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Malaria remains a public health problem in Thailand, especially along its borders where highly mobile populations can contribute to persistent transmission. This study aimed to determine resistant genotypes and phenotypes of 112 Plasmodium falciparum isolates from patients along the Thai-Cambodia border during 2013–2015. The majority of parasites harbored a pfmdr1-Y184F mutation. A single pfmdr1 copy number had CVIET haplotype of amino acids 72–76 of pfcrt and no pfcytb mutations. All isolates had a single pfk13 point mutation (R539T, R539I, or C580Y), and increased % survival in the ring-stage survival assay (except for R539I). 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. Collectively, the emergence of these resistance patterns in Thailand near Cambodia border mirrored the reports of dihydroartemisinin-piperaquine treatment failures in the adjacent province of Cambodia, Oddar Meanchey, suggesting a migration of parasites across the border. As malaria elimination efforts ramp up in Southeast Asia, host nations militaries and other groups in border regions need to coordinate the proposed interventions.

Malaria remains a public health problem in Thailand, especially along its borders where highly mobile populations can contribute to persistent transmission. This study aimed to determine resistant genotypes and phenotypes of 112 Plasmodium falciparum isolates from patients along the Thai-Cambodia border during 2013-2015. The majority of parasites harbored pfmdr1-Y184F mutation. A single pfmdr1 copy number, had CVIET haplotype of amino acids 72-76 of pfcrt and no pfcytb mutations. All isolates had a pfk13 point mutation (R539T, R539I, and C580Y), and increased ring-stage survival assay (except R539I). Multiple copies of pfpm2 and pfcrt-F145I were first 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 the elevated IC90 values of piperaquine. Antimalarials were detected in 21 samples (18.8%) by mass spectrometer, and 12 samples (10.7%) by bioassay. Collectively, the emergence of these resistance patterns mirrored the reports of dihydroartemisinin-piperaquine treatment failures in the adjacent province of Cambodia, Oddar Meanchey, suggesting a migration of parasites across the border. As malaria elimination efforts ramp up in Southeast Asia, host nations militaries and other groups in border regions must be a focus for interventions

Background High rates of dihydroartemisinin–piperaquine (DHA–PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. The genetic markers plasmepsin 2 (pfpm2), exonuclease (pfexo) and chloroquine resistance transporter (pfcrt) genes are associated with PPQ resistance and are used for monitoring the prevalence of drug resistance and guiding malaria drug treatment policy. Methods To examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia, and the presence of E415G-Exo and pfcrt mutations (T93S, H97Y, F145I, I218F, M343L, C350R, and G353V) as well as pfpm2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. Isobolographic analysis of several drug combinations for standard clones and newly cloned P. falciparum Cambodian isolates was also determined. Results The characterization of culture-adapted isolates revealed that the presence of novel pfcrt mutations (T93S, H97Y, F145I, and I218F) with E415G-Exo mutation can confer PPQ-resistance, in the absence of pfpm2 amplification. In vitro testing of PPQ resistant parasites demonstrated a bimodal dose–response, the existence of a swollen digestive vacuole phenotype, and an increased susceptibility to quinine, chloroquine, mefloquine and lumefantrine. To further characterize drug sensitivity, parental parasites were cloned in which a clonal line, 14-B5, was identified as sensitive to artemisinin and piperaquine, but resistant to chloroquine. Assessment of the clone against a panel of drug combinations revealed antagonistic activity for six different drug combinations. However, mefloquine-proguanil and atovaquone–proguanil combinations revealed synergistic antimalarial activity. Conclusions Surveillance for PPQ resistance in regions relying on DHA–PPQ as the first-line treatment is dependent on the monitoring of molecular markers of drug resistance. P. falciparum harbouring novel pfcrt mutations with E415G-exo mutations displayed PPQ resistant phenotype. The presence of pfpm2 amplification was not required to render parasites PPQ resistant suggesting that the increase in pfpm2 copy number alone is not the sole modulator of PPQ resistance. Genetic background of circulating field isolates appear to play a role in drug susceptibility and biological responses induced by drug combinations. The use of latest field isolates may be necessary for assessment of relevant drug combinations against P. falciparum strains and when down-selecting novel drug candidates.

Additional file 7: Table S6. In vitro drug combination assay for 9 combinations in asexual stages of different P. falciparum strains. DHA, dihydroartemisinin; CQ, chloroquine; MQ, mefloquine; PPQ, piperaquine; ATQ, atovaquone; PG, proguanil; TQ, tafenoquine; PND, pyronaridine. ΣFIC50 (50% Fractional Inhibitory Concentrations), synergism when ΣFIC50 ≤ 0.5; toward synergism when ΣFIC50 1; antagonism when ΣFIC50 ≥ 2 to 4. The values show the mean ± S.D of 3 independent assays for each parasite line.

Additional file 3: Table S3. Mean ± S.D of % Survival rate of Ring-stage Survival Assay (RSA) and Piperaquine Survival Assay (PSA).

Additional file 1: Table S1. The oligonucleotides of each primers P. falciparum for drug-resistant markers testing, including pfkelch13 (covering amino acid positions 539 to 580), pfexo (amino acid position 415), and pfcrt (at amino acid positions 93, 97, 145, 218, 343, 350, and 353) performed by T100™ Thermal Cycler.

Additional file 4: Table S4. IC50s of asexual stage P. falciparum parasites for different antimalarial drugs (mean ± S.D). N/A is not-applicable.

Additional file 6: Table S5. Comparison of the in vitro susceptibility of Plasmodium falciparum Cambodian isolate 14 before and after cloning as well as P. falciparum 3D7 and W2 exposed to established antimalarial agents. IC50 values report the mean ± S.D from at least 3 experiments. Statistically significant difference relative to 3D7 is indicated with one asterisk (0.05 > p > 0.01). N/A is non-applicable.

Additional file 2: Table S2. The oligonucleotides of each primers for copy number variation of P. falciparum multidrug resistance gene 1 (pfmdr1) and P. falciparum plasmepsin 2 (pfpm2) with β-tubulin, the reference gene, performed by ABI 7500 Real-time PCR system.