Blood Screening for HBV DNA

ELSEVIER

Journal of Clinical Virology 36 Suppl. 1 (2006) $30 $32 www.elsevier.com/locate/j cv

Blood Screening for HBV DNA C h y a n g T. Fang Biomedical Researeh and Development, American Red Cross, Rockville, MD, USA

Keywords." HBV DNA; HBsAg; anti-HBc; NAT; Blood safety

The safety of the blood supply has been greatly improved in the past two decades owing to continuous improvement in the technology and sensitivity of laboratory testing, better donor education and selection, and tighter quality control on blood bank operations. Despite these efforts and achievements, however, the risk of acquiring blood-borne infections, mainly human immunodeficiency virus (HIV), hepatitis C virus (HCV) and hepatitis B virus (HBV), as a result of transfusion remains a public concern. For the American Red Cross Blood Services (ARC), which collects approximately 6.5 million units of blood annually, the seroprevalence rates for these three pathogens among blood donations in a 12-month period from April 2004 to March 2005 are shown in Table 1. In 1999, nucleic acid amplification testing (NAT) on pools of 16 or 24 donor samples (MP-NAT) was implemented to provide a way to directly detect HIV-1 and HCV viral RNA which may be present in blood donated by donors during the early stage of seronegative window period (Stramer et al., 2004). While its cost-effectiveness is debatable (Jackson et al., 2003; Marshall et al., 2004), the introduction of HIV-1 and HCV NAT undoubtedly further improves blood safety. As shown in Table 1, 1.1% of HCV- and 2.8% of HIV-1contaminated blood donations identified within the ARC program were seronegative and could only be detected with MP-NAT. Dodd and colleagues (2002) of the ARC reported that, as a result of MP-NAT implementation, the residual window-period risks have been reduced to an estimate of 1:2135000 for HIV and 1:1935000 for HCV among donations from repeat donors. Furthermore, the introduction of NAT technology for blood screening led to the rapid and successful implementation of laboratory testing to reduce transfusion-transmission of West Nile virus (WNV) in 2003 (Stramer et al., 2005a). Two HBV-specific serologic markers, hepatitis B surface antigen (HBsAg) and antibodies to hepatitis B core antigen * Corresponding author. Transmissible Diseases Department, American Red Cross Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855, USA. Tel.: +1 301 738 0645; fax: +1 301 738 0495 E-mail address." [email protected] (C.T. Fang). 1590-8658/ $

see front matter 9 2006 Elsevier B.V. All rights reserved.

Table 1 Prevalence rates (per 10 000) among blood donations to the American Red Cross, April 2004 March 2005 Type of donor

Number of donations a HBV

HBsAg

HCV

Antibody

HIV

Prevalence rate (per 10000) First time

Repeat

All

1 140247

4997231

6 137478

6.542

0.184

1.365

18.294

0.342

3.677

RNA

0.096

0.028

0.041

Total

18.391

0.370

3.718

Antibody

1.017

0.104

0.274

RNA

0.018

0.006

0.008

Total

1.035

0.110

0.282

a Includes whole blood and plasmapheresis donations from volunteer and directed donors.

(anti-HBc) are routinely tested as a part of blood screening in the USA. Testing for HBsAg was first introduced in 1971. The technology has been evolved from agar gel electrophoresis, haemagglutination, radioimmunoassay, to enzyme immunoassay. The sensitivity of these assays has also been improved along the way from greater than 10 ng/mL to less than 1 ng/mL of HBsAg for the tests currently licensed by the U.S. Food and Drug Administration (FDA) and less than 0.1 ng/mL for newer, unlicensed tests (Biswas et al., 2003). The prevalence of HBsAg among contemporary donors in the USA remains high at 1.365 per 10 000 (0.184 and 6.542 per 10 000 among repeat and firsttime donors, respectively) for the ARC program (Table 1). Anti-HBc testing was implemented in 1986/1987 as a surrogate for non-A, non-B hepatitis, but was later licensed for further prevention of transfusion-associated HBV infection. With these two tests, the residual windowperiod risk of post-transfusion HBV infection was estimated at 1:488000 to 1:205000 (Dodd et al., 2002). However, according to the U.S. Centers for Disease Control and Prevention (CDC), of 7381 cases of acute hepatitis B investigated in 2003, only 10 cases were confirmed as acute hepatitis B corresponding to the time of transfusion; and

C. 77 Fang~Journal of Clinical Virology 36 Suppl. 1 (2006) $30 $32

of these 10, only one could be associated with a single infected donor (Stramer, 2005b). The differences between the estimated transfusion risk and actual observed clinical cases may be due to the following reasons: (1) duration of the HBV window period used for risk assessment may not be entirely infectious, particularly for the first 10 days after infection (Yoshikawa et al., 2005); (2) a significant portion of recipients die of their underlying disorders within the first six months post transfusion; and (3) most HBV infections in adults are either sub-clinical or with mild symptoms and hence are not reported. For these reasons, HBV NAT was not implemented along with HIV-1 and HCV in 1999; nevertheless, it has been intensively debated since 2001 (Kleinman and Busch, 2001; Stramer et al., 2001; Biswas et al., 2003; Stramer, 2005b). In this issue of JCV, scientists at Gen-Probe Incorporated (San Diego, CA) report the findings of studies using the Procleix| Ultrio | assay that allows the simultaneous detection of HIV-1 RNA, HCV RNA and HBV DNA in human plasma samples (McCormick et al., 2006). The technology of this product is based upon transcriptionmediated amplification of viral-specific nucleic acids. The same technology has been applied to its previously licensed Procleix multiplex assay for HIV-1 and HCV RNA (distributed by Chiron Corporation, Emeryville, CA), which is currently used by many blood bank laboratories around the world. According to the data presented in this paper, the authors claimed that the Procleix Ultrio assay maintained a comparable sensitivity as the Procleix multiplex assay for HIV-1 and HCV RNA, while providing an additional sensitive detection of HBV DNA for a 95% detection rate of 15 IU/mL. This product is designed for large-scale blood screening on donor plasma samples either in minipools (up to 16 samples) or individually (ID-NAT). The study was conducted with the use of its enhanced semi-automated instrument system. However, it can be applied to GenProbe's fully automated system, TIGRIS | , with claimed equivalent performance characteristics (Mimms, 2005). The window period between HBV infection and the time when HBsAg can be detected with currently licensed screening assays is believed to be approximately 59 days (Schreiber et al., 1996), comparing to 70 days for HCV and 16 days for HIV-1 (Dodd et al., 2002). The doubling time of HBV DNA is estimated at a mean of 2.565.84 days (Busch, 2001; Biswas et al., 2003; Yoshikawa et al., 2005), which is significantly longer than that for HCV or HIV-1. In other words, during the early stage of infection, HBV replicates rather slowly and reaches a viral load of 100 1100 IU/mL (based upon WHO International Standard, NIBSC, Hertfordshire, UK) or 720 11 500 geq/mL (based upon HBV seroconversion panels) before HBsAg can be detected serologically (Biswas et al., 2003). It has been shown that there was a good linear correlation between HBsAg concentration and viral load during the early ramp-up phase (Biswas et al., 2003; Yoshikawa et al., 2005). The Abbott PRISM | HBsAg assay, which is yet to be

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licensed in the USA, represents probably the most sensitive HBsAg assay at less than 0.1 ng/mL (Stramer, 2005b) and was found to have a detection limit equivalent to 102 IU/mL of HBV DNA (Biswas et al., 2003). Therefore, in order to gain advantage over a highly sensitive HBsAg serologic assay, mathematically, one must perform NAT on individual samples or pools of less than eight samples. Using seroconversion panels, comparing to the currently USlicensed HBsAg assays, the extent to which window periods could be shortened by various newer laboratory tests were: more sensitive HBsAg assays, 2-9 days; MP-NAT, 9-11 days; and ID-NAT, 25-36 days (Biswas et al., 2003). These numbers further illustrate that implementation of NAT on minipools of eight or more donor samples will provide a marginal gain over a highly sensitive HBsAg assay. Recently, a multicenter study conducted in Europe testing 15 seroconversion panels (Koppelman et al., 2005) demonstrated that the Procleix Ultrio NAT assay had a 95% detection rate at 11 IU/mL (WHO International Standard) and had an earlier detection by an average of 3 days with pools of 16, 6 days with pools of 8 and 14 days with individual samples, over the PRISM HBsAg assay. As mentioned above, blood screening for anti-HBc was implemented in the USA as a surrogate for non-A, non-B hepatitis in 1986/1987. Since the discovery of HCV, this serologic test has been retained to prevent transmission of HBV from transfusion of blood donated at a time when HBsAg declines to undetectable levels and antibodies to HBsAg (anti-HBs) have yet to appear. Although the currently licensed anti-HBc assays are not highly specific and a confirmatory test does not exist, most anti-HBc-true-positive but HBsAg-negative samples are also positive for anti-HBs. However, a small percentage (depending on the population prevalence) of these samples contains HBV DNA at a level that can be detected only with the ID-NAT (Yoshikawa et al., 2005). The potential infectivity of these types of samples is unknown and may depend upon whether the amount of anti-HBs is sufficient enough to prevent infection. Satake et al. (2005) of the Japanese Red Cross recently reported that 2.5% of blood containing HBsAg-negative, anti-HBc-positive and low-level HBV DNA (ID-NAT positive/MP-NAT negative) could transmit infection, although this transmission rate was less than one tenth of the frequency of infection from blood containing low-level HBV DNA in the early window phase (HBsAg negative/anti-HBc negative). Hence, antiHBc testing provides an additional layer of blood safety against HBV It has been suggested that implementation of MP-NAT for HBV might lead to discontinuation of HBsAg testing but not anti-HBc (Kleinman et al., 2005), even though only 67-74% of HBsAg confirmed positive samples were positive with MP-NAT in a study conducted by the German Red Cross (Roth et al., 2002). However, in high-prevalence countries where anti-HBc blood screening will cause deferral of a significant percentage of donors, HBV NAT may represent the only option to further prevent

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HBV transmission in addition to HBsAg testing (De Felice et al., 2005). Furthermore, in rare cases, HBV DNA may be the only marker present in blood and/or liver tissue. For this situation, the viral load is usually low (<500IU/mL) and, hence, sensitive HBV NAT will be needed for detection (Allain, 2004). Routine NAT blood screening on minipools of 16-24 donor samples for HIV-1 and HCV RNA was implemented in 1999 in the USA (Stramer et al., 2004). The same approach was successfully applied to the blood screening for WNV RNA starting in 2003 (Stramer et al., 2005a). It is generally recognized that HBV NAT using the same minipool testing strategy will have a marginal gain over the most sensitive HBsAg assay. However, ID-NAT, when compared to current HBsAg tests, was expected to prevent 30-35 transfusions containing HBV per year (Biswas et al., 2003) in the USA. ID-NAT for WNV using a fully automated system (Gen-Probe's TIGRIS instrument) has been implemented under an FDA-approved investigational protocol in 2004 at a few blood testing laboratories in the USA with successful outcomes (Stramer et al., 2005a), and data have been submitted to the FDA for biological license consideration. It is reasonable to expect that NAT on individual samples or in smaller pools for HBV together with HIV-1 and HCV may be applied to the same system in the future in order to further enhance the safety of the blood supply.

Acknowledgement Data in Table 1 were compiled by Edward R Notari IV

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