CHAPTER 1: ITRODUCTION

The selection of
healthy and safe donors has always been a serious concern for blood transfusion
centers worldwide to reduce transfusion-transmitted infections (TTI) for more
safety blood supply. Hepatitis B virus is the most frequent
transfusion-transmitted viral infection (Candotti & Allian, 2009).

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In Malaysia 2.4
million people are carriers of hepatitis B (Malaysian Liver Foundation, 2005).
Hepatitis B vaccination has been implemented in Malaysia since 1989 (Raihan et
al, 2017).  According to a study that
analysed the trend and estimation of Hepatitis B infection cases in Malaysia
(2003-2012), the highest number of Hepatitis B infection cases was reported in
Sabah (Rajamoorthy et al, 2016).

Hepatitis B
remains a major risk of transfusion transmitted viral infection. Hepatitis B
virus (HBV) infection is a major cause of liver disease such as cirrhosis and
hepatocellular carcinoma (Ng et al, 2013). To minimize the risk of the
transmission of hepatitis infection through route of transfusion, all donated
blood should screened for evidence of presence of infection prior to the
release of blood and blood components for clinical use.

The serology of
hepatitis B virus is complex. Different serological marker will develop during
the course of infection, including hepatitis B surface antigen (HBsAg) and
hepatitis B core antibody (anti-HBc). Other than that, HBV DNA can be detected
in the majority of hepatitis B infection cases. Hepatitis B surface antigen
(HBsAg) test are recommended by world health organization (WHO) for all donated
blood.

 

 

 

1.1  Objective of the study

This study
presents data on the prevalence rate of Hepatitis B infection among blood donor
in Hospital Queen Elizabeth II from September 2017 to December 2017. The aim of
study is determine the prevalence of Hepatitis B virus infection among blood
donors and their demography in department of transfusion medicine, Hospital
Queen Elizabeth II.

·        
Determine the infected donor background i.e. gender,
age, donor status, birth year- born before mass hepatitis B vaccination (before
1989) or bon in the year of 1989 and after, occupation, distribution of
population from different ethnicity – improving donor selection.

·        
Determine the number of confirmed Hepatitis B cases
among blood donor – Monitoring safety blood supply and donor screening
effectiveness.

 

 

 

 

 

 

 

 

 

 

 

CHAPTER
2: LITERATURE REVIEW

2.1       Hepatitis
B virus (HBV)

            2.1.1    Agent

Hepatitis
B remains a major chronic viral illness worldwide. The causative agent of
hepatitis is Hepatitis B virus (HBV) which is an enveloped DNA virus. Hepatitis
B virus (HBV) is a member in the virus family Hepadnaviridae (Shepard et al,
2006). Hepatitis B virus is transmissible by the parenteral route and through
transfusion of blood or blood product. (Ocama et al, 2005). The infectious
virus is 42-47 nm in diameter and the concentrations of HBV circulate in blood
as high as 108 virions per ml of blood. (Shepared et al, 2006).
Hepatitis B virus is double –stranded DNA virus with the inner core of the
virus contained Hepatitis core antigen HBcAg, Hepatitis B envelope antigen
HBeAg and DNA polymerase with reverse-transcriptase. Hepatitis B surface
antigen HBsAg is found on the surface of hepatitis B virus. (Shepard et al,
2005). Hepatitis B virus travels to the liver via the bloodstream and
replicates in hepatocytes. In hepatocytes, the virus uncoated and enter the
nucleus as covalently closed circular DNA. A template of RNA was produce. RNA
are subsequently encapsulated and transcribed in the cytoplasm of cell.
Positive DNA strands that acquire the hepatitis B envelope antigen HBsAg and
leave the cell. HbsAg in form of spheres and rods are secreted.

Figure 1: Structure of Hepatitis B virus

            2.1.2    Disease and transmission

“Hepatitis”
is inflammation of the liver, when inflame or damage occur in the liver, the
function of liver can be affected. Hepatitis is most caused by a viral
infection. Most common types of viral hepatitis are Hepatitis A, Hepatitis B
and Hepatitis C (Centers for Disease Control and Prevention, CDC, 2016). Infection
with Hepatitis B virus can be a serious liver disease. Person infected with HBV
may also develop chronic infection and has a higher probability of progressing
to liver cirrhosis and hepatocellular carcinoma. (Shepard et al, 2006). According
to World Health Organization (WHO), transmission of HBV can through childbirth
and from family member to a child in the early childhood, this route is
vertical transmission. Another route is horizontal transmission, such as
through occupation exposure, sexual contact, transfusion and the use of
infected needles. (WHO, 2009)

 

 

 

 

2.2       Hepatitis
in Malaysia

2.2.1    Current
situation

Malaysia
was an intermediate endemicity country, the Hepatitis surface antigen (HBsAg)
prevalence is 5% – 7% (Raihan et al, 2017). According to Malaysian Liver
Foundation, 2.4 million people are carriers of hepatitis B in Malaysia. An
estimated 1 million people are chronically infected with HBV in Malaysia
(Raihan et al, 2017). Sabah was the highest number of hepatitis B infection
cases reported, followed by Pahang and Wilayah Persekututan, perlis was the
lowest cases reported (Rajamoorthy et al, 2016). According to the study of
Rajamoorthy et al 2016, the estimated Hepatitis B infection cases indicate the
number of case and incidence rates will increase in future. Public awareness
program are needed to lower the incidence of hepatitis B infection in the
general population.

 

2.2.2    Expanded
Program on Immunization (EPI)

Hepatitis
B vaccine was available in 1982. Hepatitis B vaccine are manufactured using
recombinant DNA technology. Malaysia National EPI was implement in 1989 (Ng et
al, 2012). In Malaysia, Hepatitis B vaccination of all newborn infants has been
started since 1989 (Raihan et al, 2017). Through the vaccination program in
1989, the rate of infection has been successfully reduced (Rajamoothy et al,
2016). Malaysia started routine hepatitis B immunization using three-dose
regime. The first dose given shortly after birth, second dose at 1 month, and the
third dose at 5 months old (Malaysia Paediatric Association MPA, 2005).
Hepatitis B vaccination to prevent newborn infants from infected of HBV (Ministry
of Health Malaysia, 2004).

 

            2.2.2
   Previous study            

An
earlier study in 1997, the prevalence of hepatitis B among the healthy
volunteers in Malaysia was 5.2% (Malaysian Liver Foundation, 2005). A study by
Yousuf et al 2007, investigated the prevalence and trends in hepatitis B
infection among blood donors in Transfusion Medical Unit at Hospital Universiti
Sains Malaysia, Kelantan (Yousuf et al, 2007). The study shows that the
prevalence of hepatitis B infection in regular and first time donors was 1.1%. According
to another study in the Faculties of Medicine and Dentistry, University of
Malaya, the HBsAg prevalence among the 2923 students was 0.62%. The prevalence
rate for those born before 1989 was 1.08% and 0.2% among those born after 1989
(Ng et al, 2013).

2.3       Screening
Assay

            2.3.1
   Types of assay

Various
types of assay have been developed for use in blood screening. The main types
of assay used for blood screening are: Enzyme Immunoassays EIA,
Chemiluminescent Immunoassays CLIA / CMIA and Nucleic Acid Amplification Technology
NAT assays.

 

            2.3.2    Enzyme Immunoassays and Chemiluminescent Immunoassays

Enzyme
and chemiluminescent immunoassays are currently most commonly used assays for
blood screening (Candotti et al, 2017). Enzyme and chemiluminescent
immunoassays are suitable for the screening of large number of samples and
require a range of specific equipment such as the ARCHITECT i200SR immunoassay
analyzer.

            Figure
2: ARCHITECT i2000SR immunoassay analyzer

            Blood samples were taken for
serological testing. HBsAg was tested using the

ARCHITECT
i2000SR immunoassay analyzer. The ARCHITECT HBsAg assay is a two-step
immunoassay, using chemiluminescent microparticle immunoassay (CMIA)
technology, with flexible assay protocols referred to as Chemiflex, for the
quantitative determination of HBsAg in human serum and plasma. In the first
step, sample and anti-HBs coated paramagnetic microparticles are combined.
HBsAg present in the sample binds to the anti-HBs coated microparticles. After
washing, acridinium-labeled anti-HBs conjugate is added in the second step.
Following another wash cycle, Pre-Trigger and Trigger Solutions are added to
the reaction mixture. The resulting chemiluminescent reaction is measured as
relative light units (RLUs). A direct relationship exists between the amount of
HBsAg in the sample and the RLUs detected by the ARCHITECT i* System optics.
The concentration of hepatitis B surface antigen in the specimen is determined
using a previously generated ARCHITECT HBsAg calibration curve. If the
concentration of the specimen is greater than or equal to 0.05 IU/mL, the
specimen is considered reactive for HBsAg.

 

            2.3.3    Nucleic Acid Amplification Aechnology Assays

Nucleic
acid amplification technology assays detects the presence of viral nucleic
acid, DNA and RNA (Candotti et al, 2017). A specific DNA or RNA segment of the
virus is amplified. Through the amplification step, amount of the specific
segment of the viral will increase to an easily detectable level. The presence
of viral nucleic acid indicates the presence of the viral itself. Detection of
HBV DNA reduce the risk of HBV transmission through transfusion of infected
blood donated during the acute window period: HBsAg Test are negative, but HBV
DNA is positive (Biswas et al).

 

2.3.4    Screening

Serology
marker – Hepatitis B surface antigen

Viral
nucleic acid – HBV DNA

Several
serological markers develop during the course of Hepatitis B infection. HBsAg
test is the first-line of blood screening for HBV (Candotti & Allain,
2009). HBsAg is the first serological maker of HBV infection. HBV produces an
excess of hepatitis B surface antigen (HBsAg) (Ocama et al, 2005), also known
as Australia antigen, which can be detected in the blood of infected
individuals. The level of the virus itself are variable, a study indicate that
some individuals that have low levels of detectable Hepatitis B surface antigen
may cause the HBsAg test negative (Gerlich et al, 2007). HBsAg test are
recommended as minimum standard for blood screening (WHO). HBsAg persists
during this acute phase and clears late in the convalescence period. Failure to
clear HBsAg within six months indicates a chronic HBsAg carrier state. HBsAg
assays are used to identify persons infected with HBV and to prevent
transmission of the virus by blood and blood products as well as to monitor the
status of infected individuals in combination with other hepatitis B
serological markersIn an infected individual, HBV DNA normally present. HBV DNA
can be detected in most of the cases, although in HBsAg negative phases of
infection the HBV DNA are generally low “window period”. Detection of HBV DNA
(NAT) reduces the risk of HBV transmission through the transfusion of infected
blood during the window period (Condotti et al, 2017).

 

 

 

 

 

 

 

 

 

 

 

 

CHAPTER 3: MATERIALS AND METHODS

3.1       STUDY
DESIGN

This was a
retrospective cross-sectional study on blood donors to examine the incidence of
hepatitis B infection in the blood donors at the Transfusion transmittable
infection (TTD) screening center for west coast Sabah, namely Queen Elizabeth
Hospital II (QEH II).

 

3.2       LOCATION
AND PERIOD OF STUDY

This study
was conducted at Transfusion Medicine Department, QEH II, Kota Kinabalu Sabah..
The study started from September 2017 to December 2017

 

3.3       STUDY
VARIABLES

3.3.1    Independent Variables

?       Sociodemographic factors of age,
sex, race, occupation and age

?       Birth year, born before mass
hepatitis B vaccination (before 1989) or born in the year of 1989 and after.

 

3.3.2    Dependent Variables

?       Hepatitis B serology screening
result

?       Hepatitis B serology confirmation result

 

 

 

3.4       STUDY
POPULATION

A Total of
2400 blood donors who donated in the west coast of Sabah were included in this
study.

3.5       INCLUSION
AND EXCLUSION CRITERIA

            3.5.1    Inclusion Criteria

                               
i.           
Eligible
blood donor based on National Blood Center Guideline

                             
ii.           
Age
from 17 to 65 years old

                           
iii.           
Hemoglobin
more than 12.5 g/dl

                           
iv.           
Normal
blood pressure

                             
v.           
Body
weight more than 45 kg

                           
vi.           
Whole
blood and apheresis blood donation

 

3.5.2 Exclusion Criteria

                               
i.           
Donor
not eligible for donation

                             
ii.           
Weak
positive screening result

                           
iii.           
Incomplete
demographic data in the blood donation form

                           
iv.           
Incomplete
hepatitis B serology result in the laboratory information system

 

 

 

 

 

3.6       SAMPLE
SIZE CALCULATION

Sample size
calculated based on sample size calculation formula (Pourhoeinholie et al., 2013) as following:

            n
= Z2 P (1-P)

                        d2

 

n
= sample size

Z = confidence interval

P = proportion of prevalence/incidence

d = precision

The
proportion was set based on the incidence of hepatitis B infection of 0.62% in
the previous study by Ng et al.
(2013), confidence interval was set at 95%, and precision was set at 0.05. The
calculated sample size was 2400.

3.7       SAMPLING
METHOD

Convenience
sampling method was used to collect data from blood donation record and
laboratory information system in the Transfusion Medicine Department, QEH II,
Sabah.

3.8       DATA
COLLECTION

Blood
donors’ demographic data and hepatitis B serology result were retrospectively
collected based on blood donation form and QEH II lab information system namely
Darah Link.

 

 

3.9       HEPATITS
B SEROLOGY TESTING FOR BLOOD DONORS

·        
Blood samples are collected from
donor.

·        
blood samples are sent to transfusion
microbiology laboratory.

·        
Perform screening test (HBsAg), using
the ARCHITECT ABBOTT i2000SR immunoassay analyser.

·        
blood samples that tested HBsAg
positive(reactive) will then proceed with confirmatory test (neutralization
test), using the ARCHITECT ABBOTT i2000SR immunoassay analyser.

·        
Information of donor and the outcome
are recorded.

 

3.10     STATISTICAL
ANALYSIS

All statistical data were analyzed by SPSS (Statistical
Package for the Social Sciences) Version 24.0 software (IBM, New York, United
States). The numerical descriptive results were presented as mean ± standard
deviation for normally distributed data, median for non normally distributed
data and percentage (%) for prevalence results. Multiple linear regression was
used to analyze relationship between demographic factors and hepatitis B
infection among blood donors. Meanwhile chi square test was used to analyze birth
year in relation to hepatitis B infection.

 

3.11     ETHICAL
ISSUES

Ethical
approval was granted by CRC QEH II, Kota Kinabalu Sabah and department approval
from JPT head of department

 

3.12     STUDY
FLOW CHART

Study population

 

Exclusion                                                                                            Inclusion

 

Sample size

 

Demographic Data
collection from donation form

 

Data collection from Laboratory information system

(Hepatitis B screening
(HbSAg) result)

 

Non
Reactive                                                                                      Reactive

 

Hepatitis
B confirmation (Neutralization test)

 

Data
collection from Laboratory Information system

 

Statistical
Analysis

 

Result
Reporting

 

 

References

1.      Candotti
D, Allain JP. Transfusion-transmitted hepatitis B virus infection. Journal of
hepatology 51(2009) 798-809.

2.      Malaysian
Liver Foundation. Hepatitis B: fact sheet for doctors. (2005)

3.      Raihan
R, Mohamed R, Hassan M, Said R, Chronic Viral Hepatitis in Malaysia: “where are
we now?”. Euroasian Journal of Hepato-Gastroenterology, January –June
2017;7(1): 65-67.

4.      Rajamoothy
Y, Taib NM, Rahim KA, Munusamy S. Trends and estimation of hepatitis cases in
Malaysia, Malaysian Journal of Public Health Medicine 2016, 16 (1): 113-120.

5.      NG
K P, Ngeow Y F, Rozainah K, Rosmawati M. Hepatitis B seroprevalence among
University of Malaya students in the post-universal infant vaccination era, Med
J Malaysia vol 68 NO  2 april 2013,
144-147.

6.      World
Health Organization. Weekly Epidemiological Record 2009;84, 405-420.

7.      Shepard
C W, Simard E P, Finelli L, Fiore A E, Bell B P. Hepatitis B Virus infection :
Epidemiology and vaccination. Johns Hopkins Bloomberg School of Public Health,
Epidermiology reviews 2006; 28: 112-125.

8.      Ocama
P, Opio C K, Lee W M. Hepatitis B virus infection: current status. The America
Journal of Medicine (2005) 118, 1414.e15-1413.e22.

9.      Centers
for Disease Control and Prevention. Hepatitis B. June 2016. Available from: http://www.cdc.gov/hepatitis
.

10.  Ministry
Of Health Malaysia. Clinical Practice Guidelines (childhood immunization),
MOH/P/PAK/81/04(Gu). December 2004. Available from http://medicaldev.moh.gov.my/casemix/files/Handbook%20For%20Recording%20Diagnosis%20and%20Procedure%20-%20Sep%202010.pdf.

11.  Malaysian
Paediatric Association (MPA). Hepatitis B immunization in current practice. 24
may 2005. Available from: http://mpaweb.org.my/article.php?aid=23

12.  Yousuf
R, Raoiaah M, Ahmed SA, Rosline H, Salam A, Selama S, Roshan TM. Trends in
Hepatitis B virus infection among blod donors in Kelantan, Malaysia: A
Retrospactive Study. Universiti Sains Malaysia. Southeath Asian J Trop Med
Puclic Health. vol 38no. 6 november 2007, 1070-1074.

13.   Candotti D, Boizeau L, Laperche S. occult
hepatitis B infection and transfusion-transmission risk. Transfusion Clinique et Biologique 24 (2017) 189-195.

14.  Biswas
R et al. comparative sensitivity of HBV NAT and HBsAg assays for detection of
acute HBV infection. Transfusion, 2003, 43(6): 788-798.

15.  Gerlich
wh et al. HBsAg non-reactive HBV infection in blood donors. Transmission and
phatogenecity. Journal of Medical Virology, 2007, s32-s36.

16.  Pourhoseingholi
MA, Vahedi M, Rahimzadeh M. Sample saiz calculation in medical studies.
Gatroenterol Hepato Bed Bench 20136(1); 14-17.

17.   

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