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Human Parvovirus B19 is a non-enveloped, single-stranded DNA virus of about 5,000 nucleotides7. Parvovirus B19 is belonging to the genus of erythrovirus within the family of Parvoviridae, subfamily Parvovirinae3,5. It is amongst the smallest DNA viruses with the virion diameter of 18 to 26 nm and mainly composed of 60 copies of protein and DNA 1,3. The parvovirus virion consist of relatively small structure, encodes two capsid proteins (VP1 and VP2) and three structural proteins NS1, P11, P7.5 2. It contains two major open reading frames, a 71-kDa non-structural protein that is encoded by the left open reading frame and overlapping structural proteins, VP1 (83 kDa), and VP2 (58 kDa) , encoded by the right of the B19 genome8.
Human Parvovirus B19 was discovered at 1975 by Cossart et al in the United Kingdom, when they were performing laboratory assay on hepatitis B virus in the blood of healthy donors9. The name of B19 was given due to the virus is found in the well B19 of a microtiter plate1.
Human parvovirus B19 is mainly associated to caused 'fifth disease', erythema infectiosum 1,5. Moreover, infections with parvovirus B19 also related to numbers of diseases, including arthritis, aplastic anemia, chronic anemia, myocarditis, vasculitis, glomerulonephritis, hydrop fetalis and congenital anemia 5. This virus often infected children and causing them to develop skin rash and formation of protective antibodies 14. But B19 prevalence may further increase during lifetime and reach values higher than 85% in elderly 4.The symptoms ranging from asymptomatically, mild to more severe illness, especially in immunocompromised patients 7.
4. Problem Statement :
Screening of blood donation is crucial and is considered as first line of defense towards reducing the risks of transfusion-transmitted infections from donors to recipients and to ensure the transfusion is safe. The risk of infection can be minimized or even eliminated if the screening of blood donation is performed correctly. Large volume of blood or blood components are given to the patients during transfusion therapy, however, during the transfusion, even a low viral load may lead to infection in the recipient.
Screening of all blood donations has been conducted and is compulsory to perform HIV screening (screening of combination of HIV antigen or HIV antibodies), Hepatitis B (screening of hepatitis B surface antigen, HBsAg) , hepatitis C (screening for either a combination of hepatitis C antigen-antibodies or hepatitis C antibodies), and syphilis (screening for specific treponemal antibodies).
In most countries, screening for human parvovirus B19 is not being done even though it can also be transmitted via blood product. In addition, testing for B19 in the donations or donors is not required according to Guidelines for the Collection of Blood and Blood Components and for the USE of Blood Products or according to the recommendations of the Council of Europe or World Health Organization (WHO) recommendation on blood and blood products5. Primarily human parvovirus B19 is spread through respiratory routes via aerosol droplet. However, the transmission may also occur through parental transmission, via infected blood or blood products12.
Since parvovirus B19 can be infected through blood product, testing of B19 DNA should be taken into consideration to further increase virus safety of blood products3. Newer information suggests that the recipient have higher risk to be exposed to low titer of B19 DNA from blood products than previously thought13.
Several methods can be done in the detection of human parvovirus B19 DNA including Polymerase Chain Reaction (PCR), enzyme-linked immunosorbent assay (ELISA), antibody capture radioimmunoassay, nucleic acid amplification technology (NAT), real-time PCR, and receptor-mediated hemagglutination (RHA). Quantification of PCR assay based on real-time PCR allows a sensitive detection of parvovirus B19 DNA and can be done in large-scale screening of blood products.
Seroprevalence of B19 infection varies in different countries. To the date, the lowest seroprevalence is 16.2% in Singapore, 32.8% to as high 80% in Japan, 75% in Belgium, 60% in England, 49% in America, 40% to 46.8% in Germany and 64% in North Africa 4. In the other hand, seroprevalence rate of B19 infection in Asian countries is higher in the younger age group as compared to B19 infection in Malaysia2.
5. Objectives :
The general objective of the research is to determine the prevalence and detection of human parvovirus B19 in the blood donors using real-time PCR.
Specific objectives include:
To study whether Malaysian blood donors from National Blood Centre have infected with human Parvovirus B19 or not.
To determine of donor blood products seroconversion of B19.
To estimate of the prevalence of plasma B19 DNA in blood donations.
Null hypothesis :
There is no prevalence of Malaysian blood donors with human parvovirus B19 detected by real-time PCR.
Alternative hypothesis :
There is prevalence of Malaysian blood donors with human parvovirus B19 detected by real-time PCR.
6. Methodology :
Study location: This study was conducted at laboratory unit, Faculty of Health Sciences, UiTM Puncak Alam.
Study population: Study population of this study is volunteer donors who donate their blood at National Blood Centre, Malaysia.
Sample size: 500 blood donors from National Blood Centre are collected in EDTA tube to be screened for real-time PCR.
Donors' blood is collected in EDTA tube.
Separate the plasma from RBCs by centrifugation.
2) Extraction of viral DNA from plasma.
Isolate viral DNA from the plasma by centrifuge using viral DNA mini-kit and perform by following the manufacturer's instruction.
Store DNA extraction at -80°C before PCR analysis.
Amplification of PCR assay.
A plasmid containing the entire genome of human parvovirus B19 is used as a template for amplification.
The primer set amplifies a 78 base pair fragment in a conserved NS1 region of B19.
25µl reaction mixture contains:
1 X PCR buffer (20 mM Tris-HCl, pH 8.5)
7 mM MgCl2
200 µM dATP, dCTP, dGTP
400 µM dUTP
300 nM forward primer
900 nM reverse primer
200 nµ wild type and internal control probe
0.5 µl Rox reference dye
1.25 U Taq DNA polymerase
0.5 U uracil-N-glycosylase
10 µl sample preparation
4) Amplification cycle:
The DNA was amplified and detected with real-time PCR systems.
Real-time quantification amplification of B19 DNA is performing according to the manufacturer's instruction using a thermocycler.
5) Statistical analysis
Using SPSS version 1.8 to evaluate the prevalence of human Parvovirus B19 in blood donations through Fisher exact test.
7. Budget :
Real-time PCR kit (Taqman or other brand available)
Comment of Project Management Committee: