Inoculum effect leads to overestimation of in vitro resistance for artemisinin derivatives and standard antimalarials: a Gambian field study

Parasitology ◽  
1999 ◽  
Vol 119 (5) ◽  
pp. 435-440 ◽  
Author(s):  
M. T. DURAISINGH ◽  
P. JONES ◽  
I. SAMBOU ◽  
L. VON SEIDLEIN ◽  
M. PINDER ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Multidrug Resistance ◽  
Field Study ◽  
Susceptibility Testing ◽  
Inoculum Size ◽  
Cross Resistance ◽  
Artemisinin Derivatives ◽  
Inoculum Effect ◽  
Measured Sensitivity

Artemisinin (QHS) and its derivatives are new antimalarials which are effective against Plasmodium falciparum parasites resistant to chloroquine (CQ). As these drugs are introduced it is imperative that resistance is monitored. In this paper we demonstrate that the inoculum size used in in vitro testing influences the measured in vitro susceptibility to QHS and its derivative dihydroartemisinin (DHA) and to mefloquine (MEF) and CQ over the range of parasitaemias routinely used in testing with the WHO in vitro microtest. An increase in parasitaemia and/or haematocrit was accompanied by a decrease in the measured sensitivity of 2 laboratory lines. In the context of a field study testing in vitro susceptibility of parasite isolates from patients with uncomplicated malaria in Fajara, The Gambia we demonstrate that failure to control for inoculum size significantly overestimates the level of resistance to QHS and DHA as well as MEF, halofantrine (HAL) and quinine (QUIN). When controlling for the inoculum effect, cross-resistance was observed between QHS, MEF and HAL suggesting the presence of a multidrug resistance-like mechanism. These studies underline the importance of inoculum size in in vitro susceptibility testing.

scholarly journals In Vitro Activity of Ferroquine Is Independent of Polymorphisms in Transport Protein Genes Implicated in Quinoline Resistance in Plasmodium falciparum

2008 ◽  
Vol 52 (8) ◽  
pp. 2755-2759 ◽  
Author(s):  
Maud Henry ◽  
Sébastien Briolant ◽  
Albin Fontaine ◽  
Joel Mosnier ◽  
Eric Baret ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Plasmodium Falciparum ◽  
Multidrug Resistance ◽  
Transport Proteins ◽  
Vitro Activity ◽  
Cross Resistance ◽  
In Vitro Activity ◽  
Highly Active ◽  
In Vitro Response

ABSTRACT The in vitro activity of ferroquine (FQ) (SR97193), a 4-aminoquinoline antimalarial compound that contains a ferrocenic nucleus, against 15 Plasmodium falciparum strains was assessed and compared with those of chloroquine (CQ), quinine (QN), monodesethylamodiaquine (MDAQ), and mefloquine (MQ). These 15 strains were genotyped for polymorphisms in quinoline resistance-associated genes such as Pfcrt, Pfmdr1, Pfmrp, and Pfnhe-1. FQ was highly active against CQ-resistant parasites or in parasites with reduced susceptibility to QN, MDAQ, or MQ. Encouragingly, we did not find a correlation between responses to FQ and those to other quinoline drugs. These results suggest that no cross-resistance exits between FQ and CQ or quinoline antimalarial drugs. Mutations in codons 74, 75, 76, 220, 271, 326, 356, and 371 of the Pfcrt gene; codons 86, 184, 1034, 1042, and 1246 of the Pfmdr1 gene; and codons 191 and 437 of the Pfmrp gene were not significantly associated with P. falciparum susceptibility to FQ. Neither the number of ms4760 DNNND or DDNHNDNHNN repeats in Pfnhe-1 nor the profile of ms4760 was significantly associated with the FQ in vitro response. These data suggest the FQ may not interact with transport proteins in quinoline-resistant parasites. The present results justify further clinical trials of FQ in multidrug resistance areas.

scholarly journals Deamplification of pfmdr1-Containing Amplicon on Chromosome 5 in Plasmodium falciparum Is Associated with Reduced Resistance to Artelinic Acid In Vitro

2010 ◽  
Vol 54 (8) ◽  
pp. 3395-3401 ◽  
Author(s):  
Nanhua Chen ◽  
Marina Chavchich ◽  
Jennifer M. Peters ◽  
Dennis E. Kyle ◽  
Michelle L. Gatton ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Multidrug Resistance ◽  
Drug Pressure ◽  
Rapid Reduction ◽  
Artemisinin Derivatives ◽  
Multiple Copies ◽  
Multidrug Resistance 1 Gene ◽  
The Stability ◽  
Increased Susceptibility

ABSTRACT Amplification of the Plasmodium falciparum multidrug resistance 1 gene (pfmdr1) has been implicated in multidrug resistance, including in vitro resistance to artelinic acid (AL). The stability and fitness of having multiple copies of pfmdr1 are important factors due to their potential effects on the resistance phenotype of parasites. These factors were investigated by using an AL-resistant line of P. falciparum (W2AL80) and clones originating from W2AL80. A rapid reduction in pfmdr1 copy number (CN) was observed in the uncloned W2AL80 line; 63% of this population reverted to a CN of <3 without exposure to the drug. Deamplification of the pfmdr1 amplicon was then determined in three clones, each initially containing three copies of pfmdr1. Interestingly, two outcomes were observed during 3 months without drug pressure. In one clone, parasites with fewer than 3 copies of pfmdr1 emerged rapidly. In two other clones, the reversion was significantly delayed. In all subclones, the reduction in pfmdr1 CN involved the deamplification of the entire amplicon (19 genes). Importantly, deamplification of the pfmdr1 amplicon resulted in partial reversal of resistance to AL and increased susceptibility to mefloquine. These results demonstrate that multiple copies of the pfmdr1-containing amplicon in AL-resistant parasites are unstable when drug pressure is withdrawn and have practical implications for the maintenance and spread of parasites resistant to artemisinin derivatives.

The in vitro antimalarial interaction of 9-hydroxycalabaxanthone and α-mangostin with mefloquine/artesunate

2014 ◽  
Vol 60 (1) ◽  
Author(s):  
Wanna Chaijaroenkul ◽  
Kesara Na-Bangchang
Keyword(s):  
Plasmodium Falciparum ◽  
Multidrug Resistance ◽  
Drug Concentration ◽  
Combination Index ◽  
Antimalarial Drugs ◽  
Antagonistic Effect ◽  
Parasite Growth ◽  
Major Health Problem ◽  
Major Health

AbstractMultidrug resistance Plasmodium falciparum is the major health problem in Thailand. Discovery and development of new antimalarial drugs with novel modes of action is urgently required. The aim of the present study was to investigate the antimalarial interaction of 9-hydroxycalabaxanthone and α-mangostin with the standard antimalarial drugs mefloquine and artesunate in chloroquine sensitive (3D7) and chloroquine resistant (K1) P. falciparum clones in vitro. Median (range) IC50 (drug concentration which produces 50% parasite growth inhibition) values of the 9-hydroxycalabaxanthone, α-mangostin, artesunate and mefloquine for 3D7 vs K1 clones were 1.5 (0.9-2.1) vs 1.2 (1.1-1.6) μM, 17.9 (15.7.0-20.0) vs 9.7 (6.0-14.0) μM, 1.0 (0.4-3.0) vs 1.7 (1.0-2.5) nM, and 13.3 (11.1-13.3) vs 7.1 (6.7-12.2) nM, respectively. Analysis of isobologram and combination index (CI) of 9-hydroxycalabaxanthone with artesunate or mefloquine showed synergistic and indifference antimalarial interaction, respectively. α-mangostin-artesunate combination exhibited a slight antagonistic effect of antimalarial interaction, whereas α-mangostin and mefloquine combination showed indifference interaction in both clones. The combination of 9-hydroxycalabaxanthone with α-mangostin showed the synergistic antimalarial interaction in both clones

scholarly journals Plasmid-Mediated Resistance to Thrombin-Induced Platelet Microbicidal Protein in Staphylococci: Role of theqacA Locus

1999 ◽  
Vol 43 (10) ◽  
pp. 2395-2399 ◽  
Author(s):  
Leon Iri Kupferwasser ◽  
Ronald A. Skurray ◽  
Melissa H. Brown ◽  
Neville Firth ◽  
Michael R. Yeaman ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Efflux Pump ◽  
Endothelial Damage ◽  
Cross Resistance ◽  
Cationic Peptide ◽  
Multidrug Resistance Gene ◽  
Phenotypic Resistance ◽  
Platelet Microbicidal Protein

ABSTRACT Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide released from rabbit platelets following thrombin stimulation. In vitro resistance to this peptide among strains of Staphylococcus aureus correlates with the survival advantage of such strains at sites of endothelial damage in humans as well as in experimental endovascular infections. The mechanisms involved in the phenotypic resistance of S. aureus to tPMP-1 are not fully delineated. The plasmid-encoded staphylococcal gene qacA mediates multidrug resistance to multiple organic cations via a proton motive force-dependent efflux pump. We studied whether the qacA gene might also confer resistance to cationic tPMP-1. Staphylococcal plasmids encoding qacA were found to confer resistance to tPMP-1 in an otherwise susceptible parental strain. Deletions which removed the region containing theqacA gene in the S. aureus multiresistance plasmid pSK1 abolished tPMP-1 resistance. Resistance to tPMP-1 in theqacA-bearing strains was inoculum independent but peptide concentration dependent, with the level of resistance decreasing at higher peptide concentrations for a given inoculum. There was no apparent cross-resistance in qacA-bearing strains to other endogenous cationic antimicrobial peptides which are structurally distinct from tPMP-1, including human neutrophil defensin 1, protamine, or the staphylococcal lantibiotics pep5 and nisin. These data demonstrate that the staphylococcal multidrug resistance geneqacA also mediates in vitro resistance to cationic tPMP-1.

scholarly journals Reversal of Mefloquine and Quinine Resistance in Plasmodium falciparum with NP30

2003 ◽  
Vol 47 (8) ◽  
pp. 2393-2396 ◽  
Author(s):  
Michelle Ciach ◽  
Kathleen Zong ◽  
Kevin C. Kain ◽  
Ian Crandall
Keyword(s):  
Plasmodium Falciparum ◽  
Multidrug Resistance ◽  
Genetic Analysis ◽  
Resistance Genes ◽  
Mammalian Cells ◽  
Point Mutations ◽  
In Vitro Assays ◽  
Drug Efflux ◽  
P Glycoprotein

ABSTRACT Quinoline resistance in malaria is frequently compared with P-glycoprotein-mediated multidrug resistance (mdr) in mammalian cells. We have previously reported that nonylphenolethoxylates, such as NP30, are potential Plasmodium falciparum P-glycoprotein substrates and drug efflux inhibitors. We used in vitro assays to compare the ability of verapamil and NP30 to sensitize two parasite isolates to four quinolines: chloroquine (CQ), mefloquine (MF), quinine (QN), and quinidine (QD). NP30 was able to sensitize (reversal, >80%) P. falciparum to MF, QN, QD, and, to a lesser extent, CQ. The presence of 2 μM verapamil had no effect on mefloquine resistance; however, the presence of verapamil modulated the activities of QN and QD in a manner parallel to that observed for CQ. Genetic analysis of putative quinoline resistance genes did not suggest an association between known point mutations in pfcrt and pfmdr1 and NP30 sensitization activity. We conclude that the sensitization action of NP30 is distinct both phenotypically and genotypically from that of verapamil.

In vitro susceptibility of Gabonese wild isolates of Plasmodium falciparum to artemether, and comparison with chloroquine, quinine, halofantrine and amodiaquine

Parasitology ◽  
1998 ◽  
Vol 117 (6) ◽  
pp. 541-545 ◽  
Author(s):  
B. PRADINES ◽  
M. MABIKA MAMFOUMBI ◽  
D. PARZY ◽  
M. OWONO MEDANG ◽  
C. LEBEAU ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Inhibitory Concentration ◽  
Narrow Range ◽  
Drug Susceptibility ◽  
Drug Uptake ◽  
Cross Resistance ◽  
In Vitro Susceptibility ◽  
Positive Correlation ◽  
Common Features

The in vitro activity of artemether against 63 African isolates of Plasmodium falciparum from Libreville, Gabon was evaluated using an isotopic drug susceptibility semi-microtest. The 50% inhibitory concentration (IC50) values for artemether were in a narrow range from 0·8 to 34·8 nm (mean IC50 5·0 nm) and the 95% confidence interval (CI95%) was 3·6–6·3 nm. In vitro decreased susceptibility or resistance were observed with artemether (14%), to chloroquine (90%), to quinine (32%). Isolate susceptibility to amodiaquine and halofantrine was high i.e. 100% and 98%, respectively. There was a significant positive correlation between responses to artemether and amodiaquine (r2=0·45, P<0·001), artemether and chloroquine (r2=0·36, P<0·001), artemether and quinine (r2=0·31, P<0·001), and artemether and halofantrine r2=0·19, P<0·01). Positive correlation between these drugs suggests in vitro cross-resistance or at least common features in drug uptake and/or mode of action or resistance.

scholarly journals Plasmodium falciparum Multidrug Resistance Proteins (pfMRPs)

2021 ◽  
Vol 12 ◽  
Author(s):  
José Pedro Gil ◽  
Cláudia Fançony
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Multidrug Resistance ◽  
Multi Drug Resistance ◽  
Multidrug Resistance Proteins ◽  
Resistance Proteins ◽  
Diverse Groups ◽  
Endogenous Substances

The capacity of the lethal Plasmodium falciparum parasite to develop resistance against anti-malarial drugs represents a central challenge in the global control and elimination of malaria. Historically, the action of drug transporters is known to play a pivotal role in the capacity of the parasite to evade drug action. MRPs (Multidrug Resistance Protein) are known in many phylogenetically diverse groups to be related to drug resistance by being able to handle a large range of substrates, including important endogenous substances as glutathione and its conjugates. P. falciparum MRPs are associated with in vivo and in vitro altered drug response, and might be important factors for the development of multi-drug resistance phenotypes, a latent possibility in the present, and future, combination therapy environment. Information on P. falciparum MRPs is scattered in the literature, with no specialized review available. We herein address this issue by reviewing the present state of knowledge.

scholarly journals Mapping the malaria parasite drug-able genome using in vitro evolution and chemogenomics

2017 ◽  
Author(s):  
Annie N. Cowell ◽  
Eva S. Istvan ◽  
Amanda K. Lukens ◽  
Maria G. Gomez-Lorenzo ◽  
Manu Vanaerschot ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Multidrug Resistance ◽  
Resistance Genes ◽  
Genome Analysis ◽  
Drug Targets ◽  
Malaria Parasite ◽  
Resistance Mechanisms ◽  
Whole Genome

AbstractChemogenetic characterization through in vitro evolution combined with whole genome analysis is a powerful tool to discover novel antimalarial drug targets and identify drug resistance genes. Our comprehensive genome analysis of 262 Plasmodium falciparum parasites treated with 37 diverse compounds reveals how the parasite evolves to evade the action of small molecule growth inhibitors. This detailed data set revealed 159 gene amplifications and 148 nonsynonymous changes in 83 genes which developed during resistance acquisition. Using a new algorithm, we show that gene amplifications contribute to 1/3 of drug resistance acquisition events. In addition to confirming known multidrug resistance mechanisms, we discovered novel multidrug resistance genes. Furthermore, we identified promising new drug target-inhibitor pairs to advance the malaria elimination campaign, including: thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This deep exploration of the P. falciparum resistome and drug-able genome will guide future drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms of the deadliest malaria parasite.One Sentence SummaryWhole genome sequencing reveals how Plasmodium falciparum evolves resistance to diverse compounds and identifies new antimalarial drug targets.

Prediction of the Effects of Inoculum Size on the Antimicrobial Action of Trovafloxacin and Ciprofloxacin againstStaphylococcus aureus and Escherichia coli in an In Vitro Dynamic Model

1999 ◽  
Vol 43 (3) ◽  
pp. 498-502 ◽  
Author(s):  
Alexander A. Firsov ◽  
Sergey N. Vostrov ◽  
Olga V. Kononenko ◽  
Stephen H. Zinner ◽  
Yury A. Portnoy
Keyword(s):  
Escherichia Coli ◽  
Dynamic Models ◽  
Antimicrobial Effect ◽  
Inoculum Size ◽  
Antimicrobial Action ◽  
Time Curve ◽  
Mean Values ◽  
E Coli ◽  
Inoculum Effect

ABSTRACT The effect of inoculum size (N 0) on antimicrobial action has not been extensively studied in in vitro dynamic models. To investigate this effect and its predictability, killing and regrowth kinetics of Staphylococcus aureus andEscherichia coli exposed to monoexponentially decreasing concentrations of trovafloxacin (as a single dose) and ciprofloxacin (two doses at a 12-h interval) were compared atN 0 = 106 and 109 CFU/ml (S. aureus) and at N 0 = 106, 107, and 109 CFU/ml (E. coli). A series of pharmacokinetic profiles of trovafloxacin and ciprofloxacin with respective half-lives of 9.2 and 4 h were simulated at different ratios of area under the concentration-time curve (AUC) to MIC (in [micrograms × hours/milliliter]/[micrograms/milliliter]): 58 to 466 with trovafloxacin and 116 to 932 with ciprofloxacin for S. aureus and 58 to 233 and 116 to 466 for E. coli, respectively. Although the effect of N 0 was more pronounced for E. coli than for S. aureus, only a minor increase in minimum numbers of surviving bacteria and an almost negligible delay in their regrowth were associated with an increase of the N 0 for both organisms. TheN 0-induced reductions of the intensity of the antimicrobial effect (IE , area between control growth and the killing-regrowth curves) were also relatively small. However, the N 0 effect could not be eliminated either by simple shifting of the time-kill curves obtained at higherN 0s by the difference between the higher and lowest N 0 or by operating withIE s determined within theN 0-adopted upper limits of bacterial numbers (IE ′s). By using multivariate correlation and regression analyses, linear relationships betweenIE and log AUC/MIC and logN 0 related to the respective mean values [(log AUC/MIC)average and (logN 0)average] were established for both trovafloxacin and ciprofloxacin against each of the strains (r 2 = 0.97 to 0.99). The antimicrobial effect may be accurately predicted at a given AUC/MIC of trovafloxacin or ciprofloxacin and at a given N 0 based on the relationship IE = a + b [(log AUC/MIC)/(log AUC/MIC)average] − c [(logN 0)/(logN 0)average]. Moreover, the relative impacts of AUC/MIC and N 0 onIE may be evaluated. Since the c/bratios for trovafloxacin and ciprofloxacin against E. coliwere much lower (0.3 to 0.4) than that for ampicillin-sulbactam as examined previously (1.9), the inoculum effect with the quinolones may be much less pronounced than with the β-lactams. The described approach to the analysis of the inoculum effect in in vitro dynamic models might be useful in studies with other antibiotic classes.

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