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Acta Trop. 2012 March ; 121(3): 267–273. doi:10.1016/j.actatropica.2011.11.008.

Malaria in India: The Center for the Study of Complex Malaria in

India
Aparup Dasa, Anupkumar R. Anvikara, Lauren J. Catorb, Ramesh C. Dhimana, Alex Eapenc,
Neelima Mishraa, Bhupinder N. Nagpala, Nutan Nandaa, Kamaraju Raghavendraa, Andrew
F. Readd, Surya K. Sharmae, Om P. Singha, Vineeta Singha, Photini Sinnisf, Harish C.
Srivastavag, Steven A. Sullivanh, Patrick L. Suttonh, Matthew B. Thomasb, Jane M.
Carltonh,*, and Neena Valechaa
Aparup Das: aparup@mrcindia.org; Anupkumar R. Anvikar: anvikar@rediffmail.com; Lauren J. Cator:
laurencator@gmail.com; Ramesh C. Dhiman: dhimanrc@icmr.org.in; Alex Eapen: alexeapen@yahoo.com; Neelima
Mishra: neelima@mrcindia.org; Bhupinder N. Nagpal: b_n_nagpal@hotmail.com; Nutan Nanda: nutanmrc@yahoo.co.in;
Kamaraju Raghavendra: kamarajur2000@yahoo.com; Andrew F. Read: a.read@psu.edu; Surya K. Sharma:
suryaksharma@gmail.com; Om P. Singh: singh@mrcindia.org; Vineeta Singh: vineetas_2000@yahoo.com; Photini
Sinnis: psinnis@jhsph.edu; Harish C. Srivastava: hcrsv_52@rediffmail.com; Steven A. Sullivan: steven.sullivan@nyu.edu;
Patrick L. Sutton: patrick.sutton@nyu.edu; Matthew B. Thomas: mbt13@psu.edu; Neena Valecha:
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neenavalecha@gmail.com
aNational Institute of Malaria Research (Indian Council of Medical Research), Sector 8 Dwarka,
Delhi, India
bCenter for Infectious Disease Dynamics and Department of Entomology, Pennsylvania State
University, University Park, PA, USA
cNational
Institute of Malaria Research Field Unit, National Institute of Epidemiology Campus,
Ayapakkam, Chennai, Tamil Nadu, India
dCentre for Infectious Disease Dynamics, Departments of Biology and Entomology, Pennsylvania
State University, University Park, PA, USA
eNational Institute of Malaria Research Field Unit, Sector 5, Rourkela, Orissa, India
fDepartment of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of
Public Health, Baltimore, MD, USA
gNational Institute of Malaria Research Field Unit, Civil Hospital, Nadiad, Gujarat, India
hCenter for Genomics and Systems Biology, Department of Biology, New York University, New
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York, NY, USA

Abstract
Malaria is a major public health problem in India and one which contributes significantly to the
overall malaria burden in Southeast Asia. The National Vector Borne Disease Control Program of
India reported ~1.6 million cases and ~1100 malaria deaths in 2009. Some experts argue that this
is a serious underestimation and that the actual number of malaria cases per year is likely between
9 and 50 times greater, with an approximate 13-fold underestimation of malaria-related mortality.
The difficulty in making these estimations is further exacerbated by (i) highly variable malaria

© 2011 Elsevier B.V. All rights reserved.

*
Corresponding author: Tel.: +1 212 992 6981; fax: +1 212 995 4266, jane.carlton@nyu.edu (J.M. Carlton).
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our
customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of
the resulting proof before it is published in its final citable form. Please note that during the production process errors may be
discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
 Das et al. Page 2

eco-epidemiological profiles, (ii) the transmission and overlap of multiple Plasmodium species
and Anopheles vectors, (iii) increasing antimalarial drug resistance and insecticide resistance, and
(iv) the impact of climate change on each of these variables. Simply stated, the burden of malaria
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in India is complex. Here we describe plans for a Center for the Study of Complex Malaria in
India (CSCMi), one of ten International Centers of Excellence in Malaria Research (ICEMRs)
located in malarious regions of the world recently funded by the National Institute of Allergy and
Infectious Diseases, National Institutes of Health. The CSCMi is a close partnership between
Indian and United States scientists, and aims to address major gaps in our understanding of the
complexity of malaria in India, including changing patterns of epidemiology, vector biology and
control, drug resistance, and parasite genomics. We hope that such a multidisciplinary approach
that integrates clinical and field studies with laboratory, molecular, and genomic methods will
provide a powerful combination for malaria control and prevention in India.

Keywords
Malaria; Plasmodium; Anopheles; India; Genomics; Epidemiology

1. Malaria in India
As the second most populous country in the world, with a population exceeding one billion
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people, India’s public health system faces many challenges including implementation of
surveillance programs to accurately estimate and control the national malaria burden.
Historically, the highest incidence of malaria in India occurred in the 1950s, with an
estimated 75 million cases and 0.8 million deaths per year (World Health Organization,
Country Office for India). The launch of the National Malaria Control Program (NMCP) in
1953 resulted in a significant decline in the number of reported cases to <50,000 and no
reported mortality, by 1961. Despite its near elimination in the mid-1960’s, malaria resurged
to ~6.45 million cases in 1976. Since then, confirmed cases have gradually decreased to 1.6
million cases and ~1,100 deaths in 2009 (Figure 1). Recently, it has been suggested that the
malaria incidence is between 9 to 50 times greater than reported (reviewed in Hay et al.,
2010), with a ~13-fold underestimation of malaria-related mortality (Dhingra et al., 2010).
Such claims reinforce the need for robust and comprehensive epidemiological surveillance
studies across the country (Singh et al., 2009) to determine the actual burden.

2. Indian malaria parasites and their vectors

2.1 India has a diverse topography, ecology and climate
India’s expansive geography and diverse climate supports ideal environments for sustaining
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malaria parasites and their vectors. The climate varies from tropical monsoon in the south of
the country to temperate in the north, but has four major climatic zones: mountain climate,
tropical wet climate, tropical dry climate, and subtropical humid climate. Such climatic
variation is due to a sharp temperature gradient caused by atmospheric changes in wind
circulation and precipitation, lending to seasonally-dependent asymmetric heating patterns
of India’s peripheral bodies of water and land. The average annual temperature in India
varies with the altitude, as the foothills and mountainous regions near the Himalayas and
Western Ghats average 20°C, while coastal areas are tropical and humid, averaging above
30°C annually. The monsoon strikes the Indian southwestern coast in June every year,
marking the beginning of the rainy season. Monsoon bursts drastically change the average
daily rainfall and shape the overall annual rainfall in most regions of India. Remarkably, it is
not uncommon for Meghalaya and the coastlines of Kerala, Karnataka, Goa, and
Maharashtra to receive >250cm of rainfall each year, while some areas in Rajasthan receive
<25cm each year. Such climatic diversity influences the distribution of vectors and species

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of malaria parasite; as a result, malaria in India takes a number of different forms, including
forest/tribal malaria, urban/slum malaria, industrial malaria, and plains malaria.
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2.2 Malaria parasite species in India

The two major human malaria species in India are Plasmodium falciparum and P. vivax; P.
malariae has been reported in the eastern India state of Orissa (Sharma et al., 2006), while
P. ovale appears to be extremely rare if not absent. Intriguingly, the two major infecting
species vary in proportion across India. For example, the southern state of Tamil Nadu
suffers from P. vivax, P. falciparum is the dominant parasite in Orissa, and mixed-species
infections are prevalent in the west (e.g., Gujarat state). Although P. falciparum and P. vivax
are unevenly distributed across India, this is not due to Anopheles vector restriction (even
though the vector species also differ in geographical distribution, see below) since both
species are transmitted by the same vectors. Historically, P. vivax has been the major
infecting species; however, over the past several years P. vivax cases have decreased: the
ratio of P. falciparum versus P. vivax malaria was 0.41 in 1985, gradually increasing to 0.60
by 1995, and shifting to 1.01 by 2010 (Singh et al., 2004a,b) (Figure 1). In states where P.
falciparum and P. vivax co-circulate, fluctuating proportions of the two species complicate
diagnosis and treatment. Treatment is based upon the primary species identified in an
infection by standard microscopy diagnosis, subjecting all species in a single host to the
same drug treatment, likely contributing to this surge in drug-resistant parasite strains.
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Approximately 65% of those at risk for becoming infected with malaria in Southeast Asia
are individuals residing in India (WHO 2010). The central and eastern regions of India
report the most malaria (Figure 2), particularly the eastern states of Orissa, West Bengal, and
Jharkhand, the central states of Chhattisgarh and Madhya Pradesh, and the western states of
Gujarat, Karnataka and Rajasthan, with the largest number of deaths reported in Orissa
(Joshi et al., 2008). Malaria cases in India are reported throughout the year, since a perfect
combination of average temperature (15–30°C), rainfall and precipitation-inducing
conditions persist across the different parts of the country over all the seasons. With
increasing ecological and man-made environmental change (e.g. urbanization, construction
of dams, agricultural intensification, deforestation) malaria in India is exhibiting general
trends from rural to urban malaria, from forest to plain malaria, and from industrial to travel
malaria (Sharma et al., 2006).

2.3 Malaria vectors in India

The diverse malaria epidemiology in India is mirrored by high diversity of malaria vector
species, most of which exist as complexes comprising several cryptic species that vary in
vectorial capacity (Dash et al., 2007; Singh et al., 2009). In fact, close to 60 morphologically
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distinct species have been described in India; six of these have been found to vector malaria
(Table 1), although An. culicifacies is responsible for 60–65% of the malaria burden
(Goswami et al., 2006; Dash et al., 2008; Singh et al., 2009). As argued by Singh et al.,
(2009), this level of diversity makes India an ideal ‘model’ to study interactions amongst
host-vector-parasite and the environment to both fully understand malaria and develop
sustainable solutions.

The geographically widespread vector, An. culicifacies, comprises a complex of five

morphologically indistinguishable sibling species: A, B, C, D, and E (Goswami et al., 2006;
Adak et al., 1999). Though these sibling species are physically indistinguishable, they differ
physiologically in terms of vectorial capacity (Adak et al., 1999). Of the three sibling
species tested, species A was found be the most susceptible to infection, as measured by
oocyst abundance and sporozoite maturation; species B was the least susceptible to
infection, bordering on being a non-vector (Adak et al., 1999). Similar complexity exists for

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the other principle malaria vectors in India. For example, An. fluviatilis s.l. exists as a
complex of four sibling species (S, T, U, and V) (Subbarao et al., 1994), An. minimus s.l. a
complex of three sibling species (Singh et al., 2010 ) (An. minimus, An. harrisoni and E) and
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An. dirus a complex of at least seven sibling species two of which, An. baimaii and An.
elegans, occur in India (Dash et al., 2008).

3. Malaria control programs in India

3.1 National Vector Borne Disease Control Programme (NVBDCP)
The National Vector Borne Disease Control Programme (NVBDCP) is the agency
responsible for the prevention and control of all vector-borne diseases in India, including
malaria. It is one of the technical departments of the Directorate General of Health Services
under the Ministry of Health and Family Welfare and is responsible for framing technical
guidelines and policies, and monitoring implementation through regular reports on malaria
control. The NVBDCP goals are to develop a well-informed and self-sustained health care
system in India with equitable access to quality health care and to ensure that the program
activities are in accord with the Millennium Development Goal of halting and reversing the
incidence of malaria and other vector-borne diseases by the year 2015.

The major strategies being pursued by the NVBDCP to help achieve its objectives are: (i)
disease management through early case detection and complete treatment, (ii) integrated
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vector management (IVM) to reduce the risk of vector-borne transmission; and (iii)
supportive interventions which include communicating behavior change, capacity building
and monitoring and evaluation of programs. To facilitate disease management, fever
treatment depots (FTDs) exist at the village level. The FTDs are diagnostic stations for the
collection of blood slides from febrile patients, with an Annual Blood Examination Rate
(ABER) target set by NVBDCP at ≥10% for screening the Indian population. An important
initiative recently introduced by the National Rural Health Mission (NRHM) is the provision
of village-based Accredited Social and Health Activist (ASHAs), personnel that have been
trained in malaria diagnosis by rapid diagnostic tests (RDTs), and antimalarial drug
administration.

The NVBDCP has introduced the use of RDTs to help facilitate early detection and also the
deployment of insecticide-treated bed nets in high-risk regions for prevention. The
NVBDCP achieves evaluation of its programs in collaboration with the National Institute of
Malaria Research (NIMR), one of the permanent institutes of the Indian Council of Medical
Research (ICMR; under the Department of Health Research, Ministry of Health and Family
Welfare, Government of India). While the NVBDCP undertakes the fortnightly domiciliary
operational surveillance of malaria across India, NIMR provides technical support to the
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national program for the control of malaria. Thus NIMR, through its ten field stations (see
below), evaluates new insecticides and diagnostic kits, conducts clinical trials, and monitors
resistance to insecticides among vectors and drug therapy among parasites. The institute has
also established quality assurance of malaria RDTs for NVBDCP.

3.2 National Institute of Malaria Research, Indian Council of Medical Research

The National Institute of Malaria Research (NIMR) was established in 1977 and is the only
research institute in India completely devoted to malaria research. The primary task of the
NIMR is to find solutions to the problems of malaria through basic, applied, and operational
field research. The institute also plays a key role in resource development through training
workshops and transfer of technologies for malaria control. Field evaluations of new
insecticides, bio-larvicides, insecticide-impregnated bed nets, antimalarial medicines and
parasite diagnostic kits are also carried out, and many of these research results are
incorporated by the NVBDCP to strengthen their control initiatives. The NIMR has a

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network of 10 field units spread across India that serve as units for testing these new
technologies (Figure 2). Each site has a different eco-epidemiological profile and can be
used to study the variety of different forms of malaria, including tribal malaria, plain
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malaria, urban/slum malaria, forest malaria and industrial malaria.

3.3. Challenges for controlling malaria in India

The three most important challenges for malaria control in India are difficulties in control
implementation and logistics, insecticide resistance, and antimalarial drug resistance. As the
world’s second most populous country, India’s challenge is to design and restructure its
health system to fulfill the needs of its large population. Several commentaries and research
reports have emerged in the last several years highlighting the ongoing struggle of adequate
infectious disease surveillance and control across India, including malaria surveillance
(Sharma 2007; Hay et al., 2007; Dhingra et al., 2010; John et al., 2011).

Chemical insecticides against adult mosquitoes are among the most effective malaria control
tools yet developed. Indoor residual spraying (IRS) with insecticides continues to be a
mainstay of malaria control, having been responsible for often spectacular reductions in
disease incidence last century and for the elimination of malaria from many countries (Trigg
et al., 1998; Shiff 2002; Mabaso et al., 2004). In India, IRS with
Dichlorodiphenyltrichloroethane (DDT) reduced cases from 75 million to just 0.1 million in
1966 (Sharma 1999). More recently, insecticide-treated bed nets (ITNs) have become a
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leading tool for malaria control (Lengeler 2004; Wakabi 2007). Because of these historic
and contemporary successes, the major international efforts currently underway to
comprehensively control and even globally eradicate malaria involve enormous up-scaling
of IRS and ITN deployment (Roberts and Enserink 2007; Feachem and Sabaot 2008; Anon
2008; Grabowsky 2008). Indoor house-spraying and insecticide-treated bed nets are among
the cheapest, most effective and best proven methods of controlling malaria globally, and in
India. Unfortunately, mosquitoes can rapidly evolve resistance to all currently approved
classes of public health insecticides. As was seen last century, one of the major challenges to
these new efforts is the evolution of insecticide resistance in Anopheles populations (Trigg et
al., 1998; Shiff 2002; Hemingway et al., 2002; N’Guessan et al., 2007; Nauen 2007; Kelly-
Hope et al., 2008). Insecticide-resistant mosquitoes were one of the main hurdles faced by
the ultimately unsuccessful Global Malaria Eradication plan in the middle of last century
(Davidson and Zahar 1973; Harrison 1978; Trigg et al., 1998; Shiff 2002; Hemingway et al.,
2002; Nauen 2007; Kelly-Hope et al., 2008), and present-day experience reconfirms this
experience.

DDT, hexachlorocyclohexane (HCH), and malathion are used to control malaria throughout
India, especially in rural areas. However, the development of insecticide resistance threatens
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to halt these once effective methods of control and prevention. In particular, growing
insecticide resistance in the predominant malaria vectors such as An. culicifacies and An.
stephensi is a major concern (Singh et al., 2009). While strategies and capacity for resistance
surveillance exist through the NIMR and the NVBDCP, insecticide resistance is increasing
in India. Increasing insecticide resistance surveillance, especially in India’s rural regions, is
a necessity to prevent the dissemination of double and triple resistant strains. Yet, even as
control programs intensify, so too will selection for resistance. Understanding how this will
impact malaria transmission across different eco-epidemiological contexts is imperative for
malaria control. Without this knowledge there is only partial insight into the sustainability of
current control programs and the utility of prospective resistance management strategies.

Malaria imposes a major Indian global health burden in large part because the Plasmodium
parasite readily evolves drug resistance. Resistance of P. falciparum to chloroquine (CQ)
was first reported in 1973, and resistance likely originated from neighboring countries

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(Thailand, Burma, Bangladesh), which reported CQ-treatment failures earlier (Shah et al.,
2011). Resistance then spread across India, particularly with migrant workers travelling to
the west and south, with treatment failures increasing significantly between 1978 and 2007.
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CQ resistance is now widespread in India, but with geographic clustering of resistance areas
(Shah et al., 2011). A national antimalarial drug policy was introduced in 1982, with
sulfadoxine/pyrimethamine (SP) established as the treatment in CQ resistant areas. In 2004,
artesunate plus SP replaced SP alone as the second-line drug for use in CQ-treatment failure.
In 2007, artesunate plus SP became the first line treatment in high-risk districts with
identified CQ resistance, and in 2010 this treatment became the recommended first-line
treatment throughout India (Shah et al., 2011).

There have been a few case reports of CQ resistant P. vivax in India (Valecha 2009), but
systematic trials from across the country have reported 100% efficacy of standard dose CQ
treatment. Thus, CQ resistant P. vivax appears not to be a serious concern in India, despite
over 50 years of chloroquine use (Shah et al., 2011). Why resistance is not present despite
selection pressure is an interesting question in its own right, not least because it is unclear
whether resistance is just a matter of time, or whether there is something different about P.
vivax in India which prevents resistance evolution, in very sharp contrast to the experience
with CQ against P. falciparum.

Thus the major threat today is the potential for resistance to arise in P. falciparum against
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artesunate or its partner drug, SP. Reduced efficacy of SP treatment was observed in some
recent surveys in the north-east (Shah et al., 2011), but molecular markers for SP resistance
are well known so that survelliance is possible. The greater challenge is surveillance for
artesunate tolerance or resistance. Less sensitive parasites have been detected in SE Asia
(Dondorp et al., 2009), and must be considered as an imminent threat to India. However,
surveillance for artesunate resistance is a challenge. Once evolution has generated enough
treatment failure to arouse suspicions of resistance, the spread of resistance is probably well
advanced in an area. For new drugs like artesunate, little can be known in advance about the
underlying resistance mechanisms. Moreover, diverse genetic mechanisms can be
responsible for resistance in different regions, against different drugs and indeed in different
malaria parasite species.

4. The Center for the Study of Complex Malaria in India

The Center for the Study of Complex Malaria in India (CSCMi) is a partnership between
Indian and U.S.-based investigators to develop the knowledge, tools and evidence-based
strategies needed to support the intervention and control programs of Indian government
organizations, and to build research capacity in India and help train the next generation of
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malaria and mosquito vector biologists. Our research focus is the analysis of mixed-species
and mixed-genotype infections (complex malaria), with respect to their eco-epidemiological
profiles, transmission capabilities, and potential impact on drug resistance. To capture the
eco-epidemiological diversity in India, three NIMR field stations have been selected as
sentinel research sites for this study (Figure 2). The Rourkela field station (Orissa), in the
east of the country, has predominantly P. falciparum in forest/riparian ecology, while the
urban Chennai field station (Tamil Nadu) in the south of the country has predominantly P.
vivax, and the Nadiad field station (Gujarat State) in the western plains has both P. vivax and
P. falciparum transmission.

4.1 A five-year malaria epidemiology survey

Disease surveillance for malaria in India is generally facilitated by passive case detection
(PCD) and active case detection (ACD) of fever cases fortnightly. However, these methods
do not account for individuals who are asymptomatic, or present symptomatically but are

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missed by active surveillance. In addition, statistics from the treatment of malaria cases in
the private sector are hard to come by. Consequently, the malaria prevalence rate in India
may be underestimated or inaccurate, making it difficult to implement effective control
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strategies. Furthermore, due to this incomplete scope of detection, malaria parasite diversity
and the distribution of malaria drug resistance on the Indian subcontinent may not be
optimally characterized.

A major component of the CSCMi is to survey the circulating Plasmodium parasite

populations in three different eco-epidemiological regions in India. Fundamental to attaining
our primary objectives of how complex malaria in India influences disease outcome, vector
transmission, and drug resistance is the collection and analysis of parasites that cause both
symptomatic and asymptomatic malaria. Towards this aim, we will be running two
interconnected epidemiological studies, a Community Study that uses active case detection
to estimate the incidence and prevalence of asymptomatic malaria within each field site, and
a Clinic Study that recruits individuals through passive case detection, focusing on
symptomatic malaria and the detection of drug resistant parasites. A powerful relational
database and data management system will facilitate data collection and storage, and
interpretation of research results.

4.2 Determining the ecological and evolutionary determinants of malaria transmission

A key objective of the CSCMi is to advance ecological and evolutionary understanding of
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diverse malaria transmission in India and use this knowledge to progress towards sustainable
management of adult mosquito vectors. By pooling the substantial expertise in entomology
and intervention trials in India with the expertise in vector and parasite ecology and
evolution in the United States, this work will contribute substantially to malaria control in
India and generate novel insights into basic biological processes applicable to disease
processes globally.

First, research will be conducted to quantify the role of environmental conditions in

determining malaria transmission intensity (risk) in different eco-epidemiological contexts,
including the future effects of climate change. The rationale behind this work is that most of
the key components of R0 (basic reproduction number) for vector borne diseases are
temperature dependent, including the rate of development of mosquitoes (and hence under
many conditions, mosquito densities) and the rate of development of malaria parasites inside
mosquitoes. However, the standard relationships used to describe the influence of
temperature on key mosquito and parasite life history traits derive from research done in the
early part of last century with a mishmash of species. Examining the interaction of key
mosquito species with relevant locally derived parasites will provide valuable insights into
precisely how temperature determines malaria transmission intensity. This entomological
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research is key for estimating malaria risk now from climate maps and also for determining
the likely impact of altered environmental conditions, particularly due to climate change.

Second, we plan to evaluate the evolutionary responses of key mosquito vectors to the
increasing adoption of insecticide-based interventions, quantifying the implications for
malaria transmission in different eco-epidemiological contexts. A first step to resistance
management is resistance surveillance. The recent introduction of long-lasting treated bed
nets into India provides an exciting opportunity to look at bed net-driven resistance
evolution, particularly since genetic resistance mechanisms are already present in Indian
mosquitoes (Singh et al., 2009). Resistance management requires an understanding of the
sensitivity of the resistance phenotypes revealed in standard surveillance to different
environmental conditions, an understanding of the fitness costs of resistance, and then
assessment of the consequences of resistance for malaria transmission and of alternative

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strategies. We aim to investigate these issues with a range of lab and field studies on
resistant and susceptible mosquito lines.
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Together these projects combine novel research and capacity building on entomology,
vector biology and ecology, and the impact of vector management and control strategies on
disease transmission. In so doing they address a need recently identified by the World
Health Organization to evaluate the effectiveness of approaches such as IVM to
simultaneously control disease, respond to ecological change and reduce pesticide use and
insecticide resistance, in different settings (WHO 2009).

4.3 Genomics and drug resistance

Malaria imposes a major global health burden in large part because the Plasmodium parasite
readily evolves drug resistance. In P. falciparum resistance has arisen against all classes of
first line antimalarial drugs and several have had to be withdrawn from use in many
countries. CQ, for instance, is no longer recommended for treatment of P. falciparum in
India, and has been replaced with the more expensive artemisinin-based combination
therapy (ACT). It is now accepted as inevitable by the World Health Organization that all
drugs will eventually fail in the face of resistance evolution. The only solution to this failure
is to shift to new therapies or reformulations of old ones when resistance becomes too
widespread.
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In the CSCMi we propose to use new technologies, including next generation genomics, to
study antimalarial drug resistance in India. The approaches we propose here are designed to
develop tools necessary for improved surveillance for resistance against drugs in use in India
now, and not least for P. vivax as well as P. falciparum. Vivax malaria is a critical part of
the Indian malaria picture, but globally, the genetic analysis of resistance in the species lags
far behind that of P. falciparum. These tools will also be applicable to the detection and
analysis of resistance to drug classes not yet in therapeutic use (e.g. ACT for P. vivax, next
generation compounds or formulations for P. falciparum). This future-proofing is an
important aspect of our aims.

First, we plan to use new generation sequencing technologies to detect evidence of the early
signs of failure of ACT in P. falciparum and chloroquine in P. vivax in India. The approach
we propose to investigate uses deep sequencing to identify the presence of ‘tolerant’ or
‘resistant’ parasite clones present in low levels in a patient – clones which are rare because
resistance is newly arisen and associated with fitness costs. [Note this is the worst case
scenario for traditional methods because if it is rare it will take weeks to be detected (and so
could look like a re-infection) and it will not affect the rate of parasite clearance
immediately post-treatment]. Deep sequencing of a polymorphic marker will enable
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detection of rare clones (Juliano et al., 2010) even if the less-susceptible clone is at a
frequency of 5 in 2000 parasites, whereas conventional PCR methods cannot, since they
struggle to find alleles at frequencies <1%. The presence of a clone which is less sensitive to
treatment than a wild-type clone is identified through a change in its frequency as a patient
transitions from an untreated to treated state. The parasites from that patient can then be
subject to standard in vitro testing, or analysis of candidate resistance markers.

A second aim of the CSCMi genomics and drug resistance project deals specifically with P.
vivax and has the goal of developing a map of common P. vivax SNPs (single nucleotide
polymorphisms) segregating in the Indian subcontinent. This will provide preliminary data
for a haplotype map that can be used for association studies of drug resistance. Vivax
malaria is a key component and critical part of the malaria picture in India, but research into
this species lags far behind that of P. falciparum, especially as far as genetic diversity and
genomic studies are concerned. This paradox is most likely due to the refractoriness of P.

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vivax to in vitro culture, and unique aspects of its biology that include low patient
parasitemia and a dormant ‘hypnozoite’ form in the liver. Currently only a single reference
genome sequence of an isolate of P. vivax from El Salvador is available for the malaria
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research community (Carlton et al., 2008). The lack of genetic sequence data from
additional P. vivax genomes undoubtedly hampers whole genome analyses and elucidation
of the unique biology associated with the species. In particular genetic diversity within P.
vivax populations contributes directly to drug resistance and antigenic variation, allowing
the parasite to evolve immunity to antimalarial compounds and to evade the host immune
response. Knowledge of this genetic variation is critical for the development of intervention
strategies including drugs and vaccines. In contrast to P. falciparum, which is thought to
have undergone one or more bottlenecks in recent history (Joy et al., 2002), P. vivax is
thought to have had a more stable demographic past (Mu et al., 2003), which could mean
that P. vivax may exhibit a greater degree of genetic polymorphism within populations and
greater divergence among populations in disparate geographic locations (Gupta et al., 2011).
This has important implications as it suggests that widespread sampling of P. vivax in
different geographical regions is necessary in order to understand the diversity and
population structure of the species in different areas. Specifically, the availability of
sequence data from Indian isolates will provide the context for understanding where
sequence conservation or divergence is critical, and provide insights into sequence/function
relationships, with the ultimate aim of generating a genetic diversity map of P. vivax in India
that can be used for association studies of drug resistance.
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As part of the CSCMi, we plan to develop a next generation sequencing core at the NIMR to
enable deep sequencing of Plasmodium parasites, for both the multiplicity of infection
studies and P. vivax whole genome sequencing described above. Precisely which NGS
platform we intend to install at NIMR remains to be decided, although several have obvious
advantages for implementing in a field location (Table 2).

5. Conclusion
Malaria is a major public health problem in India, the world’s largest democracy and its
second most populous country. We intend to leverage the existing strong relationship
between Indian and U.S scientists to develop a Center for the Study of Complex Malaria in
India. The CSCMi is a collaborative scientific research center working towards the goal of
enhancing malaria intervention and control programs in India.

Acknowledgments
We thank the previous NIMR Directors Professor A. P. Dash and Dr. V. K. Dua for facilitating the CSCMi, and the
NIH-PA Author Manuscript

Director General of the Indian Council of Medical Research, Dr. V. M. Katoch, for project permissions and
encouragement. We are also indebted to Dr. Hema Joshi who was central to the project before her untimely death in
March 2010. We also thank CSCMi Project Coordinator Dr. Lalitha Ramanathapuram and Dr. A.C. Dhariwal,
Director of the National Vector Borne Disease Control Programme, Government of India, for input and comments.
This work was supported by National Institute of Allergy and Infectious Diseases, National Institutes of Health
(NIH) grant U19AI089676. P.L.S. was supported by a Fogarty International Center/NIH U.S. Global Health
Postdoctoral Scientist Fellowship 3D43TW007884-03S1. The content is solely the responsibility of the authors and
does not necessarily represent the official views of the Fogarty International Center or the National Institutes of
Health.

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Figure 1.
Malaria cases and changing species pattern in India during the years 1995–2010. The total
number of reported malaria cases has decreased since 1995, primarily through a reduction in
the number of reported P. vivax cases. Currently, there is a 1:1 ratio of P. falciparum to P.
vivax cases.

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 Das et al. Page 14
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Figure 2.
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Malaria endemicity in India. State boundaries are color-coded according to total malaria
endemicity (see key). Data taken from the National Vector Borne Disease Control
Programme (http://www.nvbdcp.gov.in/) for year 2010. NIMR field stations are indicated as
red dots. The three NIMR field stations incorporated as part of the CSCMi are: Nadiad
(Gujarat), Rourkela (Orissa) and Chennai (Tamil Nadu), each with different eco-
epidemiological profiles as shown. EIR: entomological inoculation rate; API: annual
parasite incidence.
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Table 1
Distribution of principal species of malaria vectors in India.

Primary vectors No. sibling species recorded No. sibling species in India Members Ecological distribution Species to be studied as part of CSCMi
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An. stephensia - - - Urban Yes

An. culicifacies s.l. 5 5 A, B, C, D, E Rural/peri-urban Yes

An. fluviatilis s.l. 4 4 S, T, U, V Foothills/forest Yes
An. minimus s.l. 3 1 An. minimus, An. harrisoni, E Northeastern states Yes
An. dirus s.l. 7 2 D, E Northeastern states Yes
An. sundaicus s.l. 3 1 New cytotype D Andaman-Nicobar Islands No

a
No description of An. stephensi sibling species is available. However, two races, i.e. the vector ‘type form’ and non-vector ‘var. mysoriensis’ are often reported. The ‘type form’ is found in urban areas,
while ‘var. mysoriensis’ is found in rural areas. Another form, ‘intermediate’ is also suggested. Adapted from Singh et al., 2009.

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Table 2
Next generation sequencing platforms and their suitability for endemic country field settings. All six platforms are currently either being sold or expected
to be sold in India shortly.
Das et al.

Platform Read type Run time Computing requirements Cost Suitability for a field setting

Roche 454 GS FLX Long read 10 hr Desktop $$$ Potential: low computational requirements, but high capital and running costs

Roche 454 GS Junior Long read 10 hr Desktop $$$$ Potential: low computational requirements, but high running costs

Illumina GAII/HiSeq Short read 5 days Cluster $ Problematic: short reads, high computational requirements, high capital cost

Illumina MiSeq Short read 26 hr Desktop $$ Promising: low cost, short run time, small footprint

Life Technologies SOLiD 4/5500 Short read 8 days Cluster $ Problematic: short reads, unusual bioinformatics analysis, computational requirements
high

Life Technologies Ion Torrent ‘318’ chip Long read 2 hr Desktop $$ Promising: low cost, short run time, simple mechanics, small footprint, long reads

Short read: sequences <150 bp; Long read: sequences >150bp. $: <$0.5/Mb; $$: ~$1/Mb; $$$: >$7/Mb.

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