|Year : 2017 | Volume
| Issue : 4 | Page : 529-534
Aetiological study of viruses causing acute encephalitis syndrome in North West India
Jitendra Kumar Tiwari1, Bharti Malhotra1, Aradhana Chauhan1, Hemant Malhotra2, Pratibha Sharma1, Farah Deeba1, Khushbu Trivedi1, Anjenya M Swamy1
1 Department of Microbiology and Immunology, Advanced Basic Sciences and Clinical Research Laboratory, ICMR Grade 1/ DHR State, Sawai Man Singh Medical College, Jaipur, Rajasthan, India
2 Department of Medicine, Sawai Man Singh Hospital, Jaipur, Rajasthan, India
|Date of Web Publication||1-Feb-2018|
Dr. Bharti Malhotra
Department of Microbiology and Immunology, Aadvanced Basic Sciences and Clinical Research Laboratory, (ICMR Grade – I Viral Diagnostics and Research Laboratory), Sawai Man Singh Medical College, Jaipur - 302 004, Rajasthan
Source of Support: None, Conflict of Interest: None
Context: Acute encephalitis syndrome (AES) is a serious public health problem, caused mainly by viruses. However, the profile of viruses causing AES in Rajasthan is not well characterised. Aims: The present study was undertaken to identify the viruses causing AES and develop diagnostic algorithm so as to help in improved diagnosis, treatment, prevention and control. Settings and Design: The present study is a hospital-based descriptive, observational study. Samples were processed at Grade-1 DHR/ICMR Viral Research and Diagnostic Laboratory at SMS, Jaipur. Subjects and Methods: Cerebrospinal fluid (CSF) samples were processed for IgM antibody detection by enzyme-linked immunosorbent assay (ELISA) for mumps virus (MPV), measles virus (MV), Rubella virus (RV), Japanese encephalitis virus (JEV), West Nile virus (WNV) and Dengue virus using commercial kits. Nucleic acid was extracted from CSF using automated extraction system. Real-time polymerase chain reaction was done using specific primers and probes for Herpes simplex virus (HSV), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) and enterovirus (EV). Statistical Analysis Used: Statistical analysis was done using ANOVA. Results: Among 3088 patients, 702 (22.7%) patients were positive for one or more viruses. HSV (261;8.45%) was the most common followed by EBV (173;5.6%), VZV (97;3.1%), CMV (68;2.2%), EV (32;1.03%), MPV (27;0.9%), DV (28;0.9%), MV (19;0.6%) and RV (6;0.2%). Conclusions: AES occurred sporadically in Rajasthan, samples should be tested first for herpes group of viruses followed by EV or/and for arboviruses depending on season or measles, mumps and RVs in children.
Keywords: Acute encephalitis syndrome, cerebrospinal fluid, enzyme-linked immunosorbent assay, herpes simplex virus, Japanese encephalitis, polymerase chain reaction
|How to cite this article:|
Tiwari JK, Malhotra B, Chauhan A, Malhotra H, Sharma P, Deeba F, Trivedi K, Swamy AM. Aetiological study of viruses causing acute encephalitis syndrome in North West India. Indian J Med Microbiol 2017;35:529-34
|How to cite this URL:|
Tiwari JK, Malhotra B, Chauhan A, Malhotra H, Sharma P, Deeba F, Trivedi K, Swamy AM. Aetiological study of viruses causing acute encephalitis syndrome in North West India. Indian J Med Microbiol [serial online] 2017 [cited 2019 Dec 10];35:529-34. Available from: http://www.ijmm.org/text.asp?2017/35/4/529/224424
| ~ Introduction|| |
Acute encephalitis syndrome (AES) is defined as the acute-onset of fever and a change in mental status and/or new-onset of seizures in a person of any age at any time of the year. AES is an emerging public health problem, claiming thousands of lives  and the disease most commonly affects children and young adults and can lead to considerable morbidity and mortality.
Although bacteria, viruses and protozoan parasites may cause encephalitis, among these the viruses are the most common and important cause of encephalitis. Cases of AES have been reported from many states of India, but the aetiological agent has been identified in only 20%–30% cases. Among all, viral encephalitis that is encountered in India, JE appears to be of greater significance during outbreaks as well as in sporadic cases. Herpes group of viruses, enterovirus (EV), measles virus (MV), mumps virus (MPV) and Rubella virus (RV) also constitute significant numbers in sporadic and outbreak cases in India. However, the profile of agents causing AES varies from place to place. Clinically and neurodiagnostic tests can usually establish the presence of encephalitis but do not necessarily establish the aetiologic cause, which often remains unknown.
There is only one report from Rajasthan on aetiology of AES cases, where only HSV was tested. Rajasthan has its own geographic characteristics, and till recently, the state has been considered to be non-Japanese encephalitis (JE) area. With better irrigation and paddy cultivation in the state, the vector for Japanese encephalitis virus (JEV) is available, and the risk has consequently increased. The present study was undertaken to identify the viruses causing AES in patients. These results are likely to help in proper diagnosis, treatment, planning prevention and control strategies and in developing diagnostic algorithm in our state. The findings of our study are also likely to help clinicians in the management of AES.
| ~ Subjects and Methods|| |
The present study is a hospital-based descriptive, observational study.
This study was carried out on patients admitted to SMS and attached group of hospitals, Jaipur.
The study period was November 2011 to December 2016.
Place of study
Samples were processed at Virology Laboratory, Jaipur. Institutional Ethics Committee permission was obtained before initiating the study.
The minimum sample size for this study was calculated as 375 with an estimated 0.42 true proportion rate  at a confidence level of 95% assuming desired precision rate of 5% using the formula from EpiTools epidemiological calculators n = (Z2 × P [1 – P])/e2 (where Z is value from standard normal distribution corresponding to desired confidence level (Z = 1.96 for 95% confidence interval), 'P' is expected true proportion and 'e' is desired precision (5%). However, to study the trends, all samples meeting criteria and tested over 5 years were included in the study.
Patients presenting with fever of <15 days duration with or without altered sensorium, seizures, headache, vomiting, abdominal pain and paralysis.
Patients suffering from bacterial meningitis, head injury, simple febrile seizures, seizure disorder, heat stroke, metabolic disorders and papilloedema.
Sample collection and transportation
A total of 3088 cerebrospinal fluid (CSF) samples were collected from patients suspected of AES. All samples were received with prior informed consent from the patients/parents/guardians. Detailed pro forma was filled; samples were labelled and sent to lab. Each sample was processed in accordance with established SOPs in the laboratory.
IgM antibody detection by enzyme-linked immunosorbent assay (ELISA) for MPV, MV, RV, JEV, West Nile virus (WNV) and Dengue virus (DV) were done in patient CSF using commercial ELISA kits [Table 1] as per manufacturer's instructions.
|Table 1: Enzyme linked immunosorbent assay kits used for the detection of viruses causing acute encephalitis syndrome|
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Polymerase chain reaction-based detection
Nucleic acid extraction
Briefly, nucleic acid (NA) was extracted by an automatic nucleic acid extractor (NucliSENS easyMAG-Biomerieux) according to the manufacturer's instructions. A total of 300 μl of each CSF sample was loaded in the instrument for on-board lysis, and 60 μl of magnetic silica were added to each specimen after 10 min incubation and mixed well. The NA was extracted in a final volume of 100 μl to be used in real-time polymerase chain reaction (PCR) and an aliquot was stored at 80°C for further use.
Standardisation of real-time polymerase chain reaction
Standardizations were done for real-time PCR by using the positive controls. The cell culture supernatant of Herpes simplex virus (HSV), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV) and EV were used as positive control which was provided by Manipal Center for Virus Research, Kasturba Medical College (KMC), Manipal.
Amplification and detection of viruses was done on Light Cycler 480 instrument (Roche) as per manufacturer's instructions using their master mix and primer probes as per previously published sequences as given in [Table 2].,,, Briefly for DNA viruses 10 μl of Light Cycler 480 Probes Master ready mix (Roche), 2 μl of primer probe mix [Table 2], 3 μl of PCR grade water and 5 μl of DNA were added, cycling profile for PCR was; preincubation at 95°C for 10 min followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 55°C for 30 s and extension at 72°C for 1 s.
|Table 2: Customized primers and probes for the detection of viruses causing acute encephalitis syndrome using real time polymerase chain reaction|
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For the detection of RNA viruses, 20 μl RNA Master Mix (Roche) was used comprising of 0.3 μl enzyme blend, 3 μl reaction buffer plus 2 μl primer-probe mix, 9.7 μl PCR grade water and 5 μl RNA. Reverse transcription was done at 50°C for 10 min. The cycling profile of PCR was initial denaturation at 95°C for 30 s, followed by 45 cycles of denaturation at 95°C for 1 s, annealing at 55°C for 20 s and extension at 72°C for 1 s. Each assay was run using a positive control and a negative control.
| ~ Results|| |
Virus identification, serologically or by molecular testing, was possible in 22.7% of AES suspected patients. The positivity for viruses in male (27.62%) and female (26.85%) was not statistically significant (P = 1.00). The positivity in children (41.10%) was significantly higher (P = 0.0001) as compared to adults (21.51%) [Table 3].
|Table 3: Positivity for viruses in patients suspected of acute encephalitis syndrome|
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Most common virus identified in AES suspects in our study was HSV (8.4%) followed by EBV (5.6%), VZV (3.1%), CMV (2.2%), EV (1.0%), MPV (0.9%), DV (0.9%), MV (0.6%) and RV (0.2%). None of the samples were found positive for JE and WNV. Coinfection was seen in nine (0.3%) patients with two viruses each, HSV and VZV in four (0.1%) patients, and HSV and EBV in five (0.16%) patients. Age- and sex-wise distribution for different viruses in positive cases is given in [Table 4]. Positivity in males was higher than females for HSV and EBV but for other viruses, it was either similar or slightly more in males. The HSV, VZV, EBV, CMV, EV and DV were found more in adults compared to children whereas MPV, MV and RV were found only in children [Table 4].
|Table 4: Sex and age group wise occurrence of different viruses in positive cases|
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The most common clinical presentation was fever followed by altered sensorium, seizures, headache, vomiting and abdominal pain [Table 5]. No seasonal variation was observed for all viruses except DV in the present study [Figure 1].
|Table 5: Clinical profile of acute encephalitis syndrome in suspected patients (n=3088)|
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| ~ Discussion|| |
There is a wide variation in the viral aetiological agents causing CNS infections across the globe and even in the same continent, and a country. For better management of patients, it is important to know the profile of viruses causing AES in the given area so that immediate action can be taken before laboratory results are available.
In the present study, viral confirmation of AES was documented in only 702 (22.73%) patients. Similarly, other Indian studies have reported positivity from 17.20% to 29.81%, at Odisha (17.2%), at Uttar Pradesh (21.83%), at West Bengal (29.81%) but other studies from India reported higher positivity from 50% to 71.92% at Uttar Pradesh (58.36%), at Karnataka (60%) and at New Delhi (71.92%) Positivity from other countries ranged from 4% to 69% in AES suspected patients.,,,,
The positivity varies from place-to-place and depends on the number of viruses included in the detection panel; samples included in the study; detection techniques; geographical location; occurrence of any epidemic/outbreak during the study period, etc.
Among the positive cases, Herpes group of viruses were the most common (84.24%) causative agent of AES in the present study. Wide variation in positivity has been reported for Herpes group in India from 13% to 59.10% at Karnataka, Eastern India, and UP , and abroad also from 7.19% to 55%.,,,,, Herpes group remains the most common causative agent in acute sporadic encephalitis cases in the developed world and in India too. All these viruses have specific antiviral therapy available (acyclovir), and early diagnosis can alert the clinician for timely initiation of specific therapy and prevent the high mortality and morbidity which can occur in absence or delay in treatment.
Additional viruses identified in positive cases in our study were EV (4.50%), DV (3.94%), MPV (3.80%), MV (2.67%) and RV (0.84%). EV-71 is an important emerging encephalitogenic virus. Similar to our study, low positivity was observed to these viruses from 0.19% to 2.6% from Eastern India, but higher positivity to all these viruses from one study from Western UP (EV 71 in 42.1%, MV in 21.1%, and MPV in 10.5%). Whereas EV was not reported in another study from UP. On the other hand, very high positivity was reported for EV 71 (35.1%) from New Delhi.
Before the start of nationwide, MMR vaccination programs, mumps and measles were most common causative agents detected in encephalitis suspected children. From developed countries, these organisms have almost disappeared; however, they continue to be most common causative agents of AES in developing countries like India, probably because of suboptimal immunisation rates.
In the present study, none of the samples were found positive for JEV and WNV. Similarly, other studies from India and abroad also did not report JEV, from Western UP, Andhra Pradesh, Taiwan, and Malaysia.
JEV is a big public health problem in India causing frequent outbreaks and high mortality ,, but Rajasthan belongs to non-JE endemic area, and even now, no case was reported in our study also. However, there may be some limitations in our study, the kit used of JE was from Inbios and not from NIV moreover majority of our samples belonged to about 22 districts of Rajasthan. There is a need to systematically test samples from all the districts of Rajasthan, especially those where there has been change in ecosystem due to buildings of dams and canals and presence of JE vector has been reported.
In the present study, co-infection was seen in nine samples with two viruses each HSV and VZV in four samples and HSV and EBV in five samples. Similarly, other studies also reported coinfection. A study from Sweden  detected coinfection in two samples with two viruses each; HSV and EBV in one sample and VZV and EBV in the other sample. Authors from Italy  reported coinfection in four samples with two viruses each HSV1 and VZV in two samples, HSV1 and HSV2 in one sample and EBV and HSV2 in one sample. Herpes group of viruses may be positive due to reactivation of latent viruses in the setting of a primary central nervous system infectious agent.
In the present study, positivity for AES was found in both children and adults, but it was significantly higher in children (P = 0.0001) as compared to adults. Similar findings have been reported in Lucknow, UP, which also showed higher positivity in children. However, many studies conducted for AES in India were limited to children only.,,,,
The MPV, MV and RV were found positive in children only; whereas other viruses occurred both in children and adults but predominantly in adults. MMR has been reported to mainly affect older teens and young adults, which include the unvaccinated population; this population has probably not developed immunity through exposure to virus because of decreased circulation of the virus after the implementation of childhood immunisation program.
In the present study, the number of males enroled was higher but positivity in both sexes was similar. Although none of the CNS infections are known to have a male predominance, this apparent male predominance can be attributed to the male-dominated social system where a sick male gets preferential medical attention.
In the present study, post-monsoon presence of DV was observed which coincides with seasonal increase in DV infection post-monsoon. However, no distinct seasonal variation was observed for other viruses. Similarly, Jain et al. from UP  observed a definite seasonality for DV and JEV in monsoon and post-monsoon seasons. While from Gorakhpur, UP  reported AES cases throughout the year, but the incidence peaked in post-monsoon period. The seasonal trends depend on the predominant agent involved in each study. Fever with altered mental state was the most common symptom complex in our patients as reported earlier.
The diagnostic algorithm which can be followed in our setting is as follows; first panel HSV, EBV and VZV; second panel CMV, EV and DV; third panel MPV, MV and RV; fourth panel JE, WNV and other viruses as per clinical history.
| ~ Conclusions|| |
AES occurred sporadically in Rajasthan, samples should be tested first for herpes group of viruses followed by EV or/and for arboviruses depending on season or measles, mumps and RVs in children.
We are grateful to the Indian Council of Medical Research (ICMR), New Delhi, for financial support for establishing Grade 1 Virology laboratory and DHR for establishing State DHR VRDL.
Financial support and sponsorship
This study was financially supported by the ICMR, New Delhi.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]