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Plasmodium falciparum phenotypic and genotypic resistance profile during the emergence of Piperaquine resistance in Northeastern Thailand.

ABSTRACT: 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.

SUBMITTER: Boonyalai N

PROVIDER: S-EPMC8238947 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHigh 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.MethodsTo 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.ResultsThe 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.ConclusionsSurveillance 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.

Project description:BackgroundThe 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.MethodsEx 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.ResultsIEV 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.ConclusionsClinical 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).

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Plasmodium falciparum phenotypic and genotypic resistance profile during the emergence of Piperaquine resistance in Northeastern Thailand.

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