Review Article:      315

Chikungunya epidemic: An Indian perspective
S. P. KALANTRI, RAJNISH JOSHI, LEE W. RILEY

ABSTRACT Chikungunya, caused by the chikungunya virus, recently emerged as an important public health problem in the Indian Ocean islands and India. In 2006, an estimated 1.38 million people across southern and central India developed symptomatic disease. The incidence of the disease may have been higher but may have been underreported due to lack of accurate reporting. First isolated in Tanzania in 1953, the chikungunya virus belongs to the family Togaviridae (single-stranded RNA alphaviruses) and has 3 distinct genotypes: East African, West African and Asian. Previous outbreaks in India (1963 and 1973) were caused by the Asian genotypes, but the 2005 epidemic in the Indian Ocean islands and the 2006 epidemic in India have been attributed to the East African genotype. The virus is transmitted to humans by the bites of mosquitoes of the species Aedes aegypti and A. albopictus. Researchers speculate that mutation of the virus, absence of herd immunity, lack of vector control, and globalization of trade and travel might have contributed to the resurgence of the infection. Chikungunya is characterized by high fever, severe arthralgia and rash. Although viral diagnostics (culture, serological tests and polymerase chain reaction tests) can be used to confirm the infection, these tests are not accessible during outbreaks to the majority of the population. The disease is a self-limiting febrile illness and treatment is symptomatic. As no effective vaccine or antiviral drugs are available, mosquito control by evidence- based interventions is the most appropriate strategy to contain the epidemic and pre-empt future outbreaks.

Natl Med J India 2006;19:315–22

INTRODUCTION
Chikungunya, until recently a disease unknown to most Indian healthcare workers, suddenly appeared as a major epidemic in India in 2006 after 32 years. Most estimates, drawn from epidemiological, geographical and demographic data, suggest that 1.38 million people in India were affected by the disease, of whom almost half were from Karnataka and a quarter from Maharashtra.1 These estimates, provided by the official noti- fication and surveillance systems, lack accuracy. The task was made impossible by the fact that a large proportion of those who became ill either consulted private healthcare professionals or sought alternative medical care such as ayurveda or homoeopathy, and few private doctors report suspected chikungunya cases to public health officials. The disease started waning in the central and southern states since October 2006, but not as rapidly as hoped, and more recently new cases were reported in northern India.1
   During the epidemic, rural hospitals and clinics, both public and private, were flooded with victims of chikungunya. Although the virus did not kill its victims, it inflicted considerable pain and misery, and caused substantial and unexpected local, regional and national financial burden towards healthcare. It took patients several weeks before they could limp back to their normal pattern of life. Hospitals, ill equipped to handle the burden of the epidemic, collapsed. Quacks thrived and makeshift hospitals in rural areas, often run by unregistered practitioners, began to proliferate. The only laboratories in the country to offer chikungunya diagnostic tests—the National Institute of Virology (NIV) and National Institute of Communicable Diseases (NICD)—were overwhelmed. Villagers’ vocabulary got enriched by a new word—chikungunya, the bended walker—from an African language. Because there are no high quality data, we cannot estimate precisely the routinely measured costs (e.g. economic burden, effects on health systems and loss of workdays) associated with the epidemic. The socio- economic impact was tremendous: school attendance dropped, productivity at work declined sharply and farmers could not tend to their crops. Festivals passed by almost unnoticed and marriages were postponed. Sufferers lost their wages, sold household items and were forced to borrow money at high interest rates.2
   The chikungunya epidemic epitomizes the classic interaction between agent, host and environment. The outbreak assumed epidemic proportions because the viral agent mutated, the vector discovered new ways to spread and the host lacked immunity to fight the disease. Modern transport systems—cars, buses, trains and aircraft—played a key role in spreading the disease and helped sustain the man–mosquito–man cycle. Indeed, the mobility of the viraemic Homo sapiens was an important factor in transmission of the disease.3
   In this review we describe the virus, its vector, the epidemiology, clinical features, laboratory findings, management and prevention of chikungunya. We also present updated information about the 2006 epidemic that appeared to have preferentially affected rural India.

SEARCH STRATEGY
We searched the following electronic databases for primary studies, review articles, editorials and letters to the editor on the chikungunya virus (CHIKV) and Chikungunya (CHIK): PubMed (1950 to December 2006), BIOSIS (1969 to November 2006), and Web of Science (1945 to November 2006). We identified published studies in the English language that reported data on the chikungunya virus, clinical descriptions, viral genotype analysis, chikungunya epidemics, serological surveys, entomological investigations, diagnostic studies and public health opinions. Our search strategy included the terms ‘Chikungunya virus’, ‘CHIKV’, ‘Chikungunya’ and ‘CHIK’. We identified additional studies by contacting experts in the field and by searching a reference list of primary studies, review articles and textbook chapters, and selected those judged to be relevant. We hand-searched the indices of Emerging Infectious Diseases, Eurosurveillance and Indian Journal of Medical Research for relevant articles.

THE VIRUS, THE VECTOR AND DISEASE TRANSMISSION
The virus
Chikungunya is caused by an RNA alphavirus belonging to the family Togaviridae. The virus encodes 9 genes, consisting of coding sequences for non-structural polyproteins (precursor for nsP1–nsP4 proteins), structural polyproteins (precursor for C, E1–3 and 6K proteins), and polyadenylation site, flanked by 5´ and 3´ sequences4 (Fig. 1). E1 protein correlates with the serological response in human hosts5 and also modulates penetration of the virus in the mosquito species.6
   Genetic analyses and historical accounts suggest that the chikungunya virus originated in tropical Africa,7 and subsequently evolved into 3 distinct genotypes—the East African, the West African and the Asian genotypes. The Asian genotypes have a high degree of nucleic acid sequence homology among themselves, but the African strains exhibit wider sequence diversity, 7 and have been shown to undergo genetic micro-evolutions even during the course of an epidemic.6 Prior to the 2006 epidemic in India, the 3 genotypes were restricted to the geographical areas denoted by their names. Experts suggest that virus strains isolated from the 2005 epidemic in the Indian Ocean islands,6 and the strains that are currently circulating in India 8 have evolved from the East African genotype. The past outbreaks in India were caused by the Asian genotypes. The African genotype, as suggested recently, arrived in India 5 years ago.8 Researchers are trying to find out how the virus sneaked in, why it lay dormant for 5 years and what made it erupt in 2006.

The vector
Chikungunya virus is transmitted by mosquitoes of the genus Aedes such as Aedes aegypti and A. albopictus in Asia 9-11 and A. frucifer, A. luetocephalus, A. taylori in Africa.12 A. albopictus was the principal vector in the outbreaks in the Indian Ocean islands and A. aegypti in the 2006 Indian epidemic.8 Regional differences in the mosquito species exist: Anopheles is a predominant circulating vector species in the rural areas of Orissa and Madhya Pradesh 13,14 and A. albopictus in Tamil Nadu 15 and Southeast Asia.16
   Several attributes make A. aegypti an efficient vector for the chikungunya virus: it is highly susceptible to the virus, prefers to live close to people, seeks a blood meal during the day time and bites—almost painlessly—several people in a short period for one blood meal.17 The mosquito, well adapted to life in urban settings, typically breeds in clean puddles of standing water and collections of water in artificial containers such as tin cans, pots, plastic containers, rain barrels, buckets and discarded tyres.18

Virus–vector interactions
Several hypotheses have been put forward to explain the 2006 epidemic in the Indian Ocean islands and India. First, French researchers have recently detected a mutation (at position 226 of the E1 gene) in 90% of viral sequences from the Indian Ocean strains.19 The mutation allows the virus to acquire an ability to invade and thrive in cells which lack cholesterol (e.g. mosquito cells).6 In addition, the virus gained the ability to infect a new vector, A. albopictus, enhancing the opportunity for transmission to humans. Second, experimental evidence suggests that mosquitoes concurrently infected with microfilaria transmit arboviruses more efficiently.20 Because a large proportion of reported cases of chikungunya from India belong to areas where the prevalence of filarial parasitic infection is high, researchers speculate that filarial parasitic infections could be modulating the re-emergence of chikungunya.21 Third, the affected population lacked herd immunity. Introduction of the virus into a non- immune population could have contributed to the present outbreak.22, 23 Figure 2 shows the epidemiological interactions between the virus, vector and host.

FIG 1. Genomic structure of chikungunya virus NTR non- terminal repeat nsP1–P4 non-structural proteins C capsid E1–3 envelope Poly A internal polyadenylation site (adapted from Khan et al.4)


















 

 

FIG 2. Epidemiological interactions leading to the 2005–06 chikungunya epidemics

Risk of spread to distant areas
As the Aedes can fly only a few hundred metres,24 flying mosquitoes are unlikely to spread the disease across a large geographical area. More than the mosquito, the mobility of people influenced by the globalization of trade and travel is largely responsible for the wide dissemination of the virus. The epidemic in the Indian Ocean islands of Comoros, Mauritius, Seychelles and Reunion lends credence to this suggestion. These islands, popular among tourists, attract about 1.5 million people every year.25 After a major epidemic broke out in these islands in 2005, several tourists returning from the islands to Italy,25 southern France26, 27 and Spain26 were found to be viraemic. Concerns have been raised that A. albopictus, already established in southern Europe, can help the virus find its way into previously uninfected territories. In addition, A. aegypti is known to multiply by laying its eggs in a puddle of water in discarded tyres. Such tyres, when transported across countries, can spread the infected Aedes species and their eggs worldwide.19 Indeed, in the past 2 decades, A. albopictus has found new homes—United States (1985), Central America and Brazil (1986–88), Southern Europe (1991) and France (1999).26

EPIDEMICS: PAST AND PRESENT
Chikungunya epidemics in various countries are listed in Table I. The first outbreak of chikungunya was reported from Makonde, Tanzania in 1952.28 Over the next 2 decades, the chikungunya virus caused outbreaks in various countries in Eastern, Southern and West Africa.53 The outbreak in Congo in 1999–2000 infected an estimated 50 000 people.7 Akin to victims of yellow fever in Africa, most African victims of chikungunya live close to forests, suggesting an epizootic cycle of chikungunya infection in Africa. Compared with the smaller geographical area involved in African outbreaks, Asian outbreaks, affecting people living in both rural and peri-urban areas, have been more extensive. The first outbreak in Asia was reported in Thailand in 1958; over the next two decades, outbreaks were reported from various countries in Southeast Asia. The disease re-emerged recently in Thailand,42 Malaysia52 and Indonesia.51 In 2004–05 islands in the southwest Indian Ocean (Seychelles, Mauritius, Madagascar and the Reunion islands) were affected by the epidemic and, within a year, a third of the population was infected.6

TABLE I. Chikungunya epidemics by country, year and virus strain

Country	Year of reported epidemics (Chikungunya virus strain)
Africa	1952–53 (TA53 Ross—East African) 7 ,28
Tanzania	1956; 1975–77 (SA76 2123—East African) 7, 29
South Africa	1958 30; 1999–2000 31
Congo	1959 32
Zimbabwe	195833; 1968 34
Uganda	1962 35
Zambia	1966 (SE 66 PM2951—West African) 7,36; 1982 (SE83 37997—West African)7,37;
Senegal	1996–97 38
Nigeria	1964 (NI 64 IBH 35—West African)7; 1969; 1974 39
Angola	1970–71 40
Asia and Indian Ocean islands
Thailand	1958; 1962 (TH 62 15561—Asian)7, 41; 1995 (TH95 C039295—Asian) 7 , 42
India	1963–64 (IN63 Gibbs—Asian) 7, 43–45 ; 1973 (IN73 PO731460—Asian)7,46 ; 2006 (IN06—various—East African)8
Cambodia	1963 47
Vietnam	1963 48
Philippines	1968; 1985–86 (PH85 H15483—Asian) 7, 49
Sri Lanka	1965 50
Indonesia	1985 (ID85 RSU1—Asian)7; 2001–0351
Malaysia	1998 (Asian); 2006 (Asian)52
Comoros islands	2005 (East African) 6
Mauritius, Reunion islands	2005–06 (RE06 OPY1—East African) 6

   In India the first outbreak occurred in 1964 in South India (Pondicherry, Chennai and Vellore),43 followed by another in 1973 in Central India (Nagpur and Barsi).46 In 2006, the disease re-emerged after 32 years.54 As of 12 December 2006, according to the National Vector Control Board, New Delhi, chikungunya had affected 197 districts in 12 states (Andhra Pradesh, Andaman and Nicobar Islands, Tamil Nadu, Pondicherry, Karnataka, Maharashtra, Rajasthan, Goa, Gujarat, Madhya Pradesh, Kerala and Delhi; Fig. 3).1 Most estimates, drawn from epidemiological, geographical and demographic data, suggest that up to December 2006, 1.38 million people were affected by the disease; Karnataka accounted for almost half and Maharashtra nearly a quarter of them. In some areas reported attack rates reached 45%. The disease was confirmed serologically in 1831 persons.1
   The results of the various chikungunya serosurveys are summarized in Table II. These surveys done during and between the epidemics provide objective evidence of prevalence of the virus in the population. In Africa, where rural areas are known to have sylvatic transmission of chikungunya virus, differences in the interepidemic seroprevalence of chikungunya have been striking: less than 1% in Kenya65 and 47% in Uganda.66 Prior to 1964, in India the antibody seroprevalence rate was low (4% in Kolkata55 and 11% in Chennai59). In Chennai, during the first chikungunya epidemic in 1964, serosurveys showed an antibody prevalence rate of 60% in febrile patients,60 and one-third of the population continued to be seropositive for up to 6 months after the onset of the outbreak.61 By contrast, in Kolkata, the antibody seroprevalence rate (26% in 1968) was similar to pre-1964 levels in 1995. In general, seroprevalence was lower in young adults compared with the older population. These surveys lend credence to temporal changes in the epidemiology of chikungunya infection. Interestingly, following the 1964 epidemic, a large proportion of samples (75%) in non-outbreak states also tested positive for anti- CHIK antibodies. Asymptomatic infections or cross-reactivity with another virus might explain this high seropositivity.62

CLINICAL FEATURES AND COMPLICATIONS
Most descriptions of chikungunya are based on data obtained during epidemics. Chikungunya (International Classification of Diseases-10, code A.92.0) is characterized by acute fever with

FIG 3. Suspected chikungunya cases in different states of India (adapted from National Vector Borne Disease Control Programme, Delhi, as on 12 December 20061). KA Karnataka MH Maharashtra AP Andhra Pradesh GJ Gujarat KR Kerala TN Tamil Nadu MP Madhya Pradesh AN Andaman and Nicobar Islands PO Pondicherry DL Delhi GA Goa RJ Rajasthan

or without chills, headache, nausea, abdominal pain, photo- phobia, conjunctival injection, skin rash and disabling arthralgia. The incubation period usually ranges between 2 and 10 days. The disease preferably affects adults (of the 333 seropositive patients for chikungunya infection in the 2006 epidemic, 299 [90%] were >15 years of age).8 Etymologically, chikungunya owes its origin to kungunyala, a word from the Makonde language of Tanzania. The word, meaning ‘that which bends up’, aptly conjures up the image of a patient who adopts a stooped posture because of severe arthritis. Typically, the wrists, hands, ankles and feet become intensely painful; large joint involvement is not uncommon. The painful back, knees and ankles can lead to diagnostic uncertainty—patients, unable to walk because of incapacitating pain, mistakenly believe, as do their physicians, that their leg weakness is neurological in origin. Although the fever and skin rash are short-lasting, the joint pains may recur or linger for a long time, sometimes for as long as 3 years after the onset of disease (Fig. 4).67 Old fracture sites begin to pain and pre-existing arthritis worsens. Tender, enlarged lymph nodes are common.68,69 Papular or maculopapular rash shows up on the arms and abdomen after 48 hours of illness; this is often attributed to adverse drug reactions.

Differential diagnosis
Dengue (fever and myalgia syndrome) and chikungunya (fever,

FIG 4. Temporal sequence of clinical features and laboratory findings in chikungunya infection

rash and arthritis syndrome),41 not only share a common vector, A. aegypti, but also several clinical characteristics (Table III). The skin rash adds to the diagnostic confusion. In the 2006 epidemic, 17 of the 989 samples (1.7%) from 3 states of India were found to be positive for anti-CHIKV and anti-dengue IGM antibodies, suggesting that the two viruses can co-exist.8 A previous study reported that of 477 patients with acute fever, 58% had chikungunya alone and 3% had co-infection with the chikungunya and dengue viruses.58 The differential diagnosis of chikungunya includes infection with other alphaviruses that cause the fever–arthritis syndrome (e.g. O.nyong nyong virus [ONN], Sindibis virus, Mayaro, Ross River and Burmah Forest viruses). In addition, chikungunya should be differentiated from hepatitis, dengue, acute rheumatic fever, primary HIV infection, malaria, typhoid, mycoplasmal infection, rickettsiosis and relapsing fever. Chikungunya infection can also be asymptomatic or may present as acute undifferentiated fever. 70 During an epidemic, the triad of fever.arthritis.rash makes the diagnosis of chikungunya most likely. 43 Although petechiae, ecchymoses and epistaxis have been described, 71 these features are far more common with other diseases such as dengue and leptospirosis. During the 2006 epidemic, we found that other competing diagnoses for acute undifferentiated fevers were malaria, dengue, leptospirosis, hepatitis and typhoid fever. In central India acute vaso-occlusive crises of sickle cell anaemia presenting with fever and polyarthritis is an important differential diagnosis.

Complications
There have been reports of neurological diseases including
TABLE II. Seroprevalence of anti-CHIK antibodies in India

*A haemorrhagic fever epidemic, presumably dengue, reported in 1963 - 65  . No known CHIK epidemic had occurred in this area, a dengue epidemic had occurred in 1968 . Ongoing dengue epidemic HI haemagglutination CF complement fixation ELISA enzyme-linked immunosorbent assay

T ABLE III. Distinguishing features of chikungunya and dengue

Frequency of finding: + = 1%.25%, ++ = 26%.50%; +++ = 51%.75%; ++++ = 76%.100%

meningoencephalitis (12 cases in the recent epidemic in the Indian Ocean islands), acute inflammatory demyelinating neuropathy, optic neuritis, encephalopathy and myocarditis associated with chikungunya. 6,58,72 Although an association between chikungunya and acute hepatitis has been suggested, alcohol appears to have confounded the association. 73 Recently, mother-to-child trans- mission 74 and 3 foetal deaths caused by mother-to-foetus trans- placental transmission before the sixteenth week of gestation have been reported; a polymerase chain reaction (reverse transcriptase [RT]-PCR) study showed viral genome in the amniotic fluid of the 3 foetuses, in the placentae of 2 and the brains of 2. 75 Researchers from Malawi described an association between chikungunya virus and endemic Burkitt lymphoma (eBL): patients with eBL were more likely to be seropositive for chikungunya virus than hospital controls (odds ratio 2.3; 95% CI: 1.3, 4.5) and community controls (odds ratio 2.3; 95% CI: 1.1, 5.1). 76 Although chikungunya is considered a self-limiting disease, until June 2006, an estimated 264 000 chikungunya infections accounted for 237 deaths in the Indian Ocean islands. 77 Most patients who died were either old or had co-morbidities. The National Vector Borne Disease Control Programme, Delhi, did not report any chikungunya- associated death from India. 1

DIAGNOSIS
Chikungunya is associated with leukopenia, anaemia and serum aminotransferase elevation; none of these laboratory features are specific for the diagnosis. The diagnosis remains clinical, as most patients cannot access the sophisticated laboratory tests needed to confirm or exclude the diagnosis. Dengue and malaria can be differentiated by serological tests and microscopy, respectively. Other alphaviruses known to cause the fever.arthritis syndrome have not been reported from India.
   The specific diagnosis of chikungunya can be obtained by serological tests, molecular methods or viral cultures. Serological tests detect anti-CHIK antibodies (suggested cut-off levels: IgM >0.15 and IgG >0.10) using IgM capture enzyme-linked immune- sorbent assay (ELISA). 69 However, anti-CHIK IgM antibodies appear only 4.5 days after the onset of fever, and fever would have subsided in most cases by then (Fig. 2). Also, paired sera (acute- and convalescent-phase serum specimens spaced at least 2 weeks apart) are required for accurate serological diagnosis. The diagnostic accuracy of chikungunya serology (with molecular methods or viral culture as reference standards) is not known, but the proportion of false-positives may be high in areas where other alphaviruses co-circulate.
   Molecular diagnosis of chikungunya by RT-PCR has come into vogue recently. Patients with chikungunya tend to have viraemia that can last up to 6 days. 78 The virus can be detected by PCR, a specific test with a turn-around time of one day. 79,80 Pfeffer et al. 79 using 26S RNA and E2 gene regions as the primer sequence in PCR reactions could detect as few as 10 viral genome equivalents. Another study, using simpler methods for highly conserved regions in E1 and nsP1, reported higher sensitivity. 81 Parida et al. 82 have reported the diagnostic accuracy of reverse transcription loop mediated isothermal amplification (RT-LAMP) assay targeting the E1 gene. Compared with traditional PCR techniques, the assay is simple to perform, obviates the need of sending the samples to specialized research laboratories and could thus be used to diagnose and monitor chikungunya during and between epidemics. PCR positivity during the 2006 epidemic ranged between 5.8% (112 / 1938) 8 and 49.3% (74/150) 80 .the higher positivity in the later study appears to be due to referral bias. Viral culture, the reference standard for the diagnosis of chikungunya infection, 80 is expensive, time-consuming and technology-intensive, and therefore cannot be used in clinical settings.
   It is important to note that 723 seropositive samples reported by the National Institute of Virology, Pune, the major specialized diagnostic laboratory in India, represent only a tiny fraction of the total. Indeed, of the 1.38 million people suspected to have had chikungunya in India during the 2006 epidemic, as of 12 December 2006, the diagnosis was confirmed in only 1831. 1 Also, diagnostic tests for chikungunya infection (serology, viral culture or PCR tests) are not available commercially or outside specialized research laboratories. The diagnostic tests at the NIV, for example, can only be done by special arrangement with local or regional healthcare departments, and are therefore not available to the vast population that needs these tests.

TREATMENT
There is no established antiviral treatment for chikungunya infection. The fever lasts no more than a few days but joint pains can be unbearable. Patients need bed rest (under mosquito nets) and pain-killers such as paracetamol or non-steroidal anti- inflammatory agents (NSAIDs). Aspirin should be avoided. Unpublished data from our district (Wardha in Maharashtra) indicates that during the 2006 epidemic most patients with suspected chikungunya received a cocktail of an antibiotic, an antimalarial, an NSAID.paracetamol combination, anti-ulcer medications, antihistaminics and multivitamins. Disproportionately large numbers of inpatients with the fever.rash.arthritis syndrome received ceftriaxone, a third-generation cephalosporin, and artemether, a drug reserved for complicated malaria. Surgeons in our hospital say that they operated on more patients with bowel perforations and peritonitis during the 2006 epidemic than they did in the previous year.a fact they attribute to the widespread use of NSAIDs and steroids for treating arthritis. We found that about 5% of patients with chikungunya had stiff, swollen and painful joints that lasted for several weeks after the onset of the disease. Although a study 83 (case series design; n=10) had demonstrated the efficacy of chloroquine therapy in post- chikungunya arthritis, the systematic and random errors in the study invalidate the conclusion.

PREVENTION
No licensed vaccine is available for chikungunya and, in the absence of research, is unlikely to be available for general use in the near future. Evidence suggests that vector control programmes that involve environmental management are highly effective in reducing the mortality and morbidity of malaria, 84 and disease transmission rates of dengue. 85 The vector control options include spraying dwellings with residual-action insecticides, biological control, larviciding, environmental management including source reduction and use of personal protection measures. A systematic review 86 has concluded that community-based vector control strategies in addition to habitat control (through biological and chemical means) could reduce the density of A. aegypti. The authors argued that multifaceted, rather than single interventions, work better because they address a larger variety of barriers for change. These data supplement the recent report from Mexico and Venezuela 85 which shows that the use of window curtains treated with insecticide alone or in combination with treated jar covers can substantially reduce the dengue vector population and potentially reduce disease transmission.
   As the dengue flavivirus and the chikungunya alphavirus share a common vector, we believe that vector control programmes which worked well in dengue should work as well in chikungunya. Implicit in the programmes is an assumption that efforts aimed at modification or manipulation of the environment coupled with education of the community can significantly reduce the population of A. aegypti. The resultant decline in the vector.host cycle can reduce transmission of the vector with a consequent decline in the incidence of the disease. 87 Since mosquitoes have been associated with 5 major diseases in India (malaria, dengue, Japanese encephalitis, chikungunya and filaria), we need integrated vector control programmes. Evidence suggests that when communities actively take part in abolishing the breeding places of A. aegypti (destruction, alteration, disposal or recycling of domestic containers), the density of larvae is significantly reduced. 88 An important caveat is that participatory programmes can succeed if they are inexpensive, simple, indigenous, convenient, effective and culturally acceptable. By contrast, interventions may not work if they are substandard, unacceptable and unsafe (DDT), unsustainable (larvicidal fishes or insecticide spraying), insensitive to bite times of the mosquito (pyrethroid impregnated bed-nets are not useful because A. aegypti is a daytime biter) or impractical (wearing protective clothing during outdoor living and activities). Personal protection measures such as applying insect repellant to the exposed skin can keep out A. aegypti, a daytime biter. Insect repellants containing 30% DEET have been shown to provide an average of 5 hours of complete protection against A. aegypti bites after a single application on the exposed skin. 89 However, in field conditions, perspiration, rain and rising temperature may make DEET less effective.

CONCLUSION AND FUTURE PROSPECTS
Future work on chikungunya should explore several questions. We need to better characterize the natural history of chikungunya. What do the virus and the vector do between epidemics. What proportion of people with acute febrile illnesses has chikungunya. Is there an epidemiological correlate of protection after an epidemic of chikungunya in a community. What are the viral biological correlates of protection. What is the diagnostic accuracy of various tests for diagnosing chikungunya infection. How best can we develop cost-effective diagnostics.and make them available. to the community at the point of care. Can well-designed randomized controlled trials help us choose the most appropriate therapy for chikungunya-associated chronic arthritis. We also need to find out why chikungunya preferentially affected small towns and villages in central and southern India and spared northern and eastern India. Finally, chikungunya needs to be considered not in isolation but along with other mosquito-borne diseases such as dengue, malaria and encephalitis. We need to implement robust evidence-based interventions to help prevent future epidemics. We hope clinicians, scientists, microbiologists, epidemiologists, public health officials and policy-makers will collaborate to mobilize support and funds for multidisciplinary research that will fill gaps in knowledge and generate an evidence base on the dynamics and determinants of disease transmission. We must ensure that the virus and the vector, possibly lurking in the dark, do not catch us unawares again.

ACKNOWLEDGEMENT
R.J. acknowledges training support from the Fogarty AIDS International Training Program (grant 1-D43-TW00003-17), National Institutes of Health, USA. This funding source had no involvement whatsoever with the content of this paper.

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