CHAPTER during the short period between infection and

CHAPTER 1

 

INTRODUCTION

 

1.1           
Study
background

Human
Immunodeficiency Virus (HIV) is a causative agent of Acquired Immunodeficiency
Syndrome (AIDS) and was first described in 1981, when previously healthy young
adults mainly men living in urban areas of the United States – began falling
ill with opportunistic infection previously unknown among this age group. HIV
belong to the family of retroviruses and it can be transmitted through sexual
transmission, blood transfusion, intravenous drug use and maternal vertical
transmission.

Human
Immunodeficiency Virus (HIV) infection is remains as an important public health
in most of the country. In 2016, UNAIDS reported that trend in new HIV
infection among adult (aged 15 years and older) had remained static between
2010 and 2015 at 1.9 million new infection per year 2015 range of 1.7 million
– 2.2 million.(Nations and Families n.d.).
In Malaysia, there are 3,397 new HIV cases reported in 2016 by Malaysian AIDS
Council and Malaysian AIDS Foundation.

Anti-HIV screening
was introduced in the blood transfusion services in Malaysia in 1986. Since
then, the development of screening method use for detection of HIV infection is
upgrade from time to time. The availability of the new techniques and also with
the increasing understanding of HIV transmission, have changed the strategies
in HIV testing. Early diagnosis is critical to prevent transmission and also to
provide effective antiviral treatment. The main type assays used to detect
infection of HIV is serological markers and viral nucleic acid. The serological
markers include anti-HIV1, including group O, and anti-HIV 2 and HIV p24
antigen (p24 Ag), meanwhile for viral nucleic acid is detected HIV RNA.

The p24 antigen
is designed to detect a protein associated with HIV infection within 7 to 15
days. The p24 antigen can be used to assist in the diagnosis HIV infection in
earlier stage. At the newly infected persons, p24 antigen are present in blood
serum. This can be detectable during the short period between infection and
serocoversion. The p24 antigen assay measures the virus capsid p24 protein in
blood that is detectable earlier than the appearance of HIV antibody. It occurs
early after infection due to the initial burst of virus replication and is
associated with high level of viremia during which the individual is highly
infectious.(Base and Response 2017).
However, p24 antigen is often no longer demonstrable when antibodies to HIV
become detectable due to antigen-antibody complexing in the blood.

 

 

1.2       Problem Statement

The window
period is a challenge stage in early diagnosis of HIV infection. The p24
antigen test can be used to reduce the diagnostic window. It is important to
evaluate the performance of p24 antigen detection before it can be implement as
an alternative Human Immunodeficiency Virus (HIV) testing in Pusat Darah
Negara.

 

1.3       Objectives

1.3.1    General objective

To develop and
evaluate a detection method for p24 antigen in Human Immunodeficiency Virus
(HIV) in Pusat Darah Negara. This assay is useful for diagnosing earlier
detection of HIV infection and reduces the diagnostic window. It is a reliable
tool for alternative diagnosis of HIV, especially in blood donors and patients
with high risk behavior.

 

1.3.2    Specific objectives

1.3.2.1      To determine of specificity and
sensitivity of the p24 antigen assay.

1.3.2.2      To determine the reproducibility of the assay.

1.3.2.3      To determine the detection limit of the
assay.

1.3.2.4      To evaluate and implement a detection
method for p24 antigen assay                      using
Cobas 6000 .

 

1.4       Hypothesis

The p24 antigen assay is suitable as an
alternative to identify an HIV infection in association with the anti-HIV
antibody screening test in persons known to be at risk of contracting an HIV
infection.

 

1.5       Study Justification

The rational to carry out this study is
to evaluate the p24 antigen assay and to be as an alternative test to identify HIV
infection for donated blood in Pusat Darah Negara. The HIV testing is important
in blood screening in order to make sure the blood supply is safe for patient
use. This is because, before the appearance of antibodies in the blood, free
viruses may be present which can only be detected by the p24 antigen test or
NAT testing.

 

CHAPTER 2

 

LITERATURE REVIEW

 

2.1       Human Immunodeficiency Virus

Human
Immunodeficiency Virus (HIV) is belong to family of retrovirus and is
transmitted through sexual contact, contamination blood transfusions, injecting
drug use, failure to observe what are now termed in medical circle ” universal
precautions”, and from mother to infant during pregnancy, delivery and
breastfeeding. The virus spread through certain body fluids and attack immune
system by destroying CD4 positive (CD4+). The damage to the immune system cause
difficulty to body to fight against infection. A
number of different groups and subtypes (clades) have been described with some
significant antigenic differences; HIV-1 and HIV-2 are the two major distinct
virus types and there is significant cross-reactivity between them.

The World Health
Organization (WHO) blood transfusion safety program provides policy guidance
and technical assistance to countries worldwide to achieve equitable access to
safe blood products and their safe and rational use.(Miranda et al. 2014).
The HIV testing technique have been introduced to detect the present of HIV
antigen or antibodies.  The HIV testing
assays can be divided into three category. First category is the test that can
be detect HIV antibodies, second is the test that can detect antigens (p24) and
third category is the test that can detect or quantify viral nucleic acid.

Capsid protein
(CA), or p24 antigen of human immunodeficiency virus type-1 (HIV-1) is the most
abundant viral protein, since each virus contains about 1500 to 3000 p24
molecules.(Tang et al. 2010).
In the early of the HIV infection, p24 antigen are present high in blood serum
due to the initial burst of virus replication. Early detection of HIV can be
done at this stage to reduce the diagnostics window. For the early detection of
HIV-1 infection, p24 antigen or anti-HIV antibody / p24 antigen combination
assay are used to reduce the seroconversion “window period” to an everage of 17
days post-infection and aid in identification of acute infection.(Botulinum and Study 2014).
The p24 antigen also can be used in identify HIV infection associated with the anti-HIV
antibodies screening test in persons known to be at risk of contracting HIV infection,
and newborns baby  Early detection of p24
is important for the HIV prevention and for the long-term clinical benefit. Detection
of early HIV infection may also facilitate behavior change, as some persons who
become aware that they are HIV-infected, including those with acute HIV
infection, report declines in unprotected sex.

 

           

           

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

CHAPTER 3

 

METHODOLOGY

 

3.1       Study design

This study is an experimental based
study and will be perform in Pusat Darah Negara Kuala Lumpur. Hundred  (100) samples will be collected and study
will be carried out in Transfusion Microbiology Laboratory (TML). Any subjects
involved will be verbally informed and given the information sheet about the
study and subjects will sign the consent form before any involvement proceeded
(if applicable).

 

 

3.2       Subject

3.2.1    Known
Positive Samples

·        
Samples will be
collected from the archive repository that tested positive with respective
serology confirmatory test i.e INNO-LIA HIV.

3.2.2    QC
Samples

·        
The Elecsys® positive controls
will be used in validating p24 antigen. Result obtain from inter-run is used to
assess the reproducibility of the assay by using mean, standard deviation (SD)
and coefficient of variation (CV).

3.2.3    Window
Period Samples

·        
Samples that screened
with PRISM ChLIA give Non Reactive results while tested using Nucleic Acid
Testing (NAT) give Reactive results.

 

3.3       Sample collection

Serum from known positive samples,
window period samples and also QC samples.

 

3.4       Sample processing

All tests will be performed according
manufacturer’s operator manual and will be performed in Pusat Darah Negara
Kuala Lumpur. All samples that initially reactive will be test in duplicate.
Samples that repeatedly reactive will be proceed with confirmatory test to
determine the true status. Serum from known positive samples, QC samples and
window period samples will be tested with Cobas e 601 using Elecsys HIV Ag
assay.

Test
Principal

1.      1st incubation : HIV p24 antigen (from 50 µL of sample), a
biotinylated monoclonal HIV p24-specific antibody and a monoclonal HIV
p24-specific antibody labelled with a ruthenium complexª react to form a
sandwich complex.

2.     
2nd incubation :
After addition of streptavidin-coated microparticles, the complex becomes bound
to the solid phase via interaction of biotin and streptavidin.

3.     
The reaction
mixture is aspirated into the measuring cell where the microparticles are
magnetically captured onto the surface of the electrode. Unbound substances are
then removed with ProCell/ProCell M. Application of a voltage to the electrode
then induces chemiluminescent emission which is measured by a photomultiplier.

4.     
Results are
determined automatically by the software by comparing the
electrochemiluminescence signal obtained from the reaction product of the
sample with the signal of the cutoff value previously obtained by calibration.

 

3.5       Data Collection

The data collection
will be collected from February 2018 until December 2018.

 

3.6       Data Analysis

The data analysis will follow the
following criteria :

1.      Specificity

2.      Sensitivity

3.      Data
transmission

 

 

 

 

 

 

CHAPTER 4

 

EXPECTED RESULTS

 

The
expected results for this research will be presented in tabulated form of :

·        
Comparison known
positive samples with INNO-LIA HIV and Elecsys HIV Ag assay.

·        
Comparison window
period samples with NAT and Elecsys
HIV Ag assay.

 

The
prediction results should be shown as below:

Samples

INNO-LIA HIV

NAT

P24 Ag

Known
Positive Samples

Positive

 

Reactive

Window
Period samples

 

Detected

Reactive

 

 

 

 

 

 

 

 

 

 

 

 

APPENDICES

 

 

ESTIMATED
BUDGET

NO

REAGENT

Price  per unit (RM)

Unit needed

Total price
(RM)

1

Elecsys HIV Ag assay kit

848

2

1698

TOTAL

RM 1698

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

GANTT
CHART

YEAR

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Selection
of title

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Literature
Review

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Progress
report

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Proposal
Defense

 

 

 

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Ethic
Committee Approval

 

 

 

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Submit
Written Proposal

 

 

 

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Data
Collection

 

 

 

 

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Lab
Work

 

 

 

 

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Data
Analysis

 

 

 

 

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Thesis
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Viva

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Thesis
Submission

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

X