Background
Malaria is a life-threatening ancient parasitic disease and causes 250-500 million clinical episodes and nearly one million deaths annually [
1]. Among the five human malaria species,
Plasmodium falciparum is the most severe form, causing malignant malaria globally, while
Plasmodium vivax is the most widespread species outside Africa, causing huge morbidity and can be severe and fatal [
2‐
7].
The worldwide spread of chloroquine (CQ) resistant strains of
P. falciparum has led to use of sulphadoxine-pyrimethamine (SP) as the first-line anti-malarial drug in Southeast Asian countries. Sulphadoxine and pyrimethamine sequentially inhibits dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) enzymes respectively in the folate biosynthesis pathway resulting synergistic anti-malarial effect [
8]. Parasite has overcome the effect of SP by evolving point mutations in the respective genes encoding enzymes involved in the folate biosynthesis pathway. The mutated DHPS and DHFR enzymes have reduced binding affinity with SP drug and thus parasite survives in the presence of drug [
9,
10].
In India, resistance to CQ was reported for the first time in 1973 in
P. falciparum from north-eastern states [
11] and later it spread throughout the country [
12]. To overcome CQ resistance problem, in 1982, SP was employed as first-line anti-malarial therapy for treatment of falciparum malaria in areas with >25% CQ resistant level, complicated malaria case, and higher malaria endemicity [
12]. Currently, according to the national anti-malarial drug policy, artimisinin-based combination therapy (ACT) (Artesunate+SP) is being used in most of the malaria endemic regions [
12].
Reduced susceptibility to CQ in
P. vivax was for the first time observed in 1989 in Australian soldiers returned from Papua New Guinea [
13]. Later, several cases from Papua New Guinea, Indonesia, New Guinea, Brazil and India were documented [
14‐
19]. Recent studies from
P. vivax ex-vivo maturation experiment showed reduction in the susceptibility to CQ in Southeast Asian counties [
20,
21]. This information indicates gradual increase in CQ resistance cases of
P. vivax.
India contributes more than 78% of total malaria cases of Southeast Asia and
P. vivax accounts for more than 50% of annual malaria cases [
22]. Point mutations in
P. vivax dhfr had been documented from different parts of India [
23‐
25], however mutation data of
dhps is yet to identified in order to understand the molecular epidemiology of anti-folate resistance. Therefore, identifying information about presence of anti-folate drug resistance related point mutations (
dhfr/dhps) in
P. vivax from Indian sub continent would be highly helpful to understand the global pattern of anti-folate drug resistance. This study aims to identify point mutations in
dhfr/dhps and the spatio temporal pattern of anti-folate drug resistance in Indian sub-continent.
Discussion
Due to emergence and widespread of drug resistant strains of human Plasmodium species against chemotherapeutic intervention agents, clinical trial for monitoring drug efficacy over regular intervals as well as monitoring mutation data in relevant genes related with anti-malarial resistance are the crucial steps in deciding scope of a drug in field. This study uncovers 1) progressive increase in the frequency of quadruple mutant dhfr genotypes, 2) limited point mutations in dhps among Indian isolates, and 3) distinct pattern of anti-folate resistance in Indian sub-continent linked with geographical regions.
Plasmodium vivax is still susceptible to chloroquine in India [
30,
31] and anti-folate drugs are not used for treatment of vivax malaria, therefore, the appearance of point mutations in
dhfr/dhps is surprising. This has been explained earlier because of the use of sulphadoxine-pyrimethamine to treat chloroquine-resistant
P. falciparum is creating selection pressure in the
P. vivax population. This has been reflected in the isolates of areas with sympatricity of
P. falciparum and
P. vivax[
23,
25], which presented a higher proportion of double and quadruple mutant
dhfr genotypes than other regions. Cotrimoxazole (sulfamethoxazole and trimethoprim), an anti-microbial drug that target bacterial
dhfr/dhps, is widely used in bacterial infection as prophylaxis, and has potential to select mutant
dhfr/dhps of malaria parasites [
32,
33]. Cotrimoxazole is being used in India to treat bacterial infection in wide spectrum [
34,
35], which suggests that it could be a potential factor to select anti-folate point mutations in the Indian subcontinent. Other factors, i.e. presumptive treatment of malaria without diagnosis and use of S/P by private practitioners, cannot be ruled out.
The comprehensive analysis of point mutations in
dhfr among Indian sub-continent revealed three distinct clusters/groups/types linked with geographical locations. North and south India revealed a prevalence of wild and double mutant
dhfr, respectively, whereas north-eastern and island regions revealed a high prevalence of quadruple mutant
dhfr genotype. Single to quintuple mutations have been reported in
dhfr but only triple to quintuple mutant alleles are known to confer high level of resistance [
36‐
38]. This strongly suggests that
P. vivax isolates from northern and southern part of India could be sensitive to anti-folate drug, whereas isolates from north-eastern state and Island regions will be resistant to anti-folate drug.
A significant association between
dhfr mutations and tandem repeat polymorphism was observed in studied isolates. The mutated
dhfr genotype was exclusively associated with Type-2 tandem repeat and is strongly supported by previous study from India [
23]. Further quadruple mutant
dhfr alleles were exclusively associated with type 1 tandem repeat and are supported by earlier studies from India [
23], Thailand [
38] and Myanmar [
39], however exception to this association was only showed by a single isolate from Myanmar [
39]. These observations speculate
dhfr allele with type 1 tandem repeat could be more prone for mutations and development of higher level of resistance conferring genotypes. Therefore, it could act as molecular marker to predict the risk of mutant/higher level resistance conferring mutant genotypes in any geographical area of Indian subcontinent.
Frequency of quadruple mutant
dhfr genotype was progressively increased in Kamrup isolates from 2005 to 2007 [
23]. The high proportion of quadruple mutant
dhfr genotype in endemic area may be: 1) because of the characteristics of mutant parasites associated with higher gametocytogenesis [
40‐
43] which favour transmission of mutant parasites compared to wild type
dhfr, and 2) north-eastern states of India are highly endemic for malaria and international borders with neighbouring countries surround the region. The borders are very porous and illegal migrations of people across the borders are very common thus, importing drug resistant strains. The higher frequency of quadruple mutant
dhfr genotypes in the isolates of Myanmar (71%) and Thailand (96%) [
39,
44], supports for inflow of quadruple mutant
dhfr genotype in north-eastern states of India.
The development and spread of drug resistant parasite strains is a major obstacle to the malaria elimination programme. Therefore, it is essential to identify drug resistance areas/regions on the basis of point mutations in order to manage the national anti-malarial drug policy. In this regard, this study revealed a higher and increasing prevalence of quadruple mutant dhfr genotype in north-eastern and Island regions, therefore, additional caution may be taken for treatment of vivax malaria in these regions to stop the flourishing of quadruple mutant dhfr genotypes in remaining part of country. The study concluded that P. vivax isolates from northern and southern part of India could be susceptible to SP except for the isolates from Car Nicobar (Island) and Assam (north-eastern region). Geographical clustering of dhfr mutant genotypes would provide a rationale for a more appropriate national anti-malarial drug policy.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SKP: Experiment design, experimental work, data analysis, manuscript writing
HJ: Conceptual design of work, data analysis, manuscript writing,
VD: Sample collection, parasite identification, and experimental design
VKD: Conceptual design of work, Overall supervision of work, and manuscript writing
All authors read and approved the final manuscript.