|Year : 2017 | Volume
| Issue : 4 | Page : 485-490
Correlation of in vitro sensitivity of chloroquine and other antimalarials with the partner drug resistance to Plasmodium falciparum malaria in selected sites of India
Supriya Sharma, Ram Suresh Bharti, Nitin Bhardwaj, Anupkumar R Anvikar, Neena Valecha, Neelima Mishra
Epidemiology and Clinical Research, Indian Council of Medical Research-National Institute of Malaria Research, New Delhi, India
|Date of Web Publication||1-Feb-2018|
Dr. Neelima Mishra
Indian Council of Medical Research-National Institute of Malaria Research, Sector - 8, Dwarka, New Delhi - 110 077
Source of Support: None, Conflict of Interest: None
Background: Antimalarial drug resistance is a potential threat for control and elimination of malaria. To ascertain the status of antimalarial drug resistance at the study sites, correlation between in vitro drug sensitivity pattern and drug resistance molecular markers in Plasmodium falciparum malaria was undertaken. Materials and Methods: Polymorphisms in P. falciparum chloroquine resistance transporter (pfcrt) K76T and pfmdr1 N86Y were studied in relation to the in vitro susceptibility of P. falciparum in culture (n = 10) and field isolates (n = 40) to chloroquine (CQ), amodiaquine (AQ), quinine (QN), mefloquine (MQ) and artemisinin (ART). The prevalence of drug resistance molecular markers, pfdhfr (codon S108N, C59R, N51I, I164 L and A16V), pfdhps (codon S436F and A437G), pfATPase6 (codon D639G and E431K) and mutation in the propeller domain of pfK13 gene were also analysed. Chi-square test and parametric Pearson correlation test were performed using SPSS version 17. Results: In vitro assay showed 18% resistance to CQ, 8% to AQ and 4% to QN. However, no resistance was observed towards MQ and ART. The mutations in pfcrt and pfmdr1 were statistically not significantly associated with susceptibility responses for antimalarials; however, increased IC50values of drugs were reflected as mutant and/or mixed isolates for both gene polymorphisms. CQ was found as independent predictor for other antimalarials, i.e., AQ, QN and ART, with r2 score 0.241, 0.241 and 0.091, respectively. Mutation in the pfATPase6 gene at codon E431K was observed in only one sample from Tripura which also had increased IC50value of 6.28 nM. However, moderate numbers of mutations at codon S108N, C59R and I164 L for pfdhfr gene and S436F and A437G for pfdhps gene were also observed. None of the samples showed mutation in propeller domain of pfK13 gene. Conclusion: The correlation between IC50and molecular markers for antimalarial drug resistance is reported for the first time through this study. A positive correlation between in vitro drug resistance with molecular markers for antimalarial drug resistance could make in vitro assay a reliable tool to predict drug efficacy which is needed for detection of emerging resistance in the country.
Keywords: Antimalarials, drug resistance, malaria, Plasmodium falciparum
|How to cite this article:|
Sharma S, Bharti RS, Bhardwaj N, Anvikar AR, Valecha N, Mishra N. Correlation of in vitro sensitivity of chloroquine and other antimalarials with the partner drug resistance to Plasmodium falciparum malaria in selected sites of India. Indian J Med Microbiol 2017;35:485-90
|How to cite this URL:|
Sharma S, Bharti RS, Bhardwaj N, Anvikar AR, Valecha N, Mishra N. Correlation of in vitro sensitivity of chloroquine and other antimalarials with the partner drug resistance to Plasmodium falciparum malaria in selected sites of India. Indian J Med Microbiol [serial online] 2017 [cited 2019 Dec 10];35:485-90. Available from: http://www.ijmm.org/text.asp?2017/35/4/485/224423
| ~ Introduction|| |
Antimalarial drug resistance is a major hurdle in the successful treatment of malaria. Plasmodium falciparum has developed resistance to almost all antimalarials currently in use. Resistance to all known antimalarial drugs including artemisinin (ART)-based combination therapy (ACTs) varies worldwide.P. falciparum resistance to ART has now been detected in five countries; in the Greater Mekong subregion, Cambodia, Lao People's Democratic Republic, Myanmar, Thailand and Viet Nam. The antimalarial drug resistance is monitored by various methods such as therapeutic efficacy studies, in vitro tests, molecular markers and measurement of drug concentrations. As compared to in vivo tests, in vitro tests provide meticulous experimental environment and fast analysis. It has relatively low cost and is simple to conduct as compared to in vivo assessments.
Histidine rich protein II (HRPII) is naturally occurring histidine and alanine-rich protein which is an excellent indicator of parasite growth and its inhibition by antimalarial drugs. In a recent study, HRPII in vitro assay has been found to be better than WHO Mark III assay for in vitro drug sensitivity test.
Molecular markers are considered as a promising public health tools for identifying drug resistance in malaria, which has a great potential value. Molecular marker studies are comparatively faster and less expensive than clinical studies. The non-synonymous mutations confer parasite resistance to drugs and are targets for molecular studies. However, the genetic mechanisms of P. falciparum drug resistance have not been completely elucidated till date.
The mutations in the P. falciparum chloroquine resistance transporter (pfcrt) gene have been linked to chloroquine (CQ) resistance and are important indicator of in vitro resistance as well as therapeutic failure., The amplification and/or polymorphisms of pfmdr 1 (P. falciparum multidrug resistance1) gene have also been shown to affect the susceptibility to antimalarial drugs such as mefloquine (MQ), artesunate (AS), lumefantrine (LF) and quinine (QN). Resistance against sulfadoxine and pyrimethamine (SP) is conferred by single or multiple mutations in the dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) genes, respectively., Mutations in plasmodium ATPase6 gene  and pfk13 propeller region are important markers for ART resistance. Recently, Kelch 13 propeller polymorphism has been established as a useful molecular marker for large-scale surveillance efforts to frame ART resistance. To provide a better understanding of resistance pattern at the study sites, the present study was planned to correlate in vitro HRPII ELISA assay with molecular markers.In vitro susceptibilities for five antimalarial drugs and molecular markers, pfcrt (codon K76T), pfmdr 1 (codon N86Y), pfdhfr (codon S108N, C59R, N51I, I164 L and A16V), pfdhps (codon S436F and A437G), pfATPase6 (codon D639G and E431K) genes and pfK13 gene have been studied.
| ~ Materials and Methods|| |
A total of 50 P. falciparum isolates were collected from five states, namely Chhattisgarh, Meghalaya, Mizoram, Odisha and Tripura of India during December 2011–September 2014, including 10 culture-adapted samples from the Malaria Parasite Bank (MPB) of National Institute of Malaria Research. The ten isolates from parasite bank include one sample each from Goa, Chhattisgarh, Jharkhand and two isolates from Odisha and Rajasthan states while three reference controls 3D7, Dd2 and NF54 were also included.
The collected field samples were revived, and in vitro HRPII ELISA protocol was followed as per previously published protocol. DNA was extracted from the dried blood spots on filter paper using Qiagen Mini DNA isolation Kit (Qiagen, Germany) as per manufactures' instructions. Genotyping was carried out for pfcrt (codon K76T), pfmdr1 (codon N86Y), pfdhfr (codon A16V, N51I, C59R, S108N and I164 L) and pfdhps (codon S436F and A437G) using PCR-RFLP as per published methods., PCR amplification was performed for pfATPase6 (codon D639G and E431K) and pfk13 propeller region and PCR products were purified and sequenced. Sequencing results were analysed using Mega 6 Software. The results of the molecular markers study were correlated with in vitro sensitivity pattern.
The IC50 was estimated by dose-response curves of non-linear regression analysis using HN-NonLinReg.analysis (Malaria.farch.net). Chi-square test was performed for categorical data, and IC50 geometric mean was calculated for continuous data. Further, parametric Pearson correlation test was performed for continuous data using SPSS version 17 (McGraw-Hill, New York, USA). Assessment of standard cross-resistance of antimalarial drugs with each other was estimated by the Pearson correlation coefficient (r). Univariate linear regression analysis was performed to identify CQ as independent predictor of other antimalarial drugs (AQ, MQ, QN and ART). Model 1 linear regression analysis was performed for predicting other antimalarial with respect to CQ as predictor. P < 0.05 was considered statistically significant.
| ~ Results|| |
Correlation among antimalarial drug sensitivities
Parametric Pearson correlation analysis of in vitro HRPII assay showed significant positive correlation between the responses of CQ versus AQ (r = 0.527, P ≤ 0.0001), CQ versus QN (r = 0.320, P = 0.023), AQ versus QN (r = 0.394, P = 0.005), AQ versus ART (r = 0.409, P = 0.003) and QN versus ART (r = 0.501, P ≤ 0.0001). Interestingly, MQ showed strong significant positive correlation with QN (r = 0.582, P ≤ 0.0001) and ART (r = 0.505, P ≤ 0.0001) [Table 1]. In univariate linear regression analysis [Table 2] for HRPII assay, CQ showed a strong significant positive correlation as independent predictor for other antimalarials, i.e., AQ and QN, with r2 score 0.278 and 0.103, respectively. The results have shown that CQ can be considered as a predictor for other antimalarial drug resistance; similar results were observed in state-wise analysis [Table 3]. In Northeast (Meghalaya, Mizoram and Tripura) samples, CQ showed significant positive correlation as independent predictor of AQ with r2 score 0.561 and 0.564, respectively. Interestingly, CQ showed significant positive correlation as independent predictor for MQ with r2 score 0.775 and QN with r2 score 0.487 only in Tripura. Similarly, in MPB samples, CQ showed significant positive correlation as independent predictor of ART with r2 score 0.473.
|Table 3: Location-wise chloroquine prediction pattern for other antimalarial drugs|
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Molecular analysis of Plasmodium falciparum chloroquine resistance transporter K76T and pfmdr1 N86Y gene
Out of 50 samples, 64% were mutant, 22% were wild and 14% were mixed type for pfcrt K76T. Similarly, 56% were mixed, 40%wild and 4% mutant for pfmdr1 N86Y. In Chhattisgarh, all samples were mutant for pfcrt K76T and 90% for pfmdr1 N86Y. Similarly, in Northeast, all samples were mutant for pfcrt K76T whereas 80% samples were mutant for pfmdr1 N86Y. In contrast, the culture isolates of MPB showed 100% mutation for pfmdr-1 N86Y and 80% for pfcrt K76T. Samples collected from Odisha showed 60% mutation for pfcrt K76T and 90% for pfmdr 1 N86Y. In Tripura, 50% samples were mutant for pfcrt K76T and 90% for pfmdr1 N86Y.
Prevalence of mutations in pfATPase6 and pfK13 gene
Out of the 50 samples, 30 samples were successfully amplified and sequenced for pfATPase6. Of these 30 samples, mutation at E431K was observed in only one sample (1/30; 3.3%) from Tripura with an increased IC50 value of 6.28 nM. None of 50 samples showed mutation for propeller region pfK13 gene.
Association among Plasmodium falciparum chloroquine resistance transporter K76T and pfmdr1 N86Y and in pfATPase6 and pfK13 gene
The polymorphism study revealed that pfcrt K76T and pfmdr1 N86Y were correlated for the mixed, mutant and wild type genotype for few isolates; however, the same was not true statistically. Similarly, there were isolates with increased IC50 value for ART which showed amplification for pfATPase6 gene, but no significant point mutation was recorded expect in one sample.
Association between antimalarial drug sensitivity with Plasmodium falciparum chloroquine resistance transporter K76T and pfmdr1 N86Y gene polymorphisms
We observed both wild and mutant genotypes in the distribution of pfcrt K76T with antimalarial drugs [Table 4]. Among all drugs, CQ showed both wild and mutant type genotypes for pfcrt K76T. CQ sensitivity correlated with pfcrt wild type pattern with K76T in 20% while CQ-resistant isolates (80%) correlated with mutant K76T genotype. However, increased IC50 of CQ did not statistically correlated with the polymorphism in K76T. Furthermore, no K76T polymorphism was observed for AQ, MQ, QN and ART.
|Table 4: Association between Plasmodium falciparum chloroquine resistance transporter K76T mutation and antimalarial drug resistance|
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The polymorphism distribution of pfmdr1 N86Y with CQ, MQ, AQ, QN and ART drug is shown in [Table 5]. Interestingly, CQ is the only drug which showed both wild (n = 1) and mix (n = 4) type pattern for pfmdr1 N86Y; however, it was not correlated significantly statistically. Likewise, no mutation was observed for AQ, MQ, QN and ART.
|Table 5: Association between pfmdr1 N86Y mutation and antimalarial drugs|
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Prevalence of mutations in pfdhfr and pfdhps gene
For pfdhfr gene, 28 samples (56%) showed mutation at S108N codon, while mutation at the C59R codon was observed in 21 samples (42%). The mutation at I164 L codon was observed in only one sample (2%) whereas A16V and N51I codon were wild type. The mutant allele of pfdhfr for codon C59R and S108N were predominant in Chhattisgarh with a prevalence of 80% each followed by 70% each in Odisha and 60% and 70%, respectively, in Northeast [Figure 1]. In pfdhps gene, mutation at A437G codon was observed in 16 samples (32%), while mutation at the S436F codon was observed in 10 samples (20%) [Figure 2].
| ~ Discussion|| |
This study reveals increasing trend of in vitro resistance in P. falciparum to CQ, AQ and QN and the correlation between reduced in vitro sensitivity of parasites to CQ, AQ and QN and the polymorphisms in the pfcrt K76T and pfmdr1 N86Y genes in the study sites. In India, for the first time, CQ resistance was reported from Karbi Anglong district, Assam in 1973. Interestingly, CQ showed significant positive correlation as independent predictor for AQ, QN and MQ. Till date, to the best of our knowledge, no such prediction analysis has been reported so far for these antimalarials, although reports of cross-reactivity between CQ and AQ are documented in malaria endemic areas. In addition, four samples were found to be resistant to QN through in vitro assay with increased IC50 value. A few case reports of QN resistance have been reported in the past, particularly from the northeastern states and Kolar district in Karnataka. In this study, AQ resistance was also recorded in four samples which is similar to the finding of decreased sensitivity to AQ and polymorphisms in pfcrt and pfmdr1 genes in Southwest Nigeria.
pfcrt K76T analysis showed that majority of the samples were found to be resistant to CQ. These findings uphold well with the previous finding. In a recent study conducted in Thai-Myanmar border, CQ-resistant isolates were found with increased IC50 value and all resistant isolates were detected with mutation in pfcrt at positions 76. Our findings also suggest that pfcrt K76T could be a useful marker for decreased susceptibility of CQ in vitro assay.
The role of pfmdr1 gene polymorphism is still unclear. However, there is lack of sufficient information to describe the SNPs' association with decreased in vitro susceptibilities to antimalarials.pfmdr1 N86Y mutant samples (60%) were observed to be associated with decreased sensitivity in Northeast; which is quite similar to recent reports of declined sensitivity in Northeast. Recent study has revealed the role of pfmdr1 in controlling the degree of MQ, QN and ART susceptibilities. Interestingly, an increased IC50 value for QN was observed in this study.
The pfmdr1 86Y allele can predict increased in vitro susceptibility to monodesethyl-amodiaquine (MDAQ). In contrast, the 86Y mutation observed in this study was not significantly related with increased susceptibility to AQ (P = 0.98), a finding similar to study conducted in Nigeria. In Benin, no difference in AQ IC50 between N86 and 86Y haplotypes has been observed. The connection of N86Y in QN resistance is quiet disputed; in a few studies, pfmdr1 N86Y mutation has been associated with increased susceptibility., The N86Y mutation was not linked with increased susceptibility to QN (P = 0.65), which is quite similar to previous study.
Regardless of ample information on mode of action of ART, till date, it is a matter of debate and research. One of the proposed mechanisms is the study of parasite interaction with sarcoplasmic reticulum Ca2+ATPase 6 (pfATPase6). However, no such association was observed between pfATPase6 and in vitro increased susceptibility in the present study, which is also reaffirmed by previous studies., This study also observed single sample of E431K point mutation. Few reports on pfATPase6 gene mutation at codon E431K are available from India., Studies with larger samples for E431K mutation analysis can confirm its role as recognized markers for the assessment of ART resistance in the country. Interestingly, no mutation was observed in Kelch13 propeller region, which is similar to reports from Kolkata. However, there are reports, which have documented non-synonymous mutations from India.,
ART-based combination therapy has been recommended by the Government of India since 2010 as the first-line therapy for the treatment of uncomplicated malaria. The point mutations in pfdhfr and pfdhps genes have been linked to the SP treatment failure. However, no in vitro study for SP could be done; attempts have been made to undertake molecular analysis of the five codons of pfdhfr and two codons of pfdhps to assess the AS + SP-ACT efficacy. A triple mutant for pfdhfr gene at codons C59R, S108N and C59R and double mutant for pfdhps at codon S436F and A437G were observed. These results are parallel to the recent publication. Antifolate resistance related to point mutations in pfdhfr and pfdhps genes were observed among the field samples of Tripura, Chhattisgarh and MPB.
| ~ Conclusion|| |
This is the first study to document the correlation between IC50 and molecular markers with respect to antimalarial drug resistance in the study sites in India. Overall, the data from the in vitro susceptibility and molecular analysis may specify the tendency of declining sensitivity of P. falciparum isolates towards CQ, AQ and QN in India; still, all the samples were found to be sensitive to ART and MQ during the study period. Surveillance study involving large sample size is required to confirm the association of in vitro susceptibility of antimalarial drugs with the molecular markers in the country. In conclusion, results of this study suggest that in vitro susceptibility of P. falci parum isolates to antimalarials along with molecular marker is a valuable and dependable tool with increased prediction efficiency for drug resistance.
The author SS would like to express her gratitude to the Indian Council of Medical Research for providing fellowship (ICMR fellowship no is - 3/1/3/JRF-2010/HRD-88), National Institute of Malaria Research for infrastructure for infrastructure support and Department of Biotechnology, Goa University for PhD registration.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]