Until 2010, no Japanese encephalitis (JE) had been reported from Delhi. Upon report of four confirmed cases of JE in September 2011, detailed investigations were carried out to determine whether the cases were imported or indigenous.
Until 2010, no Japanese encephalitis (JE) had been reported from Delhi. Upon report of four confirmed cases of JE in September 2011, detailed investigations were carried out to determine whether the cases were imported or indigenous.
Entomological surveys were carried out and all mosquito pools were tested for the detection of JE virus by ELISA method using specific monoclonal antibody. Human blood samples from contacts of the patients were tested by IgM-captured ELISA method. Pig's blood samples were also tested for the detection of JE virus.
Culex tritaeniorhynchus, Culex vishnui and Culex pseudovishnui mosquitoes were found. In contrast to rural areas, their breeding habitats were different in the city. 19 pools were tested. JE virus was detected in two pools of Cx. tritaeniorhynchus females reared from field-collected larvae, indicating vertical transmission. One pool of Cx. vishnui was also positive. This is the first report for the detection of JE virus in mosquitoes from Delhi. JE IgM antibodies in five contacts/residents indicate recent infection. JE virus was also detected in pigs.
Present analysis shows that of four reported JE cases, three were confirmed indigenous, indicating that the virus is multiplying in the city. Mapping of infected JE vector mosquitoes in the cities is required for preventive measures to contain further spread of the disease.
Jusqu'en 2010 l'encéphalite japonaise (EJ) n'était pas signalée à Delhi. Sur base d'un report de quatre cas confirmés d'EJ en septembre 2011, des enquêtes détaillées ont été menées pour déterminer si les cas étaient importés ou indigènes.
Des enquêtes entomologiques ont été effectuées et tous les pools de moustiques ont été testés pour la détection du virus de l'EJ par la méthode ELISA utilisant des anticorps monoclonaux spécifiques. Des échantillons de sang des contacts humains des patients ont été testés par la méthode ELISA des IgM capturées. Des échantillons de sang de cochons ont également été testés pour la détection du virus de l'EJ.
Les moustiques Culex tritaeniorhynchus, Culex vishui et Culex pseudovishnui ont été trouvés. Contrairement aux zones rurales, leurs habitats de reproduction étaient différents dans la ville. 19 pools ont été testés. Le virus de l'EJ a été détecté dans 2 pools de femelles Culex tritaeniorhynchus élevées à partir de larves prélevés sur le terrain, indiquant une transmission verticale. Un pool de Culex vishui était également positif. Il s'agit ici du 1er rapport sur la détection du virus de l'EJ dans des moustiques de Delhi. La présence d'anticorps IgM de l'EJ chez 5 contacts/résidents indique une infection récente. Le virus de l'EJ a également été détecté chez les cochons.
La présente analyse montre que sur les 4 cas d'EJ rapportés 3 ont été confirmés indigènes, ce qui indique que le virus se multiplie dans la ville. La cartographie des moustiques vecteurs infectés par le virus de l'EJ dans les villes est utile aux mesures de prévention pour contenir la propagation de la maladie.
Hasta el 2010 no se habían reportado casos de Encefalitis Japonesa (EJ) en Delhi. Después de un informe de cuatro casos confirmados de EJ en Septiembre del 2011, se llevaron a cabo investigaciones detalladas para determinar si los casos eran importados o nativos.
Se llevaron a cabo estudios entomológicos y todos las muestras de mosquitos fueron evaluadas para detectar la presencia del virus de la EJ mediante el método de ELISA utilizando un anticuerpo monoclonal específico. Las muestras de sangre humana de los contactos de los pacientes fueron examinadas mediante el método de ELISA de captura de IgM. También se examinaron muestras de sangre de cerdos para determinar la presencia del virus de la EJ.
Se hallaron mosquitos Culex tritaeniorhynchus, Culex vishui y Culex pseudovishnui. A diferencia de las áreas rurales, sus hábitats de reproducción eran diferentes en la ciudades. Se evaluaron 19 grupos de muestras. Se detectó el virus de la EJ en 2 grupos de hembras de Culex tritaeniorhynchus nacidas de larvas recolectadas en el campo, indicando una transmisión vertical. Un grupo de Culex vishui también era positivo. Este es el primer reporte sobre la detección del virus de la EJ en mosquitos de Delhi. La presencia de anticuerpos IgM frente a EJ en 5 contactos/residentes eran indicativo de una infección reciente. El virus de EJ también se detectó en cerdos.
Este análisis muestra que de 4 casos de EJ reportados, 3 fueron confirmados como nativos, indicando que el virus se está multiplicando en la ciudad. Es necesario mapear los mosquitos vectoriales infectados con el virus de EJ en las ciudades, con el fin de planificar medidas preventivas para contener la futura dispersión de la enfermedad.
Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in South-East Asia, affecting more than 50 000 patients annually (Mackenzie et al. 2007). JE is now an emerging viral disease with international importance because it is invading the previously known non-endemic areas. From Asia, it has stretched to Papua New Guinea and Torres Strait of northern Australia (Hanna et al. 1996; Mackenzie et al. 1997). In India, the first human case was reported from Tamil Nadu, South India, in 1955 (Webb & Pereira 1956). Until 1973, the disease was confined to southern parts of India with low prevalence; subsequently, the disease has spread to various other parts of India. In 2011, the Directorate of National Vector Borne Disease Control Programme reported 1214 cases and 181 deaths due to JE from 15 states/UTs. In north-eastern India, JE has been reported repeatedly from Uttar Pradesh, Bihar and Assam since 1978. In the north-western part of India, in year 1990, the first JE outbreak was reported in Haryana State (Kar & Saxena 1998). Later, in 2003, the disease was also reported from district Saharanpur further west. JE is mainly known as rural disease, found in areas associated with rice fields (Reuben et al. 1992; Kanojia 2007). Until 2010, no indigenous JE case was reported from Delhi. After reported JE cases in urban areas of Delhi in September 2011, detailed investigations were carried out for the first time to check whether the cases were ‘imported’ or ‘indigenous’ and whether the virus was multiplying in the city. Results of the study are presented in the present paper.
The National Capital Territory (NCT) of Delhi is the largest metropolis by area and the second-largest metropolis by population in India. It occupies 1485 km2, of which 900 km2 is classified as urban. Delhi has a population of 16.7 million (2011 Census) and receives 611 mm of rainfall on an average annually, mainly from July to September. The highest monthly average maximum temperature is 41 °C in May, and the lowest monthly average minimum temperature is 7 °C in January.
The National Centre for Disease Control (NCDC) reported for the first time on 20.9.2011 four cases of JE from Delhi. Line listing of cases showed that three cases were from North Delhi: two cases from Jahangirpuri, two cases from Bawana and two cases from the central part of the city at Gole Market. The following investigations were carried out in all three localities from 25.9.2011 to 16.11.2011.
All potential breeding sites for JE vectors in the affected localities were checked thoroughly daily until 30.9.2011, then weekly from October to mid-November 2011. All collected larvae and pupae were reared in a laboratory for species identification.
Adult mosquito collections inside human dwellings were performed with the help of an aspirator tube and torch light and outdoors using the sweep and Hope Cage method applied to grass, bushes, pit shelters, etc. All mosquitoes were identified and abdominal conditions such as unfed (UF), fully fed (FF), semi-gravid (SG), gravid (G) were noted. To detect the source of the blood meal, abdomens of freshly FF mosquitoes were used to prepare blood smear on the filter paper. These samples were tested by enzyme-linked immunosorbent assay (ELISA) method using bovine, porcine and human antisera.
All mosquitoes were identified and species-, locality-, date- and sexwise pools were made for the detection of JE virus by antigen-capture ELISA using monoclonal antibody (MAb), 6B4A-10 as capture antibody and MAb–peroxidase conjugate MAb 6B6C-1 as detector antibody (Gajanana et al. 1995). Mosquito pools were considered positive for JE virus antigen if their optical density (OD) was greater than or equal to mean ± 4 standard deviation (SD) of the OD of the normal laboratory mosquito pools. The virus infection rate in mosquitoes was expressed as minimum infection rate (MIR) per 1000 females tested (Chiang & Reeves 1962).
The surveys consisted of investigations of the patients' households and surroundings by door-to-door surveys eliciting patient's movements during the month before onset of the disease and the number of fever cases and domestic pigs in the locality. Information was collected for determining the movements of the patient's relatives and whether any member of the household had been ill prior to, during or subsequent to the patient's illness. The animals reared or present in the immediate vicinity of the household were also noted. Particular attention was paid to determine whether there was a ‘link’ or amplifier host and reservoir present.
Pigs' blood samples were collected through Municipal Cooperation of Delhi from those localities, from where con?rmed JE cases were reported and sent to National Research Centre of Equines, Hisar, Haryana, India, for the detection of JE virus. Samples were tested by haemagglutination inhibition (HAI) to confirm the JE virus in pig using specific antibody against JE virus.
Human blood samples collected in the field from contacts of the patients with fever/no fever and residents from surrounding areas after reporting of confirmed JE cases were tested using IgM-capture ELISA for the detection of JE virus antibodies.
All four JE patients were hospitalised and the following details were documented. Case 1 was a 13-year-old girl from 1353 E Block Jahangirpuri. Onset of fever was on 6.9.2011. A cerebrospinal fluid (CSF) sample received on 13.09.2011 tested positive for JE antibodies. She had not been outside Delhi for the last four months before the onset of symptoms. She was very weak and unable to speak clearly, with no neurological problems on 16.9.2011. She lives in a small house and her family is poor. Later, a blood sample was collected from her father, who was also positive for JE.
Case 2 was a 35-year-old woman from C3/182 Kali Marg, Gole Market. Her CSF sample was received by NCDC on 13.09.2011 from Dr. Ram Manohar Lohia Hospital and tested positive for JE. The patient is HIV positive and lives in small hut (Jugghi) in the central part of Delhi.
Case 3 was a 10-year-old boy from KB 552 Jahangirpuri, hospitalised on 14.09.2011. Blood and CSF samples tested positive for JE IgM antibodies. The patient had not visited outside Delhi for the last three months. His family is poor and lives in Jugghi.
Case 4 was a 9-year-old boy from D. 912, J.J. Colony Bawana, hospitalised in unconscious condition on 14.9.2011. His CSF sample was found positive for JE. Later, the patient had recovered. He had come to Delhi from Mujaffarpur, 7 months before the onset of symptoms. The child is from a poor family and lives in Jugghi.
The details of positive breeding sites and distances from the JE patients' houses (Table 1) show that in Jahangirpuri, breeding Cx. tritaeniorhynchus and Cx. vishnui were found in ponds with aquatic vegetation (Figure 1) and ground water pools in the grass field/park. Culex mosquitoes did not breed inside the houses, only Aedes aegypti did, in plastic containers. However, breeding Cx. quinquefasciatus were found in drains, pits and stagnant water in the locality.
|Distance from JE +ve house||Breeding sites||Density/dip larvae||Density/dip pupae||Species identified|
|Locality : Jahangirpuri KB Block in and around house of JE-positive case|
|About 50 m||Ground water pool||16.4||2.4||Cx. tritaeniorhynchus|
|About 100 m||Drain||14.8||1.8||Cx. quinquefasciatus|
|About 200–300 m||Drain near police station||10.4||1.0||Cx. quinquefasciatus|
|About 200–300 m||Plastic containers in fire station campus||4.2||0||A. aegypti|
|About 300 m||Plastic drums||1.8||0.5||A. aegypti|
|About 500 m||Ground water pool in the park||5.4||1.0||Cx. tritaeniorhynchus|
|Locality : Jahangirpuri E Block in and around house of JE-positive case|
|About 50 m||Small ground water collection near residential area (with grass)||5.8||1.6||Cx. tritaeniorhynchus|
|About 100 m||Pits in the park||2.2||0||Cx. quinquefasciatus|
|About 100 m||Stagnant pool of water in the field||1.2||0|| |
|About 50–100 m||Pond with grasses at vicinity of houses||18.8||1.33|| |
|House E-1351||Plastic containers||1.8||0||A. aegypti|
|House E-1836||Flower pots||0.6||0||A. aegypti|
|Jahangirpuri back side of E and KB Blocks|
|About 1 km from +ve case||Pond no.1 towards National High way with water hyacinth||16.8||1.5||Cx. tritaeniorhynchus|
|About 200–300 m||Pond no. 2 with aquatic vegetation in a big park||25||3.3||Cx. tritaeniorhynchus|
|About 200–300 m||Pond no 3 with small grasses in a big park||14.1||0||Cx. tritaeniorhynchus|
|About 400–500 m||Pond no 4 with small grasses/aquatic plants||10.2||1.2||Cx. tritaeniorhynchus|
|Locality : Bawana|
|About 50 m–100 m||Polluted water drainage around the houses||9.0||2.4||Cx. quinquefasciatus|
|About 200–300 m||Pond no.1||8.0||2.4||Cx. tritaeniorhynchus|
|About 400–600 m||Pond no. 2||12.0||3.7||Cx. tritaeniorhynchus|
|About 200–300 m||Ground Pools of water collection||0||0|
|About 50–100 m||Drains||9.3||0||Cx. quinquefasciatus|
|About 400–600 m||Ground water collection||7.8||3.2||Cx. tritaeniorhynchus|
|About 800–900 m||Ditches behind temple||2.0||0||Cx. tritaeniorhynchus|
|About 900–1500 m||Canal||4.4||1.2||Cx. tritaeniorhynchus|
In Bawana, only Cx. tritaeniorhynchus was breeding in ponds, ditches, water pools in the grass field and in a canal near the JE patient's house. Aquatic vegetation such as grass was present in all positive ponds.
The adult mosquito collections from different resting habitats (Table 2) gathered a total of 52 wild-caught adult Cx. vishnui mosquitoes. Cx. tritaeniorhynchus was the predominant species, significantly more common outdoors than indoors, while Cx. pseudovishnui and Cx. vishnui were more common indoors. A good proportion of fed and gravid Cx. tritaeniorhynchus was found near the breeding sites, indicating their preference for outdoor resting. However, most Cx. quinquefasciatus preferred indoor resting. After the report of a confirmed JE case, adulticides and larvicides were deployed to kill adult mosquitoes as well as larvae; therefore, it was rather difficult to find mosquitoes there.
|Locality||Mosquito species||Type of sites||No caught||Total|
|Indoor adult collection|
|Jahangirpuri||Cx. vishnui||HD||1||3 (UF, SG, FF)||4|
|Cx. pseudovishnui||HD||2||7 (6 UF, 1 SG)||9|
|Cx. tritaeniorhynchus||HD||2||3 (3 UF)||4|
|Bawana||Cx. tritaeniorhynchus||HD||2||4 (3 UF, 1 FF)||6|
|Outdoor adult collection|
|Jahangirpuri||Cx. tritaeniorhynchus||grass/bushes||5||15(7 UF, 2 SG, 4 FF, 2 G,)||20|
|Cx. vishnui||grass/bushes||0||1 (FF)||1|
|Bawana||Cx. tritaeniorhynchus||grass/bushes||3||4 (3 UF, 1 SG)||7|
Entomological surveys were also carried out in Gole Market. Neither JE vector mosquitoes nor potential breeding sites of JE vectors were detected in the vicinity. However, A. aegypti and Culex quinquefasciatus were breeding nearby. No pigs were seen nearby.
Five blood meal samples of JE vectors collected from outdoor resting sites from Jahangirpuri were tested. None was positive for human antiserum, while one C. vishnui was found positive for pig antiserum and two C. tritaeniorhynchus were positive for bovine antisera. Two were non-reactive.
In Jahangirpuri, JE virus was detected in one pool of Cx. tritaeniorhynchus female mosquitoes reared from field-collected larvae and one pool of Cx. vishnui (wild-caught adult female from human dwelling) (Table 3). In Bawana, there were four pools of Cx. tritaeniorhynchus, one positive for JE virus antigen. In total, 16.66% of Cx. tritaeniorhynchus and 16.66 % of Cx. vishnui harboured JE virus. Presence of JE virus in the mosquitoes reared from field-collected larvae showed vertical transmission (adults to eggs). MIR of Cx. tritaeniorhynchus was 34.09%; for Cx. vishnui, it was 125%.
|Locality||Mosquito species||No. of pools tested||No. of mosquitoes tested||No. pools +ve with JEV||Location|
|Jahangirpuri||Cx. tritaeniorhynchus||8||57 (39 M, 18 F)||1 (12 F)||Immature collected on 28.9.2011 from ponds|
|Cx. vishnui||6||8 (2 M, 6F)||1 (1 F)||Adult caught from human dwelling on 5.10.2011|
|Cx. pseudovishnui||1||1(1 F)||0||0|
|Bawana||Cx. tritaeniorhynchus||4||22 (13 M, 9 F)||1 (3 F)||Immature collected on 28.9.2011 from ponds|
Total of 81 pig serum samples tested, 17 samples were found positive with JE virus in Delhi, showing its circulation in domestic pigs. Later in 2011, con?rmed JE cases were also reported from Shahadra South and SP Zones. It is interesting to highlight that in Shahadra South Zone, JE virus could be detected in vector mosquitoes as well as in domestic pigs.
Of the 42 field-collected samples from Jahangirpuri after getting confirmed JE cases from that locality, four were positive for the presence of IgM antibodies to JE. In Bawana, 13 blood samples were tested; one tested positive with IgM antibodies to JE. Presence of JE IgM antibodies indicates recent infection with JE. A human sero-survey in Karol Bagh of 15 blood samples from residents showed that none was positive for JE.
All four patients had no travel history outside Delhi during the last 2–3 months before the onset of symptoms. All cases were reported from low socio-economic strata. Pigs were also reared in the nearby locality of patients. Pigsties/ shelters were found in their small houses to keep pigs during night-time.
Of these first four cases (two females) from Delhi, three were children and one was an HIV-infected woman aged 35 years. However, in serological survey (asymptomatic) in the areas, IgM antibodies for JE were detected in four adults (between 28 and 47 years). Of those, three were males, including the father of a patient. All were the residents of Delhi. None of the cases had history of travelling to JE endemic areas in India. No death was reported. However, in Bawana, one contact was found positive with IgM antibodies to JE, who was a woman aged 25 years old from same family of JE patient from Bawana.
Pigs were reared in the vicinity of patients' houses and migratory birds (Egret; local name - Bagula) from Family Ardeidae were spotted near breeding sites of Cx. vishnui mosquitoes along with herd of domestic pigs and piglets (Figure 1).
Culex vishnui subgroup mosquitoes comprising Cx. tritaeniorhynchus, Cx. vishnui and Cx. pseudovishnui have been implicated as major vectors of JE in India (Reuben et al. 1994; Kanojia 2007) and in many countries of South-East Asia (Leake et al. 1986; Amersinghe et al. 1988). In Delhi, after reporting of first four confirmed cases of JE, immature and adult stages of all three species of C. vishnui subgroup were collected, in spite of extensive use of insecticides in the localities during the period under report. Although these mosquitoes prefer to rest outdoors, they were found both indoors and outdoors in Delhi. Cx. tritaeniorhynchus, the major and incriminated vector of JE in India, was also reported resting indoors in large numbers during JE transmission season (Kanojia 2007). In view of its substantially high population density during the JE occurrence period and the largest number of positive pools, Cx. tritaeniorhynchus is likely to be a vector of JE in Delhi. However, Cx. vishnui may also play an important role in JE transmission as is evident from its preference of resting in human dwellings and infection with JE virus; MIR was significantly higher (125%). Compared to the most prevalent Cx. tritaeniorhynchus, even a low density of Cx. vishnui could be more dangerous.
Culex tritaeniorhynchus was the most abundant JE vector in India. Rice fields are their most productive breeding sites, and its population dynamics has been closely associated with paddy cultivation in rural areas (Reuben et al. 1992; Sunish & Reuben 2001; Kanojia 2007). However, in urban areas in Delhi, JE vectors were breeding profusely in ponds and ground water collections in the parks. Therefore, such types of JE vector breeding sites should be targeted for vector control in cities. In urban areas of Kurnool, India, high Cx. tritaeniorhynchus density was also noticed compared to rural areas (Murty et al. 2010).
Thus, in the capital city of India, prevalence of infected Cx. tritaeniorhynchus and Cx. vishnui and other eco-epidemiological conditions were conducive for JE transmission. Presence of domestic pig and piglet populations along with Arbeid egrets in the vicinity of breeding sites of JE vector mosquitoes explained the possible route of JE transmission. A fairly high proportion of cattle egrets and pond herons possess neutralising antibodies to JEV, indicating that these birds are the natural reservoir of JEV in India (Rodrigues et al. 1981; Banerjee et al. 1988). The pigs are considered as ‘amplifiers’ of the virus. Pigs get infection from migratory birds, and this virus gets amplified 400 times when it enters the pigs' bodies. So mosquitoes biting pigs can be dangerous for humans. The virus tends to spill over into human populations when infected mosquito populations build up explosively and the human biting rate increases. Therefore, there is a possibility that pigs reared in Delhi could have been infected through water birds through mosquito bites. Human populations in such areas might be susceptible for JE virus. The fact that JE virus was detected in vectors, in domestic pigs, contacts and residents of Delhi who did not travel during the past 2–3 months suggests indigenous transmission of JE.
In urban areas of Can Tho City, Vietnam (Lindahl et al. 2012), keeping pigs increased the number of mosquitoes competent as vectors for JEV and pigs were associated with increased numbers of Cx. tritaeniorhynchus.
In Delhi, JE was associated with low socio-economic status. A recent study in West Bengal 2005–2010 indicates that socio-economic status and environmental conditions were statistically significant contextual risk factors for serologically diagnosed JE; 65.7% cases were from low-income groups (Sarkar et al. 2012). Also, JE risk was significantly associated with rural residents living in close proximity to irrigated rice fields and pig-rearing places. However, in the urban areas in our investigation, JE cases were reported much closer to the ponds and stagnant ground water pools with vector breeding. Control measures should focus on these habitats.
The present analysis shows that of four reported JE cases, three were confirmed as indigenous, indicating that virus is multiplying in the city. Thus, JE is not restricted to rural areas associated with rice fields; it can also spread in urban areas in suitable ecological conditions. Mapping of infected JE vector mosquitoes in cities is therefore required for planning preventive measures to contain further spread of the disease.
We thank Mohamad Mujib, Priya Singh and Narender Singh Rawat at the Centre for Medical Entomology in National Centre for Disease Control for technical assistance. We also acknowledge with thanks, the cooperation extended by the staffs and officers of the Directorate of National Vector Borne Disease Control Programme and Municipal Corporation of Delhi for the study. Zoonosis Division of NCDC and National Research Centre of Equines, Hisar, Haryana, are thanked for laboratory work.