Optimizing the HRP-2 in vitro malaria drug susceptibility assay using a reference clone to improve comparisons of Plasmodium falciparum 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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Evaluation of an In Vitro Assay System for Drug Susceptibility of Field Isolates of Plasmodium Falciparum from Southern Thailand

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.1988.38.19 ◽

1988 ◽

Vol 38 (1) ◽

pp. 19-23 ◽

Cited By ~ 5

Author(s):

G. E. Childs ◽

T. Wimonwattrawatee ◽

N. Pooyindee

Keyword(s):

Plasmodium Falciparum ◽

Drug Susceptibility ◽

In Vitro Assay ◽

Assay System ◽

Vitro Assay ◽

Field Isolates ◽

Southern Thailand

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Antifolate screening using yeast expressing Plasmodium vivax dihydrofolate reductase and in vitro drug susceptibility assay for Plasmodium falciparum

Molecular and Biochemical Parasitology ◽

10.1016/j.molbiopara.2007.07.009 ◽

2007 ◽

Vol 156 (1) ◽

pp. 89-92 ◽

Cited By ~ 8

Author(s):

Liselotte Yimga Djapa ◽

Leonardo K. Basco ◽

Ruth Zelikson ◽

Andre Rosowsky ◽

Joseph Allico Djaman ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Plasmodium Vivax ◽

Dihydrofolate Reductase ◽

Drug Susceptibility ◽

In Vitro Drug Susceptibility ◽

Susceptibility Assay

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Antimalarial Activity of Tigecycline, a Novel Glycylcycline Antibiotic

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00312-09 ◽

2009 ◽

Vol 53 (9) ◽

pp. 4040-4042 ◽

Cited By ~ 11

Author(s):

P. Starzengruber ◽

K. Thriemer ◽

R. Haque ◽

W. A. Khan ◽

H. P. Fuehrer ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Confidence Interval ◽

Antimalarial Activity ◽

Drug Susceptibility ◽

Clinical Isolates ◽

In Vitro Drug Susceptibility ◽

Antibacterial Spectrum ◽

Susceptibility Assay ◽

Broad Antibacterial Spectrum

ABSTRACT Tigecycline is a novel glycylcycline antibiotic with a broad antibacterial spectrum. Tigecycline was tested with 66 clinical isolates of Plasmodium falciparum from Bangladesh using the histidine-rich protein 2 in vitro drug susceptibility assay. The 50% and 90% inhibitory concentrations of tigecycline were 699 (95% confidence interval, 496 to 986) and 5,905 nM (4,344 to 8,028). Tigecycline shows no activity correlation with traditional antimalarials and has substantial antimalarial activity on its own.

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Plasmodium falciparum and Plasmodium vivax: Lactate-Dehydrogenase Activity and Its Application for in Vitro Drug Susceptibility Assay

Experimental Parasitology ◽

10.1006/expr.1995.1032 ◽

1995 ◽

Vol 80 (2) ◽

pp. 260-271 ◽

Cited By ~ 46

Author(s):

L.K. Basco ◽

F. Marquet ◽

M.M. Makler ◽

J. Lebras

Keyword(s):

Plasmodium Falciparum ◽

Lactate Dehydrogenase ◽

Plasmodium Vivax ◽

Dehydrogenase Activity ◽

Drug Susceptibility ◽

Lactate Dehydrogenase Activity ◽

In Vitro Drug Susceptibility ◽

Susceptibility Assay

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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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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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