ReducedIn VitroDoxycycline Susceptibility in Plasmodium falciparum Field Isolates from Kenya Is Associated with PfTetQ KYNNNN Sequence Polymorphism

ABSTRACTDoxycycline is widely used for malaria prophylaxis by international travelers. However, there is limited information on doxycycline efficacy in Kenya, and genetic polymorphisms associated with reduced efficacy are not well defined.In vitrodoxycycline susceptibility profiles for 96Plasmodium falciparumfield isolates from Kenya were determined. Genetic polymorphisms were assessed inP. falciparummetabolite drug transporter (Pfmdt) andP. falciparumGTPasetetQ(PftetQ) genes. Copy number variation of the gene and the number of KYNNNN amino acid motif repeats within the protein encoded by PftetQwere determined. Reducedin vitrosusceptibility to doxycycline was defined by 50% inhibitory concentrations (IC50s) of ≥35,000 nM. The odds ratio (OR) of having 2 PfTetQ KYNNNN amino acid repeats in isolates with IC50s of >35,000 nM relative to those with IC50s of <35,000 nM is 15 (95% confidence interval [CI], 3.0 to 74.3;Pvalue of <0.0002). Isolates with 1 copy of the Pfmdtgene had a median IC50of 6,971 nM, whereas those with a Pfmdtcopy number of >1 had a median IC50of 9,912 nM (P= 0.0245). Isolates with 1 copy of PftetQhad a median IC50of 6,370 nM, whereas isolates with a PftetQcopy number of >1 had a median IC50of 3,422 nM (P< 0.0007). Isolates with 2 PfTetQ KYNNNN motif repeats had a median IC50of 26,165 nM, whereas isolates with 3 PfTetQ KYNNNN repeats had a median IC50of 3,352 nM (P= 0.0023). PfTetQ sequence polymorphism is associated with a reduced doxycycline susceptibility phenotype in Kenyan isolates and is a potential marker for susceptibility testing.

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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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pfmdr1Amplification Is Related to Increased Plasmodium falciparum In Vitro Sensitivity to the Bisquinoline Piperaquine

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.06350-11 ◽

2012 ◽

Vol 56 (7) ◽

pp. 3615-3619 ◽

Cited By ~ 21

Author(s):

M. I. Veiga ◽

P. E. Ferreira ◽

M. Malmberg ◽

L. Jörnhagen ◽

A. Björkman ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Copy Number ◽

Statistical Significance ◽

Resistance Locus ◽

Content Type ◽

Enhanced Sensitivity ◽

In Vitro Sensitivity ◽

Artemisinin Combination Therapies

ABSTRACTThe 4-aminoquinoline bisquinoline piperaquine is an important partner drug in one of the presently recommended artemisinin combination therapies. Recent clinical trials have confirmed its high efficacy in combination with dihydroartemisinin. Resistance to piperaquine alone has, however, been documented. Amplification in copy number of thePlasmodium falciparummultidrug resistance locus on chromosome 5, containing thepfmdr1gene, has been shown to confer resistance to structurally unrelated antimalarials. Through the determination of the 50% inhibitory concentrations (IC50s) and IC90s for piperaquine and chloroquine in a set of 46 adaptedP. falciparumcultures originating from the Thai-Burmese border, we have characterized the regions around thepfmdr1gene and identified a significant association between the presence ofpfmdr1duplications and enhanced sensitivity to piperaquine (P= 0.005 for IC50andP= 0.002 for IC90) and chloroquine, reaching statistical significance at IC90s (P= 0.026). These results substantiate the potential importance ofpfmdr1copy number amplifications in the efficacy of the combination therapy piperaquine-dihydroartemisinin. It supports the rational use of 4-aminoquinolines and artemisinin-based compounds, as they independently select for mutually incompatible combinations of mutations.

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Altered Plasmodium falciparum Sensitivity to the Antiretroviral Protease Inhibitor Lopinavir Associated with Polymorphisms in pfmdr1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01949-16 ◽

2016 ◽

Vol 61 (1) ◽

Cited By ~ 3

Author(s):

Ebere Sonoiki ◽

Christian Nsanzabana ◽

Jennifer Legac ◽

Kirthana M. V. Sindhe ◽

Joseph DeRisi ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Protease Inhibitor ◽

Genome Sequencing ◽

Copy Number ◽

Hiv Protease ◽

Parasite Development ◽

Whole Genome ◽

Aspartic Proteases ◽

Content Type

ABSTRACT The HIV protease inhibitor lopinavir inhibits Plasmodium falciparum aspartic proteases (plasmepsins) and parasite development, and children receiving lopinavir-ritonavir experienced fewer episodes of malaria than those receiving other antiretroviral regimens. Resistance to lopinavir was selected in vitro over ∼9 months, with ∼4-fold decreased sensitivity. Whole-genome sequencing of resistant parasites showed a mutation and increased copy number in pfmdr1 and a mutation in a protein of unknown function, but no polymorphisms in plasmepsin genes.

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Decreasingpfmdr1Copy Number Suggests that Plasmodium falciparum in Western Cambodia Is RegainingIn VitroSusceptibility to Mefloquine

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05163-14 ◽

2015 ◽

Vol 59 (5) ◽

pp. 2934-2937 ◽

Cited By ~ 25

Author(s):

Pharath Lim ◽

Dalin Dek ◽

Vorleak Try ◽

Sokunthea Sreng ◽

Seila Suon ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Combination Therapy ◽

Molecular Marker ◽

Copy Number ◽

Artemisinin Combination Therapy ◽

Clinical Isolates ◽

Content Type

ABSTRACTDihydroartemisinin-piperaquine is the current frontline artemisinin combination therapy (ACT) forPlasmodium falciparummalaria in Cambodia but is now failing in several western provinces. To investigate artesunate plus mefloquine (AS+MQ) as a replacement ACT, we measured the prevalence of multiplepfmdr1copies—a molecular marker for MQ resistance—in 844P. falciparumclinical isolates collected in 2008 to 2013. Thepfmdr1copy number is decreasing in Western Cambodia, suggesting thatP. falciparumis regainingin vitrosusceptibility to MQ.

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Differential Association of Plasmodium falciparum Na+/H+Exchanger Polymorphism and Quinine Responses in Field- and Culture-Adapted Isolates of Plasmodium falciparum

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00477-11 ◽

2011 ◽

Vol 55 (12) ◽

pp. 5834-5841 ◽

Cited By ~ 12

Author(s):

Stéphane Pelleau ◽

Lionel Bertaux ◽

Sébastien Briolant ◽

Michael T. Ferdig ◽

Véronique Sinou ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Drug Testing ◽

Inhibitory Concentration ◽

Cumulative Effect ◽

Differential Association ◽

Microsatellite Sequence ◽

Content Type ◽

Field Isolates ◽

Selection Of

ABSTRACTPlasmodium falciparumisolates with decreased susceptibility to quinine are increasingly being found in malaria patients. Mechanisms involved in this resistance are not yet understood. Several studies claim that alongside mutations in the Pfcrtand Pfmdr1genes, the Pfnhe-1Na+/H+exchanger polymorphism plays a role in decreasing susceptibility. However, conflicting results on the link between the Pfnhe-1gene and quinine resistance arise from field- and culture-adapted isolates. We tested the association between Pfnhe-1, Pfcrt, and Pfmdr1polymorphisms in field- and culture-adapted isolates from various countries with theirin vitrosusceptibility to quinine. Field isolates presented a higher diversity of the Pfnhe-1microsatellite sequence than culture-adapted isolates. In culture-adapted isolates but not in field isolates, mutations in the Pfcrtand Pfmdr1genes, as well as a higher number of DNNND repeats in the Pfnhe-1gene, were associated with a higher 50% inhibitory concentration (IC50) of quinine. Furthermore, most of the culture-adapted isolates with more than one DNNND repeat in the Pfnhe-1gene also harbored mutated Pfcrtand Pfmdr1genes with an apparent cumulative effect on quinine susceptibility. This study supports the involvement of the Pfnhe-1gene in the modulation of thein vitroquinine response when associated with mutated Pfcrtand Pfmdr1genes. Culture adaptation could be responsible for selection of specific haplotypes of these three genes. Methods used for drug testing might thus influence the association between Pfnhe-1polymorphism and quinine susceptibility. However, we do not exclude the possibility that in particular settings, Pfnhe-1polymorphism can be used as a molecular marker for surveillance of quinine resistance.

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Molecular Characterization of Acquired Enrofloxacin Resistance in Mycoplasma synoviae Field Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00203-13 ◽

2013 ◽

Vol 57 (7) ◽

pp. 3072-3077 ◽

Cited By ~ 20

Author(s):

I. Lysnyansky ◽

I. Gerchman ◽

I. Mikula ◽

F. Gobbo ◽

S. Catania ◽

...

Keyword(s):

Amino Acid ◽

Molecular Typing ◽

Clear Correlation ◽

Amino Acid Substitutions ◽

Mycoplasma Synoviae ◽

Content Type ◽

Field Isolates ◽

Clear Cut

ABSTRACTThein vitroactivity of enrofloxacin against 73Mycoplasma synoviaefield strains isolated in Israel and Europe was determined by broth microdilution. Decreased susceptibility to enrofloxacin was identified in 59% of strains, with the MICs ranging from 1 to >16 μg/ml. The estimated MIC50and MIC90values for enrofloxacin were 2 and 8 μg/ml, respectively. Moreover, this study showed that 92% of recent Israeli field isolates (2009 to 2011) ofM. synoviaehave MICs of ≥2 μg/ml to enrofloxacin. Comparison of the quinolone resistance-determining regions (QRDRs) inM. synoviaeisolates revealed a clear correlation between the presence of one of the amino acid substitutions Asp79-Asn, Thr80-Ala/Ile, Ser81-Pro, and Asp84-Asn/Tyr/His of the ParC QRDR and decreased susceptibility to enrofloxacin (MIC, ≥1 μg/ml). Amino acid substitutions at positions GyrA 87, GyrB 401/402, and ParE 420/454 were also identified, but there was no clear-cut correlation with susceptibility to enrofloxacin. Comparison ofvlhAmolecular profiles revealed the presence of 9 different genotypes in the IsraeliM. synoviaefield isolates and 10 genotypes in the European isolates; only onevlhAgenotype (type 4) was identified in both cohorts. Based on results ofvlhAmolecular typing, several mechanisms for emergence and dissemination of Israeli enrofloxacin-resistantM. synoviaeisolates are suggested.

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

10.21203/rs.3.rs-22444/v2 ◽

2020 ◽

Author(s):

Nonlawat Boonyalai ◽

Brian A Vesely ◽

Chatchadaporn Thamnurak ◽

Chantida Praditpol ◽

Watcharintorn Fagnark ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Copy Number ◽

Antimalarial Activity ◽

Drug Susceptibility ◽

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 arteminsinin 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 harboring 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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Kenyan Plasmodium falciparum Field Isolates: Correlation between Pyrimethamine and Chlorcycloguanil Activity In Vitro and Point Mutations in the Dihydrofolate Reductase Domain

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.1.164 ◽

1998 ◽

Vol 42 (1) ◽

pp. 164-169 ◽

Cited By ~ 65

Author(s):

A. Nzila-Mounda ◽

E. K. Mberu ◽

C. H. Sibley ◽

C. V. Plowe ◽

P. A. Winstanley ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Dihydrofolate Reductase ◽

Drug Response ◽

Point Mutations ◽

Distinct Group ◽

Wild Type ◽

Field Isolates ◽

Vitro Data ◽

In Vitro Data

ABSTRACT Sixty-nine Kenyan Plasmodium falciparum field isolates were tested in vitro against pyrimethamine (PM), chlorcycloguanil (CCG), sulfadoxine (SD), and dapsone (DDS), and their dihydrofolate reductase (DHFR) genotypes were determined. The in vitro data show that CCG is more potent than PM and that DDS is more potent than SD. DHFR genotype is correlated with PM and CCG drug response. Isolates can be classified into three distinct groups based on their 50% inhibitory concentrations (IC50s) for PM and CCG (P< 0.01) and their DHFR genotypes. The first group consists of wild-type isolates with mean PM and CCG IC50s of 3.71 ± 6.94 and 0.24 ± 0.21 nM, respectively. The second group includes parasites which all have mutations at codon 108 alone or also at codons 51 or 59 and represents one homogeneous group for which 25- and 6-fold increases in PM and CCG IC50s, respectively, are observed. Parasites with mutations at codons 108, 51, and 59 (triple mutants) form a third distinct group for which nine- and eightfold increases in IC50s, respectively, of PM and CCG compared to the second group are observed. Surprisingly, there is a significant decrease (P < 0.01) of SD and DDS susceptibility in these triple mutants. Our data show that more than 92% of Kenyan field isolates have undergone at least one point mutation associated with a decrease in PM activity. These findings are of great concern because they may indicate imminent PM-SD failure, and there is no affordable antimalarial drug to replace PM-SD (Fansidar).

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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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