Occult hepatitis B infections among blood donors in Lao PDR

Vox Sanguinis (2014) 106, 31–37

ORIGINAL PAPER © 2013 International Society of Blood TransfusionDOI: 10.1111/vox.12073

Occult hepatitis B infections among blood donorsin Lao PDRP. Jutavijittum,1 I. E. Andernach,2 A. Yousukh,1 B. Samountry,3 K. Samountry,3 T. Thammavong,4 J. Keokhamphue,4

K. Toriyama5 & C. P. Muller2,61Department of Pathology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand2Institute of Immunology, Centre de Recherche Public de la Sant�e/Laboratoire National de Sant�e, Luxembourg, Grand-Duchy of Luxembourg3Department of Pathology, Faculty of Medicine, University of Health Sciences, Vientiane, Lao PDR4National Blood Transfusion Center, Lao Red Cross, Vientiane, Lao PDR5Department of Pathology, Japanese Red-Cross Nagasaki Atomic Bomb Hospital, Nagasaki, Japan6Institut Pasteur du Laos, Vientiane, Lao PDR

Received: 4 February 2013,revised 1 July 2013,accepted 2 July 2013,published online 12 August 2013

Background and Objectives In Lao People’s Democratic Republic, hepatitis Bvirus is highly endemic. However, blood donations are only screened for HBsAg,leaving a risk of transmission by HBsAg-negative occult infected donors. Here,we characterized first-time blood donors to assess prevalence of hepatitis B virusinfections and occult infected donors.

Materials and Methods Sera were screened for HBsAg, HBeAg and anti-HBs,anti-HBc and anti-HBe antibodies. Occult HBV infections (OBIs) were assessed inHBsAg-negative sera by PCR, and sera of HBsAg positive and occult infecteddonors were phylogenetically characterized.

Results 9�6% of the donors were HBsAg positive, and 45.5% were positive for atleast one of the hepatitis B virus serum markers. More than 40% HBsAg carrierswere HBeAg positive, with HBeAg seroconversion occurring around 30 years ofage. Furthermore, 10�9% of HBsAg-negative, anti-HBc and/or anti-HBs-positivedonors were occult infected with hepatitis B virus. Thus, at least 3�9% of blooddonations would potentially be unsafe, but hepatitis B virus DNA copy numbersgreatly varied between donors.

Conclusion In Lao People’s Democratic Republic, a sizable proportion of HBsAg-negative and anti-HBc antibody-positive blood donations are potentially DNApositive and infective for hepatitis B.

Key words: chronic hepatitis B infection, hepatitis B virus, occult hepatitis Binfection, seroclearance, serological markers.

Introduction

More than one-third of the world’s population has been

infected with hepatitis B virus (HBV) at some point during

their life, and 75% of the estimated 360 million chronic

carriers worldwide live in the Asia-Pacific region alone

[1, 2]. Chronically infected individuals with detectable

HBV surface antigen (HBsAg) represent a significant risk

of virus transmission by blood donation. The Asian geno-

types B and C are often found at higher viral loads in the

blood (>107–108 copies/ml) [3], whereas other genotypes,

such as genotype E in Africa, are associated with HBeAg

seroconversion before the age of 15 or 16 and lower viral

loads [3, 4]. An additional risk for transfusion-transmitted

infections (TTI) results from occult HBV infections (OBI),

defined as the presence of HBV DNA in the blood or liver

without detectable HBsAg. The patho-physiological basis

of OBI is still poorly understood, but is probably due to

an incomplete immune control of the infection. Most

individuals with OBI have antibodies against HBV core

antigen (anti-HBc) possibly reflecting an ongoing chronic

Correspondence: Claude P. Muller: Institute of Immunology, Centre deRecherche Public de la Sant�e/Laboratoire National de Sant�e, 20A rueAuguste Lumi�ere, L-1950 Luxembourg, Grand-Duchy of LuxembourgE-mail: [email protected]

31

infection with HBsAg below detection limits. This is par-

ticularly common in endemic countries where individuals

are infected during early childhood. OBI with antibodies

against HBV surface antigen (anti-HBs), with or without

anti-HBc antibodies, sometimes occur during the recovery

phase of the infection. Rare sero-negative cases of OBI

occur in individuals with no markers of HBV infection

other than HBV DNA. Individuals with chronic hepatitis

who have mutated HBsAg that is not detected by diag-

nostic assays also contribute to OBI. While OBI infections

are generally characterized by very low levels of HBV

DNA in the blood (usually <1000 copies/ml or <200 IU/

ml [5]), they nevertheless are a concern for recipients of

blood donations [6–8].

In Western countries with a low prevalence of HBV

infections, anti-HBc antibody testing is included in the

screening of blood donations for TTI to exclude individu-

als with past exposure to HBV (including the vast major-

ity of OBI). However, in highly endemic countries, this

approach would exclude a large proportion of healthy

donors that have cleared HBV infections, severely under-

mining the blood supply [9, 10]. In these settings, screen-

ing for TTI commonly includes HBsAg testing [6, 10].

However, this strategy does not detect OBI or acute infec-

tions during the initial infection period. Thus, nucleic acid

testing (NAT) has been proposed and tested as a highly

sensitive technique for HBV DNA detection in OBI blood

donors. However, in low-income countries, NAT may not

be possible due to limited resources [6, 9–11].

In Lao PDR, HBsAg is highly endemic, with a preva-

lence of 8.7% in Lao blood donors [12]. As in its neigh-

bouring countries Cambodia and Vietnam [10], blood

donations in Lao PDR are screened for HBsAg only, with

the risk of TTI by HBsAg-negative blood donors with OBI.

In this study, we determined the serological profile of Lao

blood donors and the prevalence of OBI. We discuss pos-

sible implications for the safety of Lao blood donations.

Materials and methods

Study group

A total of 906 first-time blood donors, including 296

females, were randomly recruited between March and

June 2006. In Lao PDR, all blood donors are routinely

asked for sexual and medical risk factors related to TTI

including clinical hepatitis in an interview and by ques-

tionnaire. Donors with previous hepatitis B, C, HIV and

syphilis were excluded. Donors are not routinely asked

whether they are vaccinated against HBV, and sera are

not routinely tested for ALT. The mean age of the donors

was 23.8 – 7.4 years (range 16–53 years), with about

80% of donors being <30 years old. Serum samples were

obtained from Vientiane municipality, Vientiane Province

and Bolikhamsay Province, either at the National Blood

Transfusion Centre of the Lao Red Cross in Vientiane or

by mobile collecting units and were stored at -20°C until

further testing.

Serological analyses

All assays were performed on an automated AxSYM�

system (Abbott, Wiesbaden, Germany). HBsAg, HBeAg

and anti-HBs, anti-HBc, anti-HBe antibodies were

detected by AxSYM� HBsAg V2, AxSYM� HBe 2.0,

AxSYM� AUSAB, AxSYM� CORE and AxSYM� Anti-HBe

2.0 assays, respectively.

Molecular and phylogenetic analyses

DNA was extracted using the QIAGEN DNA Blood Mini

Kit (QIAGEN, Venlo, the Netherlands), according to the

manufacturer’s instructions. The HBV genome was

amplified as four overlapping fragments (preS, S, X and

C) as described previously [13]. The sensitivity of the

PCR reactions for preS, S, X and C was 1424, 1009,

2240 and 950 copies per reaction (or 285, 202, 448 and

190 IU/ml), respectively. Phylogenetic analyses were per-

formed using MEGA v.5 (Tempe, AZ, USA) [14] and the

neighbour-joining method of the Kimura 2-parameter

model with 1000 bootstrap replicates. Genotyping was

performed by analysing at least one of the four frag-

ments. For detection of OBI, DNA was extracted from

200 ll of serum from HBsAg-negative, anti-HBc and/or

anti-HBs-positive donors. The DNA was eluted in 60 ll,and 5 ll was used in quantitative real-time PCR. The

PCR was performed using forward primer 5′-ACTCA-

CCAACCTCTTGTCCT-3′ (nt 333–352 in relation to the

EcoR1 site), reverse primer 5′-GACAAACGGG CAACA-

TACCT-3′ (nt 476–457) and TaqMan Probe (5′-FAM

and 3′-TAMRA) 5′-TATCGCTGGATGTGTCTGCGGCGT-3′

(nt 368–391). The reaction was performed at 50°C for

2 min, 95°C for 10 min and 50 cycles of 95°C for 15 s

and 60°C for 60 s [15] and compared with a HBV

plasmid dilution series ranging from 18.5 to

1.85 9 1010 copies in the PCR corresponding to 1110 to

111 9 1010 copies/ml or 222 to 22 9 1010 IU/ml serum.

Although viral DNA can be detected below the linear

range, they were not quantifiable. Therefore, values

below this linear range (CT values above 34) are

expressed as ‘<1110 copies/ml’. The estimated limit of

detection using our in-house plasmid was about

110 copies/ml or approximately 20 IU/ml. Samples with

CT values above 37 were considered negative. Negative

controls were included in all extractions and PCR

experiments.

� 2013 International Society of Blood TransfusionVox Sanguinis (2014) 106, 31–37

32 P. Jutavijittum et al.

Statistical analyses

A computerized data sheet was used for record keeping;

all data were evaluated with SPSS 17.0 for Windows statis-

tic software package (SPSS Inc., Chicago, IL, USA) or

SigmaPlot 12.0 (Systat Software Inc., San Jose, CA, USA).

Parameters were compared by chi-square test or Fisher’s

exact test where appropriate.

Ethical statement

This study was approved by the competent Research

Ethics Committee of the Faculty of Medicine of Chiang

Mai University, Thailand, and the Ethics Committee of the

Faculty of Medical Sciences, National University of Laos,

Lao PDR.

Results

HBsAg, anti-HBs and anti-HBc antibodies

In the 906 blood donors, the overall HBsAg prevalence

was 9.6% (87/906), while anti-HBc and anti-HBs antibod-

ies were detected in 43.7% (396/906) and 27.7% (251/

906) of donors, respectively. Anti-HBc and anti-HBs anti-

body prevalence increased with age with significantly

higher percentages in the oldest compared to the youn-

gest age group (P < 0.005 and P < 0.05, respectively),

while HBsAg was less frequent in the older age groups,

with a peak prevalence of 11.6% at 20–29 years of age

(Fig. 1, Table 1). Overall, males were more frequently

infected with HBV than females (48.7% and 38.9%,

respectively; P < 0.05). 86 of the 87 HBsAg-positive

donors were also positive for anti-HBc, while one (a 22-

year-old male) was repeatedly negative for both anti-HBc

and anti-HBs, suggestive of recent infection. One of the

HBsAg-positive samples was also positive for anti-HBs

(Table 1).

Four hundred and ninety four of the 819 HBsAg-nega-

tive sera were negative for all serum markers, while the

remaining 325 sera were positive for either anti-HBc anti-

bodies (n = 75; 9.2% of HBsAg-negative sera), anti-HBs

antibodies (n = 15; 1.8%) or both (n = 235; 28.7%)

(Table 1).

HBeAg and anti-HBe antibodies

Eighty five HBsAg-positive carriers, excluding two sam-

ples with insufficient serum volumes, were further

screened for HBeAg and anti-HBe antibodies. HBeAg and

anti-HBe antibodies were mutually exclusive and were

found in 44.7% (38/85) and 55.3% (47/85) of HBsAg-

positive donors, respectively (Table 2). At a younger age

(<30 years), approximately half of the carriers were posi-

tive for either HBeAg or anti-HBe. In contrast, all 10

donors older than 30 years were positive for anti-HBe

(Table 2).

Occult infections

To determine the prevalence of OBI, 320 of the 325

HBsAg-negative, anti-HBc and/or anti-HBs-positive sera

were screened with a highly sensitive in-house TaqMan

PCR. In 35 of the 320 samples (10.9%), HBV DNA was

detected, with 32 of these having <1110 copies/ml. All 35

TaqMan positive samples were found to be anti-HBc posi-

tive, with 23 of these also positive for anti-HBs (Table 3).

HBV viral load ranged from below 1110–17 550 copies/

ml (222–3510 IU/ml), with the highest concentration

observed in anti-HBc positive/anti-HBs negative.

HBV genotypes

Of the 85 HBsAg-positive sera that were screened for

HBeAg and anti-HBe, 43 were PCR positive on at least

one of the four HBV genotyping fragments. Most HBeAg-

positive sera (33/38) could be genotyped but as a result

of reduced viral load after seroconversion, only few anti-

HBe-positive samples (10/47) were PCR positive and could

be genotyped. Phylogenetic analyses revealed that strains

belonged to genotypes B (19/43), C (21/43) and I (3/43),

with similar rates of genotypes B and C throughout the

different age groups (Table 4).

Additionally, the 35 TaqMan PCR-positive OBI sera

were subjected to preS and S-gene genotyping PCRs. 11

HBsAg +

anti-HBc +

anti-HBs +

0

10

20

30

40

50

60

70

16-19 20-29 30-39 40-53

% o

f ser

um

mar

kers

wit

hin

ag

e g

rou

p

Age group

Fig. 1 Percentage of HBsAg, anti-HBs and anti-HBc positives among

age groups. Prevalence of hepatitis B virus (HBV) serum markers accord-

ing to age group. Prevalences for HBsAg (blue), anti-HBc (red) and

anti-HBs (green) are calculated based on the whole study group (906

individuals).


Occult hepatitis B infections in Laos 33

of these, with viral copies ranging from less than 1110

to 17 550 per ml (222–3510 IU/ml), were found to be

positive in at least one of the PCRs. Phylogenetic anal-

yses revealed a predominance of HBV genotype C (8/

11), while one strain each was attributed to genotypes

B and I. Another strain was suspected to be mixed

infected with multiple genotypes and was not further

analysed. While the proportion of genotype C infections

in the occult group was significantly higher than in the

HBsAg-positive group (P < 0.05), this is likely to be a

selection bias, since HBV/C infections tend to have

higher viral loads than HBV/B infections. Thus, OBI

with HBV/B variants is more likely to remain unde-

tected.

Table 2 Seroprevalence of HBeAg and anti-HBe in HBsAg-positive sera according to age groups

Serological markers No. samples (% of age group)

TotalHBeAg Anti-HBe 16–19 20–29 30–39 40–53

+ - 16 (51.6%) 22 (50.0%) 0 (0%) 0 (0%) 38 (44.7%)

- + 15 (48.4%) 22 (50.0%) 6 (100%) 4 (100%) 47 (55.3%)

Table 3 DNA positivity among HBsAg-negative, anti-HBc and/or anti-HBs-positive donors

DNA


TotalAnti-HBc Anti-HBs 16–19 20–29 30–39 40–53

+ + - 3 (3.2%) 3 (2.3%) 5 (7.4%) 1 (3.6%) 12 (3.8%)

+ + + 10 (10.8%) 8 (6.1%) 2 (2.9%) 3 (10.7%) 23 (7.2%)

+ - + 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)

- + - 12 (12.9%) 28 (21.4%) 16 (23.5%) 8 (28.6%) 64 (20.0%)

- + + 63 (67.7%) 89 (67.9%) 40 (58.8%) 14 (50%) 206 (64.4%)

- - + 5 (5.4%) 3 (2.3%) 5 (7.4%) 2 (7.1%) 15 (4.7%)

Table 1 Seroprevalence of HBsAg, anti-HBc and anti-HBs according to age groups


TotalHBsAg Anti-HBc Anti-HBs 16–19 20–29 30–39 40–53

+ - - 0 (0.0%) 1 (0.2%) 0 (0.0%) 0 (0.0%) 1 (0.1%)

+ + + 1 (0.3%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 1 (0.1%)

+ + - 31 (9.0%) 44 (11.4%) 6 (4.7%) 4 (8.0%) 85 (9.4%)

- + - 17 (5.0%) 29 (7.5%) 20 (15.8%) 9 (18.0%) 75 (8.3%)

- + + 75 (21.9%) 101 (26.1%) 42 (33.1%) 17 (34.0%) 235 (25.9%)

- - + 5 (1.5%) 3 (0.8%) 5 (3.9%) 2 (4.0%) 15 (1.7%)

- - - 213 (62.3%) 209 (54.0%) 54 (42.5%) 18 (36.0%) 494 (54.5%)

Table 4 Prevalence of hepatitis B virus (HBV) genotypes B and C according to age groups and HBeAg/anti-HBe status in 11 HBsAg-positive sera

Genotype


TotalHBeAg Anti-HBe 16–19 20–29 30–39 40–53

B + - 5 (29.4%) 10 (41.7%) 0 (0%) 0 (0%) 15 (34.9%)

- + 3 (17.6%) 0 (0%) 1 (50%) 0 (0%) 4 (9.3%)

C + - 7 (41.2%) 10 (41.7%) 0 (0%) 0 (0%) 17 (39.5%)

- + 2 (11.8%) 1 (4.2%) 1 (50%) 0 (0%) 4 (9.3%)

I + - 0 (0%) 1 (4.2%) 0 (0%) 0 (0%) 1 (2.3%)

- + 0 (0%) 2 (8.3%) 0 (0%) 0 (0%) 2 (4.7%)



Discussion

In healthy first-time blood donors, we found a high

prevalence of HBV serum markers. We determined that

45.5% of the blood donors were positive for any of the

HBV markers, suggesting that almost half of the Lao

population has been in contact with HBV at one point

during their lives. 9.6% of the donors were HBsAg

positive. This is consistent with the 8.7% HBsAg

prevalence reported before from Lao PDR [12] as well as

from neighbouring countries such as Cambodia (7.7%

HBsAg and 58.6% anti-HBc prevalence) and Vietnam

(11.4% HBsAg and 51.7% anti-HBc) [10].

The presence of anti-HBc and also anti-HBs antibodies

in the investigated cohort increased with age and was

significantly higher in the oldest age group, while HBsAg

positivity had a tendency to decrease in the higher age

groups. This is likely to represent spontaneous HBsAg

seroclearance during late stages of HBV infections and is

consistent with clearance rates of up to 45% observed in

endemic regions [16]. In Lao PDR, a country with a high

prevalence of chronic HBV carriers, most infections

are acquired during early life, perpetuating chronic

infections. Anti-HBs antibodies were found in 27.7% of

all donors, and only 6.1% of these were anti-HBc

negative and therefore potentially vaccinated. This is in

accordance with the low HBV vaccine coverage in adults

in Lao PDR where HBV vaccine was only introduced in

2001 for infants. Furthermore, 8.3% of donors were

anti-HBc positive, but negative for HBsAg and anti-HBs,

indicating high numbers of later stage chronic HBV

infections with loss or reduced levels of HBsAg, or

resolved infections (HBV DNA negative) with anti-HBs

antibodies below the detection limit (Fig. 1 and Table 1).

Interestingly, one sample was repeatedly positive for

HBsAg as well as for anti-HBc and anti-HBs antibodies.

While this may be a case of exposure to heterologous

HBsAg serotypes, we have no further basis for such

speculations [17]. This serological profile may also be

indicative of acute HBV infection or the case of a chronic

carrier with severe liver disease, although the latter is

unlikely as blood donors are normally healthy volunteers.

More than 40% (38/85) of HBsAg carriers in this study

were HBeAg positive. Interestingly, while about half of

the carriers below 30 years of age were positive for either

HBeAg or anti-HBe, all 10 donors older than 30 years

were positive for anti-HBe but not HBeAg (Table 2). This

suggests that seroconversion from HBeAg to anti-HBe, an

indicator of sustained remission, occurs at around

30 years of age in Lao PDR. Although previous studies

indicated earlier HBeAg seroconversion in patients

infected with HBV genotype B than for those infected

with HBV genotype C [18, 19], we could not observe a

difference in the prevalence of HBV B and C genotypes in

the different age groups (Table 4).

In our study, 10.9% (35/320) of HBsAg-negative, anti-

HBV antibody-positive donors were HBV DNA positive in

a highly sensitive real-time PCR. Thus, at least 3.9% of

all 906 investigated donors were occult infected with

HBV (OBI). When a less sensitive commercial assay was

used, only four samples, including the three with the

highest DNA levels, were DNA positive (data not shown).

This would correspond to an OBI prevalence of 1.3% of

HBsAg-negative, anti-HBV antibody-positive donors and

0.4% of all donors. It is possible that the individuals

detected with the commercial assay represent the greatest

risk of onwards HBV transmission. This apparent differ-

ence in prevalence highlights the difficulty of comparing

OBI prevalence data from studies using different detection

techniques. Previous studies in highly endemic regions

also showed high prevalences of OBI. In Indonesia, where

9.4% of the population is chronically infected, a study

using nested PCR found 8.1% of blood donors to be

occult infected [20]. Similarly, in a highly endemic popu-

lation in India, an in-house PCR detected OBI in 30% of

anti-HBc antibody-positive donors [21]. This is in contrast

to data from regions where HBV is not endemic, such as

the USA, where OBI can be as low as 3.7% of anti-HBc

antibody-positive donors [22]. While the prevalence of

OBI is dependent on epidemiological aspects such as the

route of infection, the clinical evolution of the disease

and interventions, the infectivity of OBI blood as well as

the determinants of this infectivity still remain unclear.

The majority of OBIs that could be genotyped in the

current study were attributed to genotype C (8/11).

Significantly, fewer HBV genotype B infections were

observed in the OBI group (1/11) than in HBsAg carriers

(19/43), where equal proportions of HBV genotype C and B

were observed. One study of OBI in individuals from

south-east China, where genotype B is dominant, also

showed that the prevalence of genotype C was signifi-

cantly higher in OBI than in HBsAg-positive individuals

[23], while others have not seen this difference [24]. Stud-

ies on larger cohorts are needed to further determine the

significance of a genotype bias in OBI.

All OBI donors were anti-HBc positive while the

prevalence of OBI was somewhat lower in anti-HBs/HBc

antibody double positive (23/229; 10%) than in anti-HBc

single positive samples (12/76; 15.8%; not statistically

significant). In other studies, OBI prevalences were also

higher in individuals with anti-HBc antibodies only and

lower in double positive or anti-HBs antibody only

donors [25]. In general, high levels of anti-HBs antibodies

in donated blood forestall HBV infections of recipients

(reviewed in [9]). Thus, recipient infection may not be

prevented in the presence of low levels of anti-HBs



antibodies (<100 IU/ml), in particular, when levels of

HBV DNA are high or when the recipients are immuno-

compromised [9]. In our cohort, OBI sera had a range of

anti-HBs levels from 0 to 768 IU/ml. Importantly, the

three donors with highest DNA levels of 1790, 12 800

and 17 550 copies per ml (358, 2560 and 3510 IU/ml)

had undetectable anti-HBs antibodies, re-emphasizing the

higher risk of TTI from these donors. Rare sero-negative

cases of OBI occur in individuals with no markers of HBV

infection other than HBV DNA. For example, in Thai

blood donors, about one in 3000 had HBV DNA and was

antibody and HBsAg negative. Approximately 20% of

these were window period cases [26, 27]. Since we only

investigated HBV DNA in antibody-positive cases, our

study would have missed DNA-positive (antibody-nega-

tive) window cases.

The significance of OBI for the donor and the recipient

is poorly understood. In particular, there are very few

studies investigating the infectivity of transfusion with

OBI blood. The few available studies seem to agree that

only subsets of recipients of OBI blood become infected.

For example, one study in Taiwan showed that two of 11

recipients of OBI blood became subsequently infected [28].

In another study from the Japanese Red Cross, OBI blood

was infectious in 19% of recipients [29]. Recently, it was

suggested that the 50% infectious dose of OBI blood prod-

ucts would be about 1000 HBV DNA copies [30]. However,

the relationship between viral DNA concentration or sero-

logy of the donor and infection of the recipient was not

determined in these studies. Thus, certainly a large propor-

tion of the OBIs observed in the current study, and espe-

cially the ‘anti-HBc only’ donors with higher serum DNA

levels, represent a considerable risk of TTI.

To eliminate the risk of HBV transmission by blood

transfusion, additional analyses besides HBsAg are

required in Lao PDR. While anti-HBc antibody screening

would largely exclude the HBsAg-negative/DNA-positive

sera, it would also unnecessarily exclude anti-HBc anti-

body-positive blood with undetectable HBV DNA, that is,

approximately 30% of all donors. In Thailand, a country

with an emerging economy, blood banks systematically

screen for HBV, hepatitis C virus and HIV-1/2 by nucleic

acid testing (NAT) since 2006. Together with a broad hep-

atitis B vaccination strategy, this has led to a reduction in

the incidence of new HBV infections in that country [26].

While this triple NAT screening may be cost-effective in

Thailand due to its high prevalence of HIV-1 infections,

its cost-benefit in resource poor Lao PDR requires careful

evaluation.

Acknowledgements

The authors would like to thank Miss Praijitr Tanan,

(Blood Bank of Maharaj Nakorn Chiang Mai Hospital and

Faculty of Medicine, Chiang Mai University) for her excel-

lent technical assistance and Antony Black (Institute of

Immunology, CRP-Sant�e, Luxembourg and Institut Pasteur

du Laos, Vientiane, Lao PDR) for his help in performing

some of the experiments and help with the manuscript.

This work was partially supported by the Faculty of

Medicine Research Fund, Chiang Mai University, Thailand

and by a grant from the Ministry of Cooperation and

Humanitarian Aid, Luxembourg. I.E.A. was supported by a

fellowship from the National Research Fund, Luxembourg

(TR-PHD-BFR07-129).

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Occult hepatitis B infections among blood donors in Lao PDR