In Vitro Monitoring of Plasmodium falciparum Drug Resistance in French Guiana: a Synopsis of Continuous Assessment from 1994 to 2005

ABSTRACT Implemented as one arm of the malaria control program in French Guiana in the early 1990s, our laboratory has since established in vitro profiles for parasite drug susceptibility to a panel of eight antimalarials for more than 1,000 Plasmodium falciparum isolates from infected patients. The quinine-doxycycline combination was introduced in 1995 as the first-line drug treatment against uncomplicated P. falciparum malaria, replacing chloroquine, and the first-line drug combination was changed to the artemether-lumefantrine combination in 2002. Resistance to chloroquine declined 5 years after it was dropped in 1995 as the first-line drug, but unlike similar situations in Africa, there was a rapid halt to this decline. Doxycycline susceptibility substantially decreased from 2002 to 2005, suggesting parasite selection under quinine-doxycycline drug pressure. Susceptibility to mefloquine decreased from 1997 onward. Throughout the period from 1994 to 2005, most isolates were sensitive in vitro to quinine, amodiaquine, and atovaquone. Susceptibility to amodiaquine was strongly correlated with that to chloroquine and to a lesser extent with that to mefloquine and halofantrine. Susceptibilities to mefloquine and to halofantrine were also strongly correlated. There were two alerts issued for in vitro artemether resistance in the period from 2002 to 2003 and again in 2005, both of which could be associated with the presence of an S769N polymorphism in the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)-type P. falciparum ATPase6 (PfATPase6) gene. Analysis of susceptibility to lumefantrine, conducted for the first time in 2005, indicates an alarming rate of elevated 50% inhibitory concentrations. In vitro monitoring of parasite drug susceptibility should be pursued to further document the consequences of specific drug policies on the local parasite population and, in particular, to establish profiles of susceptibility to individual components of drug combinations to provide early warning signs of emerging parasite resistance.

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Discordant Temporal Evolution ofPfcrtandPfmdr1Genotypes and Plasmodium falciparum In Vitro Drug Susceptibility to 4-Aminoquinolines after Drug Policy Change in French Guiana

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05280-11 ◽

2012 ◽

Vol 56 (3) ◽

pp. 1382-1389 ◽

Cited By ~ 20

Author(s):

Eric Legrand ◽

Joséphine Yrinesi ◽

Marie-Thérèse Ekala ◽

Julie Péneau ◽

Béatrice Volney ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Drug Policy ◽

French Guiana ◽

Target Genes ◽

Gene Copy Number ◽

Drug Susceptibility ◽

Gene Copy ◽

Poor Correlation ◽

Content Type

ABSTRACTAnalysis of the evolution of drug target genes under changing drug policy is needed to assist monitoring ofPlasmodium falciparumdrug resistance in the field. Here we genotypePfcrtandPfdmr1of 700 isolates collected in French Guiana from 2000 (5 years after withdrawal of chloroquine) to 2008, i.e., the period when the artemether-lumefantrine combination was progressively introduced and mefloquine was abandoned. Gene sequencing showed fixation of the 7G8-typePfcrtSMVNT resistance haplotype and near fixation of the NYCDYPfdmr1haplotype.Pfdmr1gene copy number correlated with 50% inhibitory concentrations of mefloquine and halofantrine (r= 0.64 and 0.47, respectively,n= 547); its temporal changes paralleled changes inin vitromefloquine susceptibility. However, the molecular parameters studied did not account for the regainedin vitrosusceptibility to chloroquine and showed a poor correlation with susceptibility to artemether, lumefantrine, or quinine. Identification of novel markers of resistance to these antimalarials is needed in this South American area.

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New InhA Inhibitors Based on Expanded Triclosan and Di-Triclosan Analogues to Develop a New Treatment for Tuberculosis

Pharmaceuticals ◽

10.3390/ph14040361 ◽

2021 ◽

Vol 14 (4) ◽

pp. 361

Author(s):

Sarentha Chetty ◽

Tom Armstrong ◽

Shalu Sharma Kharkwal ◽

William C. Drewe ◽

Cristina I. De Matteis ◽

...

Keyword(s):

Multidrug Resistant ◽

Protein Crystal ◽

First Line ◽

Isoniazid Resistance ◽

Structure Based Drug Design ◽

Extensively Drug Resistant ◽

New Treatment ◽

Flexible Ligands ◽

Line Drug

The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has reinforced the need for the development of new anti-TB drugs. The first line drug isoniazid inhibits InhA. This is a prodrug requiring activation by the enzyme KatG. Mutations in KatG have largely contributed to clinical isoniazid resistance. We aimed to design new ‘direct’ InhA inhibitors that obviate the need for activation by KatG, circumventing pre-existing resistance. In silico molecular modelling was used as part of a rational structure-based drug-design approach involving inspection of protein crystal structures of InhA:inhibitor complexes, including the broad spectrum antibiotic triclosan (TCS). One crystal structure exhibited the unusual presence of two triclosan molecules within the Mycobacterium tuberculosis InhA binding site. This became the basis of a strategy for the synthesis of novel inhibitors. A series of new, flexible ligands were designed and synthesised, expanding on the triclosan structure. Low Minimum Inhibitory Concentrations (MICs) were obtained for benzylphenyl compounds (12, 43 and 44) and di-triclosan derivative (39), against Mycobacterium bovis BCG although these may also be inhibiting other enzymes. The ether linked di-triclosan derivative (38) displayed excellent in vitro isolated enzyme inhibition results comparable with triclosan, but at a higher MIC (125 µg mL−1). These compounds offer good opportunities as leads for further optimisation.

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Assessment of the Drug Susceptibility of Plasmodium falciparum Clinical Isolates from Africa by Using a Plasmodium Lactate Dehydrogenase Immunodetection Assay and an Inhibitory Maximum Effect Model for Precise Measurement of the 50-Percent Inhibitory Concentration

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00367-06 ◽

2006 ◽

Vol 50 (10) ◽

pp. 3343-3349 ◽

Cited By ~ 61

Author(s):

Halima Kaddouri ◽

Serge Nakache ◽

Sandrine Houzé ◽

France Mentré ◽

Jacques Le Bras

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Lactate Dehydrogenase ◽

Active Metabolite ◽

Inhibitory Concentration ◽

Drug Susceptibility ◽

Maximum Effect ◽

Malaria Parasites ◽

Effect Model

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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Modulating effects of plasma containing anti-malarial antibodies on in vitro anti-malarial drug susceptibility in Plasmodium falciparum

Malaria Journal ◽

10.1186/1475-2875-9-326 ◽

2010 ◽

Vol 9 (1) ◽

pp. 326 ◽

Cited By ~ 3

Author(s):

Preeyaporn Monatrakul ◽

Mathirut Mungthin ◽

Arjen M Dondorp ◽

Srivicha Krudsood ◽

Rachanee Udomsangpetch ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Drug Susceptibility

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Mutation in Plasmodium falciparum BTB/POZ domain of K13 protein confers artemisinin resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01320-21 ◽

2021 ◽

Author(s):

Lucie Paloque ◽

Romain Coppée ◽

Barbara H. Stokes ◽

Nina F. Gnädig ◽

Karamoko Niaré ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Artemisinin Resistance ◽

Parasite Clearance ◽

West African ◽

Whole Genome Sequence ◽

Drug Pressure ◽

Synonymous Mutations ◽

Survival Assay ◽

One Year

Partial artemisinin resistance, defined in patients as a delayed parasite clearance following artemisinin-based treatment, is conferred by non-synonymous mutations in the Kelch beta-propeller domain of the Plasmodium falciparum k13 ( pfk13 ) gene. Here, we carried out in vitro selection over a one-year period on a West African P. falciparum strain isolated from Kolle (Mali) under a dose-escalating artemisinin regimen. After 18 cycles of sequential drug pressure, the selected parasites exhibited enhanced survival to dihydroartemisinin in the ring-stage survival assay (RSA 0-3h = 9.2%). Sanger and whole-genome sequence analyses identified the PfK13 P413A mutation, localized in the BTB/POZ domain, upstream of the propeller domain. This mutation was sufficient to confer in vitro artemisinin resistance when introduced into the PfK13 coding sequence of the parasite strain Dd2 by CRISPR/Cas9 gene editing. These results together with structural studies of the protein demonstrate that the propeller domain is not the sole in vitro mediator of PfK13-mediated artemisinin resistance, and highlight the importance of monitoring for mutations throughout PfK13.

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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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Growth, Drug Susceptibility, and Gene Expression Profiling of Plasmodium falciparum Cultured in Medium Supplemented with Human Serum

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057107310638 ◽

2007 ◽

Vol 12 (8) ◽

pp. 1109-1114 ◽

Cited By ~ 14

Author(s):

Kshipra Singh ◽

Ameeta Agarwal ◽

Shabana I. Khan ◽

Larry A. Walker ◽

Babu L. Tekwani

Keyword(s):

Gene Expression ◽

Plasmodium Falciparum ◽

Human Serum ◽

Global Gene Expression ◽

Drug Susceptibility ◽

Growth Cycle ◽

In Vitro Cultivation ◽

Human Malaria Parasite ◽

In Vitro Drug Susceptibility

In vitro cultivation of Plasmodium falciparum has been extremely useful in understanding the biology of the human malaria parasite as well as research on the discovery of new antimalarial drugs and vaccines. A chemically defined serum-free medium supplemented with lipid-rich bovine serum albumin (AlbuMAX I) offers the following advantages over human serum-supplemented media for the in vitro culture of P. falciparum: 1) improved growth profile, with more than a 2-fold higher yield of the parasites at any stage of the growth cycle; 2) suitability for in vitro antimalarial screening, as the parasites grown in AlbuMAX and human serum-supplemented media show similar sensitivity to standard and novel antimalarials as well as natural product extracts in the in vitro drug susceptibility assays; and 3) DNA microarray analysis comparing the global gene expression profile of sorbitol-synchronized P. falciparum trophozoites grown in the 2 different media, indicating minimal differences. ( Journal of Biomolecular Screening 2007:1109-1114)

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Impact of the first-line treatment shift from dihydroartemisinin/piperaquine to artesunate/mefloquine on Plasmodium vivax drug susceptibility in Cambodia

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa092 ◽

2020 ◽

Vol 75 (7) ◽

pp. 1766-1771 ◽

Cited By ~ 2

Author(s):

Camille Roesch ◽

Mélissa Mairet-Khedim ◽

Saorin Kim ◽

Dysoley Lek ◽

Jean Popovici ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Vivax ◽

Drug Susceptibility ◽

Single Mutation ◽

First Line ◽

The Past ◽

Drug Assays ◽

Number Variation ◽

In Vitro Response

Abstract Background Cambodia is the epicentre of the emergence of Plasmodium falciparum drug resistance. Much less is known regarding the drug susceptibility of the co-endemic Plasmodium vivax. Only in vitro drug assays can determine the parasite’s intrinsic susceptibility, but these are challenging to implement for P. vivax and rarely performed. Objectives To evaluate the evolution of Cambodian P. vivax susceptibility to antimalarial drugs and determine their association with putative markers of drug resistance. Methods In vitro response to three drugs used in the past decade in Cambodia was measured for 52 clinical isolates from Eastern Cambodia collected between 2015 and 2018 and the sequence and copy number variation of their pvmdr1 and pvcrt genes were analysed. pvmdr1 polymorphism was also determined for an additional 250 isolates collected in Eastern Cambodia between 2014 and 2019. Results Among the 52 cryopreserved isolates tested, all were susceptible to the three drugs, with overall median IC50s of 16.1 nM (IQR 11.4–22.3) chloroquine, 3.4 nM (IQR 2.1–5.0) mefloquine and 4.6 nM (IQR 2.7–7.0) piperaquine. A significant increase in chloroquine and piperaquine susceptibility was observed between 2015 and 2018, unrelated to polymorphisms in pvcrt and pvmdr1. Susceptibility to mefloquine was significantly lower in parasites with a single mutation in pvmdr1 compared with isolates with multiple mutations. The proportion of parasites with this single mutation genotype increased between 2014 and 2019. Conclusions P. vivax with decreased susceptibility to mefloquine is associated with the introduction of mefloquine-based treatment during 2017–18.

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