Criteria for hepatitis B virus screening and validation of blood components in Italy: the position of the SIMTI HBV working group

Introduction

The screening strategies for the detection of blood donors potentially able to transmit an infection to the recipient of their blood or blood products have always included clinical, behavioural and laboratory criteria in an attempt to construct a "multi-barrier" system that most effectively guarantees transfusion safety¹. These criteria are necessarily modified over time as further knowledge is acquired and new laboratory tests become available.

Hepatitis B virus (HBV) causes the blood-borne viral infection for which serological markers for screening have been available for the longest time. Indeed, the introduction of a test to detect the hepatitis B surface antigen (HBsAg) at the beginning of the 1970s certainly contributed to a drastic reduction in the number of HBV transfusion-transmitted infections. However, despite continuous improvements in the sensitivity of this test, transfusion-related transmission of hepatitis B has not been completely eliminated and in some countries an additional serological investigation (the search for antibodies against HBV core antigen, anti-HBc) was introduced. Testing for these antibodies was initially used as a "surrogate marker" for the detection of non-A, non-B hepatitis in the mid-1980s, but after the availability of a specific test for hepatitis C virus (HCV), it returned to being of specific relevance in the screening for HBV infection.

More recently, a test to detect HBV DNA was introduced for the screening of blood donors. Crucially, this test can detect an acute HBV infection earlier than HBsAg and anti-HBc tests, thus reducing the so called "window period" during which an infected donors may harbour large amounts of infectious viral particles in the absence of serological markers and/or signs and symptoms of an ongoing infection². Furthermore, DNA testing allows the identification of a number of donors -including periodic donors-characterised by the absence of HBsAg in the presence of HBV DNA with or without anti-HBs, anti-HBc and/or anti-HBe antibodies. These latter serological profiles are defined as "occult HBV infection (OBI)" in agreement with the Consensus Conference held in Taormina in 2008³. In these cases, the amount of HBV DNA -when detectable- is usually very low (<100–200 IU/mL). On the basis of the serological profile, OBI can be distinguished into seropositive (anti-HBc and/or anti-HBs positive) or seronegative (anti-HBc and anti-HBs negative) types³.

The tests that, at present, are mandatory in Italy for the prevention of transfusion-related transmission of HBV include HBsAg (using several commercially available "last-generation" immunoassays kits) and HBV DNA detection (50% of blood units are tested in pools of 6 samples by Roche Cobas Taqscreen MPX on the Cobas s 201 platform [Roche Instruments Center, Rotkreuz, Switzerland] and 50% in single tests by Novartis Ultrio [Emerville, CA] with comparable analytical and clinical sensitivity^4,5). In addition, other serological tests are performed, with a heterogeneous distribution across the country, in part as an additional criterion for the validation of blood components (i.e. anti-HBc) and in part as an evaluation of the immunological status of the donor (i.e. anti-HBs).

A national survey organised by the Italian Society of Transfusion Medicine and Immunohaematology (SIMTI) in 2009⁶ found that 78 (43%) of 181 Transfusion Services (TS) that replied to a specific questionnaire routinely performed anti-HBc and that 53 of these 78 (68%) TS were located in Northern Italy Regions. In most of the TS (81%) participating in the study, this test was carried out in first-time donors only, while in the remaining (19%) it was carried out in all donors. Fifty percent of the TS that performed this test used a positive result for anti-HBc as a criterion to exclude antibody-positive donors from donating while the remaining TS did not.

Thus, there are profound differences in the behaviour of TS, resulting from the persistence of criteria based on serological parameters and criteria derived from the introduction of the new molecular biology tests. In the light of this, SIMTI set up a Working Group charged with the task of defining the screening tests for HBV capable of guaranteeing the highest levels of safety compatible with the possibility of ensuring timely and appropriate transfusion therapy in the specific Italian epidemiological context, with the currently available technology.

Epidemiological notes

HBV infection still has a strong impact on the world's population: it has been estimated that over two billion subjects are infected, 350 million people are the chronic carriers, 4.5 million new infections occur each year and that 600,000 people die yearly due to the consequences of acute or chronic hepatitis B⁷.

The world can be divided into three major geographical areas on the basis of the prevalence of HBsAg carriers:

• - high endemicity areas with over 8% of carriers in the general population (sub-Saharan Africa, South-east Asia, the Amazon Basin, Central Asian Republics, parts of the Middle East and some countries in Eastern Europe);

• - intermediate endemicity areas with 2 to 8% of a given population being HBV carriers (South Europe, some Mediterranean countries);

• - low endemicity areas in which the carrier rate is less than 2% (USA, North and Western Europe, Australia, and parts of South America).

These areas also differ with regards to the main routes of infection^8,9.

In Italy, chronic HBV infection is still a leading cause of cirrhosis and primary hepatocellular carcinoma. The epidemiological trend in Italy has changed dramatically in recent years following the introduction in 1991 of universal vaccination in neonates and in 12-year old adolescents. Following this, young people aged between zero and 30 years old are now protected against HBV^10,11. According to the data monitored by the Italian National surveillance system (SEIEVA-Rome) the yearly incidence rate of acute hepatitis B is around 1 per 100,000 inhabitants, being somewhat higher in males than in females and higher in the Northern-Central Regions than in the Southern regions and in the Islands. Most of such infections occur in unvaccinated adults, and sexual exposure to multiple partners is the most important risk factor for acquiring HBV. Intravenous drug use, nosocomial exposure to the virus as well as beauty treatments (piercing, tattoos, manicure) also play a crucial role in the spread of the virus¹². Approximately 700,000 people (or nearly 1% of the Italian population) are carriers of HBsAg and most of them are over 40–50 years old. The higher prevalence of carriers found among the elderly is likely to be due to a cohort effect - most infections having been acquired in the past. About 5% of HBsAg carriers are co-infected with HCV. The most common HBV genotypes in Italian-born carriers are genotype D followed by genotype A, while genotypes B, C, E and F are usually found in immigrants of Asian or African origin¹³.

Surveillance of transfusion-transmissible diseases in Italy

According to data collected by the National Blood Centre, the prevalence and incidence of markers of HBV infection in Italian donors have not changed substantially over the last 15 years¹⁴. Also in Lombardy -where a surveillance system for transfusion-transmissible diseases was established in 1996- the incidence and prevalence of HBV infection in donors have shown minor fluctuations over time, as reported in Table I (institutional and unpublished data).

Table I.

Incidence and prevalence rates (x10⁵) of HBV infection in blood donors in Lombardy. The incidence is calculated in periodic donors as the number of positive subjects divided by the number of blood donations/year. The prevalence is calculated as the number of positive first-time donors divided by the total number of first time donors/year.

	1996	1997	1998	1999	2000	2001	2002	2003	2004	2005	2006	2007	2008	2009
Incidence	1.4	1.4	1.6	1.7	1.9	2.5	3.4	1.4	1.6	0.6	1.5	1.3	1.4	1.4
Prevalence	244.7	345.3	200.5	254.6	222.0	263.4	229.8	262.7	187.1	249.8	253.5	247.6	226.1	142.1

The SIMTI studies

1. In 2006 the Research & Development sector of SIMTI promoted, in collaboration with Abbott Laboratories and Roche Diagnostics, a national study aimed at estimating the prevalence of HBV infection in first-time blood donors in Italy and determining the presence of HBV DNA in subjects positive for HBsAg and in those negative for HBsAg but positive for anti-HBc. The results can be summarised as follows¹⁵:

   • - 41,670 first-time donors were enrolled in the study;

   • - the mean age of the subjects studied was 42 years, range 26–59;

   • - 30% of this population had been vaccinated against HBV;

   • - the mean prevalence of any serological markers of HBV among unvaccinated individuals was 8.3%;

   • - the rate of positivity for HBV DNA (determined using a COBAS AmpliScreen HBV Test -Roche, with the 1-mL extraction method, 95% lower limit of detection, 5 IU/mL) among HBsAg-negative/anti-HBc-positive subjects was 0.69% (15 out of 2,186).

     This finding was similar to the rates reported for other populations of blood donors^16–24. Higher prevalences have been found in a limited number of studies^25–27.

2. Another independent study in which donors were screened using nucleic acid amplification technology (NAT), yielded the following data concerning HBV⁴:

   • - between 2004 and 2009, overall 485 HBV DNA positive/HBsAg negative donors were found after testing 9,600,000 units;

   • - in 25 cases, the HBV infection was in the acute window phase (1:384.000), whereas in the remaining 460 cases (1:21.000), blood was collected from donors with occult HBV³.

     The NAT methods used during the study period were different: single tests and pools of 8 samples when using Novartis Ultrio reagents and pools of 20 to 6 with different Roche kits.

When can HBV be transmitted by transfusion?

HBV infection can be transmitted by transfusion when the donor has an acute infection or when the donor has a picture of a past HBV infection, without evidence of chronic hepatitis, but with infective virions (HBV DNA positivity) in the circulation (i.e., the donor has OBI). Thus, transmission of HBV to the recipient can occur if the two above-mentioned conditions are not intercepted by the screening tests used to validate the blood units. There are, however, other aspects that can affect the transmission of a viral infection, such as the viral load, the phase of infection of the donor and the immunological status of the recipient.

Screening tests

The tests carried out for the biological validation of blood and blood components with respect to HBV can be divided into three groups:

• - tests targeted to detect a component of the virus (HBsAg, HBV DNA),

• - tests that search for an antibody as a marker of exposure to the virus (anti-HBc),

• - surrogate tests that can be indirectly indicative, even though less specific, of a contact with the virus which caused damage to the liver (alanine aminotransferase).

The screening strategies

Throughout the world, the screening test used for the longest time and still considered of primary importance by the World Health Organization (WHO)²⁸ is HBsAg.

In some countries with a low prevalence of HBV (generally those countries with a prevalence of anti-HBc around 1% or less: Northern Europe, the USA, Canada^20,23,29) detection of anti-HBc has been added to HBsAg. The rationale for using anti-HBc in the screening is the following:

• - it is a serological marker of recent or past infection and persists over time;

• - it can be the only serological marker present in the acute phase, after the disappearance of HBsAg;

• - the number of anti-HBc–positive subjects excluded from donation is limited because of the low endemicity;

• - it can be an alternative method (less specific, but cheaper) to NAT for the detection of HBV infection.

However, it must be underlined that the specificity of this test is not optimal (albeit recently improved^23,30–32), and that an algorithm to requalify anti-HBc false-positive donors has been adopted in the recent years in some countries, including Germany^23,33 and the USA³⁴.

To limit the number of discarded units, a quite complicated screening algorithm, in which anti-HBc and anti-HBs titres are determined and blood units negative for HBV DNA and with anti-HBs <200 IU are considered usable for transfusion, is currently employed in Japan³⁵.

In countries with medium/high endemicity for HBV infection (i.e. South and East Europe, Asia, South Africa)^36–41 besides HBsAg, tests for HBV DNA have been implemented, usually together with equivalent tests for HCV RNA and HIV RNA. The additional value of introducing NAT testing for HBV can be summarised as follows:

• - it is a direct marker of the virus;

• - it reduces the window period of the acute infection;

• - it can identify subjects with OBI;

• - it identifies only those subjects who are potentially infectious for HBV who must be excluded from blood donation, limiting the unjustified deferral of blood donors and their management costs.

Furthermore, following the introduction of NAT testing for HBV DNA in the screening of blood donors, crucial information has been gained. For example:

• - the persistence of low levels of HBV DNA in the context of acute infections in which HBsAg is no longer detectable;

• - the biological dynamics and oscillations of viral load in the chronic phase of infection with levels of HBsAg below the limit of detection by currently used tests;

• - the identification of infections due to mutated HBV with altered HBsAg epitopes not detectable by common screening tests^42–44.

In Germany, a country with low HBV endemicity, both anti-HBc (since 2005) and HBV DNA assays are currently carried out on a voluntary basis and in pools of 96 samples⁴⁵. Recently most blood collection agencies in the USA and in France have begun performing HBV DNA tests in addition to those for anti-HBc⁴⁴.

Considerations on self-sufficiency of the blood supply in Italy

Italy is considered a country of intermediate/low endemicity for HBV infection²⁹ and, on the basis of the data reported above¹⁵, the use of an anti-HBc test for the purpose of selecting only new donors would lead to the exclusion of about 8% of such subjects and, in some Regions, this number would be even double. This must be put in the context of the documented finding of HBV DNA in only 0.69% of anti-HBc positive/HBsAg negative subjects.

Furthermore, it should be considered that at least 30% of new donors have already been vaccinated and that this percentage is increasing every year given the continuing practice of vaccination against HBV at birth. Thus, if a selection criterion based on the reactivity to the anti-HBc test were to be applied to the whole population of Italian donors -also including periodic donors, until now not selected by this marker- it would lead to the exclusion of such a large number of subjects as to be incompatible with the national self-sufficiency programmes. The WHO does not recommend the introduction of anti-HBc in blood screening as a routine test on the basis of similar considerations²⁸.

Considerations on transfusion safety

There have been reports in the past of some cases of post-transfusion hepatitis B due to blood products from HBsAg-negative/anti-HBc-positive donors. Actually, in these cases the search for HBV DNA was either not performed or was carried out using tests with a much lower sensitivity than those currently employed^{20,29,43,46–52}.

Recent reviews of cases of post-transfusion hepatitis B published in the literature document that the HBV infection was transmitted to the recipient through units of blood components from donors who were found, by look-back studies, to be anti-HBc positive/HBV DNA-positive (i.e. with OBI) or anti-HBc-negative/HBV DNA-positive or negative for both markers⁵³ (subjects in the initial acute phase of infection). These studies did not report recipients who acquired infection from donors who were positive for anti-HBc but HBV DNA-negative^29,54.

Two other aspects that still have to be clarified are the probability with which subjects with OBI transmit infection and the role played by the presence of anti-HBs antibodies⁵⁵. A systematic look-back study carried out by the Japanese Red Cross showed that units of anti-HBc-positive/HBV DNA-positive blood from subjects with OBI were 15-fold less capable of transmitting infection compared to units collected from subjects during the window period of infection⁵⁶. Furthermore, there is evidence that units from donors with OBI which also contained anti-HBs were unlikely to transmit infection, whereas units without anti-HBs (with only anti-HBc or anti-HBc in the presence of anti-HBe) were more infectious⁵⁷. However, the transmission of HBV from donors with low loads of HBV DNA and the presence of anti-HBs has been documented recently giving relevance to the issue of immunodeficient recipients who are more prone to be infected even when the blood they receive contains a very low number of viral particles⁵².

The molecular tests currently authorised in Italy for the screening of blood components have an analytic sensitivity of 3.7–10.4 IU/mL⁵ and a SIMTI study showed a comparable clinical sensitivity of the two tests currently on the Italian market (Roche in pools of 6 and Novartis in single specimens)⁴.

In our epidemiological situation, the scientific evidence supports the use of the two direct markers of HBV -HBsAg and HBV DNA- in order to maximise the safety of the selection of donors as already foreseen by the current legislation in Italy. In conclusion, in our epidemiological context, the following recommendations can be made concerning the validation of blood components in order to ensure the recipients' safety.

Testing for HBsAg and HBV DNA

At present, screening of blood units with these two direct tests (HBsAg and HBV DNA) is the safest choice for reducing the residual risk of transmitting HBV through transfusion because these tests:

• - enable the selection of blood components with the lowest probability of transmitting HBV infection in the window period of an acute infection, in chronic phase infection and in OBI;

• - can limit the exclusion of anti-HBc-positive subjects from blood donation to only those potentially able to transmit HBV infection.

Testing for anti-HBc antibody

The implementation of the anti-HBc test together with the two tests already mandatory by law (HBsAg and HBV DNA) does not seem to add any documented advantage with regards to increasing the safety of the blood supply. In addition, given the epidemiological pattern of HBV exposure in Italy, the use of anti-HBc tests for blood screening would lead to an unnecessary exclusion of a large proportion of potentially valid donors in this country.