Correlation ofCorrelation ofHBsAg and HBV DNAHBsAg and HBV DNA

Michael Chudy, Paul-Ehrlich-InstitutSoGAT XVIII, Bethesda MD

24-25 May 2005

PEI

HBsAg and HBV DNA

- Most sensitive HBsAg assay detects0.01 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 43:788-798 (2003)

Virus and HBsAg Particles

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)

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?

HBsAg and HBV DNA

• HBV DNA: reverse transcription from 3.35 kb pre-genomic mRNA

• HBsAg forms translated fromLHBs 2.4 kb mRNAMHBs/SHBs 2.1 kb mRNA

• Level of regulation- Transcription- Post-transcription

e.g. Transport of unspliced mRNAs from nucleus to

cytoplasm- Cellular factors

Ratio of total HBsAg to Virion-HBsAgat different HBV stages

• Early period of HBV infection– Genotype A– Genotype G

• Chronic HBV infection

Total HBsAg/Virion-HBsAg ratio in samples of SC panel PHM911 (BBI)#

21 d

#data performed in 1995

1:2,200

1:730

1:1,100

1:200

1:490

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 ofTransfusion Medicine and Immunohematology, GRC,JW Goethe University, Frankfurt (WK Roth and co-workers)

HBV transmission exclusively by genotype G-virus

Sample from R3 (2003-09-23) 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

Virion-HBsAg/total HBsAg ratio in genotype G

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 44,1332-1339)

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 >20 5x– s/co 15-20 65x– s/co 10-15 9x– s/co 5-10 7x– s/co <5 3x

• No correlation to s/co values of HBsAg test (Prism)

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 0,04

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 0,04

96 170,96 8,74 1,14 0,05

111 0,62 0,43 0,40 0,06

112 51,98 3,89 0,80 0,04

120 2,12 0,62 0,44 0,05

122 0,45 0,45 0,45 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 0,08

173 1,08 0,60 0,43 0,33

184 0,44 0,43 0,45 0,06

Summary: Correlation ofHBsAg 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