- Email: [email protected]
Identification of low HBV-DNA levels by nucleic acid amplification test (NAT) in blood donors
Journal of Infection (2009) 59, 128e133
www.elsevierhealth.com/journals/jinf
Identification of low HBV-DNA levels by nucleic acid amplification test (NAT) in blood donors Stefano Dettori a, Angela Candido a, Loreta A. Kondili a, Paola Chionne a, Stefania Taffon a, Domenico Genovese a, Paola Iudicone b, Michelina Miceli b, Maria Rapicetta a,* a
Viral Hepatitis Unit, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy b Department of Transfusional Medicine, Hospital S. Camillo-Forlanini, Rome, Italy Accepted 5 June 2009 Available online 18 June 2009
KEYWORDS HBV-DNA; Blood donors; NAT; HBV; HBsAg
Summary Objective: To evaluate the presence of HBV-DNA in 22,765 consecutive blood donors, who donated blood in the period from January 2006 to August 2007 at a transfusion centre in Lazio, a region in central Italy with low HBV endemicity. Methods: Each donation was individually tested using immunoenzymatic assays and nucleic acid amplification technologies (NAT). Samples that were reactive to generic NAT, Procleix Ultrio Assay were tested for HBV-DNA, HCV-RNA and HIV1-RNA by Discriminatory Procleix Ultrio NAT Assay. In samples that were reactive to generic NAT and negative for HBsAg, HCV-RNA and HIV1-RNA, HBV-DNA was further tested using Cobas TaqMan and an in-house nested PCR following an ultracentrifugation step. Sequence analysis confirmed HBV-DNA positivity. Results: Generic NAT identified 31 (0.13%) reactive sera. HBV-DNA discriminatory NAT identified 15 positive sera; HBsAg was positive in 12 sera. Of the 5 generic NAT-reactive/discriminatory NAT-negative/HBsAg-negative sera and of the 3 HBsAg-negative/HBV-DNA discriminatory NAT-positive sera, 7 were positive to Cobas TaqMan or the in-house PCR after ultracentrifugation. The overall HBV-DNA positivity was 0.083% [19 of 22,765 donors: 12 HBsAg-positive (HBVDNA range 102e104 IU/mL), 7 HBsAg-negative/anti-HBc positive (HBV-DNA < 6 IU/mL)]. Conclusions: For blood transfusion safety, the significance of the finding of very low HBV-DNA levels should be further investigated. Our data indicate that in areas with a low HBV endemicity, single NAT assays may not always identify blood donations with very low HBV-DNA levels. ª 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction * Corresponding author. Tel.: þ39 06 49903233; fax: þ39 06 49902662. E-mail address: [email protected] (M. Rapicetta).
Because of improved economic conditions and the HBV vaccination program introduced in 1991, Italy has become a country with low endemicity for infection with the
0163-4453/$36 ª 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2009.06.007
Low HBV-DNA levels in blood donors hepatitis B virus (HBV). In young persons, the prevalence of markers of a previous HBV infection has been observed to be low, whereas in persons over 50 years of age, because of a cohort effect, the prevalence of markers of a previous HBV infection is high. In this epidemiological context, the main risk factors for HBV transmission remain syringe exchange, sexual exposure, nosocomial exposure and, more rarely, blood transfusion. Regarding the transfusion-associated risk, sensitive assays for detecting HBV-DNA have revealed that healthy HBsAg-negative donors who are positive for anti-HBc may harbour an ‘‘occult’’ HBV infection and maintain HBV-DNA sequences in their liver and blood.1,2 However, the risk of transmitting occult HBV through blood transfusion remains controversial. Several studies have reported a potential risk of transmission in certain categories of individuals, such as children and immunosuppressed persons,3e5 whereas others have indicated that there is no risk.6,7 The extent to which the anti-HBc screening of blood donations and NAT for HBV-DNA detection are used varies by country, given that sensitive HBsAg assays are considered to be sufficient for the safety of the blood supply. In Italy, in addition to HBsAg screening of blood donations, HBV-DNA screening of donations using NAT became mandatory by law on March 27, 2008. The objective of the present study was to evaluate the presence of HBV-DNA using NAT among blood donors at a transfusion centre in central Italy.
Methods Study population The study population consisted of 22,765 blood donors, of whom 13,688 (60%) were repeat donors and 9077 (40%) were first-time donors. These donors consecutively donated 26,787 blood units at the transfusion centre of the hospital ‘‘Azienda Ospedaliera S. Camillo e Forlanini’’ in Rome (Lazio Region, Italy) in the 18-month period from January 2006 to August 2007, which is prior to the date when NAT for HBV-DNA became mandatory.
Serological assays and NAT screening of blood donations All donations were individually tested for HBsAg (AXSYM HBsAg, Abbott Laboratories, Abbott Park, IL, USA), antiHCV (AXSYM anti-HCV version 3.0, Abbott Laboratories, Abbott Park, IL, USA), and anti-HIV1/2 (Vironostika HIV1/2 Ab/Ag, bioMerieux). All donations were also individually tested for HBV-DNA, HCV-RNA, and HIV1-RNA using the NAT Generic Procleix Ultrio Assay (Chiron, Emeryville, CA, USA). The blood samples that showed reactivity to the Generic Procleix Ultrio Assay were further tested for HBV-DNA (HBV discriminatory assay, Chiron Corporation, Emeryville, CA, USA), HCV-RNA (HCV discriminatory assay, Chiron Corporation, Emeryville, CA, USA) and HIV1-RNA (HIV discriminatory assay Chiron Corporation, Emeryville, CA, USA) using the single NAT assays, which have the following detection limits: 7.42 IU/mL, 1.95 IU/mL and 17.84 IU/mL, respectively. The serum samples that were reactive for HBV-DNA
129 were tested for anti-HBc (AXSYM Core, Abbott Laboratories, Abbott Park, IL, USA), anti-HBe (AXSYM anti-HBe 2.0, Abbott Laboratories, Abbott Park, IL, USA), HBeAg (AXSYM HBe 2.0, Abbott Laboratories, Abbott Park, IL, USA) and anti-HBs (AXSYM AUSAB, Abbott Laboratories, Abbott Park, IL, USA).
HBV-DNA quantification by real time PCR Blood samples that were reactive to the Generic Procleix Ultrio Assay, negative for HCV-RNA and HIV1-RNA by the Discriminatory Procleix Ultrio NAT Assay and negative for HBsAg were further tested by a real time PCR (Roche COBAS TaqMan 48 HBV Test; Roche Molecular System, Branchburg, NJ, USA; detection limit 6 IU/mL). The High Pure System Viral Nucleic Acid Kit (Roche Molecular Systems, Branchburg, NJ, USA) was used for RNA/DNA extraction. To increase sensitivity in terms of HBV-DNA detection, 7.5 mL of plasma samples was ultracentrifugated (27,500g for 90 min); 7 mL of the supernatant was discarded and the remaining 500 mL of concentrated serum was further subjected to DNA extraction and amplification. The ultracentrifugation step was validated by applying the serial dilutions of the World Health Organisation (WHO) HBV-DNA 96/790 International standard. The results were confirmed with an in-house nested PCR on the polymerase/pre-S/S-region of HBV-DNA.8 Briefly, the first amplification primers were: PS104 (sense primer 2313 nt) and PA100 (antisense primer 249 nt); the second amplification primers were PIS104 (sense prime r-2330 nt) and PIA100 (antisense primer 222 nt). The PCR reactions were carried out in a total volume of 50 mL containing 5 mL or 2 mL DNA templates from the first and the second amplification steps, respectively. The thermal profile of both amplification steps was: one cycle at 94 C for 40 , 35 cycles at 94 C for 4000 , 48 C for 500 , and 72 C for 20 3000 and a final extension at 72 C for 100 . The sensitivity of the in-house nested PCR was calculated using a panel of serial dilutions (from 100 IU/mL to 2.5 IU/mL) of WHO HBV-DNA 96/790 International standard with a sensitivity limit of 5 IU/mL.
HBV-DNA sequencing and genotype determination To confirm the HBV-DNA specificity, the amplified products, obtained from the polymerase/pre-S/S-region, were purified on a 100,000 kDa Centricon column (Millipore, Billerica, Mass) to remove the excess primers; sequence analysis was then performed in part of the HBV-DNA polymerase region, using the previously described primers PIS104 (2330 nt) and SQA107 (2880 nt).8 Nucleotide sequences were obtained by direct sequencing, using Big Dye Terminator Chemistry, version 3.1 (Applied Biosystems, Foster City, CA) and ABI Prism 373 Genetic Analyzer (Applied Biosystems Foster City, CA). Nucleotide sequences and reference genotype sequences from Genebank (accession numbers: AF090842, X02763, X51970 for HBV genotype A; AF100309, D00329, AB033554 for HBV genotype B; AB014381, M12906, X04615 for HBV genotype C; X65259, M32138, X85254 for HBV genotype D; X75657, AB032431 for HBV genotype E; AB036910, AF223965, X69798 for HBV genotype F; AF160501,
130
S. Dettori et al.
AF405706, AB064310 for HBV genotype G; and AY090454, AY090457, AY090460 for HBV genotype H) were aligned and manually adjusted using the BioEdit program.9 Genotype determination was performed by phylogenetic analysis using the MEGA4 program. The neighbour-joining method with 1000 bootstrap replications was applied.
Results The results of the screening for HBsAg, anti-HCV and antiHIV at the time of blood donation are shown in Table 1. The prevalence of HBsAg and of anti-HCV was higher in firsttime donors, compared to repeat donors, among whom these markers were not found. The NAT Generic Procleix Ultrio Assay, applied in parallel with immunometric screening, identified 31 reactive blood units (0.13%). The results of the tests performed by the Discriminatory Procleix Ultrio Assay for HBV-DNA, HCV-RNA and HIV1-RNA detection are reported in Table 2. Overall, 15 units were positive for HBV-DNA, 8 for HCV-RNA, and 3 for HIV1-RNA. Among donors younger than 40 years of age, HBV-DNA was only positive in HBsAg-positive first-time donors (see Tables 1 and 2). Among donors older than 40 years, HBVDNA was positive in 7 of the first-time donors (of whom 2 were HBsAg-negative) and in 1 of the repeat donors (who was HBsAg-negative). Overall, 3 serum samples tested HBsAg-negative and HBV-DNA-positive by the Discriminatory Procleix Ultrio Assay. Five serum samples from the repeat donors, which were reactive to the Generic Procleix Ultrio Assay, were undetermined (negative for HIV1-RNA, HCV-RNA and HBVDNA) by the Discriminatory Procleix Ultrio Assay; these 5 samples were also HBsAg-negative. Overall, 8 serum samples (2 from first-time donors and 6 from repeat donors) were HBsAg-negative and reactive to the Generic Procleix Ultrio Assay (but negative for both HCVRNA and HIV1-RNA); of these, 3 were HBV-DNA-positive according to the Discriminatory Procleix Ultrio Assay, whereas the remaining 5 were HBV-DNA-negative and were
Table 1
classified as undetermined NAT. The results of NAT testing of these 8 HBsAg-negative samples are shown in Table 3. In 6 of the 8 samples (75%), the Generic Procleix Ultrio Assay, which was repeated an additional two times for each sample, was not reactive in at least one repeat. These 8 samples were also tested using the Roche COBAS TaqMan 48 HBV test and the in-house PCR. In all cases, an enrichment step (ultracentrifugation of 7.5 mL plasma) prior to the HBV-DNA extraction was applied in parallel. Overall, before and after the ultracentrifugation, 7 of the 8 samples were positive by COBAS Roche TaqMan or the in-house PCR. Two samples were positive only after the ultracentrifugation step. In all cases, the HBV-DNA viral load was less than 6 IU/mL. Regarding the other HBV viral markers, of these 8 samples, 4 tested positive only for anti-HBc, 2 tested positive for anti-HBc and anti-HBs and 2 tested positive for anti-HBc, anti-HBe and anti-HBs (Table 3). The phylogenetic analysis identified genotype D in 5 serum samples and genotype A in 1 sample; in the remaining sample, it was not possible to determine the genotype. Overall, the HBV-DNA positivity was 0.083% (19 HBVDNA-positive serum samples from 22,765 donors, of whom 12 were HBsAg-positive and 7 were HBsAg-negative). Considering all 26,787 blood donations, 0.045% were positive for both HBsAg and HBV-DNA, whereas 0.026% were negative for HBsAg yet positive for HBV-DNA.
Discussion Italy has a low endemicity for HBV infection. In the general population, there are an estimated 1.2e1.5 million chronic HBV carriers, most of whom are over 50 years of age, and according to the Italian Surveillance System for Acute Hepatitis (SEIEVA), the incidence of acute HBV infection is 1.3 per 100,000 person-years.10,11 Among the Italian blooddonor population, the HBsAg incidence has been reported to be 2.7 per 100,000 person-years; prior to the introduction of HBV NAT in transfusion centres, the residual risk of HBV transmission through transfusion was an estimated 12.9e15.78 per 1 million units.12,13
Prevalence of HBsAg, anti-HCV, and anti-HIV, by age group, in first-time and repeat donors.
Blood donors
<40 years
%
>40 years
%
Total
%
First time HBsAg Anti-HCV Anti-HIV
No Z 4534 7 5 0
0.15 0.11 0
No Z 4543 5 9 0
0.11 0.20 0
No Z 9077 12 14 0
0.13 0.15 0
Periodic HBsAg Anti-HCV Anti-HIV
No Z 7104 0 0 1
0 0 0.014
No Z 6584 0 0 2
0 0 0.03
No 13688 0 0 3
0 0 0.02
Total HBsAg Anti-HCV Anti-HIV
No Z 11638a 7 5 1
0.06 0.04 0.01
No Z 11127b 5 9 2
0.04 0.08 0.02
No 22765c 12 14 3
0.05 0.06 0.01
a b c
Total 14,263 donations (1.2 rate/18 months). Total 12,524 donations (1.13 rate/18 months). Total 26,787 donations (1.2 rate/18 months).
Low HBV-DNA levels in blood donors
131
Table 2 Detection of HBV-DNA, HCV-RNA, and HIV1-RNA by discriminatory NATs in 31 serum samples reactive to Generic Procleix Ultrio Assay. Blood donors
<40 years
%
>40 years
%
Total
%
First time HBV-DNA HCV-RNA HIV-RNA Undetermined
No Z 4534 7 3 0 0
0.15 0.07 0 0
No Z 4543 7a 5 0 0
0.15 0.01 0 0
No Z 9077 14 8 0 0
0.15 0.09 0 0
Periodic HBV-DNA HCV-RNA HIV-RNA Undetermined
No Z 7104 0 0 1 0
0 0 0.01 0
No Z 6584 1b 0 2 5c
0.02 0 0.03 0.08
No 13688 1 0 3 5
0.01 0 0.02 0.04
Total HBV-DNA HCV-RNA HIV-RNA Undetermined
No Z 11638 7 3 1 0
0.06 0.03 0.01 0
No Z 11127 8d 5 2 5c
0.07 0.04 0.02 0.04
No 22765 15 8 3 5
0.07 0.04 0.01 0.02
a b c d
Two of 7 samples tested HBsAg-negative. One sample that tested HBsAg-negative. Five samples that tested reactive by Procleix Ultrio, negative by discriminatory NATs; all HBsAg-negative. Three of 8 samples tested HBsAg-negative.
In our study, which was conducted before NAT became mandatory in transfusion centres, the overall HBV-DNA positivity was 0.083%: of the 22,765 donors, 19 were HBVDNA-positive, of whom 12 were HBsAg-positive and 7 were HBsAg-negative. It should be stressed that the ultracentrifugation steps were applied to each unit that was generic NAT-reactive and negative for HCV-RNA, HIV1-RNA, and HBsAg. The Discriminatory Procleix Ultrio NAT Assay (region S of HBV) first detected the presence of HBV-DNA in HBsAgnegative blood donors; HBV-DNA positivity was then confirmed using an in-house nested PCR able to amplify part of the polymerase and preS/S region and by a real time PCR kit (COBAS TaqMan) which amplifies the pre-core/core region. Moreover, the HBV-DNA specificity was confirmed by sequence analysis in the polymerase region. Of the 19 HBV-DNA-positive serum samples, 12 (63%) were from first-time donors who tested positive for HBsAg.
Table 3
HBV-RNA detection in 8 HBsAg-negative serum samples. In-house PCR HBV markers HBV-DNA viral load (IU/ML) TaqMan Pol/pre-S/S HBcAb HBeAb HBeAg HBsAb Generic NAT- Discriminatory pre-core/core region positive/time HBV-DNA NAT region tested
Blood donor
Gender Age Procleix Ultrio Assay
Periodic Periodic First time First time Periodic Periodic Periodic Periodic
Male Male Female Male Male Female Male Male
a
The remaining 7 samples, which tested negative for HBsAg, were from donors older than 40 years of age, 5 (71%) of whom were repeat donors. The follow-up analysis of these 7 cases showed that the HBV infection was not in the window period. These data suggest that very sensitive methods for genetic virus detection could more frequently detect low HBV-DNA levels in HBsAg-negative donors older than 40 years of age. However, in 6 (75%) of the 8 samples that were HBsAgnegative and reactive to the Generic Procleix Ultrio Assay (but negative for both HCV-RNA and HIV1-RNA), the HBVDNA reactivity by the Discriminatory Procleix Ultrio Assay fluctuated, possibly because of the very low viral load in these samples. Moreover, of the 7 repeat donors whose serum samples were HBV-DNA-positive and HBsAg-negative, 5 had donated blood both before and after the implementation of HBV NAT in the transfusion centre. These data show
50 55 49 48 57 52 59 58
3/3 2/3 1/3 1/3 1/3 1/3 3/3 1/3
e e þ þ e þ e e
<6a <6 <6 <6 negative <6a <6 <6
Samples tested HBV-DNA-positive only after the enrichment procedures.
þ þ þ e e þ þ þ
þ þ þ þ þ þ þ þ
e e e e e e þ þ
e e e e e e e e
e e e e þ þ þ þ
132 that the lack of reactivity to the Procleix Ultrio Assay does not indicate HBV-DNA negativity in 100% of cases. In fact, in all of the donors who were HBV-DNA-positive and HBsAgnegative, the HBV-DNA levels were very low (less than 6 IU/mL), compared to the levels of HBsAg-positive donors observed during the study period (range: 102e104 IU/mL) (data not shown), and in 2 of the 8 cases HBV-DNA was only detectable after the ultracentrifugation step. The results of this study indicate that despite the fact that NAT testing was performed on individual blood donations, very low HBV-DNA levels (less than 6 IU/mL) were not always detected. Moreover, testing these samples in mini-pools could further reduce the chances of detecting HBV-DNA, which needs to be considered by transfusion centres that still use mini-pools to screen blood donations for HBV NAT. Although it was not the aim of this study to determine the prevalence of anti-HBc among donors, and for the purposes of ensuring blood safety it is not necessary to test for anti-HBc when adopting NAT for HBV-DNA detection, we found that all blood donations that tested HBV-DNApositive and HBsAg-negative carried anti-HBc and that about 50% of these donations were also anti-HBs-positive. These data confirm that very sensitive molecular methods for virus detection could reveal the presence of HBV-DNA in healthy individuals such as blood donors, who are supposed to have completely cleared previous HBV infection.14,15 There is no clear evidence that the so-called ‘‘occult’’ HBV infection with viral loads below 100 IU/mL in the presence of anti-HBc/anti-HBs is infectious.16e18 However, we did not perform look back of the blood recipients, though according to SEIEVA no acute HBV infection was reported after these blood donations. In Italy, prior to the introduction of HBV NAT, the estimated risk of transmitting HBV through transfusion was reported to be possibly higher than the real risk, 10e12 given that the estimated risk does not take into consideration such determinants as the younger generations being protected by vaccination and the protection of older persons because of past infection. The risk of transfusiontransmitted HBV is expected to have decreased even further since the introduction of HBV NAT in transfusion centres. Nevertheless, further investigation of the pathogenic role of very low HBV-DNA levels in blood donations is necessary, in particular for children and immunocompromised persons, in whom acute HBV infection may be severe.3,5,12,16,19 In blood donors with serological markers of past HBV infection and very low HBV-DNA levels, the results of liver function tests performed prior to transfusion were within normal ranges (data not shown), and no risk of liver disease was reported for these individuals. However, the use of ALT and HBV-DNA levels to define an inactive carrier state or ‘‘occult’’ infection may not be sufficient for the histological diagnosis of liver disease. Whether a blood donor with occult HBV can transmit the virus through transfusion and whether HBV genome persistence contributes to liver disease are critical issues which need to be further investigated. In conclusion, the significance of the finding of very low HBV-DNA levels for the safety of blood transfusion should be further investigated. Our data indicate that, at least in areas with low HBV endemicity, single NAT assays may not always identify all blood donations that contain very small quantities of HBV-DNA.
S. Dettori et al.
Acknowledgements We wish to thank Romina Tomasetto and Giacinta Antonini for providing secretarial and editorial assistance. We also thank Dr. Pietro Pedrotti and Dr. Roberto Silvi of Roche Diagnostic S.p.A. for their helpful support in providing COBAS TaqMan Roche kits and advice. This work was partially supported by a grant for the project ‘‘Integrated National Project for the Study, Prevention and Treatment of Chronic Hepatitis’’ (IRCCS Ospedale Maggiore di Milano and Istituto Superiore di Sanita ` RF02.188) and the project ‘‘Genomic and proteomic profiles of chronic viral hepatitis: identification of molecular markers for diagnosis and prognosis of clinical outcomes (cirrhosis and hepatocellular carcinoma) and of response to therapy’’ (FIRB Project Prot. RBNE013PMJ D.M. 199).
References 1. Raimondo G, Navarra G, Mondello S, Costantino L, Colloredo G, Cucinotta E, et al. Occult hepatitis B virus in liver tissue of individuals without hepatic disease. J Hepatol 2008;48:743e6. 2. Allain JP. Occult hepatitis B virus infection and transfusion. J Hepatol 2006;44:617e8. 3. Liu CJ, Lo SC, Kao JH, Tseng PT, Lai MY, Ni YH, et al. Transmission of occult hepatitis B virus by transfusion to adult and pediatric recipients in Taiwan. J Hepatol 2006;44:39e46. 4. Minuk GY, Sun DF, Greenberg R, Zhang M, Hawkins K, Uhanova J, et al. Occult hepatitis B virus infection in a North American adult hemodialysis patient population. Hepatology 2004;40:1072e7. 5. Awerkiew S, Da ¨umer M, Reiser M, Wend UC, Pfister H, Kaiser R, et al. Reactivation of an occult hepatitis B virus escape mutant in an anti-HBs positive, anti-HBc negative lymphoma patient. J Clin Virol 2007;38:83e6. 6. Prati D, Gerosa A, Porretti L. Occult HBV infection and blood transfusion. J Hepatol 2006;44:818. 7. O’Brien SF, Fearon MA, Yi QL, Fan W, Scalia V, Muntz IR, et al. Hepatitis B virus DNA-positive, hepatitis B surface antigen-negative blood donations intercepted by anti-hepatitis B core antigen testing: the Canadian Blood Services experience. Transfusion 2007;47:1809e15. 8. Chaudhuri V, Tayal R, Nayak B, Acharya SK, Panda SK. Occult hepatitis B virus infection in chronic liver disease: full-length genome and analysis of mutant surface promoter. Gastroenterology 2004;127:1356e71. 9. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Res 1999;41:95e8. 10. Zanetti AR, Romano ` L, Zappa ´ A, Velati C. Changing patterns of hepatitis B infection in Italy and NAT testing for improving the safety of blood supply. J Clin Virol 2006;36:S51e5. 11. Mele A, Tosti ME, Mariano A, Pizzuti R, Ferro A, Borrini B, et al. Acute hepatitis B 14 years after the implementation of universal vaccination in Italy: areas of improvement and emerging challenges. Clin Infect Dis 2008;46:868e75. 12. Tosti ME, Solinas S, Prati D, Salvaneschi L, Manca M, Francesconi M, et al. An estimate of the current risk of transmitting blood-borne infections through blood transfusion in Italy. Br J Haematol 2002;117:215e9. 13. Coste J, Reesink HW, Engelfriet CP, Laperche S, Brown S, Busch MP, et al. Implementation of donor screening for infectious agents transmitted by blood by nucleic acid technology: update to 2003. Vox Sang 2005;88:289e303.
Low HBV-DNA levels in blood donors 14. Manzini P, Girotto M, Borsotti R, Giachino O, Guaschino R, Lanteri M, et al. Italian blood donors with anti-HBc and occult hepatitis B virus infection. Haematologica 2007;92:1664e70. 15. Brojer E, Grabarczyk P, Liszewski G, Mikulska M, Allain JP, Letowska M. Characterization of HBV DNAþ/HBsAg- blood donors in Poland identified by triplex NAT. Hepatology 2006;44: 1666e74. 16. Reesink HW, Engelfriet CP, Henn G, Mayr WR, Delage G, Bernier F, et al. Occult hepatitis B infection in blood donors. Vox Sang 2008;94:153e66.
133 17. Gerlich WH. Breakthrough of hepatitis B virus escape mutants after vaccination and virus reactivation. J Clin Virol 2006;36: S18e22. 18. Satake M, Taira R, Yugi H, Hino S, Kanemitsu K, Ikeda H, et al. Infectivity of blood components with low hepatitis B virus DNA levels identified in a look back program. Transfusion 2007;47: 1197e205. 19. Busch MP. Prevention of transmission of hepatitis B, hepatitis C and human immunodeficiency virus infections through blood transfusion by anti-HBc testing. Vox Sang 1998;74:147e54.
www.elsevierhealth.com/journals/jinf
Identification of low HBV-DNA levels by nucleic acid amplification test (NAT) in blood donors Stefano Dettori a, Angela Candido a, Loreta A. Kondili a, Paola Chionne a, Stefania Taffon a, Domenico Genovese a, Paola Iudicone b, Michelina Miceli b, Maria Rapicetta a,* a
Viral Hepatitis Unit, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy b Department of Transfusional Medicine, Hospital S. Camillo-Forlanini, Rome, Italy Accepted 5 June 2009 Available online 18 June 2009
KEYWORDS HBV-DNA; Blood donors; NAT; HBV; HBsAg
Summary Objective: To evaluate the presence of HBV-DNA in 22,765 consecutive blood donors, who donated blood in the period from January 2006 to August 2007 at a transfusion centre in Lazio, a region in central Italy with low HBV endemicity. Methods: Each donation was individually tested using immunoenzymatic assays and nucleic acid amplification technologies (NAT). Samples that were reactive to generic NAT, Procleix Ultrio Assay were tested for HBV-DNA, HCV-RNA and HIV1-RNA by Discriminatory Procleix Ultrio NAT Assay. In samples that were reactive to generic NAT and negative for HBsAg, HCV-RNA and HIV1-RNA, HBV-DNA was further tested using Cobas TaqMan and an in-house nested PCR following an ultracentrifugation step. Sequence analysis confirmed HBV-DNA positivity. Results: Generic NAT identified 31 (0.13%) reactive sera. HBV-DNA discriminatory NAT identified 15 positive sera; HBsAg was positive in 12 sera. Of the 5 generic NAT-reactive/discriminatory NAT-negative/HBsAg-negative sera and of the 3 HBsAg-negative/HBV-DNA discriminatory NAT-positive sera, 7 were positive to Cobas TaqMan or the in-house PCR after ultracentrifugation. The overall HBV-DNA positivity was 0.083% [19 of 22,765 donors: 12 HBsAg-positive (HBVDNA range 102e104 IU/mL), 7 HBsAg-negative/anti-HBc positive (HBV-DNA < 6 IU/mL)]. Conclusions: For blood transfusion safety, the significance of the finding of very low HBV-DNA levels should be further investigated. Our data indicate that in areas with a low HBV endemicity, single NAT assays may not always identify blood donations with very low HBV-DNA levels. ª 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction * Corresponding author. Tel.: þ39 06 49903233; fax: þ39 06 49902662. E-mail address: [email protected] (M. Rapicetta).
Because of improved economic conditions and the HBV vaccination program introduced in 1991, Italy has become a country with low endemicity for infection with the
0163-4453/$36 ª 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2009.06.007
Low HBV-DNA levels in blood donors hepatitis B virus (HBV). In young persons, the prevalence of markers of a previous HBV infection has been observed to be low, whereas in persons over 50 years of age, because of a cohort effect, the prevalence of markers of a previous HBV infection is high. In this epidemiological context, the main risk factors for HBV transmission remain syringe exchange, sexual exposure, nosocomial exposure and, more rarely, blood transfusion. Regarding the transfusion-associated risk, sensitive assays for detecting HBV-DNA have revealed that healthy HBsAg-negative donors who are positive for anti-HBc may harbour an ‘‘occult’’ HBV infection and maintain HBV-DNA sequences in their liver and blood.1,2 However, the risk of transmitting occult HBV through blood transfusion remains controversial. Several studies have reported a potential risk of transmission in certain categories of individuals, such as children and immunosuppressed persons,3e5 whereas others have indicated that there is no risk.6,7 The extent to which the anti-HBc screening of blood donations and NAT for HBV-DNA detection are used varies by country, given that sensitive HBsAg assays are considered to be sufficient for the safety of the blood supply. In Italy, in addition to HBsAg screening of blood donations, HBV-DNA screening of donations using NAT became mandatory by law on March 27, 2008. The objective of the present study was to evaluate the presence of HBV-DNA using NAT among blood donors at a transfusion centre in central Italy.
Methods Study population The study population consisted of 22,765 blood donors, of whom 13,688 (60%) were repeat donors and 9077 (40%) were first-time donors. These donors consecutively donated 26,787 blood units at the transfusion centre of the hospital ‘‘Azienda Ospedaliera S. Camillo e Forlanini’’ in Rome (Lazio Region, Italy) in the 18-month period from January 2006 to August 2007, which is prior to the date when NAT for HBV-DNA became mandatory.
Serological assays and NAT screening of blood donations All donations were individually tested for HBsAg (AXSYM HBsAg, Abbott Laboratories, Abbott Park, IL, USA), antiHCV (AXSYM anti-HCV version 3.0, Abbott Laboratories, Abbott Park, IL, USA), and anti-HIV1/2 (Vironostika HIV1/2 Ab/Ag, bioMerieux). All donations were also individually tested for HBV-DNA, HCV-RNA, and HIV1-RNA using the NAT Generic Procleix Ultrio Assay (Chiron, Emeryville, CA, USA). The blood samples that showed reactivity to the Generic Procleix Ultrio Assay were further tested for HBV-DNA (HBV discriminatory assay, Chiron Corporation, Emeryville, CA, USA), HCV-RNA (HCV discriminatory assay, Chiron Corporation, Emeryville, CA, USA) and HIV1-RNA (HIV discriminatory assay Chiron Corporation, Emeryville, CA, USA) using the single NAT assays, which have the following detection limits: 7.42 IU/mL, 1.95 IU/mL and 17.84 IU/mL, respectively. The serum samples that were reactive for HBV-DNA
129 were tested for anti-HBc (AXSYM Core, Abbott Laboratories, Abbott Park, IL, USA), anti-HBe (AXSYM anti-HBe 2.0, Abbott Laboratories, Abbott Park, IL, USA), HBeAg (AXSYM HBe 2.0, Abbott Laboratories, Abbott Park, IL, USA) and anti-HBs (AXSYM AUSAB, Abbott Laboratories, Abbott Park, IL, USA).
HBV-DNA quantification by real time PCR Blood samples that were reactive to the Generic Procleix Ultrio Assay, negative for HCV-RNA and HIV1-RNA by the Discriminatory Procleix Ultrio NAT Assay and negative for HBsAg were further tested by a real time PCR (Roche COBAS TaqMan 48 HBV Test; Roche Molecular System, Branchburg, NJ, USA; detection limit 6 IU/mL). The High Pure System Viral Nucleic Acid Kit (Roche Molecular Systems, Branchburg, NJ, USA) was used for RNA/DNA extraction. To increase sensitivity in terms of HBV-DNA detection, 7.5 mL of plasma samples was ultracentrifugated (27,500g for 90 min); 7 mL of the supernatant was discarded and the remaining 500 mL of concentrated serum was further subjected to DNA extraction and amplification. The ultracentrifugation step was validated by applying the serial dilutions of the World Health Organisation (WHO) HBV-DNA 96/790 International standard. The results were confirmed with an in-house nested PCR on the polymerase/pre-S/S-region of HBV-DNA.8 Briefly, the first amplification primers were: PS104 (sense primer 2313 nt) and PA100 (antisense primer 249 nt); the second amplification primers were PIS104 (sense prime r-2330 nt) and PIA100 (antisense primer 222 nt). The PCR reactions were carried out in a total volume of 50 mL containing 5 mL or 2 mL DNA templates from the first and the second amplification steps, respectively. The thermal profile of both amplification steps was: one cycle at 94 C for 40 , 35 cycles at 94 C for 4000 , 48 C for 500 , and 72 C for 20 3000 and a final extension at 72 C for 100 . The sensitivity of the in-house nested PCR was calculated using a panel of serial dilutions (from 100 IU/mL to 2.5 IU/mL) of WHO HBV-DNA 96/790 International standard with a sensitivity limit of 5 IU/mL.
HBV-DNA sequencing and genotype determination To confirm the HBV-DNA specificity, the amplified products, obtained from the polymerase/pre-S/S-region, were purified on a 100,000 kDa Centricon column (Millipore, Billerica, Mass) to remove the excess primers; sequence analysis was then performed in part of the HBV-DNA polymerase region, using the previously described primers PIS104 (2330 nt) and SQA107 (2880 nt).8 Nucleotide sequences were obtained by direct sequencing, using Big Dye Terminator Chemistry, version 3.1 (Applied Biosystems, Foster City, CA) and ABI Prism 373 Genetic Analyzer (Applied Biosystems Foster City, CA). Nucleotide sequences and reference genotype sequences from Genebank (accession numbers: AF090842, X02763, X51970 for HBV genotype A; AF100309, D00329, AB033554 for HBV genotype B; AB014381, M12906, X04615 for HBV genotype C; X65259, M32138, X85254 for HBV genotype D; X75657, AB032431 for HBV genotype E; AB036910, AF223965, X69798 for HBV genotype F; AF160501,
130
S. Dettori et al.
AF405706, AB064310 for HBV genotype G; and AY090454, AY090457, AY090460 for HBV genotype H) were aligned and manually adjusted using the BioEdit program.9 Genotype determination was performed by phylogenetic analysis using the MEGA4 program. The neighbour-joining method with 1000 bootstrap replications was applied.
Results The results of the screening for HBsAg, anti-HCV and antiHIV at the time of blood donation are shown in Table 1. The prevalence of HBsAg and of anti-HCV was higher in firsttime donors, compared to repeat donors, among whom these markers were not found. The NAT Generic Procleix Ultrio Assay, applied in parallel with immunometric screening, identified 31 reactive blood units (0.13%). The results of the tests performed by the Discriminatory Procleix Ultrio Assay for HBV-DNA, HCV-RNA and HIV1-RNA detection are reported in Table 2. Overall, 15 units were positive for HBV-DNA, 8 for HCV-RNA, and 3 for HIV1-RNA. Among donors younger than 40 years of age, HBV-DNA was only positive in HBsAg-positive first-time donors (see Tables 1 and 2). Among donors older than 40 years, HBVDNA was positive in 7 of the first-time donors (of whom 2 were HBsAg-negative) and in 1 of the repeat donors (who was HBsAg-negative). Overall, 3 serum samples tested HBsAg-negative and HBV-DNA-positive by the Discriminatory Procleix Ultrio Assay. Five serum samples from the repeat donors, which were reactive to the Generic Procleix Ultrio Assay, were undetermined (negative for HIV1-RNA, HCV-RNA and HBVDNA) by the Discriminatory Procleix Ultrio Assay; these 5 samples were also HBsAg-negative. Overall, 8 serum samples (2 from first-time donors and 6 from repeat donors) were HBsAg-negative and reactive to the Generic Procleix Ultrio Assay (but negative for both HCVRNA and HIV1-RNA); of these, 3 were HBV-DNA-positive according to the Discriminatory Procleix Ultrio Assay, whereas the remaining 5 were HBV-DNA-negative and were
Table 1
classified as undetermined NAT. The results of NAT testing of these 8 HBsAg-negative samples are shown in Table 3. In 6 of the 8 samples (75%), the Generic Procleix Ultrio Assay, which was repeated an additional two times for each sample, was not reactive in at least one repeat. These 8 samples were also tested using the Roche COBAS TaqMan 48 HBV test and the in-house PCR. In all cases, an enrichment step (ultracentrifugation of 7.5 mL plasma) prior to the HBV-DNA extraction was applied in parallel. Overall, before and after the ultracentrifugation, 7 of the 8 samples were positive by COBAS Roche TaqMan or the in-house PCR. Two samples were positive only after the ultracentrifugation step. In all cases, the HBV-DNA viral load was less than 6 IU/mL. Regarding the other HBV viral markers, of these 8 samples, 4 tested positive only for anti-HBc, 2 tested positive for anti-HBc and anti-HBs and 2 tested positive for anti-HBc, anti-HBe and anti-HBs (Table 3). The phylogenetic analysis identified genotype D in 5 serum samples and genotype A in 1 sample; in the remaining sample, it was not possible to determine the genotype. Overall, the HBV-DNA positivity was 0.083% (19 HBVDNA-positive serum samples from 22,765 donors, of whom 12 were HBsAg-positive and 7 were HBsAg-negative). Considering all 26,787 blood donations, 0.045% were positive for both HBsAg and HBV-DNA, whereas 0.026% were negative for HBsAg yet positive for HBV-DNA.
Discussion Italy has a low endemicity for HBV infection. In the general population, there are an estimated 1.2e1.5 million chronic HBV carriers, most of whom are over 50 years of age, and according to the Italian Surveillance System for Acute Hepatitis (SEIEVA), the incidence of acute HBV infection is 1.3 per 100,000 person-years.10,11 Among the Italian blooddonor population, the HBsAg incidence has been reported to be 2.7 per 100,000 person-years; prior to the introduction of HBV NAT in transfusion centres, the residual risk of HBV transmission through transfusion was an estimated 12.9e15.78 per 1 million units.12,13
Prevalence of HBsAg, anti-HCV, and anti-HIV, by age group, in first-time and repeat donors.
Blood donors
<40 years
%
>40 years
%
Total
%
First time HBsAg Anti-HCV Anti-HIV
No Z 4534 7 5 0
0.15 0.11 0
No Z 4543 5 9 0
0.11 0.20 0
No Z 9077 12 14 0
0.13 0.15 0
Periodic HBsAg Anti-HCV Anti-HIV
No Z 7104 0 0 1
0 0 0.014
No Z 6584 0 0 2
0 0 0.03
No 13688 0 0 3
0 0 0.02
Total HBsAg Anti-HCV Anti-HIV
No Z 11638a 7 5 1
0.06 0.04 0.01
No Z 11127b 5 9 2
0.04 0.08 0.02
No 22765c 12 14 3
0.05 0.06 0.01
a b c
Total 14,263 donations (1.2 rate/18 months). Total 12,524 donations (1.13 rate/18 months). Total 26,787 donations (1.2 rate/18 months).
Low HBV-DNA levels in blood donors
131
Table 2 Detection of HBV-DNA, HCV-RNA, and HIV1-RNA by discriminatory NATs in 31 serum samples reactive to Generic Procleix Ultrio Assay. Blood donors
<40 years
%
>40 years
%
Total
%
First time HBV-DNA HCV-RNA HIV-RNA Undetermined
No Z 4534 7 3 0 0
0.15 0.07 0 0
No Z 4543 7a 5 0 0
0.15 0.01 0 0
No Z 9077 14 8 0 0
0.15 0.09 0 0
Periodic HBV-DNA HCV-RNA HIV-RNA Undetermined
No Z 7104 0 0 1 0
0 0 0.01 0
No Z 6584 1b 0 2 5c
0.02 0 0.03 0.08
No 13688 1 0 3 5
0.01 0 0.02 0.04
Total HBV-DNA HCV-RNA HIV-RNA Undetermined
No Z 11638 7 3 1 0
0.06 0.03 0.01 0
No Z 11127 8d 5 2 5c
0.07 0.04 0.02 0.04
No 22765 15 8 3 5
0.07 0.04 0.01 0.02
a b c d
Two of 7 samples tested HBsAg-negative. One sample that tested HBsAg-negative. Five samples that tested reactive by Procleix Ultrio, negative by discriminatory NATs; all HBsAg-negative. Three of 8 samples tested HBsAg-negative.
In our study, which was conducted before NAT became mandatory in transfusion centres, the overall HBV-DNA positivity was 0.083%: of the 22,765 donors, 19 were HBVDNA-positive, of whom 12 were HBsAg-positive and 7 were HBsAg-negative. It should be stressed that the ultracentrifugation steps were applied to each unit that was generic NAT-reactive and negative for HCV-RNA, HIV1-RNA, and HBsAg. The Discriminatory Procleix Ultrio NAT Assay (region S of HBV) first detected the presence of HBV-DNA in HBsAgnegative blood donors; HBV-DNA positivity was then confirmed using an in-house nested PCR able to amplify part of the polymerase and preS/S region and by a real time PCR kit (COBAS TaqMan) which amplifies the pre-core/core region. Moreover, the HBV-DNA specificity was confirmed by sequence analysis in the polymerase region. Of the 19 HBV-DNA-positive serum samples, 12 (63%) were from first-time donors who tested positive for HBsAg.
Table 3
HBV-RNA detection in 8 HBsAg-negative serum samples. In-house PCR HBV markers HBV-DNA viral load (IU/ML) TaqMan Pol/pre-S/S HBcAb HBeAb HBeAg HBsAb Generic NAT- Discriminatory pre-core/core region positive/time HBV-DNA NAT region tested
Blood donor
Gender Age Procleix Ultrio Assay
Periodic Periodic First time First time Periodic Periodic Periodic Periodic
Male Male Female Male Male Female Male Male
a
The remaining 7 samples, which tested negative for HBsAg, were from donors older than 40 years of age, 5 (71%) of whom were repeat donors. The follow-up analysis of these 7 cases showed that the HBV infection was not in the window period. These data suggest that very sensitive methods for genetic virus detection could more frequently detect low HBV-DNA levels in HBsAg-negative donors older than 40 years of age. However, in 6 (75%) of the 8 samples that were HBsAgnegative and reactive to the Generic Procleix Ultrio Assay (but negative for both HCV-RNA and HIV1-RNA), the HBVDNA reactivity by the Discriminatory Procleix Ultrio Assay fluctuated, possibly because of the very low viral load in these samples. Moreover, of the 7 repeat donors whose serum samples were HBV-DNA-positive and HBsAg-negative, 5 had donated blood both before and after the implementation of HBV NAT in the transfusion centre. These data show
50 55 49 48 57 52 59 58
3/3 2/3 1/3 1/3 1/3 1/3 3/3 1/3
e e þ þ e þ e e
<6a <6 <6 <6 negative <6a <6 <6
Samples tested HBV-DNA-positive only after the enrichment procedures.
þ þ þ e e þ þ þ
þ þ þ þ þ þ þ þ
e e e e e e þ þ
e e e e e e e e
e e e e þ þ þ þ
132 that the lack of reactivity to the Procleix Ultrio Assay does not indicate HBV-DNA negativity in 100% of cases. In fact, in all of the donors who were HBV-DNA-positive and HBsAgnegative, the HBV-DNA levels were very low (less than 6 IU/mL), compared to the levels of HBsAg-positive donors observed during the study period (range: 102e104 IU/mL) (data not shown), and in 2 of the 8 cases HBV-DNA was only detectable after the ultracentrifugation step. The results of this study indicate that despite the fact that NAT testing was performed on individual blood donations, very low HBV-DNA levels (less than 6 IU/mL) were not always detected. Moreover, testing these samples in mini-pools could further reduce the chances of detecting HBV-DNA, which needs to be considered by transfusion centres that still use mini-pools to screen blood donations for HBV NAT. Although it was not the aim of this study to determine the prevalence of anti-HBc among donors, and for the purposes of ensuring blood safety it is not necessary to test for anti-HBc when adopting NAT for HBV-DNA detection, we found that all blood donations that tested HBV-DNApositive and HBsAg-negative carried anti-HBc and that about 50% of these donations were also anti-HBs-positive. These data confirm that very sensitive molecular methods for virus detection could reveal the presence of HBV-DNA in healthy individuals such as blood donors, who are supposed to have completely cleared previous HBV infection.14,15 There is no clear evidence that the so-called ‘‘occult’’ HBV infection with viral loads below 100 IU/mL in the presence of anti-HBc/anti-HBs is infectious.16e18 However, we did not perform look back of the blood recipients, though according to SEIEVA no acute HBV infection was reported after these blood donations. In Italy, prior to the introduction of HBV NAT, the estimated risk of transmitting HBV through transfusion was reported to be possibly higher than the real risk, 10e12 given that the estimated risk does not take into consideration such determinants as the younger generations being protected by vaccination and the protection of older persons because of past infection. The risk of transfusiontransmitted HBV is expected to have decreased even further since the introduction of HBV NAT in transfusion centres. Nevertheless, further investigation of the pathogenic role of very low HBV-DNA levels in blood donations is necessary, in particular for children and immunocompromised persons, in whom acute HBV infection may be severe.3,5,12,16,19 In blood donors with serological markers of past HBV infection and very low HBV-DNA levels, the results of liver function tests performed prior to transfusion were within normal ranges (data not shown), and no risk of liver disease was reported for these individuals. However, the use of ALT and HBV-DNA levels to define an inactive carrier state or ‘‘occult’’ infection may not be sufficient for the histological diagnosis of liver disease. Whether a blood donor with occult HBV can transmit the virus through transfusion and whether HBV genome persistence contributes to liver disease are critical issues which need to be further investigated. In conclusion, the significance of the finding of very low HBV-DNA levels for the safety of blood transfusion should be further investigated. Our data indicate that, at least in areas with low HBV endemicity, single NAT assays may not always identify all blood donations that contain very small quantities of HBV-DNA.
S. Dettori et al.
Acknowledgements We wish to thank Romina Tomasetto and Giacinta Antonini for providing secretarial and editorial assistance. We also thank Dr. Pietro Pedrotti and Dr. Roberto Silvi of Roche Diagnostic S.p.A. for their helpful support in providing COBAS TaqMan Roche kits and advice. This work was partially supported by a grant for the project ‘‘Integrated National Project for the Study, Prevention and Treatment of Chronic Hepatitis’’ (IRCCS Ospedale Maggiore di Milano and Istituto Superiore di Sanita ` RF02.188) and the project ‘‘Genomic and proteomic profiles of chronic viral hepatitis: identification of molecular markers for diagnosis and prognosis of clinical outcomes (cirrhosis and hepatocellular carcinoma) and of response to therapy’’ (FIRB Project Prot. RBNE013PMJ D.M. 199).
References 1. Raimondo G, Navarra G, Mondello S, Costantino L, Colloredo G, Cucinotta E, et al. Occult hepatitis B virus in liver tissue of individuals without hepatic disease. J Hepatol 2008;48:743e6. 2. Allain JP. Occult hepatitis B virus infection and transfusion. J Hepatol 2006;44:617e8. 3. Liu CJ, Lo SC, Kao JH, Tseng PT, Lai MY, Ni YH, et al. Transmission of occult hepatitis B virus by transfusion to adult and pediatric recipients in Taiwan. J Hepatol 2006;44:39e46. 4. Minuk GY, Sun DF, Greenberg R, Zhang M, Hawkins K, Uhanova J, et al. Occult hepatitis B virus infection in a North American adult hemodialysis patient population. Hepatology 2004;40:1072e7. 5. Awerkiew S, Da ¨umer M, Reiser M, Wend UC, Pfister H, Kaiser R, et al. Reactivation of an occult hepatitis B virus escape mutant in an anti-HBs positive, anti-HBc negative lymphoma patient. J Clin Virol 2007;38:83e6. 6. Prati D, Gerosa A, Porretti L. Occult HBV infection and blood transfusion. J Hepatol 2006;44:818. 7. O’Brien SF, Fearon MA, Yi QL, Fan W, Scalia V, Muntz IR, et al. Hepatitis B virus DNA-positive, hepatitis B surface antigen-negative blood donations intercepted by anti-hepatitis B core antigen testing: the Canadian Blood Services experience. Transfusion 2007;47:1809e15. 8. Chaudhuri V, Tayal R, Nayak B, Acharya SK, Panda SK. Occult hepatitis B virus infection in chronic liver disease: full-length genome and analysis of mutant surface promoter. Gastroenterology 2004;127:1356e71. 9. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Res 1999;41:95e8. 10. Zanetti AR, Romano ` L, Zappa ´ A, Velati C. Changing patterns of hepatitis B infection in Italy and NAT testing for improving the safety of blood supply. J Clin Virol 2006;36:S51e5. 11. Mele A, Tosti ME, Mariano A, Pizzuti R, Ferro A, Borrini B, et al. Acute hepatitis B 14 years after the implementation of universal vaccination in Italy: areas of improvement and emerging challenges. Clin Infect Dis 2008;46:868e75. 12. Tosti ME, Solinas S, Prati D, Salvaneschi L, Manca M, Francesconi M, et al. An estimate of the current risk of transmitting blood-borne infections through blood transfusion in Italy. Br J Haematol 2002;117:215e9. 13. Coste J, Reesink HW, Engelfriet CP, Laperche S, Brown S, Busch MP, et al. Implementation of donor screening for infectious agents transmitted by blood by nucleic acid technology: update to 2003. Vox Sang 2005;88:289e303.
Low HBV-DNA levels in blood donors 14. Manzini P, Girotto M, Borsotti R, Giachino O, Guaschino R, Lanteri M, et al. Italian blood donors with anti-HBc and occult hepatitis B virus infection. Haematologica 2007;92:1664e70. 15. Brojer E, Grabarczyk P, Liszewski G, Mikulska M, Allain JP, Letowska M. Characterization of HBV DNAþ/HBsAg- blood donors in Poland identified by triplex NAT. Hepatology 2006;44: 1666e74. 16. Reesink HW, Engelfriet CP, Henn G, Mayr WR, Delage G, Bernier F, et al. Occult hepatitis B infection in blood donors. Vox Sang 2008;94:153e66.
133 17. Gerlich WH. Breakthrough of hepatitis B virus escape mutants after vaccination and virus reactivation. J Clin Virol 2006;36: S18e22. 18. Satake M, Taira R, Yugi H, Hino S, Kanemitsu K, Ikeda H, et al. Infectivity of blood components with low hepatitis B virus DNA levels identified in a look back program. Transfusion 2007;47: 1197e205. 19. Busch MP. Prevention of transmission of hepatitis B, hepatitis C and human immunodeficiency virus infections through blood transfusion by anti-HBc testing. Vox Sang 1998;74:147e54.