several epidemiological characteristics of acute encephalitis syndrome suspected to be caused by banna virus in some provinces of vietnam

In Vietnam, in 2003 and 2005, viruses of the same group with Banna virus were isolated from patients in Thanh Hoa and Gia Lai provinces.. Participating in monitoring, diagnosis, treatmen

LIST OF ACRONYMS

AES Acute encephalitis syndromee

MAC-ELISA IgM antibody capture – enzyme linked

immunosorbent assay RT-PCR Reverse Transcription Polymerase Chain

Reaction

NIHE National Institute of Hygiene and

Epidemiology

INTRODUCTION

Acute encephalitis syndromee (AES) suspected to be caused by viruses has many different causes There is no specific treatment for this disease (except for Herpes simplex virus), with high mortality and severe neurological sequela Currently, there are about 100 virus types identified causing AES

In 1987 and 1992, Banna virus was isolated from AES and unidentified fever patients’ serum in China Banna virus was isolated

from Aedes dorsalis mosquitoes in China According to some studies

in Indonesia, Banna virus was transmitted by two types of

mosquitoes: Anopheles and Culex

In Vietnam, in 2003 and 2005, viruses of the same group with Banna virus were isolated from patients in Thanh Hoa and Gia Lai

provinces Besides, Banna virus was isolated from Culex mosquitoes

from Ha Tay (now part of Ha Noi) and Quang Binh provinces in

2002

Participating in monitoring, diagnosis, treatment and prevention

of AES suspected to be caused by Banna virus, the study “Some epidemiological characteristics of acute encephalitis syndrome suspected to be caused by Banna virus in some provinces of Vietnam” was conducted with three following specific goals:

1 Describe some epidemiological characteristics, clinical syndrome of acute encephalitis syndrome suspected to be caused by Banna virus in some provinces of Vietnam, 2002-

THESIS’ PRACTICAL IMPLICATIONS AND NEW

CONTRIBUTIONS

- New contributions:This is the first study in Vietnam identifying

incidence rate, clinical charateristics of patients with AES caused by Banna virus, infection rate of Banna virus among mosquito populations

in some provinces and bio-molecular characteristics of Banna virus

identified in Vietnam

- Practical implications: The research provides completely new

characteristics of AES caused by Banna virus data for scientific communities of Vietnam and the world Research results can be applied

in monitoring, diagnosis and prevention of AES caused by Banna virus, with strong implication in teaching as well as research and production

THESIS STRUCTURE The thesis is 113 pages long (not including references and appendixes), including 4 chapters, 30 tables, 13 figures, 1 picture Introduction is 2 pages long, Chapter 1: Overview (28 pages); Chapter 2: Subject, material and study methodologies (22 pages); Chapter 3: Study results (33 pages); Chapter 4: Discussions (22 pages); Conclusion is 3 pages long; Suggestions/proposals is 1 page; List of published work is 2 page References: 102 referenced work; 2 appendixes

Chapter I OVERVIEW 1.1 Characteristics of Banna virus

Banna virus, belonging to Seadornavirus genus, Reoviridae family, has genetic material as 12 double-stranded RNA segments The first Banna virus strain was isolated from cerebrospinal fluid of AES patients and blood samples of unidentified fever patients in Yunnan province, China; and later, isolated in different provinces from patients and mosquitoes in China, Indonesia and Vietnam 1.2 Clinical characteristics of AES caused by Banna virus

Banna virus causing acute infectious disease damaging central nervous system or unidentified fever was recorded Typical cases can

be described as following: Onset period: lasts 1-2 days but difficult to

identify when patients do not remember sudden high fever, chills,

headaches, arthralgia, and anorexia symptoms Full-fledge period: after

3-6 days, patients showing symptoms of high fever, derilium, autonomic disorder, indifference to surrounding including coma, dyspnea, photophobia, loss of appetite, nausea Symptoms of peripheral nerve injuries include paralysis, chorea Sub-acute progression period: from days 7-9 of the disease, symptoms reduced such as milder fever, stable pulse temperature, reduced central and peripheral nervous syndromee However, in this period, there are notable complications from laying for extended period such as pneumonia, sores, constipation Recovery period: patient only has mild fever, regaining conciousness, recovered appetite, remaining only sequelae depending

on the severity of the disease such as paralysis, hemorrhage, myocarditis, pericarditis, reduced memory

1.3 Epidemiological characteristics of AES caused by Banna virus Some studies in Vietnam and around the world showed that Banna virus exist in mosquitoes, there is clear evidence of Banna virus transmission among animals (pigs) from virus isolation results Moreover, Banna virus exist in some migratory birds and the migration

of these birds enables the virus to spread to other areas Mosquito has been confirmed as Banna virus transmission vector in a number of

Asian countries from isolation results of Culex tritaeniorhynchus, Culex

vishnui, Culex fuscocephalus, Anopheles vagus, Aedes albopictus and Aedes dorsalis mosquitoes

Human is the infection target of Banna virus, the study of Liu, et al (2010) of Banna virus in China from 1987 to 2007 showed that Banna virus appeared in areas with Japanese Encephalitis (JE) outbreak and

where Culex tritaeniorhynchus mosquito act as the main vector The

virus is infected through the skin from mosquito bite, once infected, the virus multiplies in the lymphatic system, virions are passively transmitted via vascular endothelia or choroid plexus, then to the central nervous system and remain in the cerebrospinal fluid Virus effectiveness peaked in the early days of the onset and decreases rapidly when neutralizing antibodies apprear After virus infection, the body may have immunizing response, neutralizing antibodies increases from day 12 of the infection, IgM antibodies have higher neutralizing effect than neutralizing antibodies IgG antibodies appear from week 3 and lower than IgM antibodies but remain for the whole lifetime 1.4 Treatment and prevention of AES caused by Banna virus Treatment: Currently, there is no specific medical treatment for AES caused by Banna virus, mainly treatment of symptoms and complications

Prevention: Banna virus is a mosquito-borne virus, a new virus discovered in the past few decades mainly in Asian region, studies of disease burden have not been mentioned, so far, there is no vaccine for the disease, the most effective prevention method to this day is to prevent mosquito as transmission vector

Research locations: Northern region (area previously Ha Tay, Bac Giang, Thanh Hoa); Central region (Quang Binh); Tay Nguyen region (Gia Lai, Kon Tum, Dak Lak, Dak Nong); Southern region (Long An, Can Tho)

- Change of mental state, or

- Nervous symptoms as meningeal signs, movement disorders

 Patients diagnosed with AES suspected to be caused by Banna virus: Cases of AES suspected to be caused by viruses detected Banna IgM antibodies from cerebrospinal fuild from positive ELISA technique

Mosquito species: Study subjects are mosquito samples collected at study sites in the Northern, Central, Southern and Tay Nguyen regions from 2001 to 2011

2.3 Contents of research

Study cases of AES: Collect samples of cerebrospinal fuilds from patients with AES suspected to be caused by viruses in Infectious diseases department in provincial hospitals Test for Banna virus IgM antibodies Investigate epidemiological characteristics, clinical symptoms of AES cases identified (+) with Banna virus antibodies, JE, ECHO30 positive isolated from retrospective medical record

Study Culex mosquito vector: Collect mosquito samples in

provinces with high number of patients with AES suspected to be caused by viruses in Northern, Central, Southern and Tay Nguyen regions, once per year in the period from March to December Mosquitoes are categorized and identified specie compositions, isolated

to identify Banna virus

Banna virus trains isolated from AES patients, from pigs and mosquitoes are genotype identified based on nucleotide gene sequence

No 12

2.4 Study methods

2.4.1 Structure of the study:

The struture of the study is cross-sectional, retrospective and prosprective epidemiologically combined with laboratory analysis

2.4.2 Determining investigation of epidemiological characteristics of AES patients

Study method and sample taking

Sample size: Take cerebrospinal fuild samples of all patients with AES suspected to be caused by viruses based on diagnostic standards above when hospitalized Choose sample based on convenient method, samples are taken in accordance with regular protocol and surveyed with pre-designed questionnaire

Study method for vector mosquito:

Sample size: Sample size is calculated based on regular protocol of NIHE; 30 household/night x 2 nights x 1 site (district/province) x 1 time/year = 60 household turn/site Mosquito investigation is conducted

at night according to regular procedure of NIHE (capture mosquitoes with CDC traps, capture female mosquitoes resting in the house and barn, from 18h00 to 22h00 in the winter and 19h00 to 23h00 in the summer

Laboratory material and testing techniques

Samples including patients' ceresbrospinal fluid samples and mosquito samples collected at study sites are analyzed in the laboratory With cerebrospinal fluid samples, use indirect ELISA technique detecting specific Banna virus antibodies With mosquito samples, use isolation technique detecting mosquito types carrying Banna virus

Isolated Banna virus strains are categorized by RT-PCR technique, collecting PCR products for product purification techniques and sequenced by sequencing machine

Data collected from the study is analyzed by bioinfomatic software such as: GraphPad, biological software DNA Star (Lasegene), MEGA 4.0

2.4.3 Data processing: Use biostatistic software Epi-info 6.04 and Stata

10 to input and process data

Chapter III STUDY RESULTS 3.1 Some epidemiological, clinical characteristics of AES caused

by Banna virus

3.1.1 Description of ratio of patients with AES caused by Banna virus Table 3.1 Identification result of Banna virus IgM antibodies in cerebrospinal fluid sample of patients with AES, 2002 – 2012

samples

Number of positive

identifying Banna virus IgM antibodies from 717 cerebrospinal fluid samples, resulting in identification of 184 (+) samples, average (+) ratio

of cerebrospinal fluid samples identified with Banna virus IgM antibodies is 25.66% (184/717), and 14.32% (184/1285) when calculated on the total cerebrospinal fluid samples of patients with AES

In 9 provinces/cities with sample specimens, identified (+) ratio ranges from 13.83% to 35.83% Province/city with highest (+) ratio is area previously Ha Tay province with 35.83%, then Ha Noi with (+) ratio of Banna virus antibodies at 34.00%; lowest identified (+) ratio with Banna virus antibodies is 13.53% in Bac Giang

According to serological surveillance, AES caused by viruses are recorded throughout the year, but cases are recorded mainly in May, June, July and August, with recorded peak of the epidemic is June with 239/717 recoded cases (33.33% of total number of cases)

Table 3.2 Ratio of cases of AES caused by viruses by age group,

In 184 cases of AES identified to be caused by Banna virus, the ratio of cases of AES caused by Banna virus in men is higher than women in all age group/

3.1.2 Clinical characteristics of patients with AES caused by Banna virus

3.1.2.1 Some signs, clinical symptoms at admission

Table 3.3 Some clinical symptoms at admission

Signs,

symptoms

BANNA virus n=103 (%)

ECHO30 virus n=43 (%)

JE virus (n=5) (%)

Ratio of BANNA and ECHO30

Ratio of Banna and

sensation are symptoms not observed from patients with AES caused

by Banna virus

When comparing clinical symptoms in patients with AES caused

by Banna virus with ECHO30 virus and JE virus, there is significant statistical difference of bulging fontanel (23.3%), stiff neck (77.45%) and Kernig sign (67,96%) appearing more than patients with AES caused by ECHO30 virus and JE virus Especially, bulging fontanel was noted primarily in patients with AES caused by Banna virus and rarely in patients with AES caused by ECHO30 virus and particularly not in patients with AES caused by JE virus

3.1.2.2 Some signs, clinical symptoms during treatment

Table 3.4 Signs, clinical symptoms after 7 days of treatment of patients infected with Banna virus comparing to infection with ECHO30 and JE

viruses

Symptoms

BANNA virus (n=103)

%

ECHO30 virus (n=43)

%

JE virus (n=59)

%

Ratio due to Banna and ECHO30 viruses

Ratio due to Banna virus and JEV

After 7 days of treatment, AES symptoms caused by different viral agents tend to reduce in all patients However, symptoms such as headache, fever (>37.5 degrees Celcius), stiff neck, Kernig sign are still observed in patients infected with Banna virus, ECHO virus and JEV Symptoms such as seizures, myalgia, joint paint, bulging fontanel, loss of sensation only observed in very few patients (1-2 patient) among the group infected with Banna virus Among them, seizures, myalgia, bulging fontanel and loss of sensation only observed from patients infected with Banna virus Joint paint only observed from patient infected with ECHO30 virus

With Kernig sign, the group infected with Banna virus still records

a high ratio of 13.59% Conversely, for the group infected with JEV this rate is 8.47% and for the group infected with ECHO30 the rate is only 4.65% With mental disorder symptom, no cases recorded in the group infected with ECHO30 virus, but the group infected with Banna virus showed a very high ratio of 55.34% while this ratio for the group infected with JEV is only 11.86%

For bradykinesia symptom, the ratio for the group infected with Banna virus is 2.91%, for the group infected with JEV is 6.78%; nausea symptom was not observed after 7 days of treatment from all patients in this study

3.1.2.3 Result after treatment of AES caused by Banna virus

Table 3.5 Average number of days of treatment of AES caused by