Real-time PCR for chloroquine sensitivity assay and for pfmdr1–pfcrt single nucleotide polymorphisms in Plasmodium falciparum

Monbrison, Frédérique de; Raynaud, Delphine; Latour-Fondanaiche, C; Staal, Anne; Favre, Sébastien; Kaiser, Karine; Peyron, François; Picot, Stéphane

doi:10.1016/s0167-7012(03)00086-1

Cited by 37 publications

(23 citation statements)

References 17 publications

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“…Between September and December 2005, fresh isolates were collected before treatment from patients hospitalized in the Lyon University Hospital for imported malaria. A diagnosis of malaria was established by microscopic examination of Giemsa-stained blood smears, and the diagnosis was confirmed by using the real-time PCR previously described by De Monbrison et al (8). Blood samples collected from the patients with confirmed cases of P. falciparum malaria were maintained at room temperature for, on average, less than 12 h until they were diluted and added to 96-well plates precoated with chloroquine and mefloquine.…”

Section: Antimalarialsmentioning

confidence: 99%

“…However, this method is time-consuming and the need for the disposal of radioactive materials has limited its widespread use, especially in countries that cannot afford the prohibitive cost of a liquid scintillation counter and where reagents are difficult, if not impossible, to obtain. Another method commonly used to determined susceptibility levels is the WHO microtest, which uses microscopic evaluation to enumerate the quantity of parasites following growth in the presence of antimalarials (8,17).…”

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confidence: 99%

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Comparison of a SYBR Green I-Based Assay with a Histidine-Rich Protein II Enzyme-Linked Immunosorbent Assay for In Vitro Antimalarial Drug Efficacy Testing and Application to Clinical Isolates

Bacon

Latour

Lucas

et al. 2007

Antimicrob Agents Chemother

107

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In vitro drug susceptibility testing with the malaria parasite has been used to assess the antimalarial activities of new compounds and to monitor drug resistance in field isolates. We investigated the validity of a SYBR green I fluorescent-based assay under various culture conditions and compared the assay results to those of previously published histidine-rich protein II (HRPII) enzyme-linked immunosorbent assay (ELISA) methods. Reference strains of Plasmodium falciparum were cultured in vitro by using standard conditions in complete medium with and without phenol red before they were dispensed into 96-well plates predosed with chloroquine, mefloquine, or quinine. Following incubation, the culture supernatants were divided and the 50% inhibitory concentrations (IC 50 s) were determined by using a SYBR green I-based method and the HRPII capture ELISA method. There were no significant differences in IC 50 values when phenol red was included in the medium. The IC 50 s and the IC 90 s of the antimalarials tested by both methods were similar or identical for each of the reference strains. Fresh clinical isolates of P. falciparum collected from imported cases of malaria in Lyon, France, were tested for in vitro resistance to chloroquine and mefloquine by using the validated SYBR green I and HRPII ELISA methods. The SYBR green I-based method was able to calculate IC 50 and IC 90 values similar or identical to those calculated by the HRPII assay with fresh clinical samples without removal of white blood cells. The SYBR green I-based method for determination of drug sensitivity levels produced results comparable to those produced by other methods, showing that this method can be used routinely to conduct surveillance for drug resistance in P. falciparum with fresh or cultured parasites.Currently, in vivo drug efficacy studies are the "gold standard" for assessment of drug resistance in malaria parasites (20). While such studies are routinely conducted on a small scale, the cost associated with in vivo drug studies with the malaria parasite prohibits sustained surveillance at the required level of vigilance. Molecular marker determination has been used to identify the single-nucleotide polymorphisms commonly associated with drug resistance in malaria parasites; however, the presence of molecular markers does not always correlate with in vivo drug resistance (25). Other molecular biology methodologies have been developed to assess the flow of drug resistance markers by using known microsatellite markers, but this technology requires specialized equipment and can be quite costly (10, 26). Although microsatellite differentiation is quite powerful and informative, it would be difficult to conduct microsatellite differentiation in the field in real time.A quick and very cost-effective alternative means to determination of the level of resistance is in vitro drug susceptibility testing (4,17). Unlike the in vivo test for drug resistance, the in vitro assay is designed to assess the parasites' responses to drugs without interf...

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Section: Antimalarialsmentioning

confidence: 99%

mentioning

confidence: 99%

Comparison of a SYBR Green I-Based Assay with a Histidine-Rich Protein II Enzyme-Linked Immunosorbent Assay for In Vitro Antimalarial Drug Efficacy Testing and Application to Clinical Isolates

Bacon

Latour

Lucas

et al. 2007

Antimicrob Agents Chemother

107

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“…[32][33][34] Among P. falciparum isolates we collected earlier (1997)(1998)(1999)(2000)(2001)(2002), Pfmdr1 codon 86 mutation (86Y) rates in two western sites (Kisumu, Kericho) were 35% and 68%, respectively (mean, 51%), whereas two eastern sites (Entosopia, Magadi) had rates > 85%. 17 This reflected CQ resistance in Kenya.…”

mentioning

confidence: 99%

Antimalarial Drug Sensitivity Profile of Western Kenya Plasmodium falciparum Field Isolates Determined by a SYBR Green I in vitro Assay and Molecular Analysis

Akala¹,

Eyase²,

Cheruiyot³

et al. 2011

The American Society of Tropical Medicine and Hygiene

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In vitro drug sensitivity and molecular analyses of Plasmodium falciparum track drug resistance. DNA-binding fluorescent dyes like SYBR Green I may allow field laboratories, proximal to P. falciparum collection sites, to conduct drug assays. In 2007–2008, we assayed 121 P. falciparum field isolates from western Kenya for 50% inhibitory concentrations (IC50) against 6 antimalarial drugs using a SYBR Green I in vitro assay: 91 immediate ex vivo (IEV) and 30 culture-adapted, along with P. falciparum reference clones D6 (chloroquine [CQ] sensitive) and W2 (CQ resistant). We also assessed P. falciparum mdr1 (Pfmdr1) copy number and single nucleotide polymorphisms (SNPs) at four codons. The IC50s for IEV and culture-adapted P. falciparum isolates were similar, and approximated historical IC50s. For Pfmdr1, mean copy number was 1, with SNPs common at codons 86 and 184. The SYBR Green I assay adapted well to our field-based laboratory, for both IEV and culture-adapted P. falciparum, warranting continued use.

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“…Other relatively lowthroughput methodologies include direct DNA sequencing, mutation-specific PCR (17), dot blot probe hybridization (18), molecular beacons (19), and single-nucleotide primer extension (20). Systems examined to provide improved throughput have included polymorphism-specific microarrays (21), melting curve analysis (22,23), quantitative PCR (24)(25)(26), and a ligase detection reaction-fluorescent microsphere (LDR-FM) assay (27,28). Each of these assays has shown success, although each has challenges, including the cost of equipment and reagents and the uncertainty of success with field samples, which are typically stored on filter paper at room temperature and commonly consist of polyclonal infections.…”

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confidence: 99%

Optimization of a Ligase Detection Reaction-Fluorescent Microsphere Assay for Characterization of Resistance-Mediating Polymorphisms in African Samples of Plasmodium falciparum

et al. 2013

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bGenetic polymorphisms in the malaria parasite Plasmodium falciparum mediate alterations in sensitivity to important antimalarial drugs. Surveillance for these polymorphisms is helpful in assessing the prevalence of drug resistance and designing strategies for malaria control. Multiple methods are available for the assessment of P. falciparum genetic polymorphisms, but they suffer from low throughput, technical limitations, and high cost. We have optimized and tested a multiplex ligase detection reaction-fluorescent microsphere (LDR-FM) assay for the identification of important P. falciparum genetic polymorphisms. For 84 clinical samples from Kampala, Uganda, a region where both transmission intensity and infection complexity are high, DNA was extracted from dried blood spots, genes of interest were amplified, amplicons were subjected to multiplex ligase detection reactions to add bead-specific oligonucleotides and biotin, fragments were hybridized to magnetic beads, and polymorphism prevalences were assessed fluorometrically in a multiplex format. A total of 19 alleles from the pfcrt, pfmdr1, pfmrp1, pfdhfr, and pfdhps genes were analyzed by LDR-FM and restriction fragment length polymorphism (RFLP) analyses. Considering samples with results from the two assays, concordance between the assays was good, with 78 to 100% of results identical at individual alleles, most nonconcordant results differing only between a mixed and pure genotype call, and full disagreement at individual alleles in only 0 to 3% of results. We estimate that the LDR-FM assay offers much higher throughput and lower cost than RFLP. Our results suggest that the LDR-FM system offers an accurate high-throughput means of classifying genetic polymorphisms in field samples of P. falciparum.

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Real-time PCR for chloroquine sensitivity assay and for pfmdr1–pfcrt single nucleotide polymorphisms in Plasmodium falciparum

Cited by 37 publications

References 17 publications

Comparison of a SYBR Green I-Based Assay with a Histidine-Rich Protein II Enzyme-Linked Immunosorbent Assay for In Vitro Antimalarial Drug Efficacy Testing and Application to Clinical Isolates

Comparison of a SYBR Green I-Based Assay with a Histidine-Rich Protein II Enzyme-Linked Immunosorbent Assay for In Vitro Antimalarial Drug Efficacy Testing and Application to Clinical Isolates

Antimalarial Drug Sensitivity Profile of Western Kenya Plasmodium falciparum Field Isolates Determined by a SYBR Green I in vitro Assay and Molecular Analysis

Optimization of a Ligase Detection Reaction-Fluorescent Microsphere Assay for Characterization of Resistance-Mediating Polymorphisms in African Samples of Plasmodium falciparum

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