|Year : 2020 | Volume
| Issue : 1 | Page : 16-21
Antimalarial drug sensitivity test: A new approach toward management of severity of Plasmodium falciparum
Gurjeet Singh1, Raksha Singh1, Anant D Urhekar2
1 Department of Microbiology, N. C. Medical College and Hospital, Panipat, Haryana, India
2 Department of Microbiology, MGM Medical College and Hospital, Navi Mumbai, Maharashtra, India
|Date of Submission||27-Feb-2020|
|Date of Acceptance||05-Mar-2020|
|Date of Web Publication||06-Jun-2020|
Dr. Gurjeet Singh
Department of Microbiology, N. C. Medical College and Hospital, Israna, Panipat 132107, Haryana.
Source of Support: None, Conflict of Interest: None
Background: Malaria despite everything represents a danger to the strength of occupants and voyagers in tropical nations. The Plasmodium species that are known to infect humans worldwide are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. Of these, P. falciparum causes high morbidity and mortality, and it is during the asexual erythrocytic stages that the majority of the indications of malaria are shown. Aim: The aim of this study was to standardize an in vitro assay for antimalarial drug sensitivity of P. falciparum isolates. Materials and Methods: This prospective study was carried out over a period of 1 year from July 2014 to June 2015. We effectively completed in vitro drug susceptibility testing on 34 isolates of P. falciparum, which were collected from the patient admitted in Mahatma Gandhi Mission’s Teaching Hospital, Navi Mumbai, Maharashtra, India. We obtained the P. falciparum 3D7 strain from Haffkine Institute for Training, Research and Testing, and Indian Institute of Technology (IIT) Bombay, Mumbai, Maharashtra, India. World Health Organization (WHO) standard in vitro micro-test (Mark III) method was used for the assessment of the response of P. falciparum to chloroquine, mefloquine, quinine, amodiaquine, sulfadoxine/pyrimethamine, and artemisinin. The method was partially modified by replacement of leukocytes with AB serum in Roswell Park Memorial Institute (RPMI)-1640 medium. Results: The development of P. falciparum 3D7 chloroquine-sensitive strain from the ring stage to schizonts (not fewer than six merozoites) in control wells containing chloroquine was 100% recorded. This level of development in the control wells empowered fast minute assurance (5min for isolate per drug) of the Minimum inhibitory concentrations (MICs) of chloroquine. The test well shows the development from the ring stage to mature schizont stages were 67.65% in well containing chloroquine, 17.65% in well containing amodiaquine, and 14.71% in well containing sulfadoxine/pyrimethamine, it means these drugs are resistant to P. falciparum, and however, there is no development of the P. falciparum from the ring stage to mature schizont (with at least six merozoites) stage in the well containing artesunate, mefloquine and quinine were equal to the control well, it means these antimalarial drugs are sensitive to the P. falciparum. Conclusion: Antimalarial drug resistance has developed as a result of prescribing this medication without having malaria. Early diagnosis of malaria and drug sensitivity testing may prevent the emergence of resistance to antimalarial drugs. Monotherapy ought to be kept up a key decent way from; the combined antimalarial drug therapy may reduce the chances of emergence of antimalarial drug resistance.
Keywords: Antimalarial drug susceptibility assay, microtiter plate, Plasmodium falciparum
|How to cite this article:|
Singh G, Singh R, Urhekar AD. Antimalarial drug sensitivity test: A new approach toward management of severity of Plasmodium falciparum. MGM J Med Sci 2020;7:16-21
|How to cite this URL:|
Singh G, Singh R, Urhekar AD. Antimalarial drug sensitivity test: A new approach toward management of severity of Plasmodium falciparum. MGM J Med Sci [serial online] 2020 [cited 2022 Sep 28];7:16-21. Available from: http://www.mgmjms.com/text.asp?2020/7/1/16/286102
| Introduction|| |
“Malaria is a protozoan disease transmitted by the bite of infected female Anopheles mosquitoes. The most important of the parasitic disease of humans, malaria, is transmitted in 91 countries containing 3 billion people and causes approximately 1200 deaths each day. The mortality rate has decreased dramatically over the past 15 years as a result of highly effective control programs in several countries. Malaria was eliminated from the United States, Canada, Europe, and Russia more than 50 years ago, but its prevalence rose in many parts of the tropics between 1970 and 2000.” “Malaria remains today, as it has been for centuries, a heavy burden on tropical communities, a threat to nonendemic countries, and a danger to travelers.” Six species of the genus Plasmodium cause nearly all malarial infections in humans. These are Plasmodium falciparum, Plasmodium vivax, two morphologically identical sympatric species of Plasmodium ovale (curtisi and wallikeri), Plasmodium malariae, and in Southeast Asia, the monkey malaria, Plasmodium knowlesi, whereas almost all deaths are caused by P. falciparum malaria; P. knowlesi and occasionally P. vivax can also cause severe illness.
According to the World Health Organization (WHO), recent worldwide malaria control efforts have produced a 29% reduction in malaria mortality globally between 2000 and 2012; however, there were still an estimated 207 million cases and 627,000 deaths in 2012. Approximately 90% of deaths occur in sub-Saharan Africa, where a close link between malaria mortality and poverty is observed. Of total deaths, 77% occur in children younger than 5 years of age. Concentrates into numerous parts of human malarial parasite science were enormously upgraded by the advancement of a technique to culture agamic blood phases of P. falciparum in vitro in 1976.
Most drug resistance tests in malaria concern P. falciparum, the most prevalent and virulent species. Initial observations of drug-resistant malaria occur most often in a clinical context, and their confirmation is frequently sought by in vivo tests. Initially, standardized by the WHO for the response of P. falciparum to chloroquine, in vivo tests have been modified several times for increased performance and assessment of other drugs. An increasing number of in vivo tests comparing drug scheme have been performed since 1996, as it was necessary to assess the response of P. falciparum to artemisinin-based combined therapy. With regard to the fast-acting artemisinin derivatives, measurement of parasite clearance times, in addition to determination of standard clinical outcomes, has been introduced.
The standard in vitro antimalarial drug susceptibility assay determines the ex vivo growth of replicating intraerythrocytic parasites from the ring stage (the only asexual P. falciparum stage found in patient peripheral blood) to the schizont stage in 24–72h in the presence of serial drug concentrations under conditions close to in vivo conditions. With regard to the standard assay, a well-suited 96-well microtiter plate format was designed using the Trager and Jensen cultivation parameters (hypoxia and buffered Roswell Park Memorial Institute [RPMI] medium with human serum), which is the basis of the most used tests.
It is also possible that drug treatment might directly affect the metabolic pathways measured, without altering the viability of parasites, which would also lead to erroneous measurements. These different situations show that metabolic activity and viability are two parameters, which can be uncoupled either naturally or in response to drug treatment and that relying on the first to measure the latter can be a possible source of artefacts. Moreover, these assays do not permit to directly measure the speed of action of antimalarial compounds on parasite viability, which is nevertheless a crucial parameter for anti-infective activities and clinical efficacy. The net effect of antimalarial compounds is primarily mediated by their ability to kill parasites and decrease parasitemia so that patient cure can be achieved and parasite recrudescence can be prevented. Killing rates can be estimated in vivo by the parasite reduction ratio (PRR), which is the ratio between parasitemia at the onset of drug treatment and 48h later, corresponding to one asexual parasite life cycle. The reduction of circulating parasitized erythrocytes generally follows a log-linear curve, and this measure can be used as a predictive therapeutic index. Beyond the killing rate itself, the effect of antimalarial drugs also depends on the type of growth inhibition involved. Cytotoxic drugs, by actively killing parasites, generally display a high level of efficacy, whereas cytostatic drugs, such as atovaquone, might not completely eliminate circulating parasites and might lead to treatment failure and resistance selection. As aforementioned, metabolism-based assays are generally poorly suited tools to discriminate between cytotoxic and cytostatic activities.
| Materials and methods|| |
Research design: Prospective and experimental.
Place of study: Department of Microbiology, MGM Medical College and Hospital, Navi Mumbai, Maharashtra, India.
Period of study: Study was carried out over a period of 1 year from July 2014 to June 2015.
Ethical clearance: The article was a part of a project, which was reviewed and examined by the Ethical Review Committee of Mahatma Gandhi Mission’s Institute of Health Sciences (Deemed University), Navi Mumbai. Consent was obtained from the patients before the start of the study.
Pre-dosed drug plates: Chloroquine phosphate was obtained from the Ipca Laboratories (Mumbai, India), mefloquine was obtained from Emcure Pharmaceuticals (Mumbai, India), quinine sulphate from Inga Laboratories (Mumbai, India), and artesunate from Zuventus Health Care (Mumbai, India). A stock solution of each drug was prepared in methanol in a sterile glass test tube. Serial dilution of each drug was prepared in methanol to obtain the drug concentration according to WHO micro-test (Mark III) method. Doubling dilutions of final antimalarial drug solutions (20 µL) were added to each well of a 96-well microtiter plate (Genetix Biotech Asia, New Delhi, India), later the microtiter plates were dried in an incubator at 37°C overnight, and then the microtiter plates were covered with Plate Sealer (HiMedia) and stored at 4°C. Randomly selected, pre-dosed plates were checked against the P. falciparum 3D7 strain before and after the study to assess drug activity.
Parasites: P. falciparum isolates were isolated from the febrile patient’s blood samples in a tertiary care hospital, Navi Mumbai, India.
Control strain: P. falciparum 3D7strain was procured from Haffkine Institute for Training, Research, and Testing, and IIT Bombay, Mumbai, Maharashtra, India.
Drug sensitivity testing: WHO standard in vitro micro-test (Mark III) method was used for the assessment of the response of P. falciparum to chloroquine, mefloquine, quinine, amodiaquine, sulfadoxine/pyrimethamine, and artemisinin. The method was partially modified by the replacement of leukocytes with AB serum in the RPMI-1640 medium. The simplest test format that has been adopted to carry out the antimalarial drug sensitivity test uses 100 µL of blood mixed with RPMI-1640 complete medium. [Figure 1] and [Figure 2]. The portion reaction information was dissected by nonlinear regression analysis (HN-NonLin [version 1.1] programming) to acquire the half inhibitory concentrations (IC50s) (geometric methods). The test was rehashed twice for each segregates [Figure 3].
|Figure 1: Plasmodium falciparum before (A) and after treatment (B) with sorbitol|
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|Figure 2: Antimalarial drug sensitivity test of Plasmodium falciparum showing 3D7 P. falciparum control strain in A and from B to H are patient samples|
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| Results|| |
Antimalarial drug susceptibility testing for 34 isolates of P. falciparum isolated from patients was done using the control strain of P. falciparum (3D7) against different dilution of antimalarial drugs such as chloroquine, quinine, mefloquine, artesunate, amodiaquine, and sulfadoxine/pyrimethamine. The development of P. falciparum 3D7 chloroquine-sensitive strain from the ring stage to schizonts (not less than six merozoites) in control wells containing chloroquine was 100% recorded. This level of development in the control wells empowered fast minute assurance (5min for isolate per drug) of the MICs of chloroquine. The test well shows the development from ring stage to mature schizont stages were 67.65% in well containing chloroquine, 17.65% in well containing amodiaquine and 14.71% in well containing sulfadoxine/pyrimethamine, it means these drugs are resistant to P. falciparum, and however, there is no development of the P. falciparum from ring stage to mature schizont (with at least six merozoites) stage in the well containing artesunate, mefloquine and quinine was equally to the control well, it means these antimalarial drugs are sensitive to the P. falciparum [Table 1], [Figure 4].,
|Figure 4: Percentages of drug resistance pattern of Plasmodium falciparum|
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| Discussion|| |
The antimalarial drug sensitivity test is done in not many labs over the world particularly for inquire about purposes. We performed antimalarial drug sensitivity testing of patients’ tests. We discovered that chloroquine obstruction in P. falciparum was stopped high at 67.65% (23 of 34). There was a development of P. falciparum (develop schizont arrangement) in 8 pmol/L medicate focus demonstrating protection from chloroquine. Our outcomes concur with those of Shrivastava et al. who announced opposition of 86.96% of P. falciparum to chloroquine in the Assam zone, Shujatullah et al. from Aligarh and Anvikar et al. from New Delhi detailed lower esteems, for example, 24.07% and 44.4%, respectively.
The clarification of high medication protection from chloroquine could be that chloroquine is given to every febrile patient in endemic regions as a prophylactic measure. This consistent indiscriminate across the board utilization of chloroquine more likely than not drove the malarial parasite to build up certain instruments to conquer the impact of chloroquine. Chloroquine opposition has been accounted for by workers in different nations likewise—Peletiri et al. (Nigeria) 88.9%, Oyedeji et al. (Nigeria) 80%, Kaddouri et al. (Mali) 60%–79%, Ringwald et al. (Cameroon) 62.18%, Olasehinde et al. (Nigeria) 51%, and Menard et al. (Central Africa) 37%. Protection from amodiaquine was seen as 17.65% (6 of 34). Anvikar et al. from New Delhi revealed opposition of 25%. Laborers from different nations revealed protection from amodiaquine running from 13% by Olasehinde et al. (Nigeria) to 44.4% by Peletiri et al. (Nigeria). Protection from sulfadoxine/pyrimethamine was seen as 14.71% (5 of 34). Menard et al. (Central Africa) and Olasehinde et al. (Nigeria) revealed obstruction of 38.3% and 5%, respectively. No opposition was found in our examination for artesunate (artemisinin), mefloquine, and quinine. Anvikar et al. from New Delhi likewise detailed comparable findings. Shrivastava et al. (Assam) and Shujatullah et al. (Aligarh) did not make reference to or study the protection from artemisinin. No protection from artemisinin was accounted for by laborers from different nations. Menard et al. (Central Africa) anyway revealed protection from mefloquine 1.6%. Multidrug obstruction (sedate protection from at least three antimalarial drugs) was not found in our investigation. Menard et al. (Central Africa) likewise announced comparable discoveries.
| Conclusion|| |
Antimalarial drug resistance has developed as a result of prescribing this medication without having malaria. Early diagnosis of malaria and drug sensitivity testing may prevent the emergence of resistance to antimalarial drugs. Monotherapy ought to be kept up a key decent ways from; the combined antimalarial drug therapy may reduce chances of emergence of antimalarial drug resistance. Proper diagnosis of malaria and drug sensitivity testing should be done routinely to prevent emergence of resistance to antimalarial drugs. Monotherapy should be avoided and combined drug therapy will reduce chances of emergence of drug resistance.
We are grateful to Dr. S.N. Kadam, former Hon’ble Vice-Chancellor, MGM Institute of Health Sciences (Deemed to be University), Navi Mumbai, Maharashtra, India, for providing cell culture laboratory and important instruments (CO2 incubator, glass desiccator, biosafety cabinet, and filtration) for research. Also, we thank Dr. Saroj Bapna, Scientific Officer, Haffkine Institute for Training, Research and Testing, Parel, Mumbai, and Dr. Swati Patankar, Professor, Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai, Maharashtra, India, for providing 3D7 ATCC strain of Plasmodium falciparum. We are additionally grateful to Ipca Laboratories, India, for providing chloroquine phosphate, primaquine phosphate, and Arteether; Emcure Pharmaceuticals, India) for mefloquine; Inga Laboratories, India, for quinine sulfate; and Zuventus Health Care, India, for artesunate. Also, we are exceptionally appreciative of Dr. Harald Noedl, Associate Professor (Director MARIB), Head Exp. Tropical Medicine and Field Research Unit, Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Austria, for providing HN-NonLin (Version 1.1) programming software for the examination of antimalarial medicate affectability testing.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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