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The distinctive features of Indian malaria parasites Aparup Das Evolutionary Genomics and Bioinformatics Laboratory, Division of Genomics and Bioinformatics, National Institute of Malaria Research, Sector 8, Dwarka, New Delhi, India

Malaria and factors driving malaria are heterogeneous in India, unlike in other countries, and the epidemiology of malaria therefore is considered ‘highly complex’. This complexity is primarily attributed to several unique features of the malaria parasites, mosquito vectors, malaria-susceptible populations, and ecoclimatic variables in India. Recent research on the genetic epidemiology of Indian malaria parasites has been successful in partly unraveling the mysteries underlying these complexities.

Malaria epidemiology in India Malaria inflicts a great socioeconomic burden on humanity, affecting 97 countries and territories in tropical and subtropical regions, primarily in sub-Saharan Africa. According to the latest estimates, 198 million cases of malaria occurred globally in 2013 and the disease led to 584 000 deaths, to which India contributed 881 730 cases and 440 deaths [World Health Organization (2014) World Malaria Report 2014 (http://apps.who.int/iris/bitstream/ 10665/144852/2/9789241564830_eng.pdf?ua=1)]. In general, India possesses enormous ecoclimatic, seasonal, and biological species diversity, and malaria is endemic there. With the exception of the states of Himachal Pradesh and Jammu and Kashmir, all Indian states are prone to this oldest infectious disease of humankind, amounting to over 70% of India’s population facing the risk of malaria infection [World Health Organization (2014) World Malaria Report 2014 (http://apps.who.int/iris/bitstream/10665/ 144852/2/9789241564830_eng.pdf?ua=1)] [1]. All northeastern states, West Bengal, Odisha, Jharkhand, Chhattisgarh, Andhra Pradesh, Maharashtra, Gujarat, and Karnataka, are high-focus malaria-prone states [1]. India is epidemiologically unique compared with other malariaendemic countries because the two most widely spread malaria parasites (Plasmodium falciparum and Plasmodium vivax) occur in almost equal proportions [1]. Traditionally, P. vivax is widely prevalent; however, in recent years P. falciparum has predominated over P. vivax in most malaria-endemic states of India [World Health Organization (2014) World Malaria Report 2014 (http://apps.who. int/iris/bitstream/10665/144852/2/9789241564830_eng. pdf?ua=1)] [1]. Further, the incidence of mixed parasitic infections due to these two species in a single patient was found to be unusually high (48%) [2]. The incidence of Corresponding author: Das, A. ([email protected]). Keywords: malaria; India; Plasmodium falciparum; Plasmodium vivax; genetic epidemiology. 1471-4922/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2015.01.006

asymptomatic P. falciparum infection in a significant proportion (8.4%) has also been recorded in a tribal population in the state of West Bengal [3] and Plasmodium knowlesi, the fifth human malaria parasite so far known to infect Southeast Asians, was recently reported to infect tribal Indians living in the Andaman and Nicobar Islands [4]. In addition, reports of the widespread prevalence of Plasmodium malariae in forest villages in the state of Madhya Pradesh have surfaced, although the incidence of infection was not as high for P. falciparum or P. vivax [5]. Combining the above reports with the prevalence of Plasmodium ovale from many Indian localities [6] informs us on the occurrence of all five species of malaria parasites infecting humans and indicates that India uniquely harbors a rich diversity of species. These features, together with the distributional prevalence of the six major malaria vectors responsible for parasite transmission [1] and the presence of malaria-susceptible populations, make malaria epidemiology in India highly complex and exceptional compared with that in other malaria-endemic countries [7]. Do malaria parasites have Indian roots? Understanding the evolutionary history of pathogens is of enormous importance for malaria interventions because ancestral populations contain high genetic diversity, requiring more stringent approaches to devising control measures. Further, knowledge about the migration pattern of parasites and the role of natural selection in adaptation of the parasite against drugs (drug resistance) and evolving host immunity (antigenic diversity) are equally important when devising novel methods of malaria control. In this regard, employing a robust multilocus approach using single nucleotide polymorphisms (SNPs), Indian P. vivax was found to have high nucleotide diversity within the population but moderate genetic differentiation between population samples [8]. Furthermore, Indian P. vivax populations seem to be loosely structured, with no correlations between geographical and/or genetic distance [8]. Also, Indian P. vivax populations maintain a stable population size and a high effective population size with a long time to the most recent common ancestor (TMRCA). All of these demographic features are hallmarks of original populations; therefore, Indian P. vivax is considered to be part of the ancestral distribution range of this species [8]. Similarly, comparing the sequence diversity of the whole mitochondrial (mt) genome of 44 Indian P. falciparum isolates with data for 96 mt genomes from other populations worldwide has provided interesting observations on evolutionary aspects of Indian isolates [9]. Specifically: (i) Indian P. falciparum populations had the highest genetic diversity compared with other isolates worldwide; and (ii) they share many Trends in Parasitology, March 2015, Vol. 31, No. 3

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Monkey Key: Representave mitochondrial genome of P. falciprum-like isolates indicang the four diagnosc single nucleode polymorphisms (SNPs) (red dots)

Plasmodium falciparum-like malaria parasites Plasmodium falciparum-like malaria parasites PfIndia* PfIndia* Indian rhesus macaque

Host-switch event Indians

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Representave mitochondrial genome of PfIndia* isolates [9] indicang the presence of one of the four diagnosc SNPs (red dot) and two unique India-specific SNPs (blue and green dots) Representave mitochondrial genome of global P. falciparum isolates

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Figure 1. A hypothetical evolutionary model of host-switch events from Plasmodium falciparum-like malaria parasites (infecting African nonhuman primates) to P. falciparum (infecting humans). Each different color-filled circle represents a different mitochondrial (mt) genome haplotype of the malaria parasites and the small dots in each circle represent SNPs present in the mt genomes. Note that Plasmodium coatneyi and Plasmodium fragile infecting Indian rhesus macaques bear identical mt genome haplotypes to Indians and are designated PfIndia* [9].

genetic features with African and, to some extent, Papua New Guinean isolates. Interestingly however, Indian isolates are separate from Asian isolates [9]. Further: (iii) Indian P. falciparum isolates contain many demographic features (high effective population size and rapid expansion in the past with the longest TMRCA), suggesting their ancestral nature [9]; and, remarkably, (iv) one of the four SNPs of the mt genome that differentiates P. falciparum from P. falciparum-like isolates (infecting African nonhuman primates; Figure 1) was found to be segregated in five (of a total of 44) Indian P. falciparum isolates (Figure 1), named PfIndia* [9]. This SNP was in tight linkage with two other novel SNPs found only in these five Indian isolates. Remarkably, all three novel India-specific SNPs were also segregated in Indian Plasmodium fragile and Plasmodium coatneyi (which infect Indian rhesus monkeys; Figure 1), providing evidence that the PfIndia* isolates (infecting Indians) possess a genetic relict of the P. falciparum-like parasites infecting African nonhuman primates (Figure 1) and therefore can be considered the ‘missing link’ between P. falciparum-like and P. falciparum isolates (Figure 1) in the process of ancestral host-switch events [9]. Unique evolutionary genetic signature at the pfcrt gene in India In addition to the unparalleled epidemiological and evolutionary features of Indian malaria parasites, evolutionary genetic patterns of resistance to the most widely used drug for malaria treatment, chloroquine (CQ), are also unique to Indian P. falciparum. Based on the distributional 84

prevalence of various haplotypes (arising from the differential occurrence of SNPs at the 72nd to 76th amino acid positions) of the pfcrt gene (which is widely known to govern resistance to CQ in P. falciparum) and rigorous population genetic analyses, it has been established that CQ-resistant (CQR) P. falciparum parasites seem to have entered India via two independent routes (Figure 2): one from Southeast Asia through the northeastern states of India via Myanmar and the other from the Papua New Guinea through Odisha state via the Andaman and Nicobar Islands [10]. Furthermore, the high observed genetic diversity and associated population genetic analyses of multiple microsatellite loci present inside the pfcrt gene compared with its flanking loci have provided evidence for the combined role of demography and natural selection in the evolution of the pfcrt gene in Indian P. falciparum [11]. These observations contrast with global patterns of evolution of the pfcrt gene and provide evidence that this gene is in the process of genetic reconstruction in Indian P. falciparum [12]. Severe P. vivax malaria: Is a highly evolved parasite in a suicidal mission? Evolutionarily, it is well established that P. vivax is an older parasite than P. falciparum. Therefore, severe and complicated malaria due to human infection by P. falciparum leading to death is explained as infection by a newly evolved parasite that has not yet ‘learned’ the mechanism of true parasitism. This is, however, not true for the rather ‘old’ parasite P. vivax, which causes mild and uncomplicated malaria in general and seldom leads to

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pfcrt-SVMNT haplotype pfcrt-CVIET haplotype pfcrt-CVMNT haplotype pfcrt-CVIDT haplotype

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Figure 2. Distributional prevalence of the four most common pfcrt haplotypes (SVMNT, CVIET, CVMNT, and CVIDT) and inferred migration routes of the two pfcrt mother haplotypes (SVMNT and CVIET) into India. With detailed DNA sequencing and thorough population genetic analyses, it was proposed that the Indian SVMNT haplotype (SagtVMNT) is not the same as that found (and which originated) in South America (StctMNT). Rather, this haplotype is of Papua New Guinean type (SagtVMNT) and has entered mainland India (to Odisha) through the Andaman and Nicobar Islands [10]. Similarly, the CVIET haplotype seems to have reached the northeastern parts of India through Myanmar from Southeast Asia, where this haplotype originated. It is further predicted that genetically hitchhiked chloroquine (CQ)-resistant (CQR) mother haplotypes (SVMNT and CVIET) reached India, underwent extensive population expansion, and migrated to various Indian P. falciparum malaria-endemic regions [11]. The pfcrt-CVMNK haplotype (which confers no protection against CQ to P. falciparum) has almost gone from India (prevalence about 2–3%), but in Odisha this haplotype is surprisingly maintained at a prevalence of about 15–20% [10,11].

fatality. Moreover, it has evolved to have an additional dormant stage (hypnozoite form) during its asexual life cycle in the human host, thereby hiding in the human liver and relapsing when favorable conditions arise (e.g., when a patient becomes immunocompromised). This general principle of the etiology of nonsevere and nonfatal P. vivax malaria is well understood. However, the first report on P. vivax-associated severe malaria came from India [13] and it has been proposed that P. vivax can cause both sequestration-related and non-sequestration-related complications of severe malaria, including cerebral malaria, renal failure, circulatory collapse, severe anemia, hemoglobinuria, abnormal bleeding, acute respiratory distress syndrome, and jaundice leading to death, which are otherwise characteristic symptoms of P. falciparum malaria [13]. Since then, similar reports from many P. vivax-endemic countries have entered the literature, implicating P. vivax in severe and fatal malaria. Unfortunately, the etiology of severe P. vivax malaria is poorly understood; therefore, the cause of fatality in P. vivaxinfected patients should be interpreted carefully. This is because similar symptoms can be caused by many other infectious agents, including bacteria, viruses, and fungal pathogens. It might be the case that, at least in certain patients who become immunocompromised (due to

another infection or disease), he or she contracts a fresh P. vivax infection or recrudescence of the existing hypnozoite form may occur. Therefore, without any solid experimental proof of the sole ability of P. vivax to cause the abovementioned fatal symptoms, the implication of P. vivax in severe and fatal malaria is unwarranted. This is because the transition from nonvirulent to virulent form is not only against the rule of true parasitism, but also against evolutionary principles. By killing its human host (which the malaria parasite utilizes for survival and asexual reproduction), P. vivax kills itself, which in principle it cannot afford to do. The way forward Clearly, malaria and the factors associated with this disease in India are unique and more complex than previously assumed [1]. Although malaria has been associated with India since ancient times, due to a lack of in-depth and basic understanding of the genetics, biology, and epidemiology of malaria in India several facts have not yet come to light regarding its complexity. It now appears that both of the most widespread malaria parasites are ancestral in India [8,9] and due to long exposure to these parasites Indians have evolved, to some extent, natural resistance against P. vivax infection by acquiring the 85

Science & Society malaria-resistant allele of the Duffy gene through evolutionary adaptation by Darwinian natural selection [14]. Such evolutionary adaptation in Indians against P. vivax infection is reflected in the epidemiological scenario there [8,14]. In view of this multifaceted epidemiology, more exhaustive field-based research directed toward understanding parasite transmission dynamics are needed to understand host–vector–parasite evolutionary interactions in specific ecological settings in the country. Such basic research could provide clues to the characteristic features of malaria parasites, vectors, and Indian populations and this knowledge, in the long term, will be helpful in devising novel, population-based malaria control measures in India. Acknowledgments The author is grateful to Professor Aditya Prasad Dash, former director, and Dr Neena Valecha, present director, of the National Institute of Malaria Research, New Delhi, India for encouragement and motivation. The work of former and current PhD students of the Evolutionary Genomics and Bioinformatics laboratory, Drs Gauri Awasthi, Jyotsana Dixit, Hemlata Srivastava, Bhavna Gupta, Anita Chittoria, Naazneen Khan, and Hueggette Gaelle Ngassa Mbenda and Ms Suchi Tyagi and Ms Kshipra Chauhan provided the baseline for this article.

References 1 Singh, V. et al. (2009) Why is it important to study malaria epidemiology in India? Trends Parasitol. 25, 452–457 2 Gupta, B. et al. (2010) High proportion of mixed-species Plasmodium infections in India revealed by PCR diagnostic assay. Trop. Med. Int. Health 15, 819–824

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Trends in Parasitology March 2015, Vol. 31, No. 3 3 Ganguly, S. et al. (2013) High prevalence of asymptomatic malaria in a tribal population in eastern India. J. Clin. Microbiol. 51, 1439– 1444 4 Tyagi, R.K. et al. (2013) Discordance in drug resistance-associated mutation patterns in marker genes of Plasmodium falciparum and Plasmodium knowlesi during co-infections. J. Antimicrob. Chemother. 68, 1081–1088 5 Bharti, P.K. et al. (2013) Emergence of a new focus of Plasmodium malariae in forest villages of district Balaghat, Central India: implications for the diagnosis of malaria and its control. Trop. Med. Int. Health 18, 12–17 6 Prakash, A. et al. (2003) Plasmodium ovale: first case report from Assam, India. Curr. Sci. 84, 1187–1188 7 Das, A. et al. (2011) Malaria in India: the center for the study of complex malaria in India. Acta Trop. 121, 267–273 8 Gupta, B. et al. (2012) Inferring the evolutionary history of Indian Plasmodium vivax from population genetic analyses of multilocus nuclear DNA fragments. Mol. Ecol. 21, 1597–1616 9 Tyagi, S. et al. (2014) New insights into the evolutionary history of Plasmodium falciparum from mitochondrial genome sequence analyses of Indian isolates. Mol. Ecol. 23, 2975–2987 10 Awasthi, G. et al. (2011) Population genetic analyses of Plasmodium falciparum chloroquine receptor transporter gene haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int. J. Parasitol. 41, 705–709 11 Chauhan, K. et al. (2013) Analyses of genetic variations at microsatellite loci present in-and-around the pfcrt gene in Indian Plasmodium falciparum. Infect. Genet. Evol. 20, 476–487 12 Das, A. and Dash, A.P. (2007) Evolutionary paradigm of chloroquineresistant malaria in India. Trends Parasitol. 23, 132–135 13 Kochar, D. et al. (2005) Plasmodium vivax malaria. Emerg. Infect. Dis. 11, 132–134 14 Chittoria, A. et al. (2012) Natural selection mediated association of the Duffy (FY) gene polymorphisms with Plasmodium vivax malaria in India. PLoS ONE 7, e45219