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

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)

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 - 1871 in Japan (Miyake, 1964)

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)

first recorded - 700 cases and 300 deaths in 1973 In West Bengal, India, first recorded - 700 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)

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)

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)

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 - 25.1% (387/1542 sera) IgM positivity rate in CSF - 13.9% (138/989 CSF) (NHL annual data, 2017)

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

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

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

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

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

Flow chart of the study

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

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

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

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

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

Figure 5. Monthly distribution of confirmed JE cases

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

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

Figure 8. Common signs and symptoms of confirmed JE cases

Figure 9. JE vaccination status of confirmed JE cases

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

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

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

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

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

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

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 14-14-2 live attenuated vaccine is between 96% and 98% after one dose (World Health Organization, 2015c)

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)

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)

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)

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

anti-JE IgM positive in CSF - 11 cases (10.2%) 2016, CSF positivity rate - 16.2% (129 JE/792 CSF) 2017, positivity rate in CSF - 13.9% (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)

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

Thank you