• View in gallery
    Figure 1.

    The effects of different antimalarials on rosetting of P. falciparum. The sample size for each experiment is shown in blue. One-way analysis of variance with Dunnett’s multiple comparison test was performed to compare the rosetting rates recorded from drug-exposed settings with that of drug-free control. (A) Chloroquine significantly increased rosetting rates at ≥ 11.69 nM (P = 0.008, 0.0001 for 11.69 nM and 23.38 nM, respectively; P < 0.0001 for 46.75–2992.34 nM). (B) Mefloquine induced significant increment of rosetting rates at 2.91 nM (P = 0.0116), 5.79 nM (P = 0.0002), and 11.56 through 370.73 nM (P < 0.0001). (C) Amodiaquine exerted significant rosette stimulation at 0.42 nM and above (P = 0.0049, 0.001, 0.002 for 0.42 nM; 0.82 nM; and 1.66 nM, respectively; P < 0.0001 for 3.32–105.8 nM). (D) Quinine exerted significant stimulation at 30.39 nM and above (P = 0.0456, 0.0204, 0.0095, 0.0044, 0.0029, 0.0025, 0.0028, 0.0014, 0.0007, 0.0006, and 0.0005 for 30.39, 60.82, 121.63, 243.23, 486.50, 973.00, 1946.00, 3891.99, 7783.98, 15567.97, and 31135.90 nM, respectively). (E) Lumefantrine (LMF) significantly increased rosetting rates at concentration points of 0.85 nM and above (P = 0.0118, 0.0108, 0.0099, 0.0078, 0.0041, 0.0022, 0.0019, 0.0007, 0.0006, 0.0005 for 0.85, 1.70, 3.40, 6.79, 13.59, 27.19, 54.35, 108.71, 217.42, and 434.83 nM, respectively).

  • View in gallery
    Figure 2.

    The effects of different antimalarials on rosetting of P. vivax. The sample size for each experiment is shown in blue. One-way analysis of variance with Dunnett’s multiple comparison test was performed to compare the rosetting rates recorded from drug-exposed settings with that of drug-free control. (A) Chloroquine significantly increased rosetting rates from concentration points of 5.85 nM and above (P = 0.0176, 0.0008, 0.0008 for 5.85, 11.69, and 23.38 nM, respectively; P < 0.0001 for 46.75–2,992.34 nM). (B) Mefloquine significantly increased rosetting rates at ≥ 0.71 nM (P = 0.0367 and 0.0008 for 0.71 and 1.45 nM, respectively; P < 0.0001 for 2.91–370.73 nM). (C) AMQ increased rosetting rates at ≥ 0.42 nM (P = 0.0412, 0.0022, 0.0004 for 0.42, 0.82, and 1.66 nM, respectively; P < 0.0001 for 3.32–105.80 nM). (D) Quinine (QN) exerted significant rosette-stimulation at ≥ 30.39 nM (P = 0.0026, 0.0015, 0.0009, 0.0005, 0.0002, 0.0001 for 30.39, 60.82, 121.63, 243.23, 486.50, and 973 nM, respectively; P < 0.0001 for 1,946–31,135.90 nM). (E) Lumefantrine induced significant changes at ≥ 0.42 nM (P = 0.0208, 0.0042, 0.0004, 0.0001 for 0.42, 0.85, 1.70, and 3.40 nM, respectively; P < 0.0001 for 6.79–434.83 nM). (F) Significant rosetting rate increment was observed with artesunate at ≥ 0.05 nM (P = 0.0373, 0.0041, 0.0005 for 0.05, 0.10, and 0.19 nM, respectively; P < 0.0001 for 0.39–49.42 nM).

  • View in gallery
    Figure 3.

    The rosette-stimulatory effect of artemisinin compounds. (A) Effect of mefloquine (MQ) on rosetting. Compared with control, a significant difference was found at drug concentration points of ≥ 28.34 nM (one-way analysis of variance [ANOVA] with Dunnett’s test P = 0.0231, 0.0071, 0.0074, 0.0035, 0.0024, 0.0011, 0.0009, and 0.0006, respectively). (B) Effect of piperaquine (PQ) on rosetting. Compared with control, a significant difference was found at drug concentration points of ≥ 154.32 nM (one-way ANOVA with Dunnett’s test P = 0.0318, 0.0197, 0.0185, 0.0136, and 0.0116, respectively). (C) Rosetting of isolates after exposure to artesunate (AS), MQ, PQ, AS + MQ, and AS + PQ. One way ANOVA with Tukey’s test was conducted. For the short AS-PCt1/2 group, rosetting rates were increased by AS, MQ, PQ, AS + MQ, and AS + PQ (P = 0.0019, 0.0014, 0.0257, 0.0002, and 0.0003, respectively). No significant difference found between AS with AS+MQ and AS + PQ (P = 0.9056 and 0.7255, respectively). For long AS-PCt1/2 group, rosetting rates were increased by AS, MQ, PQ, AS + MQ, and AS + PQ (P < 0.0001 for all). P = 0.0162 and 0.9761 for AS vs AS + PQ and AS vs. AS + MQ, respectively. No significant difference was found between AS + PQ and AS + MQ (P = 0.0762).

  • 1.

    Lee WC, Russell B, Rénia L , 2019. Sticking for a cause: the falciparum malaria parasites cytoadherence paradigm. Front Immunol 10: 1444.

  • 2.

    Lee WC et al.2020. Plasmodium-infected erythrocytes induce secretion of IGFBP7 to form type II rosettes and escape phagocytosis. eLife 9: e51546.

  • 3.

    Albrecht L et al.2020. Rosettes integrity protects Plasmodium vivax of being phagocytized. Sci Rep 10: 16706.

  • 4.

    Lee WC, Russell B, Lee B, Chu CS, Phyo AP, Sriprawat K, Lau YL, Nosten F, Rénia L , 2021. Plasmodium falciparum rosetting protects schizonts against artemisinin. EBioMedicine 73: 103680.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Borlon C, Russell B, Sriprawat K, Suwanarusk R, Erhart A, Renia L, Nosten F, D’Alessandro U , 2012. Cryopreserved Plasmodium vivax and cord blood reticulocytes can be used for invasion and short term culture. Int J Parasitol 42: 155160.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Lee WC, Russell B, Lau YL, Fong MY, Chu C, Sriprawat K, Suwanarusk R, Nosten F, Renia L , 2013. Giemsa-stained wet mount based method for reticulocyte quantification: a viable alternative in resource limited or malaria endemic settings. PLoS One 8: e60303.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Lee WC, Rénia L , 2020. Microscopy-based methods for rosetting assay in malaria research. Bio Protoc 10: e3665.

  • 8.

    Phompradit P, Muhamad P, Wisedpanichkij R, Chaijaroenkul W, Na-Bangchang K , 2014. Four years’ monitoring of in vitro sensitivity and candidate molecular markers of resistance of Plasmodium falciparum to artesunate-mefloquine combination in the Thai–Myanmar border. Malar J 13: 23.

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  • 9.

    Childs GE, Pang L, Wimonwattrawatee T, Pooyindee N, Nanakorn A, Limchitee S, Webster HK , 1987. In vitro mefloquine resistance of Plasmodium falciparum isolated from the burmese border region of Thailand. Southeast Asian J Trop Med Public Health 18: 438443.

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    Barends M, Jaidee A, Khaohirun N, Singhasivanon P, Nosten F , 2007. In vitro activity of ferroquine (ssr 97193) against Plasmodium falciparum isolates from the Thai–Burmese border. Malar J 6: 81.

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Rosetting Responses of Plasmodium-infected Erythrocytes to Antimalarials

Wenn-Chyau Lee
Department of Parasitology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia;

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Bruce RussellInfectious Diseases Labs (ID Labs), Agency for Science, Technology and Research (A*STAR), Singapore;

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Yee-Ling Lau

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Francois NostenDepartment of Microbiology and Immunology, University of Otago, Dunedin, Otago, New Zealand;
Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medical Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Tak, Thailand;

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Laurent RéniaDepartment of Parasitology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia;
Nuffield Department of Medicine, University of Oxford, United Kingdom;
Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore;

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ABSTRACT.

In malaria, rosetting is a phenomenon involving the cytoadherence of uninfected erythrocytes to infected erythrocytes (IRBC) harboring the late erythrocytic stage of Plasmodium spp. Recently, artesunate-stimulated rosetting has been demonstrated to confer a survival advantage to P. falciparum late-stage IRBC. This study investigated the rosetting response of P. falciparum and P. vivax clinical isolates to ex vivo antimalarial treatments. Brief exposure of IRBC to chloroquine, mefloquine, amodiaquine, quinine, and lumefantrine increased the rosetting rates of P. falciparum and P. vivax. Furthermore, the ex vivo combination of artesunate with mefloquine and piperaquine also resulted in increased the rosetting rates. Drug-mediated rosette-stimulation has important implications for the therapeutic failure of rapidly cleared drugs such as artesunate. However, further work is needed to establish the ramifications of increased rosetting rates by drugs with longer half-lifves, such as chloroquine, mefloquine, and piperaquine.

In malaria, the rosetting phenomenon refers to a cytoadherence event in which a Plasmodium late-stage (trophozoite–schizont)-infected erythrocyte (IRBC) stably adheres to several uninfected erythrocytes (URBC).1 Rosettes protect the IRBC from phagocytosis.2,3 Recently, we reported that the late-stage IRBC of artesunate (AS; an artemisinin [ART] derivative)-resistant P. falciparum, rapidly formed more rosettes upon AS exposure, which conferred a survival advantage to the late stages, particularly the schizonts.4 Here, the effects of brief exposure of several commonly used antimalarials on the rosetting machinery of clinical isolates from the northwestern region of Thailand are reported.

Plasmodium spp.–infected blood samples were collected in the northwestern part of Thailand by Shoklo Malaria Research Unit under ethical guidelines: OxTREC 04-10 (University of Oxford) and TMEC 09-082 (Mahidol University). Adult participants provided informed written consent, whereas for each juvenile participant, a guardian provided informed written consent on his or her behalf. Information on the reagents and tools used is available in Supplemental Table 1. Experiments were conducted on ex vivo matured-parasite suspensions with ≥ 70% of the parasite population at late stages. Drug incubation was conducted with parasite suspension of 2% hematocrit, 1% parasitemia, in 20% human AB serum-enriched RPMI 1640 medium under in vitro cultivation conditions (37°C, > 90% humidity, gas mixture of 5% CO2, 5% O2, 90% N2) for 1 hour (defined as “brief drug exposure”). Cryopreserved clinical isolates were thawed using the 12% sodium chloride method.5 Rosetting assay was conducted using the Giemsa-wet mount method.6,7

The concentration range of drug compounds was set according to the geometric means of IC50 for the P. falciparum clinical isolates from Thailand.812 Chloroquine (CQ) and amodiaquine (AMQ) were dissolved in double distilled water. Mefloquine (MQ), AS, and quinine (QN) were dissolved in 70% ethanol. Lumefantrine (LMF) was dissolved in a mixture of Triton X-100, linoleic acid, and absolute ethanol (in a ratio of 1:1:1). Piperaquine (PQ) was dissolved in 0.5% lactic acid. Drug suspensions were transferred to 96-well flat-bottom plates and air dried under sterile condition.

P. falciparum isolates were exposed to CQ (0–2,992.34 nM), MQ (0–370.73 nM), AMQ (0–105.8 nM), QN (0–3,1135.90 nM), and LMF (0–434.83 nM) briefly before the rosetting assay. The experiments were repeated with P. vivax, with an additional drug candidate, AS (0–49.42 nM). A set of P. falciparum isolates with known K13 single nucleotide polymorphism status and AS-parasite clearance half-life (AS-PCt1/2, i.e., time estimated for AS to decrease the patient’s parasitemia by half during the log-linear phase of parasite clearance after administration of AS4) was used. Before the rosetting assay, the parasites were briefly exposed to MQ (0–3627.22 nM; higher concentrations than the earlier experiments were used due to the rapid development of MQ resistance among the P. falciparum isolates in the area under study11,13) and PQ (0–2469.10 nM). The changes in rosetting rates by AS (49.42 nM) in combination with its partner drugs were evaluated by using the highest concentration point of respective drugs.

Analyses were performed with GraphPad Prism 9.0. Normality of dataset was evaluated using Shapiro-Wilk test. Multiple comparisons of normally distributed data sets were performed using one-way analysis of variance with Dunnett’s multiple comparison test (comparison against drug-free control) and Tukey’s multiple comparison test (cross-group comparisons).

Rosetting rates of P. falciparum were significantly increased by CQ, MQ, AMQ, QN, and LMF (Figure 1A–E). A similar trend was found with P. vivax (Figure 2A–E). The rosetting rates of P. vivax were significantly increased after brief AS exposure (Figure 2F). For the assessment of partner drugs involved in ACT, P. falciparum isolates experienced a significant increase in rosetting rates after exposure to MQ (Figure 3A) and PQ (Figure 3B). The concentration range of MQ in Figure 3A was higher than that of Figure 1B. Nevertheless, the findings from both sets of experiments were in agreement. The combination of AS with MQ and PQ did not prevent the rosette-stimulating effect on P. falciparum (Figure 3C). However, there were differences in the degrees of rosette-stimulation between the short and long AS-PCt1/2 groups. For P. falciparum isolates with short AS-PCt1/2 (i.e., AS sensitive), no significant difference in the degree of rosette-stimulation was found between the settings with combined drug exposure (AS + MQ and AS + PQ) and those with single drug exposure (AS, MQ, or PQ). For the long AS-PCt1/2 (i.e., AS-resistant) group, the degree of rosette stimulation by brief AS exposure was higher than that of MQ but not significantly different from that of PQ. When AS was added with the partner drugs, the rosette-stimulating effect was higher than the conditions with single drug exposure to either of the compounds under study.

Figure 1.
Figure 1.

The effects of different antimalarials on rosetting of P. falciparum. The sample size for each experiment is shown in blue. One-way analysis of variance with Dunnett’s multiple comparison test was performed to compare the rosetting rates recorded from drug-exposed settings with that of drug-free control. (A) Chloroquine significantly increased rosetting rates at ≥ 11.69 nM (P = 0.008, 0.0001 for 11.69 nM and 23.38 nM, respectively; P < 0.0001 for 46.75–2992.34 nM). (B) Mefloquine induced significant increment of rosetting rates at 2.91 nM (P = 0.0116), 5.79 nM (P = 0.0002), and 11.56 through 370.73 nM (P < 0.0001). (C) Amodiaquine exerted significant rosette stimulation at 0.42 nM and above (P = 0.0049, 0.001, 0.002 for 0.42 nM; 0.82 nM; and 1.66 nM, respectively; P < 0.0001 for 3.32–105.8 nM). (D) Quinine exerted significant stimulation at 30.39 nM and above (P = 0.0456, 0.0204, 0.0095, 0.0044, 0.0029, 0.0025, 0.0028, 0.0014, 0.0007, 0.0006, and 0.0005 for 30.39, 60.82, 121.63, 243.23, 486.50, 973.00, 1946.00, 3891.99, 7783.98, 15567.97, and 31135.90 nM, respectively). (E) Lumefantrine (LMF) significantly increased rosetting rates at concentration points of 0.85 nM and above (P = 0.0118, 0.0108, 0.0099, 0.0078, 0.0041, 0.0022, 0.0019, 0.0007, 0.0006, 0.0005 for 0.85, 1.70, 3.40, 6.79, 13.59, 27.19, 54.35, 108.71, 217.42, and 434.83 nM, respectively).

Citation: The American Journal of Tropical Medicine and Hygiene 106, 6; 10.4269/ajtmh.21-1229

Figure 2.
Figure 2.

The effects of different antimalarials on rosetting of P. vivax. The sample size for each experiment is shown in blue. One-way analysis of variance with Dunnett’s multiple comparison test was performed to compare the rosetting rates recorded from drug-exposed settings with that of drug-free control. (A) Chloroquine significantly increased rosetting rates from concentration points of 5.85 nM and above (P = 0.0176, 0.0008, 0.0008 for 5.85, 11.69, and 23.38 nM, respectively; P < 0.0001 for 46.75–2,992.34 nM). (B) Mefloquine significantly increased rosetting rates at ≥ 0.71 nM (P = 0.0367 and 0.0008 for 0.71 and 1.45 nM, respectively; P < 0.0001 for 2.91–370.73 nM). (C) AMQ increased rosetting rates at ≥ 0.42 nM (P = 0.0412, 0.0022, 0.0004 for 0.42, 0.82, and 1.66 nM, respectively; P < 0.0001 for 3.32–105.80 nM). (D) Quinine (QN) exerted significant rosette-stimulation at ≥ 30.39 nM (P = 0.0026, 0.0015, 0.0009, 0.0005, 0.0002, 0.0001 for 30.39, 60.82, 121.63, 243.23, 486.50, and 973 nM, respectively; P < 0.0001 for 1,946–31,135.90 nM). (E) Lumefantrine induced significant changes at ≥ 0.42 nM (P = 0.0208, 0.0042, 0.0004, 0.0001 for 0.42, 0.85, 1.70, and 3.40 nM, respectively; P < 0.0001 for 6.79–434.83 nM). (F) Significant rosetting rate increment was observed with artesunate at ≥ 0.05 nM (P = 0.0373, 0.0041, 0.0005 for 0.05, 0.10, and 0.19 nM, respectively; P < 0.0001 for 0.39–49.42 nM).

Citation: The American Journal of Tropical Medicine and Hygiene 106, 6; 10.4269/ajtmh.21-1229

Figure 3.
Figure 3.

The rosette-stimulatory effect of artemisinin compounds. (A) Effect of mefloquine (MQ) on rosetting. Compared with control, a significant difference was found at drug concentration points of ≥ 28.34 nM (one-way analysis of variance [ANOVA] with Dunnett’s test P = 0.0231, 0.0071, 0.0074, 0.0035, 0.0024, 0.0011, 0.0009, and 0.0006, respectively). (B) Effect of piperaquine (PQ) on rosetting. Compared with control, a significant difference was found at drug concentration points of ≥ 154.32 nM (one-way ANOVA with Dunnett’s test P = 0.0318, 0.0197, 0.0185, 0.0136, and 0.0116, respectively). (C) Rosetting of isolates after exposure to artesunate (AS), MQ, PQ, AS + MQ, and AS + PQ. One way ANOVA with Tukey’s test was conducted. For the short AS-PCt1/2 group, rosetting rates were increased by AS, MQ, PQ, AS + MQ, and AS + PQ (P = 0.0019, 0.0014, 0.0257, 0.0002, and 0.0003, respectively). No significant difference found between AS with AS+MQ and AS + PQ (P = 0.9056 and 0.7255, respectively). For long AS-PCt1/2 group, rosetting rates were increased by AS, MQ, PQ, AS + MQ, and AS + PQ (P < 0.0001 for all). P = 0.0162 and 0.9761 for AS vs AS + PQ and AS vs. AS + MQ, respectively. No significant difference was found between AS + PQ and AS + MQ (P = 0.0762).

Citation: The American Journal of Tropical Medicine and Hygiene 106, 6; 10.4269/ajtmh.21-1229

The phenomenon of drug-exposure-induced rosetting is not specific to ART and its derivatives. An earlier study suggested that as soon as antimalarial drugs enter the IRBC, they trigger an immediate shock-like signal to the intracellular parasite, which leads to the induction of rapid rosette formation mediated by the parasite’s protein trafficking machinery from the IRBC cytoplasm to the surface of IRBC.4 Such a response is probably conserved across species, at least for P. falciparum and P. vivax, based on our findings. Notably, all drug compounds in this study have good membrane permeability at physiologic pH condition,14 which may contribute to the observed rapid rosette-stimulation by the parasites. In fact, most of the antimalarials available on the market have good membrane permeability.14 Nevertheless, we do not expect all drugs to stimulate Plasmodium spp. rosette formation. Drug compounds with low lipophilicity may not be readily membrane permeable and hence are unlikely to induce rosette-stimulation. Furthermore, drug compounds that can rapidly rigidify IRBC may destroy all cytoadherence properties of IRBC.

The prevalence of rosetting rates varies with geographic origin. For example, the prevalence of rosetting is higher in cerebral malaria samples from some African countries.15,16 Outside Africa, rosetting phenomenon is relatively common for P. falciparum and P. vivax (both severe and uncomplicated malaria) from Thailand and Papua New Guinea.1719 Interestingly, the Greater Mekong Subregion (GMS) of Southeast Asia has been the epicenter of treatment-resistant malaria against several antimalarials such as ART and its derivatives CQ and MFQ.8 Of note, the rosetting ligand of P. vivax has yet to be determined. On the other hand, the expression of P. falciparum rosetting ligand PfEMP1 among the Southeast Asian P. falciparum isolates was suggested to be associated with the natural selection process of a resistance phenotype against ART and other antimalarial compounds.20 Drug-mediated rosette stimulation may be a reflection of this selection process in the parasite population of this geographic area. The ability to rosette more upon drug (threat) encounter may facilitate the parasite population to select genotypes that give rise to more specific and efficient strategies against a particular drug. The “priming” by different drugs drives better and faster adaptation of these parasites to new treatment regimens introduced to this area.

Drug-mediated rosetting is a relatively common feature in P. falciparum and P. vivax isolates from Thailand. The reflex-like response by the parasites upon drug exposure may help them to survive a brief encounter with a harmful environment. More studies are needed to evaluate the potential of a drug-mediated rosetting assay as an economic method to monitor or predict drug resistance development in the parasite population.

ACKNOWLEDGMENTS

We thank the Shoklo Malaria Research Unit staff members who assisted in the management of this study. This research was funded in part, by the Wellcome Trust Thailand/Laos Major Overseas Program Renewal (grant no. 106698). For the purpose of open access, the authors have applied a CC-BY public copyright license to any author accepted manuscript version arising from this submission, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are credited. The funding bodies had no role in the design of the study; the collection, analysis, or interpretation of data; or the writing of the manuscript. Parts of the work were presented in Lee WC, PhD dissertation 2014, Universiti Malaya, Malaysia.

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    Lee WC et al.2020. Plasmodium-infected erythrocytes induce secretion of IGFBP7 to form type II rosettes and escape phagocytosis. eLife 9: e51546.

  • 3.

    Albrecht L et al.2020. Rosettes integrity protects Plasmodium vivax of being phagocytized. Sci Rep 10: 16706.

  • 4.

    Lee WC, Russell B, Lee B, Chu CS, Phyo AP, Sriprawat K, Lau YL, Nosten F, Rénia L , 2021. Plasmodium falciparum rosetting protects schizonts against artemisinin. EBioMedicine 73: 103680.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5.

    Borlon C, Russell B, Sriprawat K, Suwanarusk R, Erhart A, Renia L, Nosten F, D’Alessandro U , 2012. Cryopreserved Plasmodium vivax and cord blood reticulocytes can be used for invasion and short term culture. Int J Parasitol 42: 155160.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 6.

    Lee WC, Russell B, Lau YL, Fong MY, Chu C, Sriprawat K, Suwanarusk R, Nosten F, Renia L , 2013. Giemsa-stained wet mount based method for reticulocyte quantification: a viable alternative in resource limited or malaria endemic settings. PLoS One 8: e60303.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 7.

    Lee WC, Rénia L , 2020. Microscopy-based methods for rosetting assay in malaria research. Bio Protoc 10: e3665.

  • 8.

    Phompradit P, Muhamad P, Wisedpanichkij R, Chaijaroenkul W, Na-Bangchang K , 2014. Four years’ monitoring of in vitro sensitivity and candidate molecular markers of resistance of Plasmodium falciparum to artesunate-mefloquine combination in the Thai–Myanmar border. Malar J 13: 23.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 9.

    Childs GE, Pang L, Wimonwattrawatee T, Pooyindee N, Nanakorn A, Limchitee S, Webster HK , 1987. In vitro mefloquine resistance of Plasmodium falciparum isolated from the burmese border region of Thailand. Southeast Asian J Trop Med Public Health 18: 438443.

    • Search Google Scholar
    • Export Citation
  • 10.

    Childs GE, Häusler B, Milhous W, Chen C, Wimonwattrawatee T, Pooyindee N, Boudreau EF , 1988. In vitro activity of pyronaridine against field isolates and reference clones of Plasmodium falciparum. Am J Trop Med Hyg 38: 2429.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Barends M, Jaidee A, Khaohirun N, Singhasivanon P, Nosten F , 2007. In vitro activity of ferroquine (ssr 97193) against Plasmodium falciparum isolates from the Thai–Burmese border. Malar J 6: 81.

    • Crossref
    • Search Google Scholar
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Author Notes

Address correspondence to Wenn-Chyau Lee, Department of Parasitology, Faculty of Medicine, Universiti Malaya, 50603 Kuala Lumpur, Malaysia, E-mail: leewc_88@hotmail.com or Laurent Rénia, A*STAR Infectious Diseases Laboratories, 8A Biomedical Grove, #03-06 Immunos, Singapore 138648, E-mail: renia_laurent@idlabs.a-star.edu.sg.

Financial support: W-C. L. and L. R. were supported by core funding from A*STAR. W-C. L. was also funded by the Open Fund-Young Individual Research grant OF-YIRG NMRC/OFYIRG/0070/2018. L. R. was also funded by A*STAR grant JCO-DP BMSI/15-800006-SIGN. B. R. was supported by a HRC e-ASIA grant (17/678) and a University of Otago Deans Bequest Grant. Shoklo Malaria Research Unit is part of the Mahidol-Oxford University Research Unit, supported by the Wellcome Trust Thailand/Laos Major Overseas Program Renewal (grant no. 106698).

Authors’ addresses: Wenn-Chyau Lee, Agency for Science, Technology and Research (A*STAR), Malaria, Singapore, E-mail: leewc_88@hotmail.com. Bruce Russel, University of Malaya, Parasitology, Kuala Lumpur, Malaysia, and University of Otago, Microbiology and Immunology, Dunedin, New Zealand, E-mail: b.russell@otago.ac.nz. Yee Ling Lau, University of Malaya, Parasitology, Faculty of Medicine, Kuala Lumpur, Malaysia, E-mail: lauyeeling@um.edu.my. Francois Nosten, Mahidol Oxford Tropical Medicine Research Unit, Shoklo Malaria Research Unit, 60/30 Bantung Road, Bangkok, Thailand, E-mail: francois@tropmedres.ac. Laurent Rénia, Agency for Science, Technology and Research (A*STAR), Singapore, Infectious Diseases Labs (ID Labs), Singapore, Nanyang Technological University, Singapore, Lee Kong Chian School of Medicine, Singapore, and Nanyang Technological University, Singapore, School of Biological Sciences, Singapore, E-mail: renia_laurent@idlabs.a-star.edu.sg.

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