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 Table of Contents  
Year : 2016  |  Volume : 6  |  Issue : 1  |  Page : 69-77  

Detection of chloroquine and artemisinin resistance molecular markers in Plasmodium falciparum: A hospital based study

1 Department of Microbiology, Jawaharlal Nehru Institute of Postgraduate Medical Education and Research, Puducherry, India
2 Department of Medicine, Jawaharlal Nehru Institute of Postgraduate Medical Education and Research, Puducherry, India
3 Department of Paediatrics, Jawaharlal Nehru Institute of Postgraduate Medical Education and Research, Puducherry, India

Date of Acceptance30-Nov-2015
Date of Web Publication28-Jan-2016

Correspondence Address:
Subhash Chandra Parija
Department of Microbiology, Jawaharlal Nehru Institute of Post-Graduate Medical Education and Research, Puducherry
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2229-5070.175110

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Introduction: Emergence of chloroquine (CQ) resistance in Plasmodium falciparum has increased the morbidity and mortality of falciparum malaria worldwide. Artemisinin-based combination therapies are now recommended by the World Health Organization as the first line treatment for falciparum malaria. Numerous molecular markers have been implicated in the CQ and artemisinin resistance. Materials and Methods: A total of 26 confirmed cases of falciparum malaria (by giemsa stained thick and thin smear, quantitative buffy coat, immunochromatographic test, or polymerase chain reaction [PCR]) were included in the study. About 5 ml of ethylenediaminetetraacetic acid blood sample was collected and stored at –20°C till use. Plasmodium DNA was extracted using QIAamp whole blood DNA extraction kit. PCR was done to amplify pfcrt, pfmdr1, pfserca, and pfmrp1 genes and the amplicons obtained were sequenced by Macrogen, Inc., Korea. Single nucleotide polymorphism (SNP) analysis was done using Bio-Edit Sequence Alignment Editor. Results: Out of the four genes targeted, we noted a SNP in the pfcrt gene alone. This SNP (G > T) was noted in the 658 th position of the gene, which was seen in 13 patients. The pfmdr1 and pfserca genes were present in 9 and 14 patients respectively. But we could not find any SNPs in these genes. This SNP in pfcrt gene was not significantly associated with any adverse outcome and neither altered disease progression. Conclusion: Presence of a single SNP may not be associated with any adverse clinical outcome. As the sample size was small, we may have not been able to detect any other known or unknown polymorphisms.

Keywords: Chloroquine, quantitative buffy coat, single nucleotide polymorphisms

How to cite this article:
Ramani S, Parija SC, Mandal J, Hamide A, Bhat V. Detection of chloroquine and artemisinin resistance molecular markers in Plasmodium falciparum: A hospital based study. Trop Parasitol 2016;6:69-77

How to cite this URL:
Ramani S, Parija SC, Mandal J, Hamide A, Bhat V. Detection of chloroquine and artemisinin resistance molecular markers in Plasmodium falciparum: A hospital based study. Trop Parasitol [serial online] 2016 [cited 2023 Feb 9];6:69-77. Available from: https://www.tropicalparasitology.org/text.asp?2016/6/1/69/175110

   Introduction Top

Chloroquine (CQ) and other quinoline-based drugs have been used for the prophylaxis and treatment of malaria for more than 50 years in all of the malaria endemic countries because of its cost effectiveness, few side effects, and easy availability. The tremendous success of CQ and its heavy use through decades eventually led to CQ resistance in Plasmodium falciparum which is responsible for fatal malaria in humans. The resistance to antimalarial drugs, especially CQ, in P. falciparum is one of the principal factors contributing to the worldwide increase in morbidity and mortality due to malaria.

Foci of resistant P. falciparum were detected in Columbia and at the Cambodia-Thailand border during the late 1950s, and then resistant strains from these foci spread throughout the world. In India, CQ resistance in P. falciparum was first reported by Manjha in the Karbi Anglong District in 1973 and from Nowgaon in 1974 in the North-Eastern state of Assam. [1] To face the challenge posed by the increasing antimalarial treatment failure due to CQ resistance, many countries have adopted artemisinin-based combination therapy (ACT). [2]

Artemisinin-based combination therapies are now recommended by the World Health Organization (WHO) as first-line treatment of uncomplicated falciparum malaria in all areas in which malaria is endemic. [3] WHO estimates that more than 90% of the 1.5-2.0 million deaths attributed to malaria each year occur in African children. Replacing ineffective, failing treatments (CQ and sulfadoxine-pyrimethamine [SP]) with artemisinin-based combination therapies has reduced the morbidity and mortality associated with malaria. Parenteral artesunate is replacing quinine for the treatment of severe malaria. Recently, there have been signs that the efficacy of ACT have declined in some parts of world. This has happened due to misuse of artemisinin as in artesunate monotherapy. Spread of artemisinin resistance would be disastrous for global malaria control.

Different approaches have been developed to monitor the extent of antimalarial drug resistance and to determine the biologic mechanisms by which the parasite has evaded the action of the drug. Laboratory strategies include in vitro drug sensitivity tests and evaluation of molecular markers associated with drug resistance. Some of these molecular markers have been strongly associated with the development of CQ resistance while some of the markers are under evaluation. In case of artemisinin, some markers have been postulated to play a role in its resistance, but no studies till date have shown any conclusive evidence. Hence, this study has been conducted to look for the proportion of CQ-resistance genetic markers in our P. falciparum isolates and also, the artemisinin resistance genetic marker in the same isolates will be detected if present.

   Materials and Methods Top

This is a descriptive study conducted in the Department of Microbiology, JIPMER, after being approved by the Institute Research and Ethics Committee. This study included single group of patients wherein the cases were recruited as per the below-mentioned inclusion and exclusion criteria. All children and adults (males and nonpregnant females) attending Pediatric and Medicine outpatient department/emergency medical services confirmed as cases of falciparum malaria or mixed infections (P. falciparum and Plasmodium vivax) were included in the study. Malaria due to other species of Plasmodium were excluded from the study. The objectives of the study were to detect the pfcrt gene, pfmdr1 gene, pfserca gene, and pfmrp1 gene by conventional polymerase chain reaction (PCR), to detect the genetic polymorphisms in these genes by DNA sequencing and to correlate the clinical response to artemisinins, with the detected mutation/s.

A total of 26 cases were included in the study period of 1 year (December 2011 - December 2012). About 5 ml of blood sample was collected in a sterile ethylenediaminetetraacetic acid (EDTA) containing bottles, from all these patients on the day of admission (0 day), 3 rd , 7 th , and 14 th day of the illness. Thick and thin smear were prepared and stained with giemsa stain. Immunochromatographic test (ICT) (NECVIPARUM, Nectar Life Sciences Ltd., Chandigarh, India) and quantitative buffy coat (QBC) test (PARASCAN, Diagnova, New Delhi, India) were also performed as per the manufacturers' instructions [Color Plates 1 and Color Plate 2]. Blood sample positive by any of these 3 methods were included in the study.

All the samples were stored at −20°C, till used. Plasmodium DNA was extracted from 200 μl of the EDTA anticoagulated blood samples using the QIAamp whole blood DNA extraction kit as per the manufacturer's instructions. All the extracted DNA were stored at −20°C. Molecular confirmation of these samples were done as given by Johnston et al [Color Plate 3].[4]

All the confirmed samples were subjected to PCR for the detection of the following resistance gene [Color Plate 4, Color Plate 5 and Color Plate 6], namely pfcrt (Pharath Lim et al.) pfmdr1 (Johnston SP), pfserca (Noedl et al.) and pfmrp1 (Pharath Lim et al.).[4],[5],[6],[7] The amplicons obtained in all the PCRs were sequenced by Macrogen, Inc., Korea. All the sequences were analyzed for the single nucleotide polymorphisms (SNP) using Bio-Edit Sequence Alignment Editor [Color Plate 7].

All the patients were observed for the clinical and parasitological response on 0 day (the day of admission), 3 rd day, 7 th day, and 14 th day of admission.

   Results Top

Out of the total 40 P. falciparum patients attended JIPMER, during the period from December 2011 to December 2012, 26 (study group = 26) patients satisfying the inclusion and exclusion criteria, were included in the study.

Distribution of Plasmodium species

During the study period, the total number of blood smears examined were 7138, out of which (28) 8% were P. falciparum and (12) 4% were mixed infections (diagnosed by combined smear, ICT, and QBC). The most common species detected was P. vivax (88%).

Age and sex distribution

The highest number of patients was found to be 14/40 (35%) between 21 and 30 years of age whereas the lowest 5/40 (12.5%) were of more than 60 years of age. No patients were recruited between 6 and 10 years of age [Figure 1]. Out of the total 40 patients, 25% were females and 75% were males.
Figure 1: Distribution of the cases according to their age and gender

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Clinical features

In our study, all the patients presented with fever (100%) [Table 1]. One patient had seizures 1/26 (3.8%). The common complication noted in our study was the acute renal failure, which was observed in 7/26 (26.9%) cases [Table 2]. The most deranged hematological parameter in our study was thrombocytopenia which was observed in 53.84% of the cases and the second common was anemia (26.9%). Mortality was seen in 2/26 (8%) of the cases in our study.
Table 1: Predominant clinical manifestations observed in the study group

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Table 2: Complications observed in the study group

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Parasitic index

Out of the total 16 smear positive samples, parasitic index was calculated using the thick smear as described by Moody. [8] The highest parasitemia noted in our study was 80,000 parasites/μl (approximately equivalent to 1.6% of infected red blood cells) [Figure 2]; 6/16 (37%) had a parasitic index <100 parasites/μl. The influence of the parasitemia on the clinical outcome is given in the [Table 3].
Figure 2: Range of parasitic load in the study group

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Table 3: Correlation between the parasitic index the clinical outcome

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Resistance genes

All the samples positive by PCR, ICT, and smear were subjected to simplex PCRs for the detection of molecular markers for CQ and artemisinin resistance. The CQ-resistant P. falciparum DNA obtained from the Regional Medical Research center was used as positive control; sterile water was used as a negative control. The positive control which we used harbored the pfcrt gene and the pfserca gene, but not the pfmdr1 or pfmrp1 gene. The gene targets selected based on the existing literature were pfcrt, pfmdr1, pfserca and pfmrp1. Out of the 26 samples, 13 (50%) were positive for pfcrt gene, 9 (34.6%) for pfmdr1 gene, and 14 (53.8%) were positive for pfserca gene. All these gene targets were found in 0, 3 rd , and 7 th day samples. None of our samples harbored the pfmrp1 gene. All the three genes were present in 7 of our samples; 9 samples harbored both the pfcrt and pfserca genes.

Single nucleotide polymorphisms

In our study, out of the 4 gene targets, SNP was detected only in the sequence of pfcrt gene in 658 th nucleotide position in the codon 220 in all the 13 samples. In the 658 th position of the pfcrt gene, G was noted instead of T. No other SNPs were noted in our study.

Clinical follow-up of the patients

Twenty-four patients were followed up on the 0, 3, 7 and 14 days after admission to the hospital. All the patients admitted were given injection artesunate 120 mg intravenous (IV) stat and then after 12 h, followed by 120 mg IV OD for 3 days along with supportive treatment. Then they received the therapy as mentioned. Tablet artemether 80 mg and tablet lumefantrine 480 mg on 0, and 8 h on 1 st day; twice daily on the second and 3 rd day.

Out of the 26 patients studied, two patients expired on the same day as that of their admission to the hospital; one patient had recrudescent malaria, who had falciparum malaria 1 month before and had now presented with fever; his peripheral blood smear showed the presence of ring forms of P. falciparum. All the others showed clinical improvement and parasitic clearance on the 3 rd day.

   Discussion Top

Malaria is a major public health problem in India. The Southeast Asia contributed only 2.5 million cases to the global burden of malaria. Out of which, India alone contributed 76% of the total case. [8] To understand the malaria situation in an area, malaria prevalence is one of the important epidemiological parameters, which can be measured either by mass blood survey or sample blood survey in the area. The region wise prevalence as per the NVBDCP, India in 2012 [9] shows a very diverse distribution. In Jammu and Kashmir, it was 3.9%, 68.6% in Assam, 36.7% in Jharkhand, 93% in Orissa, 13.7% in Gujarat, 20.1% in Maharashtra, 3.05% in Tamil Nadu, and 1.4% in Puducherry.

In the present study, a total of 26 (65%) cases infected with P. falciparum (including mixed infection with both P. falciparum and P. vivax) were studied out of 40; the remaining 14 were not included as they were lost to follow-up. However, out of the total 40 cases, 75% were males, and the maximum number of cases was found to be in the age group between 20 and 30 years.

The significantly higher malaria positivity rate recorded in males compared to that in females could be due to a difference in their exposure to the vector which are governed by the dressing pattern of females who fully cover their bodies and sleep indoors. Males are mostly involved in outdoor-related activities during evening hours, and they are likely to get more mosquito bites, as a result of which the chances of man-vector contact is high in males compared to females.

There is very limited information on gender-specific malaria prevalence in different paradigms in the country and most of the available data is arbitrary. [9],[10] In a recent epidemiological study in Sonitpur district of Assam, it was shown that overall malaria prevalence (slide positivity rate) was higher among males (43.2%) than females (34.5%). [11]

It is well known that all age groups can be affected by P. falciparum though complicated malaria is classically seen in the extremes of age. The youngest case in our study was aged 1 year 6 months and the eldest member to be affected with this parasite was 65 years of age. The disease burden was notably higher in the extremes of ages as seen in our study. This has also been documented elsewhere. [10]

There is very limited information on the specific seasonal prevalence of malaria in different regions in India. Transmission also depends on climatic conditions that may affect the number and survival of mosquitoes, such as rainfall patterns, temperature and humidity. [12] In many places, transmission is seasonal, with the peak during and just after the rainy season. In our study, we found an increased occurrence of falciparum malaria cases during the September to December months after the rains.

Clinical manifestations of falciparum malaria are protean, the most common being fever. All our patients (100%) had fever whereas chills with fever were noted in 19.2%. We did not find any asymptomatic or afebrile malaria case in our study group. Severe malaria was noted in 7/26 (26.9%).

The complications of severe malaria were noted in the forms of acute renal failure, anemia, cerebral malaria (a headache, vomiting, seizures and impaired consciousness) and jaundice. One of the patients developed circulatory failure and disseminated intravascular coagulation (DIC) resulting from acute renal failure, he later succumbed to the illness. Another patient had developed circulatory collapse resulting from anemia and jaundice, fortunately, he recovered. A third patient presented with high-grade fever which could not be controlled and finally she developed renal failure followed by circulatory collapse, from which she could not be revived.

Hematological abnormalities are common. Thrombocytopenia and anemia have been reported. Thrombocytopenia was found to occur in 60-80% and anemia in 25% in an earlier study. [13] Finding of thrombocytopenia with anemia is an important clue to the diagnosis of malaria in patients with acute febrile illness. In this study, we found thrombocytopenia in 53.84% of patients suffering from falciparum malaria while, 26.9% were found to be anemic.

Both thrombocytopenia and anemia were seen in 19.2% (5/26) of our cases studied. These figures are comparable to studies done by other investigators as 71% [14] and 58.97%, [15] 69.18%. [13] Thrombocytopenia is considered to be an important predictor of severity in childhood falciparum malaria. Bashwari et al., from Saudi Arabia had reported anemia in 60% and thrombocytopenia in 53% of cases. [16] Thrombocytopenia is seen in patients with acute febrile illness due to viral causes such as dengue, but its presence is considered an important diagnostic clue for malaria as well in endemic areas as suggested by previous investigators and particularly so when associated with anemia. [17] Mild to moderate thrombocytopenia is a common association of malaria, but it is rarely associated with hemorrhagic manifestations or a component of DIC. [18]

The cause of thrombocytopenia is poorly understood, but the immune-mediated lysis, sequestration in the spleen and a dyspoietic process in the marrow with diminished platelet production have all been postulated. Abnormalities in platelet structure and function have been described as a consequence of malaria, and in rare instances, platelets can be invaded by malarial parasites themselves. [13] Tumor necrosis factor and interleukin-10 have been implicated in the development of P. falciparum malaria-induced anemia, but the role of these cytokines has not been studied in the development of thrombocytopenia in patients with acute malaria. [13]

Deaths due to falciparum malaria were reported from Assam (0.06%), Jharkhand (0.02%), Orissa (0.03%), Gujarat (0.28%), Maharashtra (0.8%), whereas in Tamil Nadu, Puducherry, and Jammu and Kashmir, no deaths were reported due to P. falciparum. [9] Mortality was noted in 8% in the present study. All of these patients had severe malaria at the time of admission to the hospital. All these cases were not residents of Puducherry. One of them was from Bihar while another one hailed from Tamil Nadu.

Medication reduces morbidity and mortality by terminating a malaria infection in a patient and restricts malaria transmission by diminishing the parasite reservoir. Anti-malarial drugs are also used as a preventive measurement, both as chemoprophylaxis for travellers to malaria endemic areas and for intermittent preventive treatment in high-risk groups such as pregnant women, infants and children. [19]

Anti-malarial drugs remain as one of the most powerful tools in the fight against malaria. So far, malaria control has relied largely on a comparatively small number of chemically related drugs belonging to 7 classes of compounds: 4-aminoquinolines, 8-aminoquinolines, arylamino alcohols, antifolate compounds, artemisinin and derivatives, and inhibitors of the respiratory chain and antibiotics.

Until recently, CQ was the most widely used drug to treat and prevent malaria infection. The success of CQ was based upon its rapid action, safety and low cost relative to other antimalarials. With the spread of CQ resistance, many countries adopted SP combination as first line antimalarial treatment. Still, resistance to SP spread more rapidly than to CQ and is now widespread in Asia and South America and is spreading in Africa. [20]

Drug combinations, rather than monotherapy, are now regarded as the best solution for treating malaria. [21] The simultaneous use of two or more drugs is a chemotherapeutic strategy for improving treatment efficacy and retarding the development of resistance to the individual drugs in the combination. The underlying principle for the impact of combination therapy on drug resistance is based on the assumption that drug resistance essentially depends on mutation. Provided that the constituent drugs administered in the combination have independent modes of action, the probability of a parasite developing resistance to both drugs simultaneously is significantly reduced compared to developing resistance to one drug. [22]

The probability that a mutant will arise that is at the same time resistant to two different antimalarial drugs is the product of the mutation rates per parasite for the individual drugs, multiplied by the number of parasites that are exposed to the drugs, in an infection. [23] In response to the increasing burden of malaria caused by P. falciparum resistance to the standard antimalarial medicines, WHO recommended the use of combination therapies, ideally those containing artemisinin derivatives in countries where P. falciparum malaria is resistant to the conventional antimalarial medicines CQ, SP, and amodiaquine.

Unfortunately, even artemisinin derivatives, the only drugs that had been fully effective against P. falciparum until very recently, seem to be losing their efficacy along the border between Cambodia and Thailand. [8],[23],[24]

Several point mutations in the coding region 76 of pfcrt gene were reported to be associated with CQ resistance. However, mutation at codon 76 (Lys to Thr) has been found in almost all the CQ-resistant parasite lines and clinical isolates. Therefore, it has been proposed as a molecular marker to monitor the CQ resistance in field isolates. While it is true that K76T mutation is associated with CQ resistance, this mutation is not absolute. Because a large number of CQ responders are also found to harbor this mutation, and it is highly prevalent in Indian isolates. This raises several issues like the involvement of host response such as the status of the immune system which can clear the parasite irrespective of its being CQ-resistant or not. Besides K76T mutation in pfcrt, mutations at codon 72, 74, 75, 97, 220, 271, 326, 356 and 371 have also been found to be associated with CQ resistance. [25]

In a study by Veiga et al., [26] sequencing of the pfcrt, pfmdr1 and pfmrp1 genes yielded the following. SNPs at positions 191, 325, 437, 572, 785, 876, 1007, and 1390 were found in the isolates with elevated occurrence in pfmdr1 gene. Increased pfmrp1 gene copy number was not detected. The pfcrt full open reading frame (ORF) of each isolate sequenced from cDNA revealed that all isolates carried the pfcrt Dd2-like ORF haplotype (C72/74I/75E/76T/ 220S/271E/326S/356T/371I).

In our study, resistance markers (pfcrt, pfmdr1 genes) were detected in 13 (59%) and 9 (41%) patients, respectively; but we could not detect the pfmrp1 gene in any of the samples. Among these, one SNP was noted in codon 220 in 658 th nucleotide position the pfcrt gene (triplet nucleotide change [GCC-TCC]; T instead of G) in all these 13 cases. We did not find K76T mutation in our study population. No polymorphism was noted in any other site in any of the other target regions of the other genes.

The SNP in the pfcrt gene which was detected in 13 samples was found on the 1 st day of admission to hospital itself. The presence of this SNP was not significantly associated with any adverse outcome and neither altered the disease progression. The commonly known polymorphism in codon 76 of pfcrt gene was not noted in any of our study samples. We need a larger population to able to study this polymorphism better. In addition, because of the limited sample size in this study we may have not been able to detect any other known or unknown polymorphisms.

Artemisinin combination therapies are the first-line treatments for uncomplicated P. falciparum malaria in most malaria-endemic countries. Recently, partial artemisinin-resistant P. falciparum malaria has been observed on the Cambodia-Thailand border. Exposure of the parasite population to artemisinin monotherapies in subtherapeutic doses for over 30 years, and the availability of substandard artemisinins have probably been the main driving force in the selection of the resistant phenotype in the region.

A multifaceted containment programme has recently been launched, including early diagnosis and appropriate treatment, decreasing drug pressure, optimising vector control, targeting the mobile population, strengthening management and surveillance systems, and operational research.

Uhlemann et al. provided strong evidence that resistance to artemisinins may depend on an SNP in the drug's putative chemotherapeutic target, the SERCA-type ATPase protein of P. falciparum (pfATPase6). [27] A subsequent study report that P. falciparum parasites from French Guiana harboring mutant forms of the pfATPase6 gene (S769N) displayed significantly increased IC50s to artemisinins, suggesting its role in artemisinin resistance. [28] However, mutations in this gene are not associated with resistance in field isolates from elsewhere.

Causal unequivocal association between mutations in the pfATPase6 gene and resistance to artemisinins has not yet been established though, whereas other genes have also been implicated in this phenotype. In this study, we could not identify any polymorphism/mutation in this gene though the pfserca gene was detected in the 7 samples which already had an SNP in the pfcrt gene.

In a recent study, a significant association between the pfcmu (clathrin mu adaptor gene) 479A genotype in Rwanda samples and the in vitro sensitivity to dihydroartemisinin was observed. [19] These new findings suggest that polymorphisms in pfcmu can be involved in P. falciparum defense mechanisms against artemisinin derivatives. Thus, further assessment of the gene in artemisinin responses is of utmost importance and needs more attention, in the context of effective surveillance of artemisinin resistance.

With the advent of artemisinin, the therapy of malaria has seen a lot of improvements in terms of outcome and control of the progression of the disease from mild to severe. However, this should not be used alone as mutations are likely to emerge. Hence, combination therapy is recommended. The presence of particular molecular markers does not necessarily directly predict treatment outcome. Mutations in the above genes contribute to drug failure, but the outcome is not certain: Some patients with "resistant" alleles clear the infection, and some patients with "sensitive" alleles failed treatment. Therefore, it is better to think in terms of mutations encoding the raising probabilities of drug failure that ultimately depends on factors such as host immune response, the drug dose taken, and variation in drug absorption and metabolism.

Malaria continues to be a major public health problem which is further enhanced by P. falciparum drug resistance. The understanding of the mechanisms of resistance to antimalarial drugs is crucial. This thesis has shed light on the complex issue of P. falciparum resistance to artemisinins and will foster new studies to understand its mechanisms.

   Conclusions Top

The study was performed to detect the resistance markers in the genes associated or implicated for encoding resistance to CQ and artemisinin in P. falciparum. In the present study, a total of 26 (65%) cases infected with P. falciparum (including mixed infection with both P. falciparum and P. vivax) were studied out of 40; the remaining 14 were not included as they were lost to follow-up.

Out of the total 40 case studied, 75% were males, and the maximum number of cases was found to be in the age group between 20 and 30 years; malaria positivity rate recorded in males was significantly higher compared to that in females. Clinical manifestations of falciparum malaria are protean, the most common being fever. All our patients (100%) had fever whereas chills with fever were noted in 19.2%. Severe malaria was noted in 7/26 (26.9%). Mortality was noted in 8% in the present study. We found thrombocytopenia in 53.84% of patients suffering from falciparum malaria while, 26.9% were found to be anemic.

The SNP in the pfcrt gene was noted in codon 220 in the 658 th nucleotide position of the pfcrt gene (T instead of G) and detected in 13 samples. The presence of this SNP was not significantly associated with any adverse outcome and neither altered the disease progression. In this study, we could not identify any polymorphism/mutation in pfserca gene, though the gene was detected in the 7 samples which already had a SNP in the pfcrt gene.

Limitations of the study

The commonly known polymorphism in codon 76 of pfcrt gene was not noted in any of our study samples. As the sample size was small, we may have not been able to detect any other known or unknown polymorphisms. We need a larger population to be able to study these polymorphisms better. Other genes that are predicted to be responsible for artemisinin resistance were not targeted.

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Conflicts of interest

There are no conflicts of interest.

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