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SummaryDuring infections with malaria parasites P. vivax, patients exhibit rhythmic fevers every 48 hours. These fever cycles correspond with the time parasites take to traverse the Intraerythrocytic Cycle (IEC) and may be guided by a parasite-intrinsic clock. Different species of Plasmodia have cycle times that are multiples of 24 hours, suggesting they may be coordinated with the host circadian clock. We utilized an ex vivo culture of whole blood from patients infected with P. vivax to examine the dynamics of the host circadian transcriptome and the parasite IEC transcriptome. Transcriptome dynamics revealed that the phases of the host circadian cycle and the parasite IEC were correlated across multiple patients, suggesting that the cycles are coupled. In mouse model systems, host-parasite cycle coupling appears to provide a selective advantage for the parasite. Thus, understanding how host and parasite cycles are coupled in humans could enable anti-malarial therapies that disrupt this coupling.

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.

Background While intensive Plasmodium falciparum multidrug resistance surveillance continues in Cambodia, relatively little is known about Plasmodium vivax drug resistance in Cambodia or elsewhere. To investigate P. vivax anti-malarial susceptibility in Cambodia, 76 fresh P. vivax isolates collected from Oddar Meanchey (northern Cambodia) in 2013–2015 were assessed for ex vivo drug susceptibility using the microscopy-based schizont maturation test (SMT) and a Plasmodium pan-species lactate dehydrogenase (pLDH) ELISA. P. vivax multidrug resistance gene 1 (pvmdr1) mutations, and copy number were analysed in a subset of isolates. Results Ex vivo testing was interpretable in 80% of isolates using the pLDH-ELISA, but only 25% with the SMT. Plasmodium vivax drug susceptibility by pLDH-ELISA was directly compared with 58 P. falciparum isolates collected from the same locations in 2013–4, tested by histidine-rich protein-2 ELISA. Median pLDH-ELISA IC50 of P. vivax isolates was significantly lower for dihydroartemisinin (3.4 vs 6.3 nM), artesunate (3.2 vs 5.7 nM), and chloroquine (22.1 vs 103.8 nM) than P. falciparum but higher for mefloquine (92 vs 66 nM). There were not significant differences for lumefantrine or doxycycline. Both P. vivax and P. falciparum had comparable median piperaquine IC50 (106.5 vs 123.8 nM), but some P. falciparum isolates were able to grow in much higher concentrations above the normal standard range used, attaining up to 100-fold greater IC50s than P. vivax. A high percentage of P. vivax isolates had pvmdr1 Y976F (78%) and F1076L (83%) mutations but none had pvmdr1 amplification. Conclusion The findings of high P. vivax IC50 to mefloquine and piperaquine, but not chloroquine, suggest significant drug pressure from drugs used to treat multidrug resistant P. falciparum in Cambodia. Plasmodium vivax isolates are frequently exposed to mefloquine and piperaquine due to mixed infections and the long elimination half-life of these drugs. Difficulty distinguishing infection due to relapsing hypnozoites versus blood-stage recrudescence complicates clinical detection of P. vivax resistance, while well-validated molecular markers of chloroquine resistance remain elusive. The pLDH assay may be a useful adjunctive tool for monitoring for emerging drug resistance, though more thorough validation is needed. Given high grade clinical chloroquine resistance observed recently in neighbouring countries, low chloroquine IC50 values seen here should not be interpreted as susceptibility in the absence of clinical data. Incorporating pLDH monitoring with therapeutic efficacy studies for individuals with P. vivax will help to further validate this field-expedient method. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-2034-2) contains supplementary material, which is available to authorized users.

Background The recent dramatic decline in dihydroartemisinin-piperaquine (DHA-PPQ) efficacy in northwestern Cambodia has raised concerns about the rapid spread of piperaquine resistance just as DHA-PPQ is being introduced as first-line therapy in neighbouring countries. Methods Ex vivo parasite susceptibilities were tracked to determine the rate of progression of DHA, PPQ and mefloquine (MQ) resistance from sentinel sites on the Thai–Cambodian and Thai–Myanmar borders from 2010 to 2015. Immediate ex vivo (IEV) histidine-rich protein 2 (HRP-2) assays were used on fresh patient Plasmodium falciparum isolates to determine drug susceptibility profiles. Results IEV HRP-2 assays detected the precipitous emergence of PPQ resistance in Cambodia beginning in 2013 when 40 % of isolates had an IC90 greater than the upper limit of prior years, and this rate doubled to 80 % by 2015. In contrast, Thai–Myanmar isolates from 2013 to 14 remained PPQ-sensitive, while northeastern Thai isolates appeared to have an intermediate resistance profile. The opposite trend was observed for MQ where Cambodian isolates appeared to have a modest increase in overall sensitivity during the same period, with IC50 declining to median levels comparable to those found in Thailand. A significant association between increased PPQ IC50 and IC90 among Cambodian isolates with DHA-PPQ treatment failure was observed. Nearly all Cambodian and Thai isolates were deemed artemisinin resistant with a >1 % survival rate for DHA in the ring-stage assay (RSA), though there was no correlation among isolates to indicate cross-resistance between PPQ and artemisinins. Conclusions Clinical DHA-PPQ failures appear to be associated with declines in the long-acting partner drug PPQ, though sensitivity appears to remain largely intact for now in western Thailand. Rapid progression of PPQ resistance associated with DHA-PPQ treatment failures in northern Cambodia limits drugs of choice in this region, and urgently requires alternative therapy. The temporary re-introduction of artesunate AS-MQ is the current response to PPQ resistance in this area, due to inverse MQ and PPQ resistance patterns. This will require careful monitoring for re-emergence of MQ resistance, and possible simultaneous resistance to all three drugs (AS, MQ and PPQ). Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1569-y) contains supplementary material, which is available to authorized users.

Additional file 1. Artemisinin and piperaquine susceptibility profiles do not suggest cross resistance.

DNA profiles were successfully typed from four latent fingerprints deposited on glass plate, glossy magazine paper, and plastic surface that were dusted with red, green, and yellow fluorescent powder. No or partial DNA profile was generated from one latent fingerprint samples. In addition, two DNA extraction methods, i.e., QIAamp ® DNA Mini Kit and Chelex ® 100 were compared. Experiments demonstrated that the absorbance measurements by spectrophotometery were interfered by colors of the fluorescent powder extracts, although these did not affect DNA profiling and detection of the PCR-amplified products by the genetic analyzer instrument. Results demonstrated that QIAamp ® DNA Mini Kit was more suitable than Chelex ® 100 for recovery of DNA from fluorescent powder dusted fingerprints. The quality of partial DNA profiles obtained from fluorescent powder dusted fingerprints was improved through the application of low copy number (LCN) typing, by increasing the number of PCR cycles from 28 to 34. Surface type had an effect on the number of loci obtained, and fluorescent fingerprint powders used had subtle effect on the profile quality obtained.

Additional file 1. Ex vivo drug susceptibility of P. vivax by years.

Additional file 1. Ex vivo drug susceptibility of P. vivax by years.