Frequency of occult hepatitis B in HBsAg seronegative blood donors in a tertiary care hospital in kerala,south India.
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1 Frequency of occult hepatitis B in HBsAg seronegative blood donors in a tertiary care hospital in kerala,south India. Cinzia Keechilot, Veena Shenoy 1,V Anil kumar 2,Lalita Biswas 3. MBBS student * Transfusion medicine 1,Microbiology 2,Molecular biology 3. Amrita institute of medical sciences, Cochin,kerala,India
2 Introduction Transmission of Hepatitis B virus infection reduced considerably with the introduction of hepatitis B surface antigen (HBsAg) screening in blood donors. In India, estimated rate of hepatitis B carrier state is 3% (37 million carriers).pankaj puri J Clin Exp Hepatol. 2014
3 HBsAg Seropositivity in India Donors Prevalance Screening Author, geographical area n= % Enhanced chemiluminescence assay on VITROS ECiQ n= % ELISA, Hepalisa J. Mitra & Co Pathak S, Asian J Transfus Sci. 2013(Delhi) H Fernandez etal,ijhbt,2010.(mangalore) n=39, % 3 rd gen ELISA,Span P Pallavi etal. Indian J Pathol Microbiol 1999 (Mysore) n= % Hepanostika ELISA- (organon teknika) n=28, % ELISA, Hepalisa J. Mitra & Co J Mathai etal. Indian J Pathol Microbiol 2002 (Trivandrum,kerala) Pahuja S etal.jpn J infect Dis 2007 ( Delhi)
4 Screening of blood for the detection of HBsAg does not rule out the risk of transmission of hepatitis B totally. Rate of HBsAg sero-positivity among multitransfused patients like thalassemia varies from 2% (Vidja etal, 2011, IJHBT) to 13.8% ( Mollah et al, 2003, J. Health Popul. Nutr. ) Blood donation in a phase during which the HBsAg cannot be detected in the blood. Detection of the antibody to the hepatitis B core antigen (anti-hbc) serves as a useful serological marker for hepatitis B infection.
5 Prevalence of Anti-HBc world wide Anti-HBc Prevalence
6 Subjects (Blood donors) Hepatitis B core antibody testing Anti HBc Screening Author Geographical area % (IgG +IgM) Monolisa Anti-HBc PLUS, BIO-RAD) Makroo etal. Asian J Transfus Sci Delhi % RADIM Italy Asim et al. IJMR, % (DiaSorin,Italy Lavanya etal. Int. J. Med. Med. Sci. Delhi Tamilnadu % Diasorin S.p.A. Saluggia, Italy AM ismail etal.bloodtransf us Vellore
7 Hepatitis B core antibody level high: even past infection with detected High discard rate of blood units. Advanced techniques : Nucleic Acid Testing Sensitive HBV DNA amplification assay is the gold standard assay for detection of occult hepatitis B infection
8 Occult infection absence of HBsAg and the presence of HBV DNA in the blood or liver tissue with or without any hepatitis B antibodies Causes pre-seroconversion early incubation period After clearance of HBsag and before anti HBs detected (diagnostic window) due to mutations in pre S2/S splice site HBsag level below detection limit.
9 Aims To determine the percentage of HBV DNA positivity among HBsAg negative blood donors at Amrita Institute of Medical Sciences, a tertiary care hospital in Kochi, Kerala To assess Anti-HBc and Anti-HBs positivity in HBV DNA positive donors.
10 Materials and methods Blood donors tested for HBsag,anti HCV & anti HIV 1 & 2 Vitros Enhanced Chemiluminescent Immunoassay (Ortho-Clinical Diagnostics,Raritan, NJ, USA). Seronegative donors NAT for HBV DNA & HIV, HCV RNA Individual donor Nucleic Acid Testing for Viral DNA & RNA using (Procleix Ultrio Plus assay, Gen-Probe). NEGATIVE POSITIVE No further testing Discriminatory NAT for HIV, HCV &HBV Positive for HBV NAT Testing for Anti-HBc, Anti-HBs & viral load by RT-PCR
11 Sensitivity of assays Assay Instrument Analytical Sensitivity Specificity & Sensitivity HBsAg Vitrose Eci 0.88 pmol/l (0.07 ng/dl) 99.88%specificity 100% sensitivity ID -NAT Procleix ultrio assay 2 IU/ml 99.97% specificity 100% sensitivity HB core total Antibody (ELFA) VIDAS 99.9% Sensitivity 99.5%specificity Anti HBs antibody (ELFA) VIDAS 4.23 miu/ml 98% sensitivity 99.6 %specificity Quantitative PCR (Real time PCR) TAQMAN assay 20 to IU/mL 1 iu=5 copies
12 Results Study period :20 months from March Total donors tested for HBsag serology:24338 M: 98% F:2% Voluntary : 88% Replacement 12% The median age of the study donors was 25 yrs (range yrs) HBsAg Serology positive: 0.2 % (Low endemicity) NAT testing in Seronegative donors 5 HBV NAT positivity in seronegative donors Prevalance of occult Hepatitis B : 0.021% 1 in 4842 donations
13 Characterization of NAT Positive Samples HBsag HBV NAAT HBc antibody (pos:<1,neg: >1.4) Anti HBs antibody(<1 0 non immune) Quantitative PCR HbeAg pos>o. 1, neg< M/ 26Y neg positive Positive (0.08) Indeterminate (10) Detected Neg 2 M/ 24Y 3 M/ 32Y 4 M/ 38Y 5 M/ 26Y neg positive Negative (2.24) neg positive Positive (0.05) neg positive Positive (0.01) neg positive Positive (0.01) Nonimmune Not detected Neg Immune(19) Positive ( 8 iu/ml, 40 copies /ml) Neg Immune(121) Not detected Neg Nonimmune Not detected Neg
14 HBV NAT reactive cases 4 out of 5 NAT Reactive cases were hepatitis B core antibody positive. Anti Hbs antibody Immune levels in 2 Non immune in 2 Indeterminate value in 1. Out of the 5 NAT reactive samples,viral load were below detection limit of quantitative PCR in three donors.
15 Discussion Prevalence of Hepatitis B DNA in HBsAg negative donors in our study: 0.02%. subjects Geographical area Prevalance Author Screening method Hbsag negative blood donors Kerala, India 0.021% 1 in 4842 Present study ID NAT Japan 328 in 17 million (1 in 51830) 0.002% Yoshikawa et al. Transfusion 2007 Pool of 50 NAT Australia 1 in % Seed CR,Vox Sanguis,2013 ID NAT
16 subjects Geographica l area Prevala nce of OHB author Screening method HBsAg neg. Anti-HBc positive subjects Vellore, India 1.1% Ismail AM et al, 2012 MAMC, New Delhi 7.5% Mohammad et al, 2010 Eastern India 10.70% Panigrahi et al, 2012 Abbott RealTime HBV Germany Orissa 30% Panigrahi et al, 2010 In-house nested PCR Kolkatta 21% Banerjee etal. 2007
17 Low Prevalance of occult B infection in the present study Prevalence of OHB depends on endemicity in the area Ours low endemic area. We use very sensitive HbsAg assay based on chemiluminescence. High literacy rate in state and socio economic status of most of donors
18 Rationale for NAT screening for occult B infection NAT will detect even low levels of viraemia in acute phase and that which continue long after clinical recovery from acute HBV infection. Screening and exclusion of anti HBc donors alone will miss this acute infectious donors Yotsuyanagi etal. Hepatology 1998 Anti-HBc negative but HBV DNA positive donors can also transmit the infection.
19 Limitations of the study Anti Hbc IgM was not done HBV Genotype could not be assessed Mutation within highly conserved regions of viral genome may not be detected by NAT All the donors could not be followed up.
20 Conclusions Use of NAT to detect HBV will provide additional safety. Although anti HBc testing has a definite role in improving blood safety, centers that have incorporated NAT testing may not derive any additional benefit by performing anti- HBc testing. We need to establish a uniform HBV screening strategy considering the prevalence of HBV,residual risk of HBV transmission,balance between blood safety and securing sufficient amounts of blood & cost-effectiveness
21 Thank you
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