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

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. 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.Resistance to currently available drugs is still a cause of therapeutic failures and of the persistence of a high level of morbidity due to malaria. Combined therapy with artemisinin derivatives plus amodiaquine or lumefantrine is now implemented in Africa. Epidemiological assessment of Plasmodium falciparum drug susceptibility is thus necessary to sustain health recommendations for malaria treatment and prophylaxis in countries where malaria is endemic. Even though molecular techniques are improving, few genes responsible for clinical resistance have been identified. The in vitro assay, which allows the phenotypic determination of P. falciparum susceptibility to antimalarials, is thus an essential tool. The multiclonal structure of clinical Plasmodium falciparum isolates is the rule, and a mixture of susceptible and resistant parasites gives a biphasic concentration response (10, 22). As malaria parasites divide every 2 days, cloning is not a workable routine before testing for drug susceptibility. Our previous experience has shown that the in vitro response of a clinical isolate to chloroquine was monotonic at a frequency of Ͼ95%. This suggested that the majority of parasites which are present in the patient had homogeneous susceptibilities. The concentration-response relationship is a classical pharmacological sigmoid response, and the correct modeling of this response may give an accurate assessment of the drug susceptibilities of malaria parasites. Two methods are in common use for assessment of the in vitro parasite growth inhibited by drugs: schizont counting on thick films, known as the WHO Schizont Maturation assay (21), and incorporation of a radiolabeled nucleic acid precursor, [8-3 H...

<i>In vitro</i> evaluation of antiplasmodial activity of extracts of <i>Acanthospermum hispidum</i> dc (Asteraceae) and <i>Ficus thonningii</i> blume (moraceae), two plants used in traditional medicine in the republic of Congo

“…Therefore, the growth inhibition value at each concentration of the plant extract or chloroquine was obtained by adjusting OD values from plant extract treated wells for HRP2 background activity. These values were then expressed as a percentage of growth inhibition value and plotted against corresponding concentrations of the plant extract using R Statistical software (www.carn.r-project.org) to generate dose-response curves from which concentrations inhibiting 50% of parasite growth (IC 50 ) were calculated (Kaddouri et al, 2006).…”

Design, synthesis and antimalarial activity of novel bis{N-[(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-aminopropyl}amine derivatives

“…Plates were incubated for 1 h at room temperature and then read on a fluorescence plate reader (Tecan, Austria) using excitation and emission wavelengths of 485 and 535 nm, respectively. Concentrations inhibiting 50% of the parasite 0 s growth (half maximal inhibitory concentration or IC 50 values) were then calculated from the obtained experimental results using a regression program available on line 40 .…”

<i>In vitro</i> and <i>in vivo</i> antiplasmodial activity of extracts from <i>Polyalthia suaveolens, Uvaria angolensis and Monodora tenuifolia</i> (Annonaceae)

“…Upon incubation, 100 μL of SYBR green I buffer [0.2 μL of 10,000 × SYBR Green I (Invitrogen) per mL of lysis buffer {Tris (20 mM; pH 7.5), EDTA (5 mM), saponin (0.008%; wt/vol), and Triton X-100 (0.08%; vol/vol)}] were added to each well and mixed gently and further incubated in the dark at 37 °C for 1 h. Fluorescence was subsequently measured using a fluorescence multi-well plate reader (Perkin Elmer) with excitation and emission at 485 and 530 nm respectively. Fluorescence counts for ART were deducted from counts in each well and a doseresponse curve was constructed by plotting fluorescence counts against the drug concentration and activity expressed as 50% inhibitory concentration (IC 50 ) using the IC Estimator-version 1.2 software (http://www.antimalarialicestimator.net/MethodIntro.htm) where estimated parasite growth in the negative control (0.4% DMSO) is 100 %, and 0% in the positive control (ART) (Le Nagard et al, 2011;Kaddouri et al, 2006).…”