|Year : 2016 | Volume
| Issue : 4 | Page : 509-512
In vitro sensitivity pattern of chloroquine and artemisinin in Plasmodium falciparum
Supriya Sharma, Kamlesh Kaitholia, Neelima Mishra, Bina Srivastava, CR Pillai, Neena Valecha, Anupkumar R Anvikar
Department of Epidemiology and Clinical Research, National Institute of Malaria Research, Sector-8, New Delhi, India
|Date of Submission||23-Dec-2015|
|Date of Acceptance||13-Oct-2016|
|Date of Web Publication||8-Dec-2016|
Anupkumar R Anvikar
Department of Epidemiology and Clinical Research, National Institute of Malaria Research, Sector-8, New Delhi
Source of Support: None, Conflict of Interest: None
Artemisinin (ART) and its derivatives form the mainstay of antimalarial therapy. Emergence of resistance to them poses a potential threat to future malaria control and elimination on a global level. It is important to know the mechanism of action of drug and development of drug resistance. We put forwards probable correlation between the mode of action of chloroquine (CQ) and ART. Modified trophozoite maturation inhibition assay, WHO Mark III assay and molecular marker study for CQ resistance at K76T codon in Plasmodium falciparum CQ-resistant transporter gene were carried out on cultured P. falciparum. On comparing trophozoite and schizont growth for both CQ-sensitive (MRC-2) and CQ-resistant (RKL-9) culture isolates, it was observed that the clearance of trophozoites and schizonts was similar with both drugs. The experiment supports that CQ interferes with heme detoxification pathway in food vacuoles of parasite, and this may be correlated as one of the plausible mechanisms of ART.
Keywords: Antimalarial resistance, artemisinin, chloroquine, malaria
|How to cite this article:|
Sharma S, Kaitholia K, Mishra N, Srivastava B, Pillai C R, Valecha N, Anvikar AR. In vitro sensitivity pattern of chloroquine and artemisinin in Plasmodium falciparum. Indian J Med Microbiol 2016;34:509-12
|How to cite this URL:|
Sharma S, Kaitholia K, Mishra N, Srivastava B, Pillai C R, Valecha N, Anvikar AR. In vitro sensitivity pattern of chloroquine and artemisinin in Plasmodium falciparum. Indian J Med Microbiol [serial online] 2016 [cited 2020 Feb 22];34:509-12. Available from: http://www.ijmm.org/text.asp?2016/34/4/509/195365
| ~ Introduction|| |
Malaria is a major health problem worldwide, and drug resistance is one of the various challenges for its control. Artemisinin (ART) and its derivatives are effective antimalarials that are extensively used worldwide as first-line therapy for falciparum malaria and thus resistance to these is a matter of great concern. It is important to know the mode of action of ART resistance, which can help in drug discovery. Multiple target models for the mechanism of action of ART have been proposed, but none has been proved., ART supposedly acts by heme-dependent activation of an endoperoxide bridge located within the parasite's food vacuole. It has been anticipated that free radicals of ART alkylate these free heme molecules which lead to interference in their detoxification. The mode of action of chloroquine (CQ) is also to prevent the polymerisation of toxic heme released during proteolysis of haemoglobin in the plasmodium digestive vacuole.Plasmodium falciparum CQ-resistant transporter (pfcrt) protein is localised in the digestive vacuole membrane of the parasite which is considered as the transporter for CQ into the food vacuole of parasites., Alteration in pfcrt protein as a result of amino acid change at codon 76 from lysine to tyrosine leads to reduce the accumulation of CQ inside the food vacuole. Thus, CQ effluxes out of food vacuole as a result of which no CQ is left for the detoxification of heme. As both drugs have a similarity in heme detoxification pathways here, we made an attempt to understand the reasonable mechanism of action of ART.
| ~ Materials and Methods|| |
In vitro assay
Two P. falciparum culture isolates from Malaria Parasite Bank, National Institute of Malaria Research (NIMR), MRC-2 and RKL-9 were revived and cultured for 4–6 days in RPMI-1640 complete medium. They were then double synchronised in a gap of 4 h, using 5% sorbitol to eliminate all the stages except the early (ring stage) trophozoites. After the second synchronisation, 96 well plates was set for the TMI assay. The sensitivity of cultured isolates to CQ and ART was assessed by a modification of the standard WHO Mark III micro-test. The 96 well plates were labelled for MRC-2 and RKL-9 with drugs CQ and ART. Each well was filled with 100 µl of RPMI-1640 incomplete medium. Drug concentrations were added in duplicates with well 'H' having the highest concentration of both, CQ – 12.8 µM and ART – 1.6 µM, other wells were filled in by serial dilution method. The first row of 96 well plates was used as control (without drug). Ten microlitres of blood mixture containing 0.5%–1% ring-stage parasites with 2% haematocrit were added to each well starting from control well. After proper mixing, the plates were incubated at 37°C in a gas mixture of 90% N, 5% CO2 for 16–18 h for modified TMI assay and for 25–30 h for modified WHO Mark III assay. Thin and thick films were prepared, fixed and stained with Jaswant Singh Bhattacharya (JSB) I and II stains. The morphology was studied under light microscope, and results were tabulated for number of infected RBCs per twenty fields in each thin film, where each field consists of 200 erythrocytes for modified TMI assay. At the end of the incubation period of 25–30 h for modified WHO Mark III assay, suspended medium was removed while the blood within each well was used to make thick smears on a glass slide. These were air-dried, fixed and stained with JSB stain and examined under microscope at ×100 magnification. The numbers of schizont with three or more nuclei against 200 asexual parasites were counted for each sample.
DNA sequencing for kelch13 and Plasmodium falciparum chloroquine-resistant transporter mutation analysis
Isolation of DNA was carried out using QIAamp mini kit (QIAGEN, Germany) according to manufacturer's protocol. This DNA was stored at −20°C until processed further. The amplification of pfcrt gene was carried out according to previously published standard protocol. The polymerase chain reaction (PCR) product from the amplification reactions was seen by electrophoresis on 1.5% agarose gel containing ethidium bromide. Ten microlitres of the nested PCR product were digested with ApoI cutsmart restriction enzyme (NEB) for 20 min at 37°C as recommended by the manufacturer. Digested product was run on 1.5% agarose gel and visualised by UV transillumination. Further, to reaffirm SNP, primary PCR products of pfcrt gene were DNA sequencing at Xcelris Labs, Ahmedabad. The Kelch13 amplification and mutation analysis were done as per previously published protocol.
Data management and statistical analysis
The drug concentration that inhibits schizogony by 50% (IC50) relative to the drug-free control samples of each P. falciparum isolate for both in vitro assay was estimated from dose-response curves by nonlinear regression analysis using HN-NonLin Reg. Analysis. The IC50 cut-off values for determining sensitivity to antimalarials were based on the WHO micro-test protocol which is 0.8 µmol/l for CQ. The cut-off IC50 value of 0.01 µmol/l for artesunate as described by Pradines et al. was considered as WHO protocol does not recommend cut-off for ART. Statistical analyses were done using SPSS software (Version II, SPSS Inc., Chicago, IL, USA). The editing and alignments of DNA sequences were done using Mega 6 software (Tamura, Stecher, Peterson, Filipski, and Kumar 2013).
| ~ Results|| |
Trophozoite and schizont maturation inhibition patterns were analysed with different range of CQ (0–12.8 [µmol/l] and ART (0–1.6 [µmol/l]) with respective CQ-resistant and sensitive isolates collected from parasite bank. Comparison of TMI and WHO Mark III assay for CQ-resistant and sensitive isolates is shown in [Figure 1].
|Figure 1: Comparison of TMI and WHO MARK III assay for CQ resistant and sensitive isolates|
Click here to view
The IC50 and IC99 values of CQ by TMI assay and WHO Mark III in CQ-sensitive isolate were 0.25 µmol/l and 2.85 µmol/l and 0.35 µmol/l and 2.85 µmol/l, respectively. The IC50 and IC99 values of CQ by TMI assay and WHO Mark III in CQ-resistant isolate were 0.80 µmol/l and 11.80 µmol/l and 1.07 µmol/l and 11.72 µmol/l, respectively. The IC50 and IC99 values of ART by TMI assay and WHO Mark III in CQ-sensitive isolate were 0.025 µmol/l and 0.23 µmol/l and 0.036 µmol/l and 0.36 µmol/l, respectively. The IC50 and IC99 values of ART by TMI assay and WHO Mark III in CQ-resistant isolate were 0.07 µmol/l and 0.24 µmol/l and 0.036 µmol/l and 0.36 µmol/l, respectively [Table 1].
|Table 1: In vitro susceptibility of CQ sensitive and resistant isolates of P. falciparum|
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CQ resistance in P. falciparum was determined by mutation in pfcrt gene at codon K76T. Mutation at codon K76T was found in RKL-9 while MRC-2 has shown wild genotype at this codon. DNA sequence evidence for haplotype analysis of the PCR products indicates that MRC-2 has CQ-sensitive haplotype (CVMNK) and RKL-9 has CQ-resistant haplotype (SVMNT). We sequence RKL-9 and MRC-2 isolates for mutation analysis in Kelch13 gene, no SNP seen in both isolates, which are correlate to delayed parasite clearance time.
| ~ Discussion|| |
ART derivatives are effective antimalarial drugs and widely employed as first-line treatment globally. Different molecular modes of action have been postulated to explain the parasiticidal effect of these compounds; however, none has been evidently accepted, and their physiological application is still questioned.
In the present study, we tried to understand how the parasite works against the drugs in vitro assays, to correlate it for action of ART, put it abreast with CQ mechanism of action by virtue of TMI and WHO Mark III assay. A very rapid parasite clearance was observed in sensitive isolates for both drugs (CQ and ART) in TMI and WHO Mark III assays. Inhibition pattern for trophozoite and schizont with CQ and ART was analogous in MRC-2 isolates. This could be due to accumulation of CQ in food vacuoles which interferes with heme detoxification pathway leading to inhibition of trophozoite and schizont maturation. Interference of heme detoxification by alkylation is one of the postulated mechanisms of action of ART. Slow parasite clearance with both CQ and ART in TMI and WHO Mark III assay in CQ-resistant isolate was observed. Further parallel maturation inhibition in trophozoite and schizont with CQ and ART drugs was observed in CQ-resistant isolate. The possible reason for this similarity could be the because of mutation in pfcrt at codon K76T leading to decreased accumulation of CQ in food vacuoles, allowing parasite to detoxify heme and survive with given normal dose. This could be true with ART as well since the isolate showed the mutation of pfcrt gene leading to lesser alkylation of heme by ART. Hence, the parasite is able to survive for a longer duration. With respect to the IC50 value, CQ-resistant isolate showed 3-fold increase for CQ whereas 2-fold increase for ART, and IC99 value were increase 3-fold and 16-fold in resistant isolate as compared to sensitive isolate with CQ and ART drug, respectively. It suggests a probable analogues mechanism of action of both drugs. Furthermore, SNP analyses for both the genes (pfcrt and K-13) have reaffirm the status of resistant and sensitive parasites. Our experiment upholds, the heme detoxifixtion pathway of parasite is interfered by CQ and ART interferes the heme detoxification through alkylation and herby it may be correlated as one of the plausible mechanisms of action of ART.
The authors SS and KK would like to express their gratitude to the Indian Council of Medical Research for their senior research fellowship, NIMR for infrastructure and overall support and Goa University for providing them PhD registration. Dr. Harald Noedl for kindly providing HN-NonLin software. This paper bears the NIMR publication screening committee approval no. 046/2015.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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