Changing patterns of hepatitis B infection in Italy and NAT testing for improving the safety of blood supply

ELSEVIER

Journal of Clinical Virology 36 Suppl. 1 (2006) $51 $55 www.elsevier.com/locate/jcv

Changing patterns of hepatitis B infection in Italy and NAT testing for improving the safety of blood supply Alessandro

R. Zanetti a' *, L u i s a R o m a n ~ ) a, A l e s s a n d r a Z a p p a a, C l a u d i o V e l a t i b

aDepartment of Public Health-Microbiology-Virology, University of Milan, Italy bDepartment of Transfusion Medicine and Immunohematology, Hospital of Sondrio, Italy

Abstract In Italy, as in most industrialized countries, the burden of hepatitis B has progressively declined in recent decades as a consequence of general improvements in hygiene and standard of living, the introduction of several public health measures, refinement in blood screening and the implementation of specific vaccination programmes. Universal hepatitis B vaccination for all infants and adolescents as well as individuals at increased risk has resulted in considerable progress towards prevention and control of HBV infection. The residual risk of transmitting blood-borne viruses through transfusion is currently very low. Nucleic acid testing can shorten the window period and, consequently, further reduce the risk of viral transmission. Blood donor screening for HCV by NAT was initiated in Italy in 2001 and became mandatory in June 2002. NAT for HIV is currently mandatory in four regions, not mandatory but almost universally performed in another thirteen regions, and not yet introduced in the remaining four regions. NAT for HBV is currently mandatory in four regions and under evaluation in the remaining. NAT for HBV may be a useful tool in detecting acute viral infections in the window phase as well as the occult infections. Its efficacy in improving the safety of blood supply is expected to be higher in countries with intermediate/high endemicity, where anti-HBc antibody screening cannot be routinely performed. There is agreement that, at present, the implementation of HBV DNA testing will not allow for discontinuation of screening for HBsAg. 9 2006 Elsevier B.V. All rights reserved. Keywords." Hepatitis B vires; Vaccination; Occult HBV infection; Safety of blood; NAT

1. Introduction Viral hepatitis B is a major worldwide public health concern, accounting for both high morbidity and mortality rates and significant personal, social and economic costs. Hepatitis B is indeed a leading cause of acute and chronic liver disease, including cirrhosis and hepatocellular carcinoma (Lee, 1997; Maynard, 1990). W H O estimates that approximately two billion people (about one third of the global population) have been infected with hepatitis B virus. Over 350 million people (or 6% of the global population) are chronically infected, a part of whom (about 15-20%) are at risk of developing life-threatening diseases, and nearly one million people die each year from acute and chronic sequelae of primary HBV infection (Kane, 1996; Zuckerman and Zuckerman, 2000). During the last decades, Italy has seen a dramatic decline in hepatitis B infection rates, as a result of social, behavioural and demographic changes (i.e. reduction in * Corresponding author. Prof. Alessandro R. Zanetti, Department of PuNic Health Microbiology Virology,Via C. Pascal 36/38, 20133 Milano, Italy. Tel.: +39 02 50315126; fax: +39 02 50315120. E-mail address." [email protected] (A.R. Zanetti). 1590-8658/ $ see front matter 9 2006 Elsevier B.V. All rights reserved.

family size), the general improvements in standard of living and hygiene, and the introduction of public health measures including use of disposable syringes and needles, implementation of universal precautions in medical settings, refinement in blood screening, and implementation of vaccination policies (Fitzsimons et al., 2004).

2. The impact of hepatitis B vaccination Vaccination is the most effective measure to reduce the impact of hepatitis B in terms of both cost-effectiveness and cost benefit ratios. As of the beginning of 2005, 168 countries in the world have implemented vaccination programmes against hepatitis B. Italy was one of the first, implementing selective vaccination targeted to high-risk groups in 1983. In 1991, Italy introduced mandatory universal vaccination for infants and 12-year old adolescents together with mandatory screening of pregnant women in order to identify babies at risk of acquiring perinatal HBV infection, in need to be treated at birth with hyperimmune gamma globulins (HBIG) and vaccine. In addition, vaccination continued to be offered

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A.R. Zanetti et al./Journal of Clinical Virology 36 Suppl. 1 (2006) $51~,55

free of charge to people at increased risk either by lifestyle or professional exposure (Piazza et al., 1988; Zanetti et al., 1993). Thanks to the high quality of vaccination delivery and to the public awareness of the disease, Italy's hepatitis B vaccination programme met with success. The coverage rate in both children and adolescents is around 95% with somewhat lower acceptance in the southern regions compared to northern Italy. Since the implementation of this vaccination programme, more than 15 million individuals (approximately 25% of the resident population), of whom at least 12 million children, have been vaccinated with a high record of safety and efficacy (Mele et al., 2002). At the beginning of 2004, the two program cohorts, those vaccinated as infants and as adolescents, merged with the result that the Italian population aged between 0 and 25 years is long-term protected against HBV even in the absence of additional booster injections of vaccine (Zanetti et al., 2005). This is an extraordinarily important goal since young people are at greater risk both of acquiring infection, either by drug injection or by sexual exposure, and of developing the chronic carrier state. Following vaccination, the incidence of acute hepatitis B has dramatically declined. The overall number of new cases notified during the period 2001 2004 dropped by more than 80% compared with data reported during the five-year period before the implementation of vaccination. This decline was even more striking in the 15-24years age group in which the morbidity rate per 100000 inhabitants declined from 17 in 1990 to less than one, in 2004 (http://www.iss.it/epid/index.php). In addition, levels of HBV markers have dropped to near zero among children and young people during the last decade. For example, the prevalence of anti-HBc antibody among recruits, which was 16.8% in 1981, decreased to less than 1% in 2001. An added benefit of the hepatitis B vaccination is that the decline in hepatitis B infections was paralleled by a significant reduction of hepatitis caused by Delta virus (HDV), a defective virus which requires the helper function of HBV for its replication (Gaeta et al., 2000). Importantly, no clinically overt hepatitis B cases have been reported so far among successfully vaccinated individuals, even though breakthrough infections documented by seroconversion to anti-HBc positivity or by transient ALT elevation can be occasionally seen. S-gene HBV mutants, as the prototype G145R in which a single amino-acid substitution of glycine to arginine occurred at amino acid 145 of the a determinant of the surface antigen have been identified in Italy and elsewhere (Carman et al., 1990, 1997, 1998; Fortuin et al., 1994; Francois et al., 2001; Harrison et al., 1991; Hsu et al., 1997; Zanetti et al., 1988). Such mutants can potentially escape neutralizing antiHBs antibody and infect vaccinated people while evading

detection by some commercially available HBsAg assays, and hence potentially entering the blood supply. There is evidence that in highly endemic countries, such as Taiwan, the proportion of mutants of the HBV a_ determinant increased significantly over the 15 years after the universal implementation of vaccination against hepatitis B. However, the overall number of such mutants and their impact remain low. In Italy, these mutants (particularly G145R or at lesser frequency P120S and P127S) have been occasionally isolated from liver-transplant patients or from children born to HBsAg positive mothers treated with HBIG plus vaccine (Carman et al., 1990; Cariani et al., 1995; Mele et al., 2001). However, at least at present, such mutants do not pose a public health threat to our established hepatitis B vaccination programme. In summary, following vaccination and the introduction of other public health measures such as use of universal precautions in medical settings and blood screening, hepatitis B has significantly declined in Italy in the past decades.

3. The current HBV epidemiological picture Despite the progress towards control and prevention of hepatitis B, the burden of this disease remains high in Italy. According to our surveillance system, approximately 1200 individuals acquire acute hepatitis B yearly. However, due to underreporting and to the fact that a considerable number of HBV infections are asymptomatic and go unnoticed to the surveillance system, we estimate that the true number of people infected with HBV each year is 5-10 times the number of reported cases. In addition, at least 1.2-1.5 million people, most over 50 years old, are chronic carriers of HBV. The sigmoidal shape of the age-dependent curve of HBsAg carrier state reflects the recent drop in level of viral spread, where the higher prevalence of carriers among the elderly is likely due to a cohort effect, with most infections acquired in the past. The prevalence of carriers shows an increasing trend from North to South. They are more likely males than females (ratio 3:1), approximately 90% of them are antiHBe antibody positive, and 5% are co-infected with HCV. Genotype D, then A, are the most prevalent HBV genotypes in Italian-born individuals while genotypes B, C, E, and F are found in immigrants of Asian and African origin. As already mentioned above, the Italian population aging between 0 and 25 years is protected against HBV, thanks to universal vaccination. Hepatitis B may still occur in unvaccinated people through injecting drugs, sexual exposure, nosocomial exposure, and more rarely through blood transfusion.

A.R. Zanetti et al./Journal of Clinical Virology 36 SuppL 1 (2006) $51~,55

4. Nucleic acid testing (NAT) and the safety of blood supply In Italy as in other industrialized countries the safety of blood supply has dramatically improved in the past decades through the implementation of several effective strategies and measures such as selection of periodic, volunteer, unpaid donors, careful evaluation of the donor's personal and medical history, confidential unit exclusion, testing of each donation for HBV, HIV, HCV, ALT, and the appropriate use of blood and blood components (Velati et al., 2002). The recent implementation of NAT testing in addition to the pre-existing antibody/antigen-based screening has further increased the safety of blood supply. NAT shortens the window phase in which an infected donor may harbour large amounts of viral particles in the absence of symptoms, particularly during the early antibody-negative phase of HCV or HIV infection, but also before HBsAg seroconversion (Koppelman et al., 2005). Blood donor screening for HCV with NAT was first introduced in Italy in 2001 and became mandatory in June 2002. At present, no specific recommendations have been issued by our National Health Authorities regarding HIV and HBV screening. However, based on Regional Health Authorities regulations, HIV NAT is currently mandatory in four regions, not mandatory but universally performed in another thirteen regions, and not yet introduced in the remaining four regions (Coste et al., 2005). HBV NAT is currently implemented in four regions and under consideration in the remaining. Two NAT procedures are in use in Italy: the Gen-Probe/ Chiron TMA Procleix Ultrio and Roche Ampliscreen, with approximately 46% of blood donation screened for HCV with the former assay and 54% with the latter. NAT screening is performed using minipools of 10-24 samples with the Roche system while 20% of tests are carried out on eight donation samples and 80% on individual donor samples (ID-NAT) with the Chiron system. Residual risk estimates using the so-called incidenc~ window phase mathematical model before implementation of NAT were 2.7 per million for HCV, 2.2 per million for HIV and 12.9 per million for HBV (Coste et al., 2005). Since the introduction of NAT screening, 3 894 894 blood units were tested for HCV with an observed NAT yield of 3.1 per million for HCV (Table 1). However, it should be stressed that 5 of the 12 donors found infected in the window phase (i.e. HCV RNA positive/anti-HCV antibody Table 1 HCV and HIV infections detected in the seronegative window phase in blood units screened in Italy during 2001 2003 Infection

No. of blood units screened

No. of positive units in window phase

Positive units • 106 donations

HCV

3 894894

12 a

3.1

HIV

2 186468

4

1.8

a Five donors had abnormal ALT values.

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negative), had abnormal values of ALT, a test that is systematically performed in Italy. This implies that the blood of such donors would have been withdrawn even in the absence of NAT screening, and therefore the true yield of HCV NAT should be estimated to be 1.79 per million donations. Following HIV NAT screening of 2 186468 blood units the observed yield was 1.8 per million (Table 1). Differences between predicted and observed yields for both HCV and HIV were not significant. All donors found HCV or HIV NAT positive but antibody negative seroconverted during follow-up. Importantly, all donors (4/4) who were detected HIV positive and 33.3% (4/12) who were detected HCV positive in the window phase admitted behavioural risk factors after communication of their infection. The efficacy of HBV NAT in a setting with the use of highly sensitive HBsAg assays is still debated, but in Italy occult HBV infections have been relatively frequently observed.

5. Occult HBV infection By definition, individuals with occult HBV infection are HBV DNA positive and HBsAg negative with or without liver disease (Cacciola et al., 1999; Kaneko et al., 1989; Raimondo, 2001; Torbersen and Thomas, 2002; Wang et al., 1991; Zhang et al., 1993). HBV genomes are usually found co-circulating with anti-HBV antibodies like anti-HBc in the presence or not of anti-HBs and anti-HBe. However, in some cases the presence of HBV DNA is not accompanied by any detectable serological marker of infection. Long-term studies revealed that after HBsAg clearance, HBV DNA may persist for decades in serum, or more frequently in the liver of individuals with or without liver disease (asymptomatic silent carriers) (Yuki et al., 2003). Occult HBV infections may be caused by S-gene mutants that are not detected with currently available HBsAg assays or may be due to a strong suppression of HBV replication and gene expression that may also involve wild-type strains (Brechot et al., 2001; Carman et al., 1993). The causes of inhibition of HBV replication are not clear, although the host immune-surveillance probably has a critical role. Since viral DNA levels in occult hepatitis B are generally low (<1000 IU/ml), identification requires the use of highly sensitive HBV DNA assays (Allain, 2004). Whether HBV genome persistence contributes to liver disease and whether a blood donor with occult HBV is infectious by transfusion are critical issues that need to be further investigated. Evidence indicates that carriers of occult infection may transmit HBV in the case of blood transfusion or organ transplantation (Chazouilleres et al., 1994; Degos et al., 1988; Hoofnagle et al., 1978; Thiers et al., 1988). AntiHBc alone with HBV DNA has been associated with infectivity of blood products or organs. Risk of transmission

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depends on the characteristics of the circulating virus in terms of viral load, whether present as free particles or complexed to antibodies during the window phase, on the immunocompetence of the recipient, and on the complex interactions between the virus and the host. The prevalence of occult infections in Europe increases with the increasing endemicity of HBV in the population, being relatively low in northern Europe and higher in Mediterranean and East European countries with a higher frequency of HBV infection. It is not fully understood if the genotype prevalent in the population plays a role in the frequency of occult HBV infection. Elsewhere in this supplement, Allain postulates that genotype D, the most prevalent genotype in Italy, could be more prone to generating occult infection (or mutations within the S gene) compared to genotypes B and C.

Based on the assumption that approximately 20% of donors screened were first-time donors and 80% repeat donors, we can estimate a crude rate of HBV DNA positivity in absence of HBsAg of about 30 per million among repeat donors and of 150 per million among first time donors. Finally, the majority of our donors with occult infections carried low levels of HBV-DNA, suggesting that the detection rate of such infections is highly dependent on the sensitivity of the NAT used for screening and confirmation. A large multicentre study coordinated by the Italian Society of Transfusion Medicine and Immunohaematology (SIMTI) is currently in progress. Preliminary results collected in 2005 seem to confirm and to extend data collected during 2004.

7. Conclusion 6. The introduction of NAT for H B V in Italy HBV NAT was first introduced in Italy on a regional basis in 2004. As of 2005, four regions have implemented such assays. During 2004, nearly 280 000 blood donations were screened for HBV DNA by NAT, approximately 45.5% using Roche technology on mini pools (MP-NAT) of 10-20 samples, and approximately 54.5% using Chiron technology on individual donors (ID-NAT). Fourteen donors or 50 per million were found HBV DNA positive/HBsAg negative (Table 2). None of them were positive for IgM anti-HBc antibody or developed HBsAg during follow-up, and therefore were classified as having occult infections. Of these, 11 were found among 151 676 donations tested by ID-NAT (72.5 per million) and 3 among 126743 donations (23.7 per million) tested by MP-NAT. Despite the three times difference in the detection rate of IDNAT compared to MP-NAT, no definitive conclusion on the relative sensitivity of these two technologies can be drawn from this study, since the two groups of donors screened were not matched. Of the 14 donors with occult infection, six were repeat donors and eight first-time donors. All donors were anti-HBc-IgG and anti-HBs antibodies positive. Median anti-HBs was 21.5 mIU/ml, range between 4 mIU/ml and 1000 mIU/ml. Four donors were also positive for anti-HBe antibody. Table 2 Occult HBV infections detected by NAT in blood units screened in Italy in 2004 Technology

No. of units tested

HBV DNA+/HBsAg No. of units

TMA PCR Total

Chiron Roche

Units • 106 donations

151676

11

72.5

126 743

3

23.7

278419

14

50.3

Italy's hepatitis B vaccination programme has resulted in considerable progress towards control and prevention of HBV infection. According to our national surveillance system the incidence of hepatitis B has dropped dramatically after the implementation of vaccination. Levels of HBV markers have also dropped significantly among children and adolescents during the past decades, and in parallel with the decline in HBV infection, hepatitis delta virus infection has also declined significantly. In addition, thanks to the universal vaccination of both infants and 12-year old adolescents implemented in 1991, the current Italian population aged between 0 and 25 years is protected against HBV infection. The residual risk of transmitting blood-borne viruses through transfusion is currently very low in Italy. However, the safety of blood supply remains a major source of public health concern and requires continuous efforts to reach zero-risk probability. Nucleic acid testing can shorten the window phase between exposure to infection and reactivity by serological assays and, consequently, further reduce the residual risk of HBV, HCV and HIV transmission. NAT is already implemented in the USA and in most European countries for the detection of HCV RNA and HIV RNA. For hepatitis B, the estimated probability of having a potentially infectious window-phase donation released in the blood supply is even higher than that expected for HCV and HIV. Our preliminary results with NAT have demonstrated a relatively high number of donors with occult HBV infection who were missed by HBsAg screening. Follow-up studies using NAT of enhanced sensitivity as well as lookback studies in recipients of blood components are further required to assess the frequency of occult HBV infections in Italian donors and to evaluate the impact of NAT implementation in reducing the risk of post-transfusion hepatitis B. The implementation of HBV NAT of enhanced sensitivity versus alternative strategies based on HBsAg and antiHBc test algorithms (as long implemented in the USA

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and France) is currently under debate in several countries. Our preliminary data indicate that there might be a benefit of NAT in preventing infection by identifying silent HBV carriers in Italy. In conclusion, there is evidence that the implementation of NAT may improve the blood safety especially in countries with high predicted residual risk for HBV infection, where anti-HBc screening is not a practical option. There is agreement that, at present, the introduction of NAT for HBV does not allow for discontinuation of screening for HBsAg.

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