In India, malaria is distributed throughout the coun- try. A large numbers of malaria cases have been reported from Northeastern (NE) states, of which asymptomatic cases shared a major proportion1. The presence of asymp- tomatic malaria cases plays a significant role in malaria transmission. These asymptomatic cases carry malaria gametocytes and act as a long-lasting parasite reservoir that transmits malaria parasites in areas of low or unstable transmission intensity2–7. However, the role of asymp- tomatic infections in the transmission of malaria para- sites with genetic changes conferring drug-resistance is not clear2–7. Many earlier studies have been carried out in India regarding the genetic polymorphisms associated with drug resistance among symptomatic malaria pa- tients8–11.However, no extensive studies were undertaken in India to find out the genetic polymorphisms associated with antimalarial resistance among asymptomatic malaria patients. Keeping this in mind, a study was carried out in few selected malaria endemic areas of Assam and Arunachal Pradesh to observe the point mutations in Plas- modium falciparum dihydrofolate reductase (Pfdhfr) and P. falciparum dihydropteroate synthase (Pfdhps) gene among the asymptomatic P. falciparum positive patients.
This study has provided significant information regard- ing the epidemiology of asymptomatic P. falciparum malaria cases and drug resistance capability.
Based upon the previous records of malaria, three malaria endemic districts (Karbi-Anglong, North Cachar Hills and Tinsukia) in Assam and two districts (Changlang and Lohit) in Arunachal Pradesh were selected to fulfill the objective of the study. The study was undertaken for a period of three years (from 2012 to 2014). A total of 128 normal healthy (not showing any symptoms of ma- laria or other disease) participants were recruited for the study irrespective of age and sex. Out of 128, 54 cases had recent malaria infection (before one month), 29 cases had malaria infection before couple of months and re- maining 45 cases had malaria infection 1–4 yr ago. The aims and objectives of the study were explained to each participant. Institutional ethical permission was obtained from ethics committee of the Regional Medical Research
Centre (ICMR), Dibrugarh, Assam. Written informed consent/assent was taken from all participants prior to recruitment in this study. After obtaining consents/
assents 2 ml of blood samples was collected from each participant. Presence of malaria parasite was confirmed by using microscopic examination of the blood smears made from collected samples. Serum was separated from the remaining blood samples and DNA extraction was done using the QIAamp DNA Mini spin columns kit (Millipore Corporation, Qiagen, Hilden, Germany). A 648 bp portion of Pfdhfr gene and 710 bp portion of Pfdhps gene were amplified by polymerase chain reaction (PCR) method in a thermal cycler (Gene Amp®PCR System 9700 and Veriti, Applied Biosystems, Foster City, CA, USA)12. The amplified products were purified and sequenced com- mercially in South Korea through Anshul Biotechnolo- gies, Hyderabad, India. The sequencing products were analyzed in BioEdit, DnaSP version v.5.10.01 and Mega 5 software for detection of single nucleotide polymor- phisms.
Among the study samples, 45.31% (58/128) cases had P. falciparum monoinfection. Of these P. falciparum posi- tive cases, 22 cases were from Karbi-Anglong, 11 cases from North Cachar Hills and five cases were from Tinsukia district in Assam; whereas 12 cases were de- tected from the collected field samples in Changlang district and eight from Lohit district of Arunachal Pradesh.
The average parasitaemia range varied from 0.1 to 11%, SD ± 2.636 with median value 2 (Table 1). Artemisinin- based combination therapy (artesunate plus sulphadoxine- pyrimethamine) was given as per prescribed protocol to the diagnosed positive patients. Among the asymptom-
J Vector Borne Dis 53, March 2016, pp. 81–83
Antifolate resistance associated point mutations among asymptomatic malaria cases
Jitendra Sharma, Prafulla Dutta & Siraj Ahmed Khan
Entomology and Filariasis Division, Regional Medical Research Centre (ICMR), Northeast Region, Dibrugarh, Assam, India Key words Arunachal Pradesh; Assam; asymptomatic; Pfdhfr ; Pfdhps; mutation
Table 1. Percentage of parasitaemia among asymptomatic P. falciparum positive patients
Percentage of Asymptomatic P. falciparum
parasitaemia positive cases
0.1– 1.9 19
2 – 6 32
7 – 11 7
Total 58
J Vector Borne Dis 53, March 2016 82
atic P. falciparum positive cases, 93.10% (54/58) have shown mutations either in Pfdhfr or in Pfdhps gene.
Double mutation C59R+S108N in Pfdhfr gene was de- tected in 77.59% (45/58) samples. Three isolates from Changlang district had shown N51I+C59R+S108N mu- tations and two isolates had shown C59R+S108N+I164L mutations in Pfdhfr gene (Table 2). However, in case of Pfdhps gene, 34.48% (20/58) samples had shown S436F mutation. Similarly, 10.34% (6/58) isolates had shown A437G+A581G mutations, 12.07% (7/58) had S436A+
A437G mutations and 27.59% (16/58) samples had S436A+A437G+K540E mutations in Pfdhps gene (Table 2). Two P. falciparum positive cases in Changlang dis- trict of Arunachal Pradesh had N51I+C59R+S108N- S436A+A437G+K540E mutations in Pfdhfr-dhps two locus genes which may to be associated with treatment failure.
From the above finding it was observed that C59R+S108N mutations in Pfdhfr gene and S436F mu- tation in Pfdhps gene are the key point mutations among the asymptomatic P. falciparum cases. Apart from this, few cases from Assam and Arunachal Pradesh had shown triple mutations either in Pfdhfr or in Pfdhps gene. It is an indication that antifolate resistance had reached at an alarming level among asymptomatic carriers. Many ear- lier studies have reported that these types of mutations are common in symptomatic malaria cases13–19. However, no information is available in case of the asymptomatic malaria cases. These asymptomatic P. falciparum cases (having such mutations) serve as a reservoir for malaria parasite having antimalarial resistance capability. Some- times malaria causing mosquitoes bite such carriers, thereby transmitting the acquired malaria parasitaemia from asymptomatic carriers to a normal healthy person.
If malaria parasite enters into a normal population with its already developed antimalarial resistance capability, it could gradually attack the entire population.
So, it is crucial to find out the carriers having asymp- tomatic malaria infection. For this, the mapping of ma- laria endemic places in each district is necessary and ac-
cordingly mass fever survey is needed in those places at frequent interval. Until and unless we could detect and treat the asymptomatic cases, malaria elimination is not possible. By detecting asymptomatic carriers at an early stage, one can predict reduction in the malaria burden from NE region of India.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the study par- ticipants for their interest and cooperation. Special thanks to Mr. Pobitra Doloi, Mr. Sanam lama and Mr. Lakhyajit Borah (Laboratory Technicians) of Regional Medical Research Centre (ICMR), Dibrugarh for assisting in different steps during the research work.
Conflict of interest
The authors declare that they have no conflict of interest.
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Correspondence to: Mr. Jitendra Sharma, Entomology and Filariasis Division, Regional Medical Research Centre (ICMR), Northeast Region, Dibrugarh–786 001, Assam, India.
E-mail: [email protected]
Received: 3 August 2015 Accepted in revised form: 4 January 2016
