5B clone. Tables 2 and S5 represent summary of drug interaction and the SFIC50s of tested fixed drug ratio combination, respectively. Three drug combinations (DHA-PPQ, CQ-CQ, and ATQ-PG) were used as validation control. DHA-PPQ revealed antagonistic interactions, as reported previously . CQ-CQ, serving as an experimental drug combination control, showed the additive interaction while ATQ-PG revealed a synergistic interaction as previously reported by Co, E-M. A et al .
Next we tested potential novel combinations with either ATQ or PG. ATG-PG, which was the combination tested in the clinical trial, showed synergism across all strains. We selected three antimalarials (PND, MQ, and TQ) as partner drugs; PND and MQ are currently being evaluated used in our ongoing clinical study in Cambodia (ClinicalTrials.gov NCT03726593), while TQ has just been approved by the U.S. Food and Drug Administration (FDA) for the prevention of malaria in patients aged 18 and older . When PND or MQ were combined with ATG, all showed antagonism/toward antagonism, except for MQ-ATG in the C2B strain. In TQ-ATQ combination, responses varied across strain although only one line, our 14-B5 clone, revealed antagonistic interaction. When combined with PG, both PND and TQ showed different responses across strains, with our clone 14-B5 also be the only strain showing antagonism between TQ-PG. All of the tested parasites revealed toward synergistic interaction against MQ-PG combination.
The last two experiments were carried out to assess the interaction of TQ and PND, an ACT-partner drug, as well as TQ-CQ, a combination which is currently the only FDA-approved TQ combination for the treatment and radical cure of P. vivax malaria . All of the parasite lines in this study showed toward antagonistic interaction against TQ-PND. In TQ-CQ combination, 3D7 and D6 had additive interactions, while W2 displayed toward antagonism. C2B, IPC-5202, and 14-B5, on the other hand, revealed antagonistic interactions against TQ-CQ combination.