1. Aye Mya Thu, Moe Yee Soe, Ommar Swe Tin,
Occurrence of Japanese encephalitis cases among clinically suspected acute encephalitis syndrome patients in Myanmar
Aye Mya Thu, Moe Yee Soe, Ommar Swe Tin,
Wah Win Htike, Synn Theingi, Theingi Win Myat

2. Introduction Japanese encephalitis (JE)
a serious vector-borne viral encephalitis
Nearly 68,000 cases - occur globally each year
with approximate deaths of 13,600 to 20,400 cases
primarily occurs among children
(World Health Organization, 2015a)

3. severe clinical disease is rare, JE is a major public health challenge
because of its high epidemic potential and high case-fatality rate (30%)
(World Health Organization, 2015a)
The first clinical case of JE in Japan
(Miyake, 1964)

4. more than 3 billion people have the risk of infection
Among 24 countries in the WHO, South-East Asia and Western Pacific region have endemic JEV transmission
more than 3 billion people have the risk of infection
(World Health Organization, 2015a)
In South-East Asia, around 50,000 cases and 10,000 deaths occurred each year
(Diagana et al., 2007)

5. first recorded - 700 cases and 300 deaths in 1973
In West Bengal, India,
first recorded cases and 300 deaths in 1973
around 6,000 cases and 1,500 deaths in UP during 2005
In Bangladesh during 2013, 71 cases were reported
(World Health Organization, 2015b)
also endemic in Thailand
with 1,500 and 2,500 cases reported annually throughout the 1970s and 1980s
(Olsen et al., 2010)

6. Considering the spread of disease burden, bird migration
certain irrigation projects
animal husbandry
global warming
rice plantations
play the crucial factors to create
favorable environment for spread of disease
(Malhotra et al., 2015)

7. Myanmar involves in endemic pattern
occurs sporadically throughout the year
In 1974, the earliest case was recorded in Tarchileik, Eastern Shan State
(Ministry of Health & Sports, Myanmar, 2016)
JE outbreak had occurred in Rakhine State in 2008
(5/80 cases, 7.14%)
(Soe-Thiha-Lwin, 2008)
During 2010
anti-JEV IgM - 17/216 cases (8%)
(Khin-Yi-Oo et al., 2013)

8. During 2015 (from January to June)
seropositivity of anti-JEV IgM - 36/211 cases (17%)
During 2016
the seropositivity rate of IgM – 24.9% (388/1559 sera)
IgM positivity rate in CSF – 16.3% (129/792 CSF)
(NHL annual data, 2016)
During 2017
seropositivity rate % (387/1542 sera)
IgM positivity rate in CSF % (138/989 CSF)
(NHL annual data, 2017)

9. AIM AND OBJECTIVES
Aim
To study the occurrence of JE cases among clinically suspected acute encephalitis syndrome (AES) patients in Myanmar
Objectives
To detect probable JE cases in clinically suspected AES patients by detecting IgM in serum by Enzyme-Linked Immunosorbent Assay (ELISA)
To determine confirmed JE cases in clinically suspected AES patients by detecting IgM in CSF by ELISA

10. MATERIALS AND METHODS The study was
laboratory based cross-sectional descriptive study
from January to October 2018
at Virology section, National Health Laboratory (NHL)
Both serum and CSF samples received from 108 clinically suspected AES patients were tested

11. The collected blood - allowed to clot at room temperature (20-25C)
centrifuged and serum was collected
If serum or CSF sample
tested within 7 days, refrigerated (4C) or
tested after 7 days, frozen (-20C) (-70C was also acceptable for serum)
Anti-JE IgM was detected by InBios ELISA test kit according to the instruction of manufacturer

12. Flow chart of the procedure of ELISA test
Received blood samples of AES patients
50l of NC in duplicate for JERA
50l of PC in duplicate for JERA
50l of PC in duplicate for NCA
50l of NC in duplicate for NCA
50l of diluted serum samples
Incubated at 37˚C for 1 hour and wash
50l of JERA is added into row A-D and
50l of NCA is added into row E-H

13. 50l of HRP conjugate was added and
Incubated at 37˚C for 1 hour and wash
150l of Enwash solution was added and
Incubated at RT for 5mins and wash
75l of liquid TMB substrate was added and
Incubated at RT in a dark place for 10mins and wash
50l of stop solution was added
Optical Density OD was read with
Microplate Reader at 450nm within 5mins

14. Flow chart of the study

15. Results Clinically suspected AES cases n = 108 Anti JE-IgM
positive cases
in serum
n = 11
Probable
JE cases
in CSF
Confirmed

16. Figure 1. Distribution of Anti-JE IgM positive cases in serum (Probable JE cases)

17. Figure 2. Distribution of Anti-JE IgM positive cases in CSF (Confirmed JE cases)

18. Figure 3. Distribution of age in confirmed JE cases
all confirmed JE cases were under 15 years of age

19. Figure 4. Gender distribution of confirmed JE cases
7 cases (63.6%) were male
4 cases (36.4%) were female

20. Figure 5. Monthly distribution of confirmed JE cases

21. Figure 6. State and Regional wise distribution of confirmed JE cases

22. Figure 7. Map showing the JE affected areas in Myanmar during 2018

23. Figure 8. Common signs and symptoms of confirmed JE cases

24. Figure 9. JE vaccination status of confirmed JE cases

25. Table 1. Detection of Anti-JE IgM in serum according to days of fever
Number of probable JE cases
Percentage (%)
Day 3 2
18.2
Day 5 1
9.1
Day 6 3
27.2
Day 7
Day 9
Day 12
Day 16
Total cases 11
100

26. Table 2. Detection of Anti-JE IgM in CSF according to days of fever
Number of confirmed JE cases
Percentage (%)
Day 3 2
18.2
Day 6
Day 7
Day 9
Day 12 1
9.1
Day 13
Day 16
Total cases 11
100

27. Discussion All confirmed JE cases were under 15 years of age
consistent with
Win-Lei-Yee-Mon (2011) study
Soe-Thiha-Lwin (2008) study
NHL data (2017)
because they are active and roam outside, play around the water logged area, near the bushes and rice fields increase risk of being bitten by the mosquitoes
And also poor immune status increase severity
also vulnerable to other causes of viral encephalitis

28. Male > female
consistent with
Win-Lei-Yee-Mon (2011) study
the study of Khin-Yee-Oo et al. (2013)
report of Ministry of Health and Sports, Myanmar (2016)
males are mostly involved in the outdoor activities than female  greater exposure to JE infected mosquitoes
exposed body parts of the males increased risk of mosquito bites

29. Mainly in rainy season
accordant with
Soe-Thiha-Lwin (2008) study
Win-Lei-Yee-Mon (2011) study
NHL data 2015, 2016 and 2017
because of changes in local temperature, rainfall and humidity alter the distribution of vector
A desirable temperature and flooded habitats during the rainy season  increase larval development and abundance of vector

30. presented with fever, seizure and change in mental status
Being the diversity of signs and symptoms without very specific clinical features for JE infection
it is vital to differentiate it from other causes of AES by proper laboratory diagnosis

31. Among 11 JE cases, only 1 case (9.1%) was vaccinated with JE vaccine
In such vaccinated cases, JE specific IgM antibody in CSF is preferred
Among JE vaccinated cases, JE infection can occur
may be due to infected with different genotype or
the vaccine efficacy of SA live attenuated vaccine is between 96% and 98% after one dose
(World Health Organization, 2015c)

32. immense epidemiological value if paired serum sample were collected
The most cases anti-JE IgM were seropositive in serum on day 6 followed by day 3 and day 7 of fever
immense epidemiological value if paired serum sample were collected
even though it is not always possible
JEV seropositivity rate - 60% within 1-4 days
all serum samples are not positive until day 13 of illness
(Kumar & Mani, 2017)

33. Anti-JE IgM was detectable in CSF as early as day 1
The earliest day of detecting anti-JE IgM in CSF was day 3 followed by 6th, 7th and 9th day of fever
Anti-JE IgM was detectable in CSF as early as day 1
JEV IgM in CSF is more effective > serum samples in the early days of the illness because IgM in CSF is intrathecal production of CSF leukocytes
(Endy & Nisalak, 2002)
IgM - 90% of CSF samples collected 1-4 days
all CSF samples were positive by day 7 of illness
(Kumar & Mani, 2017)

34. Anti-JE IgM was detected in serum of 11 cases (10.2%)
consistent with
Win-Lei-Yee-Mon (2011) study
Soe-Thiha-Lwin (2008) and NHL (2017)
Mustapa et al (2013)

35. High seropositivity rate may be due to cross infection or concurrent non-CNS infection of JE virus or vaccine derived antibodies
Cross-infection of JE infection should be considered in the regions where two or more flaviviruses co-circulate such as Dengue (IgM is detected in serum, dengue IgM antibody is need to be excluded)
Being the vaccine preventable disease, vaccine-derived antibody should be considered if diagnosis is based on serum alone

36. anti-JE IgM positive in CSF - 11 cases (10.2%)
2016, CSF positivity rate % (129 JE/792 CSF)
2017, positivity rate in CSF % (138 JE/989 CSF)
(NHL data, 2017)
declined in 2018
may be due to mass vaccination programme during 2017
Infection with JEV without encephalitis or vaccinated patients result in increased IgM in the sera but not in the CSF
(Burke et al., 1985)

37. Conclusion Occurred mainly in children More in male population
Mainly in rainy season
Declined in 2018 may be due to vaccination program
detection of anti-JEV IgM in CSF is beneficial to confirm specific JEV infection as well as useful in vaccinated patients
And early detection of IgM antibody in CSF can help to some extent in proper treatment and prevent neurological deficit and sequelae

38. Thank you