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Year : 2018  |  Volume : 8  |  Issue : 2  |  Page : 70-76  

Drug susceptibility testing methods of antimalarial agents

Department of Microbiology, Calcutta School of Tropical Medicine, Kolkata, West Bengal, India

Date of Web Publication27-Dec-2018

Correspondence Address:
Dr Ardhendu K Maji
Department of Microbiology, Calcutta School of Tropical Medicine, 108, C. R. Avenue, Kolkata - 700 073, West Bengal
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2229-5070.248695

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Resistance to almost all available antimalarial agents, particularly for Plasmodium falciparum, is an important issue throughout all malaria endemic countries. Recently, Plasmodium vivax also showed resistance to chloroquine in some part of the World. Monitoring efficacy of used antimalarial drugs plays an important role to determine the emergence of resistant power by the prevailing parasite population of a geographical area if any. There are four different methods for antimalarial drug efficacy study. In vivo therapeutic efficacy study remains the gold standard and provides primary data for formulating antimalarial drug policy by the National Malaria Control Programmes. Several in vitro assay methods for assessing antimalarial drug susceptibility have been developed during past few decades. These assay methods are simple, easier to handle and allow early detection of drug-resistant parasites and also for the screening of different newly developed agents for their antimalarial activity. Approaches of different assay methods for testing the susceptibility of antimalarial agents and their limitations are discussed in this review article.

Keywords: Antimalarial agents, drug susceptibility assay, in vitro culture methods, Plasmodium falciparum

How to cite this article:
Maji AK. Drug susceptibility testing methods of antimalarial agents. Trop Parasitol 2018;8:70-6

How to cite this URL:
Maji AK. Drug susceptibility testing methods of antimalarial agents. Trop Parasitol [serial online] 2018 [cited 2023 Mar 25];8:70-6. Available from: https://www.tropicalparasitology.org/text.asp?2018/8/2/70/248695

   Introduction Top

Malaria still remains as a major public health problem in tropics and subtropics. More than 214 million cases of malaria were reported in 2014.[1] Very recently, World Health Organisation[2] introduced “Global Technical strategy for malaria 2016–2030” with the vision of a world free of malaria that provides a comprehensive framework for countries to develop customized programs for accelerating toward malaria elimination. The emergence of new drug-resistant strains of Plasmodium against almost all available antimalarial agents makes the situation more worsen and challenging.[3],[4] So monitoring the efficacy of different antimalarial drugs in a regular interval and identification of new antimalarial agents are two important aspects of fight against malaria. There are four different methods to determine the antimalarial drug efficacy – in vivo therapeutic efficacy studies, in vitro tests, molecular marker studies, and measurement of drug concentrations. The principle, use, advantages and disadvantages of four methods are summarized in [Table 1].
Table 1: Methods for monitoring antimalarial drug efficacy and drug resistance

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The in vivo therapeutic efficacy study still remains as the gold standard for determining antimalarial drug efficacy and its results are used as primary data by the National Malaria Control Programmes to formulate antimalarial drug policy. Therapeutic efficacy study determines both clinical and parasitological responses of antimalarial drugs either with monotherapy or in combination therapy.

Molecular marker studies have been used to identify the single-nucleotide polymorphisms in target genes associated with drug resistance in malaria parasites; however, the presence of polymorphisms in marker genes does not always correlate with in vivo drug resistance.[5] Pharmacokinetic studies measure the concentration of antimalarial drugs and/or active metabolites in whole blood, plasma or serum that used to distinguish between treatment failure due to antimalarial drug resistance and suboptimal drug exposure. It helps to define the true drug resistance to different antimalarial agents. However, it requires a number of blood samples from the patients under treatment.

In vitro susceptibility assays are more rapid and accurate for detection of reduced susceptibility of Plasmodium sp. to individual antimalarials and allows the action to be taken before clinical failure of a combination becomes established. In vitro assays are used to monitor the susceptibility of the parasite by measuring the intrinsic sensitivity to different antimalarial drugs. Parasites are directly exposed to known concentrations of drugs and examined for inhibition of growth and maturation into schizonts. These methods are completely free from host related factors such as host immunity, poor absorption, biotransformation, concentration in certain tissues, and rapid clearance.[6] In vitro methods are risk-free for the patients and do not involve any intervention. There are several methods for in vitro susceptibility assays of antimalarial drugs that differ from each other depending upon the methods used for interpretation of results. Different aspects of drug susceptibility methods for antimalarial drugs are discussed in this review.

   In Vitro Culture of Malarial Parasite Top

Susceptibility testing methods of antimalarial agents are primarily based on in vitro culture of the parasite. There are two approaches for in vitro culture of malarial parasite, short-term culture and continuous culture. Short-term culture method is simple, requires little laboratory equipment and can be easily performed in field conditions. Continuous culture method is more complicated needs well equipped laboratory, technical and logistic supports. It is an essential tool in working on malaria drug development, efficacy studies and basic research. The first short-term culture method for Plasmodium falciparum was described in early 20th century.[7],[8] The method of in vitro culture underwent a drastic modification in 1976 when a new method for continuous culture of P. falciparum was developed by Trager and Jensen.[9] The essential components of the complete blood-medium mixture are Roswell Park Memorial Institute (RPMI) 1640 medium buffered with N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) and sodium bicarbonate, human serum (or serum substitutes, including animal sera and lipid-enriched bovine albumin) and P. falciparum-infected human erythrocytes. This technical improvement led to development of several in vitro susceptibility assay systems during late 1970s and 1980s such as morphological assay based on microscopy, radioisotope microtest method, an enzyme-linked immune sorbent assay (ELISA) with monoclonal antibodies directed against either plasmodial lactate dehydrogenase (pLDH) or histidine-rich protein II (HRP II) and a fluorometric assay with DNA-binding fluorescent dyes.

   Methods Used in Susceptibility Test for Antimalarial Drugs Top

World health organisation microtest

The in vitro microtest was developed by Rieckmann et al.[10] based on culture methods of Trager and Jensen.[9] Microtiter plates precoated with different concentrations of chloroquine and two drug-free control wells per assay are used. 5 μl whole capillary blood and 50 μl buffered RPMI 1640 medium added to each well and incubated in a candle jar at 38–39°C for 24–30 h. Immediately after incubation, thick blood films are prepared from each well, and the number of schizonts is counted against 500 leukocytes. The response of the drug expressed as the percentage of the number of schizonts with each drug concentration, compared with the number of schizonts in drug-free controls. This method is also used to evaluate the in vitro response of malarial parasite to pyrimethamine and sulfadoxine-pyrimethamine in combination, with standard RPMI 1640 containing 1 mg/l folic acid and 1 mg/l para-amino benzoic acid.[11],[12] This method has important advantages of requiring only a small quantity of capillary blood and involving simplified in vitro procedures.

This is a laborious procedure and not very popular with microscopists. Parasites that grow from ring to late trophozoite stage do not reach the schizont stage within 24 h.

Drugs such as sulfadoxine and pyrimethamine that exert their action primarily during later stages of parasite development might be difficult to measure by this 24 h of incubation assays.

After the development of continuous in vitro cultivation method of P. falciparum incubation period increased to 48–96 h for several antimalarial drug susceptibility assays based on the measurement of the increase in parasitemia during a culture.[13],[14] However, interpretation of test results remains time-consuming and laborious.

Radioisotope assays

To address the disadvantages of microscopy based in vitro antimalarial drug susceptibility assays, the tritiated hypoxanthine radioisotope assay was described by Desjardins et al., 1979[15] and was considered the gold standard for a considerable time.[6] The basic principle behind the method is that in in vitro culture, uninfected red blood cells and platelets do not synthesize DNA, RNA, proteins or membranes; human leukocytes do not multiply and tend to disintegrate over a few days. The only dividing cells are the parasites. Plasmodium spp. use exogenous purines. Radiolabeled (3H) hypoxanthine, a DNA precursor can easily be incorporated into malarial nucleic acids and is the preferred radioisotope for in vitro drug sensitivity assays. The incorporation of (3H) hypoxanthine is directly proportional to the number of P. falciparum-infected erythrocytes under the usual conditions of in vitro drug sensitivity assays.

For drug assays, cultures are diluted in complete RPMI 1640 medium with uninfected erythrocytes to a final erythrocyte volume fraction of 1.5% and a starting parasitemia of 0.25–0.5%. Two-fold serial dilutions of drug solutions in complete RPMI 1640 medium (25 μl per well) are prepared in duplicate in 96-well microtiter plates with an automatic diluter (rows B–H; row A consists of drug-free control wells).

The parasite suspension, 200 μl per well plus 25 μl drug solution, i.e. a final volume of 225 μl, is distributed in each well. The plates are incubated at 37°C for 24 h in a 5% O2, 5% CO2, 90% N2 gas mixture. (3H) hypoxanthine (25 μl per well of 20 μCi/ml solution in RPMI 1640, i.e. 0.5 μCi per well) is added to each well after the first 24-h incubation period. After an additional 18 h incubation, during which (3H) hypoxanthine incorporation occurs essentially in developing mature trophozoites and schizonts of the first blood cycle (parasites in ring stage at the beginning of the assay), the assay is terminated. Incorporation of (3H) hypoxanthine is quantified with a liquid scintillation counter.

Since the late 1970s, the handling of radioactive material has been considerably restricted, and the disposal of radioisotopes is very difficult. Required equipment, such as liquid scintillation counters and harvesting machines are costly. A relatively high parasite density required for this test limits its application to the use of culture-adapted parasite strains or field samples with adequately low parasitemia.

Colorimetric based enzymatic assay (plasmodial lactate dehydrogenase)

pLDH is a terminal enzyme in the glycolysis of malaria parasite.[16] LDH of Plasmodium spp. is different from that of human by electrophoretically, immunologically, kinetically and also by its amino acid composition.[17] The levels of pLDH are directly proportionate to the parasite density.[16] The activity of pLDH is distinguishable from host LDH activity by using the 3-acetyl pyridine adenine dinucleotide (APAD), instead of NAD by human LDH. On this basis Makler et al. 1993[18] developed a drug sensitivity assay by measuring the enzymatic activity of pLDH. Pyruvate is formed from L-lactose in the presence of pLDH and APAD coenzyme. This reaction results in the formation of reduced APAD that in turn reduces blue tetrazolium, forming a blue formazan product that can be measured by spectrophotometry. Colorimetric pLDH based antimalarial drug susceptibility assays can be performed with laboratory-adapted strains and fresh clinical isolates of P. falciparum and Plasmodium vivax in 24- or 96-well culture plates, like the radioisotope method, except the addition of (3H) hypoxanthine. After incubation, the plates are frozen and thawed. The hemolyzed suspension from each well (10 μl) is transferred to another 96-well microtiter plate and mixed with 100 μl of MalstatR solution, consisting of APAD coenzyme and the l-lactate substrate. Nitro blue tetrazolium (240 μmol/l), which turns blue in reduced form, and phenazine ethosulfate (33 μmol/l) are added to measure the formation of the reduced form of APAD at 650 nm with a spectrophotometer. The raw data are expressed as optical density (OD). The OD is plotted against the logarithm of drug concentrations to determine the IC50. The main limitation of the enzymatic assay is its relatively low sensitivity for detecting malarial LDH.

   Enzyme-Linked Immune-Sorbent Assay Based Susceptibility Test for Antimalarial Drugs Top

Two different ELISA methods based on pLDH and HRP II is used for testing of antimalarial drug susceptibility.

   Plasmodial Lactate Dehydrogenase Top

Plasmodium LDH specific monoclonal antibodies are used for the diagnostic purpose in an antigen captured immune-chromatographic assay.[18] Antibodies immobilized on a dipstick, bind to the malaria antigen with high specificity. This technique is very simple and reliable for diagnosis of parasitemia without microscopic examination of blood smears.[19],[20],[21],[22] Using this principle, an ELISA-based in vitro drug sensitivity assay system called the “double-site enzyme-linked lactate dehydrogenase immune-detection” (DELI) assay was described by Druilhe et al., 2001.[23]

Parasitized blood either from continuous culture or infected patients are exposed to different concentration of antimalarial drugs in a microtiter plate and incubated at 37°C for 24 h in a 5% O2, 5% CO2, 90% N2 gas mixture as described earlier. At the end of incubation, the test plates are frozen and thawed 3 times to ensure complete hemolysis and liberation of LDH. The lysate in each well is diluted 1/20–1/200 in Phosphate Buffered Solution for a starting parasitaemia >0.01%. This is the key step, which requires adjustment to obtain the desired OD reading from drug-free control wells. The diluted lysate is transferred to another 96-well microtiter plate precoated with the monoclonal pLDH antibody. The subsequent steps are same to that of a typical ELISA assay. The color reaction is quantified with a spectrophotometer at 450 nm. The sensitivity of DELI assay is very high. It is cheap and required minimum instrumentation.

   Histidine-Rich Protein II Top

The recent development in the field of in vitro drug susceptibility assays for P. falciparum is based on the measurement of an HRP II produced by P. falciparum during the course of its growth and multiplication using the same method to that of DELI assay.[24],[25],[26] It is 10 times more sensitive than the isotopic assay. HRP II levels are closely associated with parasite density and development.[27],[28] The HRP II assay differs from DELI method by its longer incubation period of 72 h that allows the testing of slow-acting drugs without changing any protocol. The method is very simple and readymade ELISA test kits are commercially available. The assay can be performed with laboratory maintained strains and field isolates.

   Nonradioactive Methods Based on Measurement of Parasitic Nucleic Acid Top

Flow cytometry

A laser-based fluorescently activated cell sorters (FACS) is used to detect and count malaria-infected erythrocytes in the presence of a DNA-targeted fluorescent dye. The intensity of fluorescence is directly proportionate with the development of trophozoites into schizonts.

The initial assay method is same as that of isotope microtest method except the use of isotope in a 96-well microtiter plates precoated with test compounds. After the incubation period, the erythrocytes are fixed with glutaraldehyde and stained with a fluorescent DNA dye. Several dyes can be used, including thiazole orange, acridine orange, ethidium bromide, etc., The stained cells are excited with laser or ultraviolet light, depending on the dye used. The fluorescence intensity of individual wells is measured, and the data are analyzed by a computer linked to the FACS. The entire procedure, including data processing and analysis, can be automated and completed within <3 h.[29],[30],[31],[32],[33]

Though the method is rapid, accurate, highly sensitive, automated, and nonradioactive but it is costly, requires a highly skilled personal and too sophisticated instruments and well-equipped laboratory.

   Fluorometric Assay Top

An FACS is used to detect and measure the DNA content of malaria-infected erythrocytes with DNA-specific fluorochromes. It is an alternative method based on the principle of DNA labeling with fluorochromes that do not require a laser-based FACS. At the end of the incubation period, the erythrocytes are lysed with distilled water or saponin. After centrifugation, the packed pellets are dissolved in guanidinium or sodium dodecyl sulfate solution and stained with DNA-binding fluorochromes like ethidium bromide or Hoechst 33358. The procedure may require an extra step of DNA extraction in phenol-chloroform to eliminate hemozoin, which can cause quenching. Use of SYBRR Green I to stain the DNA is advantageous as lysing of erythrocytes, and washing is not necessary.[34] The fluorescence intensity, which reflects the amount of DNA in an individual well, is measured with a mini-fluorometer, fluorescence spectrophotometer or fluorescence-activated microplate reader.[35],[36] The advantages, limitations and principles of all methods are summarized in [Table 2].
Table 2: Comparison of different methods for antimalarial drug susceptibility assays

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   Conclusion Top

All susceptibility assay methods for antimalarial drugs are primarily based on development and maturation of parasites in-vitro culture medium with different known concentration of antimalarial agents. The sensitivity, feasibility, costs of each method differs from others. The radioisotope assay method was considered as gold standard for a long time, but the hazards associated with disposal of radioactive materials limited its wide use. Methods assaying with DNA-specific fluorochromes – flowcytometry and fluorometric assay are rapid, accurate, highly sensitive, highly DNA-specific, objective and automated but it these require costly instrument and the presence of leukocytes results in high background noise. Hence, more studies are needed with SYBRR Green I fluoroassays for field isolates. Two newly developed ELISA-based colorimetric assays (DELI and HRP II assays) may overcome these disadvantages and currently be the choice of methods for susceptibility assay. These are more sensitive compared with other methods[37],[38] and very simple, easy to perform at field level, do not require highly specialized equipment.

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Conflicts of interest

There are no conflicts of interest.

   References Top

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  [Table 1], [Table 2]

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