Occult hepatitis B virus infection in hematopoietic stem cell donors in a hepatitis B virus endemic area

Journal of Hepatology 42 (2005) 813–819 www.elsevier.com/locate/jhep

Occult hepatitis B virus infection in hematopoietic stem cell donors in a hepatitis B virus endemic area Chee-kin Hui1,2, Jian Sun3, Wing-yan Au1, Albert K.W. Lie1, Yui-hung Yueng1,2, Hai-ying Zhang1,2, Nikki P. Lee2, Jin-ling Hou3, Raymond Liang1, George K.K. Lau1,2,* 1

Department of Medicine, Queen Mary Hospital, The University of Hong Kong, 102 Pokfulam Road, Hong Kong SAR, China 2 Centre For the Study of Liver Diseases, The University of Hong Kong, Hong Kong SAR, China 3 Department of Infectious Diseases, Namfang Hospital, Guangzhou, China

Background/Aims: The acquisition of hepatitis B virus (HBV) infection following organ transplantation from donors with occult HBV infection is an important cause of morbidity and mortality. The aim of this study is to determine the prevalence of occult HBV in allogeneic hematopoietic stem cell (HSC) transplantation donors. Methods: We performed a retrospective study on 124 consecutive hepatitis B surface antigen negative HSC donors. Their serum samples were analyzed by PCR for the pre-S/S, pre-core/core and X regions of the virus. Samples reactive by at least two PCR assays were considered HBV-DNA positive. Results: Nineteen of the 124 HSC donors (15.3%) had occult HBV infection. Sixteen of these 19 donors with occult HBV infection (84.2%) tested positive for hepatitis B core antibody while 78 of 105 subjects (74.3%) without occult HBV infection were also positive (PZ0.56). Fourteen of the 19 donors (73.7%) with occult HBV infection tested positive for hepatitis B surface antibody while 67 of the 105 subjects without occult HBV infection were also positive (PZ0.45). Conclusions: The prevalence of occult HBV infection among HSC donors in Hong Kong is high. Anti-HBc and antiHBs status had no significant correlation with the presence of occult HBV infection. q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Occult HBV; Hematopoietic stem cell transplantation; Prevalence of occult HBV

Recovery from acute hepatitis B virus (HBV) infection is usually associated with loss of HBV DNA from serum, hepatitis B e antigen (HBeAg) seroconversion, hepatitis B surface antigen (HBsAg) seroconversion and normalization of serum aminotransaminases. The loss of HBsAg usually predicts sustained disease remission. However, clinical observations have shown that reactivation of HBV infection may occur either spontaneously or after immunosuppression in these patients [1–3]. Studies have also shown that the immune response to HBV remains vigorous long after the episode of acute HBV infection. Furthermore, HBV Received 8 December 2004; received in revised form 28 January 2005; accepted 28 January 2005; available online 31 March 2005 * Corresponding author. Address: Department of Medicine, Queen Mary Hospital, The University of Hong Kong, 102 Pokfulam Road, Hong Kong SAR, China. Tel.: C852 2818 4300; fax: C852 2818 4030. E-mail address: [email protected] (G.K.K. Lau).

DNA may also be detectable by polymerase chain reaction (PCR) assays in serum, liver and peripheral blood mononuclear cells more than a decade after an apparent recovery from HBV infection with the loss of HBsAg [4,5]. The detection of low levels of HBV DNA in HBsAg negative subjects implies persistent HBV infection in these patients [6]. Geographical variability in the prevalence of occult HBV infection has not been studied in detail. A single multinational investigation has reported a prevalence of 11% in Italy, 6.8% in Hong Kong and 0% in the United Kingdom [7]. However, data from individual studies are difficult to interpret as the study populations had different clinical factors, which may affect the prevalence of occult HBV infection. Most of these studies have been on patients with chronic liver disease or liver abnormalities [8–21]. One study showed that occult HBV infection is present in 4% of patients undergoing hemodialysis [22]. There is, however,

0168-8278/$30.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2005.01.018

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very little data on the prevalence of occult HBV infection in healthy subjects, especially in an HBV endemic area. With the advent and increased use of organ or tissue transplantation, there is a pressing need to determine the actual prevalence of occult HBV infection in the general population who may become potential organ donors. Acquisition of HBV infection following organ or tissue transplantation is an important cause of morbidity and mortality. De novo HBV infection may occur as the result of reactivation of occult HBV infection in either the recipients or the donors [23,24]. Patients who are HBsAg negative but hepatitis B core antibody (anti-HBc) positive are usually regarded as having occult HBV infection. They may transmit HBV infection to HBsAg negative recipients after organ or tissue transplantation at an incidence rate of 33–78% [25–27]. De novo HBV infection after organ or tissue transplantation is a cause of great concern as it is associated with a higher morbidity and mortality [28–30]. This has resulted in debates on whether anti-HBc positive donors should be avoided or whether specific antiviral prophylaxis is necessary when their tissues or organs are used for transplantation [31,32]. The present study aims to determine the prevalence of occult HBV infection in a group of hematopoietic stem cell (HSC) donors in an HBV endemic area and whether serological markers of HBV infection may be used to predict occult HBV infection.

1. Patients and methods One hundred and twenty-four consecutive HBsAg negative and hepatitis C virus (HCV) negative HSC donors from May 2002 to May 2004 at the Bone Marrow Transplantation Unit, University Department of Medicine, Queen Mary Hospital, Hong Kong SAR were studied retrospectively. They were all screened for HBsAg, hepatitis B surface antibody (anti-HBs) and anti-HBc (Abbott Laboratories, North Chicago, Ill, USA) and anti-HCV with the second-generation enzyme-linked immunosorbent assay (ELISA) (Ortho Diagnostics System, Raritan, NJ). All of them had a complete physical examination and blood was tested for liver biochemistry (alanine aminotransaminase, aspartate aminotransaminase, alkaline phosphatase, gamma-glutamyl transferase, albumin and bilirubin), prothrombin time, activated partial thromboplastin time and renal biochemistry.

HBV DNA was performed on all the 124 donors using nested PCR with three independent sets of primers as previously described [21]. Briefly, the HBV DNA was extracted from sera using a Qiagen Mini Blood Kit (Qiagen, Hilden, Germany). Prior to amplification, the concentration of DNA was determined by measuring the absorption at 260 nm (A260) in a spectrophotometer. Twenty nanogram of DNA extracts from each serum of each subject was analyzed for the presence of HBV genomes by performing three different nested PCR amplification assays to detect pre-S/S (S), precore/core (Core) and X viral regions. Amplification was performed for 35 cycles, each consisting of denaturing for 30 s at 94 8C, annealing for 45 s at 56 8C and extension for 1.5 min at 72 8C. Serum samples reactive by at least two of the three PCR assays were considered HBV DNA positive and diagnosed as occult HBV infected. The sequences of the primers are shown in Table 1. Ten asymptomatic HSC donors with chronic HBV infection (HBsAg positive) were included as positive controls. Ten negative controls consisting of samples both from blood donors and controls without nucleic acids but with the complete reaction mixture were included in the assay. The sensitivity of the PCR assay was 10 copies/ml as determined by serial 10-fold dilutions of cloned HBV DNA with known amount 8 (10 copies/ml) as described previously [21]. There was no difference in sensitivity between S, Core and X gene PCR. In the 10 HBsAg positive control donors, all specimens were positive for the S, Core and X genes. None of the negative controls were positive for either the S, Core or X gene.

1.1. Quantitative real time PCR for quantification of HBV DNA Quantitative real time analysis of donors with occult HBV infection was performed with the DyNAmoe HS SYBRw Green qPCR kit (Finnzymes Oy, Finland). Briefly, 25 ml of 2X master mix, 10 ml of 5X (1.5 mM each) primer mix consisting of upstream and downstream primers targeted to the Core or X regions of HBV, 10 ml of extracted DNA (20 ng of DNA/reaction) and 5 ml of H2O making a total of 50 ml for each reaction. Quantitative PCR on every set of reaction contained an internal negative control comprising of the master mix and internal positive controls using our previously cloned HBV DNA. For quantification, the OptiQuante HBV DNA panel (AccroMetrix, Benicia, CA, USA) was used as standards. Since the panel contained intact, encapsulated viral particles, the panel members were extracted using Qiagen Mini Blood Kit (Qiagen, Hilden, Germany) in order to release the viral DNA for amplification and hybridization. Quantitative real time PCR of the reactions was performed with an MJ Research Chromo 4 PTC200 (MJ Research Inc., MA, USA). After 2 min of incubation at 50 8C and 10 min of incubation at 95 8C for initial denaturation, the samples were further denatured for 15 s at 95 8C followed by annealing for 1 min at 57 8C. Data acquisition was performed at this stage to minimize the interference of primer–dimers with quantification. The samples were then incubated for 1 s at 75 8C and amplified for 39 cycles. Each cycle was 95 8C for 15 s and 57 8C for 1 min. A final extension at 95 8C for 1 min was performed to ensure that all amplification products were in a double stranded form before the melting curve step. A melting curve from 65 to 90 8C was used to check the specificity of the amplified products. Quantification of HBV DNA was performed with the Opticon Analysis Software version 2.03.5 (MJ Research Inc, MA, USA). The linear range of this quantification assay was

Table 1 Oligonucleotides used as PCR primers, hybridization probes and sequencing primers Sense primers

Antisense primers

Name

Nucleotide sequence

Position

Name

Nucleotide sequence

Position

Primer set designation

HBV 1 HBV 3a HBV 5

5 0 -GGTCACCATATTCTTGGGAA-3 0 5 0 -AATCCAGATTGGGACTTCAA-3 0 5 0 -GCCTTAGAGTCTCCTGAGCA-3 0

2817–2836 2967–2986 2023–2042

HBV 2 HBV 4a HBV 6

5 0 -AATGGCACTAGTAAACTGAG-3 0 5 0 -CCTTGATAGTCCAGAAGAAC-3 0 5 0 -GTCCAAGGAATACTAAC-3 0

690–671 459–440 2464–2448

Pre-S/S

HBV 7a HBV 13 HBV 15a

5 0 -CCTCACCATACTGCACTCA-3 0 5 0 -CCATACTGCGGAACTCCTAGC-3 0 5 0 -GCTAGGCTGTGCTGCCAACTG-3 0

2046–2068 1268–1288 1382–1402

HBV 8a HBV 14 HBV 16a

5 0 -GAGGGAGTTCTTCTTCTAGG-3 0 5 0 -CGTTCACGGTGGTCTCCAT-3 0 5 0 -CGTAAAGAGAGGTGCGCCCCG-3 0

2385–2366 1628–1608 1540–1520

a

Applied in the second round of amplification of the nested PCR.

Pre-core/ core X

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815

determined by analyzing a dilution series of the OptiQuante HBV DNA quantification panel (AccroMetrix, Benicia, CA, USA) ranging from 10 to 108 copies/ml. According to these results, the linear quantification range is from 103 to 108 copies/ml but it can still detect HBV DNA down to 10 copies/ml.

Table 2 Demographic data between subjects with and without occult hepatitis B virus infection. Continuous variables expressed in median (range)

1.2. HBV-related hepatitis in the recipients after HSCT

Age, years Sex, M:F Anti-HBc Positive Negative Anti-HBs Positive Negative Anti-HBc/antiHBs C/C C/K Bilirubin, mmol/L Alkaline phosphatase, U/L Alanine aminotransaminase, U/L Aspartate aminotransaminase, U/L Gamma glutamyltransferase, U/L

The records of the recipients after HSCT were retrospectively reviewed for development of HBV-related hepatitis until the time of analysis, which is December 2004. Clinical hepatitis was defined as more than a three-fold elevation of serum alanine aminotransaminase (ALT) on two consecutive tests 5 days apart, in the absence of clinical features suggestive of venoocclusive disease, graft-versus-host disease or superinfection with cytomegalovirus or herpes simplex virus. Hepatitis was defined as acute if the duration was less than 6 months and chronic when the duration was more than 6 months. Hepatitis was defined as HBV-related hepatitis if it was preceded by an elevation of serum HBV DNA to more than 10 times that of the preexacerbation baseline in a patient who remained HBV DNA positive, if the serum HBV DNA turned from negative to positive, or if the HBsAg became positive and remained so for two consecutive tests five days apart.

1.3. Statistical analysis All statistical analyses were performed using the Statistical Program for Social Sciences (SPSS 10.0 for windows, SPSS Inc., Chicago, IL). Mann– Whitney U test was used for continuous variables with skewed distribution and chi-square with Yates’ correction factor or Fisher’s exact test for categorical variables. A regression analysis was used to define the relationship between the serum HBV DNA results using the Core and X primers in donors with occult HBV infection. Continuous variables were expressed as median (range). Statistical significance was defined as P!0.05 (two-tailed).

2. Results Of the 124 donors, 94 (75.8%) were anti-HBc positive. Among these 94 subjects, 77 (81.9%) were anti-HBs positive as well. Four donors (3.2%) were anti-HBs positive but anti-HBc negative. Fifty donors (40.3%) were positive for the X gene, 26 donors (21%) were positive for the Core gene and eight donors (6.5%) were positive for the S gene. Nineteen donors (15.3%) had occult HBV infection with at least two viral genes detected in their sera. Fourteen of these 19 donors (73.7%) with occult HBV infection were positive for the X and Core genes while five of these 19 donors (26.3%) were positive for the X and S genes. 2.1. Demographics The demographic data of the donors with and without occult HBV infection are shown in Table 2. Sixteen of the 19 donors (84.2%) with occult HBV infection, compared with 78 of 105 donors (74.3%) without occult HBV infection, were anti-HBc positive (PZ0.56). Fourteen of the 19 donors (73.7%) with occult HBV infection and 67 of the 105 donors (63.8%) without occult HBV infection were anti-HBs positive (PZ0.45) [Tables 2 and 3]. Occult HBV infection was detected in 14 of the 63 anti-HBc and anti-HBs positive donors (22.2%), compared

Occult HBV positive (nZ19)

Occult HBV negative (nZ105)

P-value

38 (24–48) 14:5

37 (28–42) 54:51

0.93 0.07 0.56

16 3

78 27

14 5

67 38

0.45

0.73 14 2 3 (1–6) 56 (6–97)

63 15 3 (3–14) 35 (33–95)

0.98 0.59

13 (6–35)

15 (6–7)

0.44

18 (4–35)

10 (8–33)

0.46

14 (2–62)

18 (8–73)

0.67

HBV, hepatitis B virus; anti-HBc, hepatitis B core antibody; anti-HBs, hepatitis B surface antibody.

with two of the 15 anti-HBc positive but anti-HBs negative donors (13.3%) (PZ0.73) [Tables 2 and 3]. Among the 94 anti-HBc positive donors, 14 of the 16 donors (87.5%) with occult HBV infection, compared with 63 of the 78 donors (80.8%) without occult HBV infection, were positive for both anti-HBc and anti-HBs (PZ0.73) (Tables 2 and 3). 2.2. Quantity of serum HBV DNA Serum HBV DNA of the subjects was quantified. The median serum HBV DNA in donors with occult HBV infection was 32 (range 16–362) copies/ml with primers for Core region and 41 (range 12–412) copies/ml with primers for the X region (PZ0.66) (Table 4). Table 3 Prevalence of occult hepatitis B virus in the serum by serological patterns

Negative for all HBV markers Positive for anti-HBc alone Positive for anti-HBc and anti-HBs Positive for anti-HBs but negative for anti-HBc

Occult HBV positive

Occult HBV negative

P-value.

3 2 14

23 15 63

0.76 1.00 0.26

0

4

1.00

HBV, hepatitis B virus; anti-HBc, hepatitis B core antibody; anti-HBs, hepatitis B surface antibody.

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Table 4 Quantification of serum HBV DNA by quantitative real time polymerase chain reaction. Continuous variables are expressed in median (range) Occult HBV positive (nZ19)

Serum HBV DNA with precore/core primers, copies/ml Serum HBV DNA with X primers, copies/ml

Occult HBV negative (nZ105) X region positive (nZ31)

Pre-core/core region positive (nZ12)

Pre-S/S region positive (nZ3)

No region positive (nZ59)

32 (16–362.00)

Not detectable

14 (11–641)

Not detectable

Not detectable

41 (12–412)

15 (11–25)

Not detectable

Not detectable

Not detectable

HBV, hepatitis B virus.

2.3. Accuracy of using anti-HBc positivity as a predictor for occult HBV infection in a HBV endemic area For the 19 donors with occult HBV infection, 16 were anti-HBc positive while only 27 of the 105 donors without occult HBV infection, were anti-HBc negative. Hence, the sensitivity and specificity of using anti-HBc in a HBV endemic area as a predictor for occult HBV infection would only be 84.2 and 25.7%, respectively. On the other hand, the positive and negative predictive value of anti-HBc as a marker for occult HBV infection would be 17.0 and 90.0%, respectively. 2.4. HBV-related hepatitis after HSCT The recipients were followed up for a median 18 (range 6–28) months. HBV-related hepatitis occurred in three of the 124 recipients (2.4%) after HSCT. The clinical features of these three patients are shown in Table 5. Two of the recipients with HBV-related hepatitis after HSCT had donors with occult HBV infection (10.5%) while the remaining one recipient with HBV-related hepatitis after HSCT received HSC from a donor without occult HBV infection (1.0%) (PZ0.06). Two of these recipients received HSC from anti-HBc positive donors (2.1%) while one received HSC from an anti-HBc negative donor (3.3%) (PZ0.57). All three recipients were treated with lamivudine therapy once they developed HBsAg positivity with normalization of serum ALT. They were still HBsAg positive at the time of analysis.

3. Discussion The prevalence of occult HBV in HSC donors in Hong Kong is 15.3% in this study. This is in addition to the 9.6% reported using the standard monoclonal antibody-based HBsAg detection [33]. This study also demonstrates that donors with occult HBV infection have a low level of viremia with a median serum HBV DNA w40 copies/ml with either primers for the Core or X regions. There is, therefore, a need for more sensitive quantitative HBV DNA assay to accurately quantify the amount of HBV DNA in those with occult HBV infection. The serum HBV DNA levels in occult HBV positive donors with the two different primers were closely correlated. We did not perform quantification with primers for the S region as only eight donors were positive for the S gene on nested PCR. In contrast to the findings from North America and Europe [21,34,35], our data do not support the correlation between HBV DNA positivity and a positive anti-HBc status. In our population, 75.8% are anti-HBc positive. The core antigen is a potent immunogen that elicits a strong antibody response and, specific antibodies are already present during acute infection and usually persist for life. Presence of anti-HBc in the absence of any other marker may, therefore, be compatible with acute, chronic or past HBV infection. 450 Serum HBV DNA with X Primers (copies/ml)

The serum HBV DNA was quantifiable in six of the 19 donors (31.6%) with occult HBV infection using primers for the Core region compared with nine of the 19 donors (47.4%) with occult HBV infection using primers for the X region (PZ0.32). HBV DNA was present but too low to be quantitated with primers for the Core region in eight of the 19 donors (42.1%) with occult HBV infection, compared with 10 of the 19 donors (52.6%) with primers for the X region (PZ0.52). A concordant result was detected in six of 19 donors (31.6%) with both sets of primers. The results of the two primers were also closely correlated (rZ0.99, P!0.001, slopeZ0.90) (Fig. 1).

400 350 300 250 200 150 100 50 0 0

50

100

150

200

250

300

350

400

Serum HBV DNA with Core Primers (copies/ml)

Fig. 1. Comparison of HBV DNA levels among six serum samples determined by real time PCR with the primers for the Core region and the X region. The line passing through the data with a slope equal to one is the hypothetical line that all data points would fall on if the two different sets of primers yielded results in complete agreement with each other.

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Table 5 Clinical features of the three recipients who developed HBV-related hepatitis after HSCT Patient

1 2 3

Recipient HBV status pre-HSCT

Donor HBV status

HBsAg

Anti-HBc

Anti-HBs

HBsAg

Anti-HBc

Anti-HBs

– – –

– C –

– – –

– – –

– C C

– – C

Donor occult HBV status

Time of HBV-related hepatitis (months after HSCT)

C – C

5.4 5.3 6.7

Recipient HBV status at the end of follow-up HBsAg

HBeAg

Anti-HBe

C C C

– C C

C – –

HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e antigen; Anti-HBe, hepatitis B e antibody; Anti-HBc, hepatitis B core antibody; anti-HBs, hepatitis B surface antibody; HBV, hepatitis B virus; HSCT, hematopoietic stem cell transplantation.

Although acute HBV infection may be excluded by the absence of IgM antibodies against the core viral antigen, the possibility of chronic or past infection remains. As the result of the high HBV carrier rate in Hong Kong, many of our donors would have had prior exposure to HBV. Therefore, the presence of anti-HBc would be unreliable in HBV endemic areas as a marker for occult HBV infection. The results of this study on HSC donors would have an important impact on the selection of donors for organ or tissue transplantation in HBV endemic areas. Although 75.8% of our HBsAg negative HSC donors were anti-HBc positive, only 17.0% of them had evidence of occult HBV infection found by PCR assay. Therefore, the exclusion of anti-HBc positive subjects as organ or tissue donors in HBV endemic areas is not recommended. The diagnosis of occult HBV infection should not be based on anti-HBc positivity alone. Instead, we recommend that more sensitive tests such as nested PCR, real-time PCR or nucleic acid testing to be used for detection of occult HBV infection if required, rather than relying on the anti-HBc positivity alone. In our current study, recipients receiving HSC from donors with occult HBV infection (10.5%) had a higher chance of developing HBV-related hepatitis after HSCT when compared with recipients receiving HSC from donors without occult HBV infection (1%). However, the difference is not statistically significant. This might be related to the small number of subjects involved in this study. On direct sequencing of the Core and X regions in one of the recipients with HBV-related hepatitis and his donor who had occult HBV infection, both the recipient and his donor had a 90% homology in the Core region and a 95% homology in the X region (unpublished data). This suggests that the de novo hepatitis after HSCT in this particular recipient might be due to transmission of HBV infection from the occult HBV infected donor. We are unable to determine the source of the HBV infection in the remaining two recipients due to the lack of serial samples. Thus, some of the HBV-related hepatitis could be due to HBV reactivation of the recipient’s HBV rather than an infection by the donor. Hence, further prospective study is required to determine the risk of transmission of HBV from HSC infusion and the source of the HBV, whether it is transmitted from the donor or a reactivation

of pre-existing occult HBV in the recipient after immunosuppression. Another important finding is that anti-HBc plus anti-HBs positivity do not seem to offer protection against occult HBV infection. This suggests that recovery from acute hepatitis B infection may not result in complete elimination of the virus. It is only the immune system keeping the virus at a very low level in the body. A decrease in the immune status may result in HBV reactivation even in individuals who are anti-HBc and/or anti-HBs positive, as has been previously reported in anti-HBs positive patients undergoing chemotherapy [36,37]. One of the key issues in detecting low level of HBV DNA, even when using PCR, is related to the specificity and reproducibility of the assay used [38]. Hence, in our current study, in addition to meticulous steps undertaken to prevent contamination of samples and inclusion of negative controls, all assays were performed in duplicate using two independent sets of HBV primers. Accordingly, those donors with only one HBV region detectable on nested PCR with the respective region primer pair were considered occult HBV negative [38–40]. In keeping with this, none of the recipients of HSC from donors with only one region of the HBV genome detected on nested PCR developed HBV-related hepatitis after HSCT. However, a larger well-designed, prospective study is currently undertaken in our center to further confirm this finding. In conclusion, the prevalence of occult HBV infection in Hong Kong is 15.3%. Neither the anti-HBc nor any other HBV serology should be utilized as reliable predictor of occult HBV infection in our circumstances. Further prospective studies are required to determine the outcome of HSCT recipients whose donor has occult HBV infection in order to determine the need for occult HBV screening in potential HSC donors.

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