Epidemiology of HBV infection in Asian blood donors: Emphasis on occult HBV infection and the role of NAT

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Journal of Clinical Virology 36 Suppl. 1 (2006) $33 $44 www.elsevier.com/locate/jcv

Epidemiology of HBV infection in Asian blood donors: Emphasis on occult HBV infection and the role of NAT Chun-Jen

L i u a,

Ding-Shinn C h e n

a'b, P e i - J e r C h e n a'b' *

aDivision of Gastroenterology, Department of Internal Medicine, and bGraduate Institute of Clinical Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan

Abstract

Hepatitis B virus (HBV) infection is endemic in many Asian countries. Among many transmission routes, transfusion is the one that should be prevented. The first major success in enhancing transfusion safety came with the implementation of hepatitis B surface antigen (HBsAg) in the early 1970s. However, the studies quoted in this review demonstrate that transmission by blood components negative for HBsAg can still occur in the acute phase of infection during the seronegative window period, or during chronic stages of infection (i.e. "occult" HBV infection, OHB). OHB is defined as the presence ofHBV DNA in blood or liver tissues in patients negative for HBsAg, with or without any HBV antibodies. Because of limitations in current blood screening practices, OHB is an overlooked source of HBV transmission. For policy development on screening for HBV infection in blood donors, it would be useful to assess the relative contribution of the above two sources of transfusion-transmitted HBV infection from HBsAg-negative donations. New screening policy should be evaluated on the basis of available data or newly designed studies. While anti-HBc screening can eliminate residual risk of occult HBV transmission by transfusion in low-endemic areas, it would not be practical in most parts of the world where the prevalence of anti-HBc is >10% as too many otherwise healthy donors will be ineligible. On the contrary, studies mentioned in this paper indicate that nucleic acid amplification test (NAT) or new HBsAg tests of enhanced sensitivity would be effective in the screening of blood donors for OHB in highly endemic countries. However, the cost-effectiveness of blood screening tests is a major concern in Asia. We therefore have systemically reviewed the literature on prevalence and infectivity of OHB in Asian countries and the possible role of NAT for identifying blood donors in the pre-HBsAg window phase or in later stages of OHB. 9 2006 Elsevier B.V. All rights reserved. Keywords." Hepatitis B vires; Transfusion; Blood donor; Occult; NAT; Anti-HBc

1. I n t r o d u c t i o n

Blood transfusion and component therapies are wellestablished and essential medical practices. However, blood collected from large populations is inevitably associated with a risk of infectious pathogen transmission (Hoofnagle, 1990; Schreiber et al., 1996). Currently, serologic screening for HBV, HCV, HIV and HTLV-I or -II is the main method used to reduce the frequency of transfusion-transmitted viral infections. In western countries, screening policies have effectively decreased transmission rates to approximately 2.5 per million units for HIM 9.1-11.1 per million units for HCV, and 2.5-15.3 per million units for HBV (Alter et al., 1972; Donahue et al., 1992; Busch, 1998; Busch et al., 2003; Kleinman and Busch, 2000). Most of these remaining infectious blood units came from donors who * Corresponding author. Prof. Pei-Jer Chen, Graduate Institute of Clinical Medicine, National TaiwanUniversity College of Medicine, 1 Chang-Te St., Taipei 100, Taiwan. Tel.: +886 2 23123456 ext.7072; fax: +886 2 23317624. E-mail address." [email protected] 1590-8658/ $

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

were in the serologically negative window period (WP). To overcome this problem, even more sensitive screening methods for detecting viral antigens or nucleic acids have been developed. Nucleic acid amplification testing (NAT) for HIV and HCV has already been implemented in USA, Europe and Japan. However in hepatitis B endemic areas, there is an emerging problem. Using NAT, investigators have discovered that among individuals with past hepatitis B infection, around 0 . 5 - 2 5 % retained viral DNA in their blood or blood cells; this is termed occult HBV infection (OHB) (Brechot et al., 2001; Hu, 2002; Torbenson and Thomas, 2003; Allain, 2004). Even among individuals positive for antiHBs, 0 . 5 - 1 5 % still tested positive for serum HBV DNA, though at a very low titre (Noborg et al., 2000; Shih et al., 1990; Yotsuyanagi et al., 1998; Matsumoto et al., 2001; Pao et al., 1991). It is imperative to know the prevalence and infectivity of OHB in the transfusion setting. Another aspect is the lack of harmonization of screening programs for HBV infection among blood donors around

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the world. The applied tests differ between countries where prevalence of HBV infection varies greatly. In lowprevalence countries, like the USA and Japan, blood donors are screened for both HBsAg and antibody to anti-HBc (Busch, 1998; Allain et al., 1999). Individuals positive for either are disqualified because of ongoing infections or possible OHB. The strategy of combined HBsAg and antiHBc screening virtually eliminates blood-transmitted HBV, with the rare exception of donations in the early phase of the window period when all serological markers are still negative (Schreiber et al., 1996; Matsumoto et al., 2001). However, such practice is feasible only in countries where the overall hepatitis B infection rate is low (less than 1%). By contrast, in many Asian countries where hepatitis B is intermediately or highly endemic, about 16-90% of adults may have either past or ongoing hepatitis B infections (Chen, 1993; Alter, 2003). Combined HBsAg and anti-HBc screening strategy would disqualify most volunteer blood donors. Therefore in several Asian countries like Taiwan, blood donors are screened only for ongoing infections by HBsAg, but not for past infections by anti-HBc. This strategy avoids unnecessary waste of donations but bears residual risk of HBV transmission, especially those caused by donors in the window period or with OHB. The incidence of acquiring HBV infection from these HBsAg-negative and anti-HBc-positive donors with OHB is likely higher than in non-endemic areas. To explore the magnitude of this problem, we reviewed the prevalence of OHB in Asian countries and the infectivity of OHB by transfusion, and discuss the possible role of NAT in this article.

2. HBV infection in Asia About three quarters of HBV infections are in Asia where hepatitis B is the leading cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) (Chen, 2000; Chen et al., 2000; Merican et al., 2000). Recent reports suggest that prevalence of persistent or past infection is high in South Asia, Southeast Asia and Mongolia where at least 8% of the population is positive for HBsAg (CDC, 2005; Alter, 2003; WHO, 2004). In these areas, 40-90% of the population generally has serological evidence of previous HBV infection. In areas of Asia with an intermediate pattern of HBV infection, including India, Pakistan, Thailand, Philippines, Iraq, Saudi Arabia, Korea and Malaysia, the prevalence of HBsAg ranges from 2 to 7% and serological evidence of past infection is found in 16-55% of the population. Areas of Asia with a low prevalence of HBV infection include Bangladesh, Iran, Israel, and Kuwait. In most developed parts of the AsiaPacific area, including Australia and Japan, the prevalence of chronic HBV infection is less than 1%, and the overall infection rate is 4-15% (Alter, 2003). Table 1 shows the prevalence of HBsAg in the general population, in patients with chronic hepatitis, and in blood donors in Asian

Table 1 Prevalence of HBsAg in Asian areasa Country (year)

General population

Blood Patients donors with chronic (first-time) hepatitis

Taiwan (2004) Philippines (before 1997) Thailand (before 1997) Korea (1970 1983) Singapore (before 1997) China (before 1997) Malaysia (1997) Indonesia (1994) Japan (before 1997) Mongolia Pakistan India Australia (Before 1993) Nepal (1990 2004) New Zealand (2005) Saudi Arabia (1997~004)

>10 5 16 >8 7.3 6 4.6 3 5 2 5 0.8 10 2.6 2 5 0.03 1.8 0.9 1.9 5.6 7.3 7

0.85

(5.3)b

2.2 1.5

0.5 2.8 2.0 0.13

75 90 65 50 45 70 78 75 25 45 36

2 9 1 2.5 <0.1 1.2

14 17 29 35 24

1.5 5.4 (3.7)

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a Valuesare listed as percentages; dashes indicate data not available. b Personal communication(2004, Taipei Blood Center, Taiwan). countries (Andre, 2000; Amirudin et al., 1991; Budihusodo et al., 1991; Chiba et al., 2004; E1-Hazmi, 2004; Khokhar et al., 2004; Luksamijarulkul et al., 2002; Nanu et al., 1997; O'Sullivan et al., 2004; Robinson et al., 2005; Surya et al., 2005; Takahashi et al., 2004; Tareen et al., 2002; WHO, 2004). Blood transfusion was once an important route of HBV transmission in both high- and low-endemic areas. A residual risk of HBV transmission is by transfusion from donors with OHB; this route is often neglected in many developing and HBV-endemic Asian countries (Brechot et al., 2001; Conjeevaram and Lok, 2001; Allain, 2004). 2.1. Forms and epidemiology o f occult H B V infection

OHB is defined as the presence of HBV DNA in blood or liver tissues without detectable HBsAg, with or without anti-HBV antibodies (Brechot et al., 2001; Hu, 2002; Torbenson and Thomas, 2003; Allain, 2004). OHB is found in several clinical conditions (Allain, 2004), including (1) recovery from past infection indicated by the presence of anti-HBs; (2) chronic hepatitis with surface gene escape mutants that are not recognized by current assays; (3) chronic carriage without any marker of HBV infection other than HBV DNA (referred to as "seronegative"); and (4) most commonly in endemic areas, chronic carriage stage with HBsAg too low to be detected and recognized by the presence of anti-HBc as the only serological marker (referred to as "anti-HBc alone" or "isolated anti-HBc"). In low-prevalence areas, no more than 5% of HBsAg(-), anti-HBc(+) blood units contain HBV DNA (Allain et al.,

C.-J Liu et aL /Journal of Clinical Virology 36 Suppl. 1 (2006) $33 $44

1999; Kleinman et al., 2003). In contrast, in high-prevalence areas (such as India and Taiwan), serum HBV DNA is found in 4-25% of the HBsAg(-) and anti-HBc(+) population (Lai et al., 1989; Wang et al., 1991; Iizuka et al., 1992; Nagaraju et al., 1992; Minuk et al., 2005). As in highly endemic countries the majority of infections are contracted perinatally or in early childhood, a higher proportion of the infected adults have late chronic HBV with undetectable HBsAg. This may account for the higher rate of OHB in anti-HBc-positive populations in these areas. However, both HBsAg and HBV DNA detection rates are dependent upon assay sensitivity. Different laboratory methods used for screening the different study populations listed in Table 2 also may have affected the proportions of HBV DNA positives. As indicated by Brechot et al. (2001), the HBV DNA detection rate is highest in subjects who are positive for anti-HBc alone, intermediate in subjects who are positive for both anti-HBc and anti-HBs, and lowest in seronegative subjects (Tables 2 and 3). Nevertheless, the prevalence of HBV DNA in seronegative subjects is only partly defined because so far, few studies have systemically screened HBV marker negative individuals for HBV DNA in either low- or high-prevalence areas (Minuk et al., 2005; Chen, 2005). Prevalence of OHB also seems to correlate with different clinical situations. There is ample evidence in cross-sectional studies demonstrating the persistence of HBV DNA in HBsAg-negative acute and chronic HBV infections. Collectively, 5-55% of HBsAg-negative subjects with chronic hepatitis with or without associated HCC were positive for serum HBV DNA (Brechot et al., 2001; Hu, 2002; Torbenson and Thomas, 2003). In HCC, 14-100% of anti-HBc-only positive individuals had OHB and 8-87% of patients without any markers of HBV infection had OHB (Paterlini et al., 1990; Thiers et al., 1993; Fukuda et al., 1996; Shintani et al., 2000). HBV DNA, in contrast, is only identified in around 10% of HBsAg-negative patients with acute fulminant hepatitis. Finally, HBV DNA persistence is not restricted to patients with chronic liver disease, as it may be observed in apparently healthy individuals with normal liver function tests, including blood and organ donors (Marusawa et al., 2000; Wang et al., 1991; Shih et al., 1990; Hennig et al., 2002). Again, the rate of HBV DNA is significantly higher in healthy individuals with anti-HBc alone (Table 2). For example, Allain (2004) reported that average HBV-DNA detection rates of 7% and 13% were observed in anti-HBcpositive subjects with or without anti-HBs respectively, and in blood donors the rates ranged from 0% to 17%.

3. Infectivity of occult HBV infection The significance of OHB in the large population of asymptomatic carriers is not well studied. In particular, the critical issue in blood safety is how often, and at

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what level, OHB is infectious through transfusion. The available studies regarding the infectivity of OHB are of three origins: retrospective studies of clinical cases of posttransfusion HBV infection; look-back studies involving recipients of blood from anti-HBc-positive donors; and systematic studies of donor-recipient pairs (Satake, 2004; Mosley et al., 1995; Allain et al., 1999; Allain, 2004). No prospective studies however have been conducted because of the very low incidence, which requires an extremely large number of patients to be examined. Clinical observations have documented the transmission of HBV genomes from HBV-seronegative donors to recipients and the association of this transmission with post-transfusion hepatitis. Similar observations were made during transmission experiments in chimpanzees (Prince et al., 1983; Wands et al., 1986). Of the various forms of OHB, the "anti-HBc alone" pattern receives much attention in that accumulated data imply that a proportion of individuals with this serological pattern are carriers of HBV and may transmit HBV by blood or organ donation to both immunocompetent and immunosuppressed recipients (Hoofnagle et al., 1978; Wachs et al., 1995; Dickson et al., 1997; Uemoto et al., 1998; Wang et al., 2002). For the majority of these individuals with "anti-HBc alone", unresolved HBV infection or a chronic HBV carriage with non- or low-productive infection is considered (Grob et al., 2000). The risk of HBV transmission is variable (0.4-90%); it is highest when livers from anti-HBc-positive donors are transplanted to seronegative recipients (Hoofnagle et al., 1978; Dickson et al., 1997; Uemoto et al., 1998; Wachs et al., 1995; Lowell et al., 1995). Few data regarding the infectivity of blood components or donated organs containing both anti-HBc and anti-HBs are available. Theoretically, if HBV particles are present in the peripheral blood of subjects with high-titre anti-HBs, the anti-HBs may neutralize the infectivity of the viral particles. Mosley et al. (1995) showed an inverse correlation between anti-HBs level and infectivity; only 10% of blood units with low-titre anti-HBs were infectious. In the look-back study by Allain et al. (1999), they examined the potential infectivity of 97 components containing anti-HBc and lower-titer anti-HBs (< 100 IU/1) transfused to 131 recipients; again, no evidence of transmission was found. The infectivity of antiHBs-containing blood components in immunosuppressed recipients, however, has not been systematically explored. Some data are available from organ donors, in particular livers for transplantation (Wachs et al., 1995; Lowell et al., 1995). In one study that included 14 organs from antiHBs-positive donors, three donated livers and one kidney transmitted HBV to recipients. Details regarding the titer of anti-HBs or the presence of detectable HBV DNA were not available. 3.1. Infectivity and viral load As for other viral infections, HBV infectivity depends on three main factors: the infectious dose, the level of

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immune complexing by neutralizing anti-HBs, and the immunocompetence of the host. Considering the volume of infectious material involved in the transfusion of whole blood or a blood component, it is generally accepted that should any HBV DNA be present, infection may occur. In chimpanzees, 10-100 virus particles appear to be the minimum infectious dose (Prince et al., 1983; Yoshizawa, 2005). Nevertheless, the discrepancy between the relatively high prevalence of HBV DNA in donors with OHB and a low incidence of post-transfusion infection (Satake et al., 2005) suggests that not all HBV DNA-containing virions are infectious because of immune-complexing or many of the particles are defective, similar to HIV Furthermore, in humans, the available data regarding the infectivity of OHB are based mainly on serological assays but not on serum viral loads. Systematic study of any correlation between viral load, transfused volume, immune complexing, escape mutations in the S protein, viral particle integrity and infectivity in susceptible recipients should be conducted. With regard to immune aspects, blood components containing anti-HBs were not infectious in immunocompetent recipients (Mosley et al., 1995), but some infectivity of anti-HBs-containing organs was noted in some immunosuppressed recipients (Wachs et al., 1995; Lowell et al., 1995).

3.2. Significance of OHB in Asia in transfusion setting Previous data in developed countries (Soldan et al., 1999) suggested that post-transfusion hepatitis B more often implicated HBsAg-negative donors at later carrier states of the virus and positive for anti-HBc (11 cases), than donors in the window period during acute HBV infection (three cases). Consequently, this is a major problem in Asia. One study from Taiwan revealed that 11 (7.5%) of 147 stored HBsAg-negative donated units available were positive for HBV DNA by PCR and anti-HBc (Wang et al., 2002). Of the 11 HBV-naive Taiwanese patients who received the HBV DNA-positive donations, one had post-transfusion acute hepatitis B and one had transient hepatitis B viremia. In another prospective study, we investigated the incidence of post-transfusion HBV infection after receiving screened blood units (HBsAg-negative and serum ALT normal) in Taiwan (Liu et al., 2006). Of 4448 blood recipients, 467 (10.5%) were anti-HBc-negative. Posttransfusion 6-month follow-up was completed for 327. We identified 5 (1.5%) who developed hepatitis B viremia 1 week after transfusion. Three (0.9%) were children who later seroconverted to anti-HBc but with normal ALT indicating subclinical acute infection, despite all having anti-HBs from previous vaccination. One had transient transfusion-transmitted HBV without seroconversion to anti-HBc and one possibly had occult HBV infection. Our findings suggested the possibility that occult HBV infection was transmissible by transfusion. As around 10% of the general recipients in our study were negative for anti-HBc, and the post-transfusion acute HBV infection occurred in

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approximately 1% of this prospective cohort, we estimated that at least 0.1% of the overall blood recipients in Taiwan might suffer transfusion-transmitted HBV infection under the current donor screening strategy. Notably, if each recipient received in average 10 units of blood component, then the risk of transfusion-transmitted HBV infection would be 0.01% per unit (100 per million units). By contrast, the risk of transfusion-transmitted HBV infection was about 2.5-19.2 per million units in western countries and Japan (Mine et al., 2003; Pillonel et al., 2004; Kleinman and Busch, 2001; Busch et al., 2003; Donahue et al., 1992). Therefore, the risk of transfusion-transmitted HBV infection in Taiwan is at least 7-40-fold higher. In hepatitis B endemic areas, the main risk of transfusiontransmitted HBV infection originates from HBsAg-negative donors with OHB (Allain, 2004). Based on the epidemiology provided above, it can be estimated that at least 24000 HBsAg-negative but HBV DNA-positive donations per million units have been used each year. Furthermore, based on our prior findings, 2-18% of HBV-na'fve recipients developed hepatitis B viremia after having received HBV DNA-positive donations indicating the infectious potential of such HBV DNA-positive blood from donors with OHB (Wang et al., 2002). Taking these data together, the number of donors with OHB and recipients developing post-transfusion HBV infection may be substantial in endemic areas. Therefore, the critical step to ensure the safety of blood transfusion in these areas is to identify and block the transfusion of blood components from donors with OHB. Sensitive screening assays for OHB such as NAT or more sensitive HBsAg tests could be considered to identify donors with OHB and so interrupt this transmission route in Asian countries.

4. Detection of HBV infections in blood donors The major source of residual risk of transfusion of HBV in low-endemic areas relates to infectious donations given in the seronegative WP of acute HBV infection. However, another important source of transfusion risk, especially in high-endemic areas, is blood or blood products donated by chronic HBV carriers with OHB. Because of the low viral load in OHB, most studies have used in-house nested PCR to detect the presence of HBV DNA (Table 3). No standardized methods exist at present, and the sensitivity of the PCR assay for detecting OHB varies from 1 to 600 copies/ml, as reviewed by Torbenson and Thomas (2003). Sample preparation is important for the sensitivity of HBV-NAT, but little information is available on the optimal DNA extraction methods, including the total amount of input DNA, the PCR primers and assay conditions that are required for optimal HBV DNA detection in low viral load carriers. Another pronounced risk of NAT is false positive results caused by contamination or non-specific amplification of other targets. These features may account for the discrepant

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Table 3 HBV load in occult HBV infection, in relation to HBV antibody patterns Author (year)

HBV DNA level a

Anti-HBr alone Noborg et al. (2000)

263 646 copies/ml c

Loeb et al. (2000)

<700 copies/ml c

Kessler et al. (2000)

400 4x106 copies/ml c

Weber et al. (2001)

800 4x105 copies/ml c

Jilg et al. (2001)

100 10000 copies/ml b

Hennig et al. (2002)

<10 1000 IU/ml c

Kleinman et al. (2003)

10 100 copies/ml c

Anti-HBe(+) and anti-HBs(+) Grethe et al. (1998)

100 1000 genome equivalents/ml c

Noborg et al. (2000)

100 711 copies/M e

Loeb et al. (2000)

10 1000 copies/M e

Weinberger et al. (2000)

< 10 000 genome equivalents/ml c

Dreier et al. (2004)

8 ~ 6 0 IU/ml c

a HBV DNA level expressed as copies, IU, or genome equivalents/ml. b In-house PCR, c commercial Idt.

results obtained in different studies on OHB using in-house PCR-based assays. Only sensitive and standardized methods should be used to screen for HBV DNA in HBsAg-negative samples. Fortunately, a variety of testing methodologies including PCR-based and transcription-mediated amplification (TMA)-based methods are now available to screen or quantify HBV DNA, but these methods need to be compared directly using viral standards representative for the low virus concentrations in OHB.

4.1. Sensitivity of licensed and investigational HBsAg assays, and minipool (MP) and individual donation (ID) HBV NAT One recent comparative study revealed that HBsAg concentrations at the enzyme immunoassay (EIA) cut-off (CO) for investigational tests ranged from 0.07 to 0.12ng/ml, compared with 0.13 to 0.62 ng/ml for licensed HBsAg tests based on the analysis of the FDA lot-release panel (Biswas et al., 2003). Based on the analysis of a dilution series of the WHO HBV DNA standard, the estimated viral load at CO ranged from 102 to 267 IU/ml for new tests, and from 363 to 1069IU/ml for already licensed tests. More relevant for the diagnostic sensitivity of NAT methods was a longitudinal regression analysis of HBV seroconversion panels. The estimated viral load at CO varies from 729 to 2763 genome equivalents/ml for new HBsAg tests, and from 1757 to 11431 genome equivalents/ml for licensed tests (Biswas et al., 2003). The sensitivity of HBsAg reverse particle hemaglutination assay (RPHA) is inferior to those of HBsAg EIA assays and detects HBsAg at an estimated HBV DNA level of ~13 500 genome equivalents/ml. In the same study, it was shown that the sensitivity of HBV NAT,

whether MP-NAT or ID-NAT, was superior to both the licensed and new HBsAg tests. The estimated 50% hit rate detection threshold of HBV DNA ranges from 100 to 1000 genome equivalents/ml for MP-NAT and from 5 to 50 genome equivalents/ml for prototype ID-NAT (Biswas et al., 2003).

4.2. Yield of HBV NAT." Benefit of NAT over licensed and investigational HBsAg assays in WP closure during acute HBV infection To evaluate the expected yield of detecting HBV infectious blood donations by HBV NAT, 100 representative samples from 10 HBV SC panels and 28 controls (FDA HBsAg lot-release panel and dilutions of the WHO HBV NAT standard) were selected in a collaborative study (Biswas et al., 2003). All 128 samples were tested by seven HBsAg tests (four licensed and three investigational tests), and by four MP-NAT and three ID-NAT assays. The results showed that new HBsAg tests detected 31-63% of early ramp-up phase samples in the 100-member seroconversion panel, while MP-NAT detected 55-71% and ID-NAT 82-99%. Compared with currently licensed HBsAg assays, WP would be reduced by 2-9 days using newer HBsAg assays; by 9-11 days using MP-NAT; and by 25-36 days using ID-NAT. The data of these seroconversion panel studies have been used for a new approach in modeling the residual risk of window-period donations and the impact of NAT to reduce this risk. For an extensive review of the incidence window period risk model we refer to the paper by Kleinman and Busch in this supplement. The findings of the seroconversion panel study of Biswas et al. are in line with the results of other recent studies that have examined WP reduction by HBV NAT assays relative to HBsAg assays for donor screening. Matsumoto et al. (2001) reported that in 10 cases of HBV WP transmission, five turned out to be HBsAg-positive when tested with a more sensitive HBsAg assay than was used in the initial screen. Of the other five cases that were negative even using the more sensitive HBsAg assay, only two would have been detected by MP-NAT, whereas ID-NAT detected two additional cases. Several contemporary studies employing HBV MPNAT to screen blood donors yielded similar findings. For example, Roth et al. (2000, 2002) reported that 42 HBV DNA-positive donations were detected following screening of 18.5 million donations by MP-NAT (96 samples per pool). In a study from Japan (Minegishi et al., 2003), 11 million donations were tested for HBV DNA by MP-NAT (50 samples per pool) . These units had originally been screened by HBsAg RPHA, as well as by anti-HBc. However, 181 HBV DNA-positive RPHAnegative donations were detected representing early-WP or OHB infections. At the same time, only 105/181 (58%) of these RPHA-negative samples were reactive by a chemiluminescent HBsAg assay (Abbott Prism) with

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sensitivity comparable to that of HBsAg assays widely employed in most other countries. Overall, these results indicate that both more sensitive HBsAg assays and MP-NAT assays are capable of detecting a proportion of potentially infectious units that are missed by currently licensed HBsAg tests. To be expected, ID-NAT and perhaps modified MP-NAT (with smaller pool sizes, enhanced sample input or concentrating procedures, or improved analytical sensitivities) could offer significant incremental WP closure and prevent a moderate number of residual HBV transmissions not detectable by HBsAg assays, but at a higher cost (Jackson et al., 2003; Allain, 2004). Role of anti-HBc tests in the detection o f OHB in the context o f H B V NAT Because of the low viral load in donations with OHB, there is also a question as to whether they could be detected by current NAT testing or by routine anti-HBc test (Biswas et al., 2003). Several large-scale studies using contemporary NAT assays demonstrated the rate of HBV DNA in anti-HBc-positive, HBsAg-negative units, and the need for anti-HBc donor screening, especially in the context of HBV MP-NAT or if no HBV-NAT is performed. In a recent study, 395 of 1231 repository specimens, that were HBsAg-EIA-negative, anti-HBc-reactive and were anti-HBs-negative or -reactive at ~<100 IU/1 (using PRISM | Ausab), were tested by a PCR assay with a ) 9 5 % detection rate of ) 5 0 copies/ml (Kleinman et al., 2003). Four anti-HBs-negative specimens were PCR-positive, with estimated HBV DNA copy number of 10-30 copies/ml in two specimens and 50-100 copies/ml in two others. The HBV DNA detection rate in anti-HBs-negative specimens was 3.7%, and the projected rate among all anti-HBcreactive specimens was 0.24%. This study suggested that the low viral load in the HBV DNA-positive samples from anti-HBc positive donors would not be detected by MP-NAT (Kleinman et al., 2003). The authors also suggested that anti-HBc screening rather than MP-NAT detected HBsAgEIA-negative donors with OHB. Very low viremic donors with anti-HBc positivity were also found by recent blood donor screening data from Japan and Germany. Sato et al. (2001) reported a 1.1% rate of HBV DNA detection in anti-HBc-positive donors: four of the 12 HBV DNA reactive donors were not detected by their standard PCR assay (95% sensitivity of 100 copies/ml) and required an increased sample input volume of 1 ml. Similarly, Roth et al. (2002) detected HBV DNA-positive specimens only after enhancing their original PCR input volume. Based on the data above, it seems that anti-HBc screening has the potential of excluding the vast majority of OHBs, leaving only the probably rare cases with HBV DNA alone undetected. This approach, however, has two main drawbacks: it does not detect the seronegative WP infections; and most importantly, it would not be practical in most parts

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of the world where the prevalence of anti-HBc is > 10%, as too many otherwise healthy donors will be ineligible. As for HBV NAT, currently available NAT assays (having a sensitivity of 20-50IU/ml) could only detect OHBs with >320 to 800 IU/ml HBV DNA when diluted in the smallest pool size of 16. Since many cases of OHB in blood donors are below that threshold, the critical issue in Asian countries is how to enhance the sensitivity of HBV NAT screening technology. Some studies adopted ultracentrifugation to improve sensitivity of MP-NAT (Sato et al., 2001; Roth et al., 2002). ID-NAT is the alternative choice and might deserve serious consideration. To be noted, automation is a prerequisite for ID-NAT, and multiplexing might reduce the additional cost attached to ID-NAT. 4.3. Benefit and cost o f H B V NAT to screen for H B V in blood donors

The following subsections summarize the experiences of using NAT to eliminate residual risk of transfusion of HBV in Japan and in other countries. 4.4. Experiences using NAT for H B V in Japan

All blood collected by the Japanese Red Cross (JRC) is assessed for screening based on a questionnaire administered by the JRC blood centers in Japan. Samples from these donations are then screened for a number of infectious agents including HBV (Otake and Nishioka, 2000; Mine et al., 2003; Minegishi et al., 2003). The JRC criteria for serological positivity for HBV is HBsAg reactivity and/or an anti-HBc cut-off agglutination titer of ) 3 2 without anti-HBs. When the titer of anti-HBs by RPHA is >16 ()200mIU/ml), donations are accepted for NAT screening, even when the HI titre of HBc antibody is )32. Serologically positive and ALT-elevated (>60 IU/1) donations are excluded from NAT screening (Mine et al., 2003; Minegishi et al., 2003). Despite this, a residual risk of HBV transmission remains in Japan (Saito et al., 1999; Otake and Nishioka, 2000; Minegishi et al., 2003). To eliminate the residual risk of HBV transmission, NAT for HBV was implemented in Japan in 1999. In brief, NAT screening is undertaken by using a highly sensitive multiplex (MPX) nucleic acid amplification system capable of detecting HBV DNA, HIV RNA and HCV RNA simultaneously. Using this MPX reagent, NAT screening for HBV, HCV and HIV-1 was introduced in July 1999, in the JRC Society, utilizing a pool size of 500. On February 1 2000, the pool size was reduced to 50. Among 11403 327 units tested using the 50 MP-NAT up to February 28 2002, 217 were HBV DNA positive, 36 were HCV RNA positive, and five were HIV RNA positive. The initial 181 donations that were serologically negative but HBV DNA positive, were further tested by using a more sensitive chemiluminescence immunoassay (CLIA) for HBsAg. Of these 181 HBV DNA-positive donations, 96 (53%) and 76 (42%) were negative by

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individual enzyme immunoassay (EIA) and CLIA testing, respectively. Similar findings were noted in a later survey (Yoshikawa et al., 2005). The JRC data demonstrate that the sensitivity of the 50-sample pool MPX NAT system was higher than that of individual HBsAg screening, even with CLIA, but probably many HBV yield samples have concentrations around the 50% detection limit of the 50-sample pool MPX NAT system. To increase the sensitivity of the test system JRC has recently reduced the pool size for screening to minipools of 20 donations. The Japanese experience suggests that highly sensitive HBV DNA detection systems can significantly improve the overall safety of the blood supply (Otake and Nishioka, 2000; Mine et al., 2003; Minegishi et al., 2003). Experiences using NAT for H B V in other countries

In Germany, the risk of undetected infection without NAT testing was estimated to be 1 in 230000 for HBV in 2001/2002, and with MP-NAT testing, 1 in 620000 (Offergeld et al., 2005). Implementation of HBV NAT has an impact on the risk of undetected infectious donations because of the shortening of the window period. Roth et al. (2002) increased their detection of HBV DNA-positive specimens by enhancing their original PCR input volume in a study of German blood donors. It should be noted that the use of assays that require higher sample input volume is not possible in routine blood donor screening applications. In the USA, as mentioned above, MP-NAT assays are capable of detecting a proportion of potentially infectious units in the seronegative window period that are missed by currently licensed HBsAg tests (Biswas et al., 2003). For the detection of HBV DNA in anti-HBc-positive, HBsAgnegative units, another study demonstrated an estimated yield of one HBV DNA-positive, anti-HBc-positive unit in 49000 units that were otherwise eligible for transfusion (95%CI: 1 in 16600 to 1 in 152600) (Kleinman et al., 2003). Recently, a preliminary study assessing the efficacy of NAT for the detection of HBV infection was conducted in Ghana, when HBV is endemic (HBsAg prevalence 16.1%) and OHB is not rare (1.4% in the donor population) (Owusu-Ofori et al., 2005). From 2002 to 2003, they screened 9372 candidate blood donors before donation for HIM HCV, and HBV serologic markers with HBsAg rapid tests (Vedalab, Alencon, France [sensitivity 5 ng/ml]; or Determine, Abbott Laboratories, Delkenheim, Germany [sensitivity 1 ng/ml]). The efficacy of this screening was then assessed by HBsAg EIA test and NAT applied to pools of 10 plasma samples from donated units with a virusspecific triplex assay. MP-NAT-reactive pools were further resolved by viral genome identification in individual plasma samples. They found that 1.3% and 3.0% of HBV DNAcontaining blood units were negative with rapid tests but were detected in individual donations with EIA and NAT, respectively. Only half of these HBV DNA-containing units were detectable by MP-NAT in pools of 10 samples. In

EIA-nonreactive samples with OHB, the viral load ranged between 14 and 277IU/ml. In another screening subset, among the 10 MP-NAT-positive pools, 13 individual samples contained HBV DNA with a viral load ranging between 6 and 954 IU per ml. Among the 61 MP-NAT-negative pools (603 samples), 16 samples contained HBV DNA (viral load range, 5-1270IU/ml). Eight of these 16 samples reacted with the EIA. Most (13/16) HBV DNA-containing samples undetected by MP-NAT but detected by ID-NAT contained DNA <100IU/ml. They thus concluded that combining pre-donation screening with rapid tests and post-donation triplex NAT may constitute a novel and effective strategy to procure safer blood in low-resource areas provided it is feasible and affordable. Their data also echoed previous findings that MP-NAT assays are capable of detecting a proportion but not all of the potentially infectious units missed by currently licensed HBsAg tests. Cost effectiveness o f H B V NAT Relative to HIV and H C V NAT: Recent studies estimated

that using MP-NAT, the overall cost of eliminating one potentially infectious donor would be US$ 2.6 million in the case of HBV infection, $ 4.0 million in the case of HIV infection, and $ 2.3 million in the case of HCV infection (Pereira, 2003; Marshall et al., 2004). Another cost-effectiveness analysis of NAT, recently reported in the USA (Busch, 2004), included consideration of the absolute and relative value of addition of HBV NAT to HIV/HCV MP- or ID-NAT, and also included the potential cost saving associated with discontinuation of one of the serological HBV assays in the context of HBV NAT. All scenarios that included HBV NAT resulted in poorer cost-effectiveness estimates than similar strategies without HBV NAT, with a lower estimate of US$ 4.9 million/QALY [2.3-8.7] for MP-HBV/HIV/HCV NAT with discontinuation of antiHBc, and a higher estimate of US$ 9.1 million/QALY [7.8-11.2] for ID-HBV/HIV/HCV NAT. This analysis assumed a very modest incremental cost of US$ 5.00 per donation for inclusion of HBV NAT (range $3-10). Of note, all scenarios involving ID-NAT, which would be needed for efficient HBV detection, resulted in costeffectiveness point-estimates exceeding US$ 7.3 million, substantially higher than for MP-NAT. Endemic versus non-endemic areas: HBV NAT costs are of course higher in the countries where the prevalence of HBV is low. The cost effectiveness of adding NAT for HBV is obviously outside the typical range for most healthcare interventions (Graham et al., 1998). In contrast, the cost effectiveness of using such sensitive assays to identify donors with hepatitis B viremia would probably be more favorable in HBV-endemic areas, as more OHB cases are expected to be discovered. For example, in Taiwan, 24000 (2.4%) HBV DNA-positive donations per million units have been estimated to be transfused each year (Shih et al., 1990; Wang et al., 1991; Chen, 1993; Kao et al., 2002a,b). Since 2.4% (~1/40) of the donations in Taiwan

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contained HBV DNA, and assuming that it would cost approximately US$ 5-50 to test each unit for HBV, the cost of identifying one potentially infectious donor with occult HBV infection would be only around US$ 200 2000, much lower than those in low endemic areas.

Screening for blood donors' in developing Asian countries': Problems, priorities and practicalities For policy development, it would be useful to assess the relative contribution of the two sources of transfusiontransmitted HBV infection, i.e. window period and OHB in each country; and the advantages and cost of HBV NAT and anti-HBc test. Overall, anti-HBc screening may be more cost-effective than NAT in areas of low HBV prevalence. However, a major undesirable effect of anti-HBc screening is the indefinite deferral of healthy donors with anti-HBc and anti-HBs or with false-positive anti-HBc results. In countries where the prevalence is high HBV NAT seems to be a more economic option; unfortunately, most of these countries cannot afford the cost for implementation of NAT without a cost reduction. On top of the implementation of advanced and expensive screening tools, several measures are essential and may be more cost effective to ensure safe blood transfusion in developing and resource-poor countries where HBV infection is endemic. The measures include thorough donor selection and questionnaire to eliminate high-risk donors; the use of cheaper, more sensitive HBV tests; avoidance of unnecessary transfusions; cost benefit analyses of blood safety procedures; and promotion of voluntary donation as a public responsibility. For example, one aforementioned study demonstrated that a new screening strategy based on pre-donation rapid serologic tests in combination with MP-NAT after donation could effectively detect donors with HBV infection in resource-poor areas like sub-Saharan Africa (Owusu-Ofori et al., 2005). Additional advantages of this strategy included: the saving of blood bags; the saving of the equipment necessary for performing EIA as well as the relatively large proportion of control wells necessary for the daily testing of a small number of samples; the storage of safe blood only in the blood bank's refrigerator, avoiding potential confusion between tested and untested blood; and the ability to inform deferred candidate blood donors of their unsuitability for blood donation in areas where communication by post or telephone is very poor. Actually, some procedures are not in place in many Asian countries and should be implemented as a first priority (Wake and Cutting, 1998; Nanu, 2001).

5. Perspectives and conclusions The possibility of HBV transmission by donors with OHB raises several questions (Brechot et al., 2001): Should blood/organ donors be screened for anti-HBc? Should blood/organs from HBsAg-negative, anti-HBcpositive donors be used? Would HBV NAT for HBV DNA

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identify infectious donors? Could HBV NAT replace HBsAg or anti-HBc test in donor screening in the future? These questions can only be answered after a thorough understanding of the infectivity of OHB in immunocompetent and immunosuppressed recipients. Many aspects need to be investigated, including the characterization of various forms of OHB in relation to the infectivity of different blood components, tissues or organs from OHB carriers and the dynamics of HBV DNA during the natural course of acute resolving and chronic HBV infection. With more sensitive HBV NAT and HBsAg assays becoming available the impact of OHB on blood safety and the benefit of either anti-HBc or HBV NAT screening may become clear in the near future. It has now been demonstrated clearly that OHB can be transmitted by blood or tissue donation. HBV infection is endemic and the number of subjects with OHB is substantial in many Asian countries. Accordingly, the critical issue in transfusion safety currently is to identify blood or tissue donors with OHB, and then to block this transmission route. It may be that the strategy to prevent transmission of HBV by OHB carriers will be different in endemic and nonendemic areas. In low-endemic areas it is still a subject of debate whether anti-HBc screening should be implemented (Zervou et al., 2001). Whether ID-NAT would eventually be able to replace HBsAg or anti-HBc testing also remains to be studied. In HBV-endemic areas, the priority is to examine the prevalence of OHB in blood donors on a large scale, and so establish the cost-effectiveness of implementing sensitive ID-HBV NAT blood screening technology in reducing the risk of HBV transmission.

Acknowledgements The study was supported by grants from the Department of Health (DOH91-DC-1051 & DOH92-DC-1017), National Science Council, Executive Yuan, Taiwan; and National Health Research Institutes, Taiwan.

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