1. Michael Chudy, Paul-Ehrlich-Institut SoGAT XVIII, Bethesda MD
Correlation of
HBsAg and HBV DNA
Michael Chudy, Paul-Ehrlich-Institut
SoGAT XVIII, Bethesda MD
24-25 May 2005
PEI

2. HBsAg and HBV DNA
Most sensitive HBsAg assay detects 0.01 ng/ml (PEI HBsAg standard; ad)
HBsAg cut-off correspond to HBV DNA
114 IU/ml (WHO DNA standard)
461 copies/ml (five SC panels; ZeptoMetrix)
Good correlation to published data from Biswas et al.; Transfusion 43: (2003)

3. Virus and HBsAg Particles

4. HBsAg reactive polypeptides
Most sensitive ELISA detects 10 pg HBsAg/ml
Virion-HBsAg
One HBV particle: 20 ag HBsAg*
10 pg HBsAg correspond to about 500,000 HBV particles
500 particles/ml (HBsAg cutoff of SC panels) vs. 500,000 HBV particles/ml
Viral surface antigen reactive polypeptides
Virion envelope
Incomplete viral forms
Excessive incomplete viral forms are present (about 1:1,000)
*Average numbers of molecules of LHBs, MHBs, and SHBs: 30, 30, and 350, respectively; Calculation correlate well with data presented by Prof. Gerlich (EPFA meeting 2002)

5. Correlation of HBsAg and HBV DNA
Is that true for all geno-/subtypes, stages … of HBV infection?
Can HBV NAT replace the HBsAg testing in blood donors?

6. HBsAg and HBV DNA
HBV DNA: reverse transcription from 3.35 kb pre-genomic mRNA
HBsAg forms translated from
LHBs kb mRNA
MHBs/SHBs 2.1 kb mRNA
Level of regulation
Transcription
Post-transcription
e.g. Transport of unspliced mRNAs from nucleus to cytoplasm
Cellular factors

7. Ratio of total HBsAg to Virion-HBsAg at different HBV stages
Early period of HBV infection
Genotype A
Genotype G
Chronic HBV infection

8. Total HBsAg/Virion-HBsAg ratio in samples of SC panel PHM911 (BBI)#
1:2,200
1:730
1:1,100
1:200
1:490
21 d
#data performed in 1995

9. HBV genotype G
Only few infections are known (all co-infections with genotype A)
First report of HBV infection and transmission based exclusively on genotype G (2003 in Germany)
No escape variant (subtype adw2)
HBeg minus variant
Studies were performed in cooperation with the Institute of
Transfusion Medicine and Immunohematology, GRC,
JW Goethe University, Frankfurt (WK Roth and co-workers)

10. HBV transmission exclusively by genotype G-virus

11. Virion-HBsAg/total HBsAg ratio in genotype G
Sample from R3 ( ) with about 9 Mio cps/ml (positive for HBsAg, negative for anti-HBc, HBeAg/anti-HBe)
Titration in Prism HBsAg test
Virus concentration at HBsAg cut-off: 24,000 copies/ml
Ratio of virion-HBsAg to total HBsAg of 1:20
vs. 1:1,000 and more in genotype A samples of early HBV infection
Significant decrease of excessive HBsAg in infection with genotype G

12. HBsAg and HBV DNA in chronic HBV infection
Lack of correlation between HBsAg and HBV DNA in HBsAg/anti-HBc positive tested samples (Kuhns et al. 2004, Transfusion 44, )

13. HBsAg and HBV DNA in chronic HBV carriers
Investigation of 106 chronic carriers tested positive for HBsAg, anti-HBc, and anti-HBe
Only 84% (89/106) HBV NAT reactive with the Procleix Ultrio Assay (Gen-Probe)
s/co > x
s/co x
s/co x
s/co x
s/co <5 3x
No correlation to s/co values of HBsAg test (Prism)

14. 17 HBsAg positive/HBV DNA negative samples of chronic HBV carriers
Sample ID#
Prism HBsAg (s/co)
Procleix Ultrio Assay
1:10
1:100
1:1.000
s/co analyte 14
0,58
0,42
0,39
0,03 15
7,39
1,01
0,45
0,07 22
99,11
0,80
0,50
0,04 63
178,51
1,57
0,47 72
218,48
14,33
1,59
0,30 74
2,08
0,51
0,43
0,06 76
27,30
2,00
0,64 96
170,96
8,74
1,14
0,05
111
0,62
0,40
112
51,98
3,89
120
2,12
0,44
122
0,08
141
37,33
3,56
0,77
0,11
161
228,31
58,59
5,76
0,15
164
13,89
2,16
0,61
173
1,08
0,60
0,33
184

15. Summary: Correlation of HBsAg and HBV DNA
Virion-HBsAg of one particle (20 ag) correspond to one copy of HBV DNA
Significant excess of HBsAg particles in the early period of HBV infection (genotype A)
Variable ratio of Virion-HBsAg to total HBsAg in the course of infection (genotype A)
Genotype G infection shows a significant decrease in synthesis of incomplete viral forms
Lack of correlation between HBsAg and HBV DNA in chronic infection
HBsAg carrier (integrative phase) can be non-reactive by HBV NAT
Results indicate caution in any consideration of dropping HBsAg screening