scholarly journals Direct comparison of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR green I fluorescence (MSF) drug sensitivity tests in Plasmodium falciparum reference clones and fresh ex vivo field isolates from Cambodia

2013 ◽  
Vol 12 (1) ◽  
pp. 239 ◽  
Author(s):  
Suwanna Chaorattanakawee ◽  
Stuart D Tyner ◽  
Chanthap Lon ◽  
Kritsanai Yingyuen ◽  
Wiriya Ruttvisutinunt ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Immunosorbent Assay ◽  
Drug Sensitivity ◽  
Ex Vivo ◽  
Sybr Green ◽  
Sybr Green I ◽  
Enzyme Linked Immunosorbent Assay ◽  
Field Isolates ◽  
Sensitivity Tests

scholarly journals Simple Histidine-Rich Protein 2 Double-Site Sandwich Enzyme-Linked Immunosorbent Assay for Use in Malaria Drug Sensitivity Testing

2005 ◽  
Vol 49 (8) ◽  
pp. 3575-3577 ◽  
Author(s):  
Harald Noedl ◽  
Jan Bronnert ◽  
Kritsanai Yingyuen ◽  
Bernhard Attlmayr ◽  
Herwig Kollaritsch ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Drug Sensitivity ◽  
Inhibitory Concentration ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sensitivity Testing ◽  
Drug Sensitivity Test ◽  
Sensitivity Tests

ABSTRACT A simple double-site sandwich enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum in vitro drug sensitivity tests based on measuring histidine-rich protein 2 (HRP2) is presented. The ELISA uses two commercial monoclonal antibodies and provides a drastically cheaper alternative to the test kits previously used in the HRP2 drug sensitivity test. The assay is simple to establish and perform. The sensitivity is comparable and the drug sensitivity results very closely match those obtained with the commercial ELISA kits (R 2 = 0.979; P < 0.001; mean log difference at the 50% inhibitory concentration = 0.07).

In-vitro culture of Plasmodium falciparum: Utility of modified (RPNI) medium for drug-sensitivity studies using SYBR Green I assay

2011 ◽  
Vol 127 (1) ◽  
pp. 318-321 ◽  
Author(s):  
Shubhra Singh ◽  
Rajeev Kumar Srivastava ◽  
Mukesh Srivastava ◽  
S.K. Puri ◽  
K. Srivastava
Keyword(s):  
Plasmodium Falciparum ◽  
In Vitro Culture ◽  
Drug Sensitivity ◽  
Sybr Green ◽  
Sybr Green I ◽  
Sensitivity Studies

scholarly journals Ex vivo drug sensitivity profiles of Plasmodium falciparum field isolates from Cambodia and Thailand, 2005 to 2010, determined by a histidine-rich protein-2 assay

2012 ◽  
Vol 11 (1) ◽  
pp. 198 ◽  
Author(s):  
Stuart D Tyner ◽  
Chanthap Lon ◽  
Youry Se ◽  
Delia Bethell ◽  
Doung Socheat ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Drug Sensitivity ◽  
Ex Vivo ◽  
Field Isolates

scholarly journals 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

2007 ◽  
Vol 51 (4) ◽  
pp. 1172-1178 ◽  
Author(s):  
David J. Bacon ◽  
Christine Latour ◽  
Carmen Lucas ◽  
Olga Colina ◽  
Pascal Ringwald ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Immunosorbent Assay ◽  
Sybr Green ◽  
Sybr Green I ◽  
Clinical Isolates ◽  
Enzyme Linked Immunosorbent Assay ◽  
Complete Medium ◽  
Phenol Red ◽  
Reference Strains

ABSTRACT 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 (IC50s) were determined by using a SYBR green I-based method and the HRPII capture ELISA method. There were no significant differences in IC50 values when phenol red was included in the medium. The IC50s and the IC90s 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 IC50 and IC90 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.

scholarly journals A modified two-color flow cytometry assay to quantify in-vitro reinvasion and determine invasion phenotypes at low Plasmodium falciparum parasitemia

2019 ◽  
Author(s):  
Ngoh Ines Atuh ◽  
Anong Damian Nota ◽  
Fru Jerome Cho ◽  
Fatoumata Bojang ◽  
Haddijatou Mbye ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasmodium Falciparum ◽  
Fluorescent Dyes ◽  
Sybr Green ◽  
Sybr Green I ◽  
Color Flow ◽  
Background Fluorescence ◽  
Field Isolates ◽  
Field Samples ◽  
Set Up

ABSTRACTTwo-color flow cytometry(2cFCM) is the most accessible method for phenotyping parasite invasion. However, current protocols require samples of field isolates at ∼1% parasitemia for assay set-up, which are becoming more uncommon in low transmission settings. Current protocols, therefore, have to be adapted for low parasitemia if the method must have continued applicability in this era of elimination. Optimizing the protocol requires addressing; interference from young uninfected RBCs background fluorescence and biased phenotypes due to limited labeled RBCs availability and/or parasite density per assay. Here, we used SYBR Green I and CTFR Proliferation fluorescent dyes to set-up invasion assays with Plasmodium falciparum 3D7, Dd2 and field isolates cultures (diluted at 0.05% to 2.0% parasitemia) against varying unlabeled to labeled RBC ratios (1:1 to 1:4). We showed that a shorter SYBR Green I staining time of 20 minutes, down from 1hour, minimized background fluorescence from uninfected RBCs (mean= 0.03% events) and allowed 2cFCM to accurately quantify reinvasion for an assay at 0.02% parasitemia. An increase in labeled target RBCs to 1:3 per assays significantly increased heterologous reinvasion (p<0.001). This resulted in a 10% greater invasion inhibition by enzyme treatments (p<0.05). Strain-specific invasion phenotype could be accurately determined for samples with as low as 0.3% parasitemia. Samples above 0.8% parasitemia were less accurate. These findings show that invasion pathway phenotypes can be obtained for field samples with low parasitemia at greater sensitivity and reproducibility by increasing the proportion of labeled RBCs per assay by at least 2-fold what is in current methods.

Associations between varied susceptibilities of PfATP4 inhibitors and genotypes in Ugandan Plasmodium falciparum isolates

2021 ◽  
Author(s):  
Oriana Kreutzfeld ◽  
Stephanie A. Rasmussen ◽  
Aarti A. Ramanathan ◽  
Patrick K. Tumwebaze ◽  
Oswald Byaruhanga ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Single Nucleotide Polymorphisms ◽  
Ex Vivo ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Single Nucleotide ◽  
Field Isolates ◽  
Frequent Mutations ◽  
Mixed Field ◽  
P Type

Among novel compounds under recent investigation as potential new antimalarial drugs are three independently developed inhibitors of the Plasmodium falciparum P-type ATPase (PfATP4): KAE609 (cipargamin), PA92, and SJ733. We assessed ex vivo susceptibilities to these compounds of 374 fresh P. falciparum isolates collected in Tororo and Busia districts, Uganda from 2016-2019. Median IC 50 s were 65 nM for SJ733, 9.1 nM for PA92, and 0.5 nM for KAE609. Sequencing of pfatp4 for 218 of these isolates demonstrated many non-synonymous single nucleotide polymorphisms; the most frequent mutations were G1128R (69% of isolates mixed or mutant), Q1081K/R (68%), G223S (25%), N1045K (16%) and D1116G/N/Y (16%). The G223S mutation was associated with decreased susceptibility to SJ733, PA92 and KAE609. The D1116G/N/Y mutations were associated with decreased susceptibility to SJ733, and the presence of mutations at both codons 223 and 1116 was associated with decreased susceptibility to PA92 and SJ733. In all of these cases, absolute differences in susceptibilities of wild type (WT) and mutant parasites were modest. Analysis of clones separated from mixed field isolates consistently identified mutant clones as less susceptible than WT. Analysis of isolates from other sites demonstrated presence of the G223S and D1116G/N/Y mutations across Uganda. Our results indicate that malaria parasites circulating in Uganda have a number of polymorphisms in PfATP4 and that modestly decreased susceptibility to PfATP4 inhibitors is associated with some mutations now present in Ugandan parasites.

scholarly journals Adaptación y optimización de un método de lectura por fluorometría en el modelo farmacológico in vitro de cultivo de Plasmodium falciparum

2016 ◽  
Vol 45 (1) ◽  
pp. 127-146 ◽  
Author(s):  
María Helena Arias Marciales ◽  
Yinneth Victoria Rodríguez Novoa ◽  
Giovanny Garavito Cárdenas
Keyword(s):  
Plasmodium Falciparum ◽  
Sybr Green ◽  
Sybr Green I

<p>El modelo farmacológico de cultivo in vitro de P. falciparum es crucial en el tamizaje inicial de sustancias o extractos de plantas con posible actividad antiplasmodial. La densidad parasitaria puede determinarse mediante variadas metodologías, sin embargo, se han descrito numerosas ventajas y desventajas asociadas a cada una de ellas. Se evaluaron el tiempo de incubación necesario para la tinción y el uso de cultivos asincrónicos o sincrónicos en busca de valores óptimos; evidenciando un tiempo óptimo de 2 h, y límites de detección y cuantificación, menores en cultivos asincrónicos. Empleando las cepas FCR3 y FCB2 se evidenció un ruido de fondo de 12% y 38% respectivamente; la linealidad mostró una buena correlación, r2 de 0,9644 (FCR3) y 0,9841 (FCB2) y una pendiente de 1761,8 y 852,4 respectivamente. Además, se comprobó había concordancia entre los métodos, fluorométrico con SYBR Green I (SYBRG I) y microscópico con Giemsa, con diferencia media de 0,00002% y 0,09109% para FCR3 y FCB2 respectivamente. Los límites de detección y cuantificación fueron 0,5% y 1,5% de parasitemia. El factor Z con FCB2 fue 0,376, en tanto que con FCR3 alcanzó 0,702. La concentración inhibitoria 50 (CI50) frente a P. falciparum FCR3, generada por cloroquina (CQ) fue 0,37 mcg/mL por microscopía y 0,35 mcg/mL por fluorometría. Nuestros hallazgos sugieren que el ensayo de fluorescencia con SYBRG I, empleando fluorómetros comúnmente disponibles en muchos laboratorios, es preciso, robusto, rápido y exacto; para la evaluación in vitro de sustancias o extractos con posible actividad antiplasmodial.</p>

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