|Year : 2017 | Volume
| Issue : 4 | Page : 311-316
Aetiology of acute encephalitis syndrome in Uttar Pradesh, India from 2014 to 2016
Parul Jain1, Shantanu Prakash1, Danish N Khan1, Ravindra Kumar Garg2, Rashmi Kumar3, Amit Bhagat1, V Ramakrishna1, Amita Jain1
1 Department of Microbiology, King George's Medical University, Lucknow, India
2 Department of Neurology, King George's Medical University, Lucknow, India
3 Department of Paediatrics, King George's Medical University, Lucknow, India
|Date of Submission||02-Sep-2017|
|Date of Acceptance||26-Oct-2017|
|Date of Web Publication||19-Feb-2018|
Department of Microbiology, King George's Medical University, Lucknow–226 003 (UP)
Source of Support: None, Conflict of Interest: None
Background & objectives: It is imperative to know the aetiology of acute encephalitis syndrome (AES) for patient management and policy making. The present study was carried out to determine the prevalence of common aetiological agents of AES in Uttar Pradesh (UP) state of India.
Methods: Serum and/or CSF samples were collected from AES patients admitted at Gandhi Memorial and Associated Hospital, King George's Medical University, Lucknow, a tertiary care centre, UP during 2014–16. Cerebrospinal fluid (CSF) and serum samples from cases were tested for IgM antibodies against Japanese encephalitis virus (anti-JEV), and dengue virus (anti-DENV) by ELISA; and for enterovirus, herpes simplex virus (HSV) and varicella zoster virus (VZV) by real-time PCR. Serum samples of cases having sufficient CSF volume, were also tested for anti-scrub typhus IgM antibodies and for Neisseria meningitides, Streptococcus pneumoniae and Haemophilus influenzae.
Results: JEV and DENV (8% each) were the most common identified aetiology from the 4092 enrolled patients. Enterovirus, HSV and VZV, each were detected in <1% AES cases. Co-positivity occurred in 48 cases. Scrub typhus (31.8%) was the most common aetiology detected. Haemophilus influenzae and S. pneumoniae were detected in 0.97 and 0.94% cases, respectively, however, N. meningitides was not detected in any of the cases. About 40% of the JEV/DENV positive AES cases were adults. The gap between the total number of AES cases and those with JEV/ DENV infection increased during monsoon and post-monsoon seasons.
Interpretation & conclusion: Scrub typhus, JEV and DENV are the main aetiological agents of AES in UP. DENV and JEV can no longer be considered paediatric diseases. The prevalence of non-JEV/DENV aetiology of AES increases in the monsoon and post-monsoon seasons.
Keywords: Acute encephalitis syndrome; aetiology; dengue virus; Japanese encephalitis virus; scrub typhus
|How to cite this article:|
Jain P, Prakash S, Khan DN, Garg RK, Kumar R, Bhagat A, Ramakrishna V, Jain A. Aetiology of acute encephalitis syndrome in Uttar Pradesh, India from 2014 to 2016. J Vector Borne Dis 2017;54:311-6
|How to cite this URL:|
Jain P, Prakash S, Khan DN, Garg RK, Kumar R, Bhagat A, Ramakrishna V, Jain A. Aetiology of acute encephalitis syndrome in Uttar Pradesh, India from 2014 to 2016. J Vector Borne Dis [serial online] 2017 [cited 2018 Aug 16];54:311-6. Available from: http://www.jvbd.org/text.asp?2017/54/4/311/225835
| Introduction|| |
Acute encephalitis syndrome (AES) is an important public health problem worldwide. Population-based studies have estimated, a global incidence of 3.5 to 7.4 for AES per 100,000 patients in an year. The disease has been reported to be associated with several complications including limb paralysis, seizures, impaired consciousness or even death. In non-fatal cases, AES may often lead to severe permanent physical, cognitive, emotional, behavioural and social difficulties in affected individuals.
Japanese encephalitis virus (JEV) is the major identified cause of AES in India, affecting 50,000 people annually and causing 8–30% mortality and 50-60% disability. The state of Uttar Pradesh (UP) is one of the worst hit states in India, where JE outbreaks occur annually in the rainy season. In 2005, JE caused a massive outbreak in UP affecting 5737 persons including 1344 deaths. Consequently, in 2006, the Government of India launched JE vaccination drive in UP, which resulted in overall decrease in the incidence of JE (7.1% of AES). But the number of AES cases has not decreased over the years resulting in an increased gap between the total AES and JE positive cases. This implies that a majority of AES cases in UP are caused by non-JE aetiology which may include other viruses, bacteria, parasites, protozoa or fungi; viral aetiology, though is considered to be the most common cause of AES.
Identifying the aetiology of AES in patients is important, not only for patient management but also to understand the epidemiology, identify appropriate targets for immunization, and for formulating public health policies. Therefore, the present study was carried out to determine the prevalence and epidemiology of the common aetiological agents of AES in UP.
| Material & Methods|| |
Study area and sample collection
This was an observational cross-sectional study done over a period of three years. Patients with a clinical diagnosis of AES admitted at Gandhi Memorial and Associated Hospital, King George's Medical University, Lucknow, India and referred to the Virology Laboratory for testing, were enrolled consecutively in the study from January 2014 to December 2016. A case of AES was clinically defined as a constellation of symptoms comprising of acute onset fever and a change in mental status (including symptoms such as confusion, disorientation, coma, or inability to talk) and/or new onset of seizures (excluding simple febrile seizures) in a person of any age at any time of the year. Cases with known non-infectious aetiology like trauma, metabolic causes, hypoxia, etc. were excluded from the study. CSF and/or serum samples were collected from the enrolled patients.
The study was approved by the Institutional Ethics Committee (No. 7139/Ethics/R.cell-15).CSF and/ or serum samples were collected after obtaining written informed consent from the patients/guardians.
The CSF sample was tested for anti-JEV IgM antibodies (MAC ELISA kit developed by the National Institute of Virology, Pune, India) as per the manufacturer's instructions and for nucleic acids of JEV (JEV-RNA), enterovirus (enterovirus RNA), herpes simplex virus (HSVDNA) and varicella zoster virus (VZV-DNA) by real-time PCR. The serum sample was tested for anti-dengue virus IgM (anti-DENV) antibodies (MAC ELISA kit—National Institute of Virology, Pune). In case CSF sample was not available, anti-JEV IgM antibodies were tested in serum sample.
Cases, for whom both CSF and serum samples were available in sufficient quantity for further testing, the serum samples were also tested for anti-scrub typhus IgM antibodies by ELISA (Inbios International, USA) following the manufacturer's instructions. Samples with an optical density (OD) >0.5 were considered positive for anti-scrub typhus IgM antibodies. CSF samples from these cases were also tested for Neisseria meningitides, Streptococcus pneumoniae and Haemophilus influenzae by real-time PCR.
Nucleic acids were extracted from 140 μl CSF samples following the manufacturer's protocol (Qiagen, Hilden, Germany). The real-time PCRs were standardised according to conditions, as mentioned in [Table 1]. For RNA detection, the reaction mixture contained 12.5 μl of 2× RT-Buffer and 1μ of 25×RT enzyme (AgPath, Life Technologies, CA, USA) and for DNA detection, the reaction mixture contained 12.5 μl TaqMan universal PCR master mix (Life Technologies, CA, USA). For each real-time PCR reaction mixture, the final volume was 25 μl containing 7.5 μl nucleic acid, 0.5 μl each primer (10 pmol), and 0.5 μl probe (5 pmol). The cycling conditions consisted of an initial denaturation of 95°C for 10 min followed by 45 cycles of denaturation at 95°C for 15 sec and annealing and extension for 60°C for 1 min for DNA pathogens and included an extra step of pre-amplification at 45°C for 10 min for RNA pathogens. The threshold cycle (Ct value) of 35 was taken as the cut-off and only the curves showing the value of <35 were considered positive.
|Table 1: Target genes, amplicon size and probes and primers used for real-time PCRs in the study|
Click here to view
All the statistical analyses were done using Graph-Pad Prism software version 5. Intergroup comparisons of categorical and continuous variables were done using Fischer's exact test and Chi-square test, respectively.
| Results|| |
In total 4092 cases of AES were enrolled during the study period (2014–16), from which 3861 CSF and 2829 serum samples were available [Table 2]. JEV (8.3%) and DENV(7.8%) were the most common aetiological identified among the enrolled cases. Enterovirus, HSV and VZV, each were detected in < 1% AES cases [Table 2]. RT PCR for JEV(JEV PCR) detected seven, 17 and eight extra cases in 2014, 2015 and 2016, respectively in anti- JEV IgM negative cases.
|Table 2: Samples available and aetiology of AES over a period of three years (2014–16)|
Click here to view
Of the anti-DENV IgM or anti-JEV IgM positives, 16, 15 and 11 cases tested positive for both the antibodies in the years 2014, 2015 and 2016, respectively. Three patients tested positive for both anti-DENV IgM and JEV PCR and one patient tested positive for anti-DENV IgM antibodies and VZV PCR [Table 2].
It was found that 31.8% tested AES cases were positive for anti-scrub typhus IgM antibodies. Haemophilus influenzae and S. pneumoniae were detected in 0.97 and 0.94% cases, respectively while N. meningitides was not detected in any case [Table 2].
When the age and sex distribution of the two most commonly detected aetiologies (JEV and DENV) were studied, it was observed that they mostly affected children between 1 and 15 yr of age [Table 3]. It was important to note that approximately 40% of the JEV/DENV positive AES cases were adults. Also 0.6% JE and 2.4% DENV positive cases were infants. Males were more commonly affected than females in both the cases.
|Table 3: Age and sex distribution of AES cases positive for JEV and DENV over a period of three years (2014–16)|
Click here to view
The seasonal distribution of AES cases showed that AES occurred throughout the year, though the number of cases increased steeply between August and October [Figure 1]. DENV showed a similar pattern, with cases occurring throughout the year mostly during August to October. JEV had a distinct seasonality with no JE positive cases during February to June followed by a sudden rise in the number of cases from August to October and occasional cases in July, November and December. The gap between the total number of AES cases and those with JEV/DENV infection also increased during August to October [Figure 1].
|Figure 1: Seasonal distribution of AES cases referred to virology laboratory and of JEV/DENV positives.|
Click here to view
| Discussion|| |
Acute encephalitis syndrome is a major health problem in the state of UP, India, because of the multiple aetiologies involved, lack of standardized case definitions and availability of limited resources. The present study was an attempt to study the prevalence of common aetiologies of AES cases from UP.
JEV was examined in the AES cases because the World Health Organization has paralleled JEV surveillance with AES surveillance in the Southeast Asian countries and the virus is also known to cause constant outbreaks in UP since 1978. DENV was examined in the AES cases, since it has increasingly been reported as a cause of AES and other neurological manifestations,. Enteroviruses were selected, since they were implicated in 2006 and 2008 AES outbreaks in eastern UP. HSV and VZV were tested, since well-established antiviral treatment is available only against these viral aetiologic agents of AES. Other known viral aetiologies like mumps, measles and rubella viruses were not tested since these viruses contribute to only few sporadic cases, due to availability of effective vaccination. Moreover, they usually have a preceding suggestive history.
Majority of the tests were carried out from CSF samples, as detection of an aetiologic agent (antibodies or nucleic acid) in CSF represents direct neuro-invasion, and hence, CSF remains the sample of choice for diagnosing AES aetiology. Anti-DENV IgM and anti-scrub typhus IgM antibodies were detected in serum as these kits are standardized only for the serum samples. The limitation of detecting these antibodies in serum is that these might be pre-existing antibodies and not the cause of present illness, since IgM antibodies may persist for up to six months after infection.
Infections by JEV, DENV and scrub typhus were diagnosed by detecting IgM antibodies because by the time patients develop encephalitis and present to the hospital, viremia usually disappears (best detected within first week of illness) and neutralizing antibodies appears,. Hence, IgM capture assays are the most sensitive tests for diagnosing recent viral/ rickettsial, infections, though few extra JE cases were detected by real-time PCR, which could have been missed if only IgM testing was done. Infections by the remaining pathogens were diagnosed by detecting nucleic acid by real-time PCR, since viruses like HSV, VZV are known to establish latent infections with intermittent reactivations and, therefore, PCR remains the gold standard test for their detection. For bacterial infections (H. influenzae, S. pneumoniae, N. meningitides) the Center for Disease Control, USA, has recommended the use of real-time PCR in CSF samples, so that targeted therapy may be initiated as soon as possible in a patient.
In this study, JEV and DENV were identified to be the most common aetiological agents of AES each contributing to approximately 8% cases. In the years 2011–12, JEV and DENV contributed to 16 and 11% AES cases, respectively. The incidence of JEV has decreased, probably as a result of mass vaccination campaigns. This decrease has also been observed from other states like West Bengal and Assam where vaccination campaigns are going on. It is interesting to note that the proportion of DENV causing AES is constantly decreasing; from 23.4% in 2008 to 11% in 2012 and to 8% in 2016,. Change in the predominantly circulating DENV serotype might be accountable for this observation.
Few cases were positive for more than one test. In a hyperendemic region, for cases that were positive for both anti-DENV IgM and anti-JEV IgM antibodies, three possibilities exist: (i) sequential arbovirus infection; (ii) cross-reacting antibodies due to the common antigenic epitopes in the major envelope (E) protein; and (iii) coinfection. Further, testing is required to resolve the issue.
Simultaneous detection of anti-DENV IgM antibodies and JEV PCR favour co-infection.
The age preference of both JEV and DENV has changed over the years. Both the viruses were earlier considered to be predominantly affecting children <15 yr of age. But probably as a result of vaccination (JEV) or long-standing endemicity (DENV), adults were observed to be majorly affected (40% of the cases were adults) in the study. Similar age shifts have been observed earlier in areas with good quality, long-standing childhood JE vaccination programmes; and from other Asian countries including Bangladesh, Indonesia, Thailand and Singapore with long-standing dengue epidemic activities.
The cases of AES occurred throughout the year, though maximum cases were observed in the monsoon and post-monsoon seasons. A gap between the number of AES cases and those positive for JEV/DENV was also observed throughout the year, which widened in the monsoon and post-monsoon seasons despite the increase in JEV/DENV cases. This indicates the presence of other aetiologies probably with a definite seasonality.
In this study scrub typhus was observed as an important cause of AES in UP contributing to about 32% of the tested cases. Scrub typhus has also been incriminated in about 30% AES cases in Tamil Nadu and was also proved to be a cause of AES in a case control study from Gorakhpur, UP. This is a treatable infection if detected on time. Therefore, it is suggested to test all AES cases for scrub typhus in UP and initiate early and appropriate treatment on an empirical basis.
| Conclusion|| |
JEV, DENV and scrub typhus remain the main aetiologies of AES in UP. Enteroviruses, herpes viruses and bacteria cause only rare sporadic infections. The age preference of DENV and JEV is shifting and adults are now equally affected. The prevalence of non-JEV/DENV aetiology of AES increases in the monsoon and postmonsoon seasons. Aetiology could not be determined in a large number of cases, which warrants further studies for detection of other agents of encephalitis.
Conflict of interest: None.
| Acknowledgements|| |
The study was supported by a financial grant (No. 5/8/7/17/2010-ECD-1) provided by the Indian Council of Medical Research, New Delhi, India. Dr Parul Jain is supported by the ICMR MD-PhD programme.
| References|| |
Granerod J, Crowcroft NS. The epidemiology of acute encephalitis. Neuropsychol Rehabil
Rayamajhi A, Singh R, Prasad R, Khanal B, Singhi S. Study of Japanese encephalitis and other viral encephalitis in Nepali children. Pediatr Int
Clarke M, Newton RW, Klapper PE, Sutcliff EH, Laing I, Wallace G. Childhood encephalopathy: Viruses, immune response, and outcome. Dev Med Child Neurol
|4.|Details of AES/JE cases and deaths from 2008–2014
. Delhi: National Vector Borne Disease Control Programme, Directorate General of Health Services, Ministry of Health & Family Welfare. Available from: http://www.nvbdcp.gov.in/Doc/je-aes-cdfeb14.pdf
(Accessed on July 1, 2017).
Parida M, Dash PK, Tripathi NK, Ambuj, Sannarangaiah S, Saxena P, et al
. Japanese encephalitis outbreak, India, 2005. Emerg Infect Dis
2006; 12(9): 1427-30.
Gore MM. Acute encephalitis syndrome in India: Complexity of the problem. J Commun Dis
2014; 46(1): 35-49.
Sen PK, Dhariwal AC, Jaiswal RK, Lal S, Raina VK, Rastogi A. Epidemiology of acute encephalitis syndrome in India: Changing paradigm and implication for control. J Commun Dis
2014; 46(1): 4-11.
|8.|Japanese encephalitis surveillance standards. From WHO-recommended standards for surveillance of selected vaccine-preventable diseases (updated January 2006)
. Geneva, Switzerland: World Health Organization. Available from: http://www. path.org/files/WHO_surveillance_standards_ JE.pdf
(Accessed on May 5, 2017).
Kumari R, Joshi PL. A review of Japanese encephalitis in Uttar Pradesh, India. WHO South East Asia J Public Health
2012; 1(4): 374-95.
Francisco Javier Carod-Arta. Neurological manifestations of dengue viral infection. Res Rep Trop Med
Kumar R, Tripathi S, Tambe JJ. Dengue encephalopathy in children in northern India: Clinical features and comparison with non-dengue. J Neurol Sci
Sapkal GN, Bondre VP, Fulmali PV, Patil P, Gopalkrishna V, Dadhania V, et al
. Enteroviruses in patients with acute encephalitis, Uttar Pradesh, India. Emerg Infect Dis
2009; 15(2): 295-8.
Koskiniemi M, Vaheri A. Effect of measles, mumps, rubella vaccination on pattern of encephalitis in children. Lancet
Roehrig JT, Nash D, Maldin B, Labowitz A, Martin DA, Lanciotti RS, et al
. Persistence of virus-reactive serum immunoglobulin M antibody in confirmed West Nile virus encephalitis cases. Emerg Infect Dis
2003; 9(3): 376-9. doi: 10.3201/ eid0903.020531.
Swami R, Ratho RK, Mishra B, Singh MP. Usefulness of RT-PCR for the diagnosis of Japanese encephalitis in clinical samples. Scand J Infect Dis
2008; 40(10): 815-20.
Blacksell SD, Tanganuchitcharnchai A, Nawtaisong P, Kantipong P, Laongnualpanich A, Day NPJ, et al
. Diagnostic accuracy of the InBios scrub typhus detect enzyme-linked immunoassay for the detection of IgM antibodies in northern Thailand. In: Rosenberg HF, editor. Clin Vacc Immunol
2016; 23(2): 148-54.
Rahi M, Gupte MD, Bhargava A, Varghese GM, Arora R. DHR-ICMR guidelines for diagnosis and management of rickettsial diseases in India. Indian J Med Res
DeBiasi RL, Tyler KL. Molecular methods for diagnosis of viral encephalitis. Clin Microbiol Rev
Bandopadhyay B, Bhattacharyya I, Adhikary S, Mondal S, Konar J, Dawar N, et al
. Incidence of Japanese encephalitis among acute encephalitis syndrome cases in West Bengal, India. Bio Med Res Int
(Article ID 896749). Avaiable from: http://dx. doi. org/10.1155/2013/896749
Jain P, Jain A, Kumar A, Prakash S, Khan DN, Singh KP. Epidemiology and etiology of acute encephalitis syndrome in north India. Jpn J Infect Dis
Borkotoki U, Borkotoki S, Barua P, Das A, Rajkhowa A. Japanese encephalitis (JE) among acute encephalitis syndrome (AES) cases— A hospital based study from Upper Assam, India. Int J Health Sci Res
2016; 6(5): 72-7.
Singh KP, Mishra G, Jain P, Pandey N, Nagar R, Gupta S. Co-positivity of anti-dengue virus and anti-Japanese encephalitis virus IgM in endemic area: Co-infection or cross reactivity? Asian Pac J Trop Med
2014; 7(2): 124-9.
Arai S, Matsunaga Y, Takasaki T, Tanaka-Taya K, Taniguchi K, Okabe N, et al
. Vaccine preventable diseases surveillance program of Japan, Japanese encephalitis: Surveillance and elimination effort in Japan from 1982 to 2004. Jpn J Infect Dis
Bhatia R, Dash AP, Sunyoto T. Changing epidemiology of dengue in South-East Asia. WHO South-East Asia J Public Health
Kar A, Dhanaraj M, Dedeepiya D, Harikrishna K. Acute encephalitis syndrome following scrub typhus infection. Int J Crit Care Med
2014; 18(7): 453-5.
Mittal M, Thangaraj J, Rose W, Verghese V, Kumar C, Mittal M, et al
. Scrub typhus as a cause of acute encephalitis syndrome, Gorakhpur, Uttar Pradesh, India. Emerg Infect Dis
2017; 23(8): 1414-6.
[Table 1], [Table 2], [Table 3]