Attenuation of Plasmodium falciparum in vitro drug resistance phenotype following culture adaptation compared to fresh clinical isolates in Cambodia

ABSTRACT The extension of drug resistance among malaria-causing Plasmodium falciparum parasites in Africa necessitates implementation of new combined therapeutic strategies. Drug susceptibility phenotyping requires precise measurements. Until recently, schizont maturation and isotopic in vitro assays were the only methods available, but their use was limited by technical constraints. This explains the revived interest in the development of replacement methods, such as the Plasmodium lactate dehydrogenase (pLDH) immunodetection assay. We evaluated a commercially controlled pLDH enzyme-linked immunosorbent assay (ELISA; the ELISA-Malaria antigen test; DiaMed AG, Cressier s/Morat, Switzerland) to assess drug susceptibility in a standard in vitro assay using fairly basic laboratory equipment to study the in vitro resistance of malaria parasites to major antimalarials. Five Plasmodium falciparum clones and 121 clinical African isolates collected during 2003 and 2004 were studied by the pLDH ELISA and the [8-3H]hypoxanthine isotopic assay as a reference with four antimalarials. Nonlinear regression with a maximum effect model was used to estimate the 50% inhibitory concentration (IC50) and its confidence intervals. The two methods were observed to have similar reproducibilities, but the pLDH ELISA demonstrated a higher sensitivity. The high correlation (r = 0.98) and the high phenotypic agreement (κ = 0.88) between the two methods allowed comparison by determination of the IC50s. Recently collected Plasmodium falciparum African isolates were tested by pLDH ELISA and showed drug resistance or decreased susceptibilities of 62% to chloroquine and 11.5% to the active metabolite of amodiaquine. No decreased susceptibility to lumefantrine or the active metabolite of artemisinin was detected. The availability of this simple and highly sensitive pLDH immunodetection assay will provide an easier method for drug susceptibility testing of malaria parasites.

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Effect of Fluorescent Dyes onIn Vitro-Differentiated, Late-Stage Plasmodium falciparum Gametocytes

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03772-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7398-7404 ◽

Cited By ~ 14

Author(s):

Tamirat Gebru ◽

Benjamin Mordmüller ◽

Jana Held

Keyword(s):

Plasmodium Falciparum ◽

Late Stage ◽

Fluorescent Dyes ◽

Clinical Symptoms ◽

Laboratory Strain ◽

Clinical Isolates ◽

Synthetic Dyes ◽

Content Type ◽

Highly Active

ABSTRACTPlasmodium falciparumgametocytes are not associated with clinical symptoms, but they are responsible for transmitting the pathogen to mosquitoes. Therefore, gametocytocidal interventions are important for malaria control and resistance containment. Currently available drugs and vaccines are not well suited for that purpose. Several dyes have potent antimicrobial activity, but their use against gametocytes has not been investigated systematically. The gametocytocidal activity of nine synthetic dyes and four control compounds was tested against stage V gametocytes of the laboratory strain 3D7 and three clinical isolates ofP. falciparumwith a bioluminescence assay. Five of the fluorescent dyes had submicromolar 50% inhibitory concentration (IC50) values against mature gametocytes. Three mitochondrial dyes, MitoRed, dihexyloxacarbocyanine iodide (DiOC6), and rhodamine B, were highly active (IC50s < 200 nM). MitoRed showed the highest activity against gametocytes, with IC50s of 70 nM against 3D7 and 120 to 210 nM against clinical isolates. All compounds were more active against the laboratory strain 3D7 than against clinical isolates. In particular, the endoperoxides artesunate and dihydroartemisinin showed a 10-fold higher activity against 3D7 than against clinical isolates. In contrast to all clinically used antimalarials, several fluorescent dyes had surprisingly highin vitroactivity against late-stage gametocytes. Since they also act against asexual blood stages, they shall be considered starting points for the development of new antimalarial lead compounds.

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In vitro efficacy of act drugs on Plasmodium falciparum clinical isolates from Kano and Katsina States, Nigeria

Bayero Journal of Pure and Applied Sciences ◽

10.4314/bajopas.v10i1.10s ◽

2018 ◽

Vol 10 (1) ◽

pp. 49

Author(s):

B.M. Aminu ◽

M.D. Mukhtar

Keyword(s):

Plasmodium Falciparum ◽

Clinical Isolates

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Identification of a distinctive multiple drug resistance phenotype in tumour cells following in vitro exposure to X-irradiation

Cancer Treatment An Update ◽

10.1007/978-2-8178-0765-2_153 ◽

1994 ◽

pp. 723-725

Author(s):

B. T. Hill ◽

R. D. H. Whelan ◽

S. McClean

Keyword(s):

Drug Resistance ◽

Multiple Drug Resistance ◽

Tumour Cells ◽

Multiple Drug ◽

Resistance Phenotype ◽

In Vitro Exposure ◽

X Irradiation

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Piperaquine resistant Cambodian Plasmodium falciparum clinical isolates: in vitro genotypic and phenotypic characterization

10.21203/rs.3.rs-22444/v3 ◽

2020 ◽

Author(s):

Nonlawat Boonyalai ◽

Brian A Vesely ◽

Chatchadaporn Thamnurak ◽

Chantida Praditpol ◽

Watcharintorn Fagnark ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Copy Number ◽

Drug Susceptibility ◽

Antagonistic Activity ◽

Drug Combinations ◽

Chloroquine Resistance ◽

Phenotypic Characterization ◽

Field Isolates

Abstract 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 atovoquone-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.

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Artemisinin-independent inhibitory activity of Artemisia sp. infusions against different Plasmodium stages including relapse-causing hypnozoites

Life Science Alliance ◽

10.26508/lsa.202101237 ◽

2021 ◽

Vol 5 (3) ◽

pp. e202101237

Author(s):

Kutub Ashraf ◽

Shahin Tajeri ◽

Christophe-Sébastien Arnold ◽

Nadia Amanzougaghene ◽

Jean-François Franetich ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Inhibitory Activity ◽

Parasite Species ◽

Artemisia Annua ◽

Combination Therapies ◽

Lead Compounds ◽

Low Concentrations ◽

Inhibitory Activities

Artemisinin-based combination therapies (ACT) are the frontline treatments against malaria worldwide. Recently the use of traditional infusions from Artemisia annua (from which artemisinin is obtained) or Artemisia afra (lacking artemisinin) has been controversially advocated. Such unregulated plant-based remedies are strongly discouraged as they might constitute sub-optimal therapies and promote drug resistance. Here, we conducted the first comparative study of the anti-malarial effects of both plant infusions in vitro against the asexual erythrocytic stages of Plasmodium falciparum and the pre-erythrocytic (i.e., liver) stages of various Plasmodium species. Low concentrations of either infusion accounted for significant inhibitory activities across every parasite species and stage studied. We show that these antiplasmodial effects were essentially artemisinin-independent and were additionally monitored by observations of the parasite apicoplast and mitochondrion. In particular, the infusions significantly incapacitated sporozoites, and for Plasmodium vivax and P. cynomolgi, disrupted the hypnozoites. This provides the first indication that compounds other than 8-aminoquinolines could be effective antimalarials against relapsing parasites. These observations advocate for further screening to uncover urgently needed novel antimalarial lead compounds.

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Correlation of in vitro Sensitivity of Chloroquine and other Antimalarials with the Partner Drug Resistance to Plasmodium falciparum Malaria in Selected Sites of India

Indian Journal of Medical Microbiology ◽

10.4103/ijmm.ijmm_17_160 ◽

2017 ◽

Vol 35 (4) ◽

pp. 485-490 ◽

Cited By ~ 3

Author(s):

Supriya Sharma ◽

Ram Suresh Bharti ◽

Nitin Bhardwaj ◽

Anupkumar R Anvikar ◽

Neena Valecha ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Falciparum Malaria ◽

Plasmodium Falciparum Malaria ◽

In Vitro Sensitivity

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Molecular Epidemiology of Metronidazole Resistance in a Population of Trichomonas vaginalis Clinical Isolates

Journal of Clinical Microbiology ◽

10.1128/jcm.38.8.3004-3009.2000 ◽

2000 ◽

Vol 38 (8) ◽

pp. 3004-3009 ◽

Cited By ~ 57

Author(s):

Lauren J. Snipes ◽

Pascale M. Gamard ◽

Elizabeth M. Narcisi ◽

C. Ben Beard ◽

Tovi Lehmann ◽

...

Keyword(s):

Drug Resistance ◽

Trichomonas Vaginalis ◽

Random Amplified Polymorphic Dna ◽

Transmitted Disease ◽

Clinical Isolates ◽

Nucleotide Position ◽

Sexually Transmitted ◽

Metronidazole Resistance ◽

High Level

Trichomonas vaginalis, the causative agent for human trichomoniasis, is a problematic sexually transmitted disease mainly in women, where it may be asymptomatic or cause severe vaginitis and cervicitis. Despite its high prevalence, the genetic variability and drug resistance characteristics of this organism are poorly understood. To address these issues, genetic analyses were performed on 109 clinical isolates using three approaches. First, two internal transcribed spacer (ITS) regions flanking the 5.8S subunit of the ribosomal DNA gene were sequenced. The only variation was a point mutation at nucleotide position 66 of the ITS1 region found in 16 isolates (14.7%). Second, the presence of a 5.5-kb double-stranded RNAT. vaginalis virus (TVV) was assessed. TVV was detected in 55 isolates (50%). Finally, a phylogenetic analysis was performed based on random amplified polymorphic DNA data. The resulting phylogeny indicated at least two distinct lineages that correlate with the presence of TVV. A band-sharing index indicating relatedness was created for different groups of isolates. It demonstrated that isolates harboring the virus are significantly more closely related to each other than to the rest of the population, and it indicated a high level of relatedness among isolates with in vitro metronidazole resistance. This finding is consistent with the hypothesis that drug resistance toT. vaginalis resulted from a single or very few mutational events. Permutation tests and nonparametric analyses showed associations between metronidazole resistance and phylogeny, the ITS mutation, and TVV presence. These results suggest the existence of genetic markers with clinical implications for T. vaginalisinfections.

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