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Journal of Clinical Virology 45 (2009) 281284

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Journal of Clinical Virology


journal homepage: www.elsevier.com/locate/jcv

Hepatitis B viral markers in banked human milk before and


after Holder pasteurization
Patricia Ribeiro de Oliveira a,1 , Aparecida Yulie Yamamoto a,1 , Cleonice Barbosa Sandoval de Souza a,2 ,
Natalia Motta de Arajo b,3 , Selma de Andrade Gomes b,3 , Anlia Ribeiro Heck c,4 ,
Jos Fernando de Castro Figueiredo d,5 , Marisa Mrcia Mussi-Pinhata a,
a

Department of Pediatrics, Faculty of Medicine of Ribeiro Preto, University of So Paulo, Av. Bandeirantes 3900, 14049-900, Ribeiro Preto, SP, Brazil
Molecular Virology Laboratory, Oswaldo Cruz Institute, Av. Brasil 4365, Manguinhos, Rio de Janeiro, Brazil
c
Human Milk Bank, University Hospital, Faculty of Medicine of Ribeiro Preto, University of So Paulo, Av. Santa Luzia, 387 Jardim Sumar, CEP 14025-090,
Ribeiro Preto, So Paulo, Brazil
d
Department of Internal Medicine, Faculty of Medicine of Ribeiro Preto, University of So Paulo, Av. Bandeirantes 3900, 14049-900, Ribeiro Preto, SP, Brazil
b

a r t i c l e

i n f o

Article history:
Received 29 December 2008
Received in revised form 14 April 2009
Accepted 19 April 2009
Keywords:
Hepatitis B virus
Human milk donor
Human milk bank
Lactating women

a b s t r a c t
Background: Blood screening for hepatitis B virus (HBV) is not universally performed for donor selection
in human milk banks.
Objectives: To evaluate the frequency of detection of HBV surface antigen (HBsAg) and HBV-DNA in
colostrum of HBV-infected nursing mothers before and after Holder pasteurization.
Study design: Forty-two concentrated breast milk samples were obtained within two postnatal weeks from
24 HBsAg-positive women (4 HBeAg-positive and 20 HBeAg-negative, anti-HBe-positive) were tested for
the presence of HBsAg and HBV-DNA before and after Holder pasteurization (30 min at 62.5 C).
Results: Before pasteurization, HBsAg and HBV-DNA were found in 14/24 (58%), and 20/24 (75%) rst
milk samples, respectively, obtained by 4 days after delivery. At least one marker was detected in 20/24
(83%) milk samples. Both markers were identied in milk of HBeAg-positive mothers, and most mothers
with anti-HBe in blood had at least one HBV marker. Once detected, viral markers were frequently found
in milk samples subsequently obtained from the same woman. Holder pasteurization did not affect the
probability of detecting HBsAg (8/18, 44%), HBV-DNA (12/18, 67%), or at least one of them (15/18, 83%).
Conclusions: Although the biological implications of these ndings remain to be determined, considering
that HBV is highly contagious and most recipients of banked human milk are preterm infants, these
ndings should be taken into account when donors are enlisted for human milk banks without serological
screening.
2009 Elsevier B.V. All rights reserved.

1. Background

Abbreviations: HBV, hepatitis B virus; HBsAg, hepatitis B virus surface antigen;


HBeAg, hepatitis B virus e antigen; HBc, hepatitis B core; HIV, human immunodeciency virus; CMV, cytomegalovirus; HTLV-1, human T lymphotropic virus type
1.
Corresponding author. Tel.: +55 16 36330136/36022479; fax: +55 16 36022700.
E-mail addresses: propat@ig.com.br (P.R. de Oliveira), yulie@fmrp.usp.br
(A.Y. Yamamoto), cleonice@fmrp.usp.br (C.B.S. de Souza), nmaraujo@ioc.ocruz.br
(N.M. de Arajo), selma@ioc.ocruz.br (S. de Andrade Gomes),
analia heck@uol.com.br (A.R. Heck), jfcgue@fmrp.usp.br
(J.F. de Castro Figueiredo), mmmpinha@fmrp.usp.br,
mussi.pinhata@pesquisador.cnpq.br, mussi.pinhata@cnpq.pesquisador.br
(M.M. Mussi-Pinhata).
1
Tel.: +55 16 36330136/36022674; fax: +55 16 36022700.
2
Tel.: +55 16 36330136/36022670; fax: +55 16 36022700.
3
Tel.: +55 2125621847; fax: +55 2122604866.
4
Tel.: +55 16 36102649.
5
Tel.: +55 16 36022468; fax: +55 16 36336695.
1386-6532/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jcv.2009.04.003

After the rst demonstration more than 30 years ago of hepatitis B virus (HBV) surface antigen (HBsAg) in the milk of a HBV
female carrier,1 this antigen was detected in none2 to 71.4%3,4 of
milk samples tested. HBV virions (Dane particles) were identied in
4 of 10 milk samples by electron microscopy.3 HBe antigen (HBeAg)
was also found in human milk.5 More recently, after the advent of
molecular biology techniques, an overall HBV-DNA-positive rate of
about 43%69 was detected in milk of HBV-infected women from
countries with a high carrier rate.
Donor selection and techniques of milk processing and storage in human milk banks are measures taken in Europe and
North America10,11 to prevent infection in recipient infants in contrast to countries with fewer resources which do not require that
milk donors be screened for HBV. Instead, lactating women are
usually only verbally screened and milk safety relies on Holder
pasteurization12 (heating to 62.5 C for 30 min followed by cooling

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P.R. de Oliveira et al. / Journal of Clinical Virology 45 (2009) 281284

to 7 C), which inactivates cytomegalovirus (CMV), HIV and type


I HTLV.13 However, there are no data demonstrating the safety of
Holder pasteurization for the inactivation of HBV in human milk.
2. Objectives
To evaluate the frequency of HBsAg and HBV-DNA detection in
colostrum of HBsAg carriers from a region with a 1% carrier prevalence rate, and to verify the effect of Holder pasteurization on the
detection of these viral markers in milk.
3. Study design
3.1. Study population
This study was conducted at the University Hospital, Faculty of
Medicine of Ribeiro Preto, University of So Paulo, Brazil. Among
7812 live births from 1999 to 2003, 58 (0.7%) mothers tested positive for HBsAg (Hepanostika HBsAg, Organon Tecnika BV, Boxtel,
The Netherlands). HIV-negative mothers who agreed to participate
and from whom it was possible to obtain at least one colostrum
sample (a minimum volume of 10 ml) within 1 week of delivery
were selected for the study. Twenty-four women were enrolled.

round of amplication was performed using sense primer PS1


(5 -CCATATTCTTGGGAACAAGA-3 , nt position 28262845) and
antisense primer SR (5 -CGAACCACTGAACAAATGGC-3 , nt position
685704). The second round of amplication was performed with
1 l of the rst round PCR product, sense primer PS1 and antisense primer PS2 (5 -GGTCCCCAGTCCTCGAGAAG-3 , nt position
124143). The protocol used for both rounds was: 94 C, 3 min;
30 cycles at 95 C (30 s), 52 C (40 s), 72 C (2 min), followed by a
nal elongation step at 72 C for 7 min. Negative and positive controls were added in both extraction and PCR procedures. The lower
limit of DNA detection by this PCR assay was determined using
serial dilutions of quantied recombinant plasmids carrying HBV
genomes, as previously described.16 This two-round PCR assay has a
sensitivity of about 10100 DNA molecules per assay. For the 539 bp
amplicon detection, an aliquot of the amplied products was submitted to electrophoresis in 2% agarose gel and visualized under UV
light after ethidium bromide-staining.
3.4. Data analysis
The proportions of the variables under study were determined
by the chi-square test with Yates correction or by the Fisher exact
test when indicated. The level of signicance was set at = 5% for
all tests.

3.2. Biological samples and testing for HBV serological markers

4. Results

Serum samples from all 24 HBsAg-positive mothers, obtained


upon admission for delivery, were stored at 20 C and retested for
HBsAg (Hepanostika HBsAg Uni-Forn II, microelisa system, Organon
Tecnika BV, Boxtel, Holanda), and tested for anti-HBc and HBe/antiHBe (Axsym, Microelisa System, Abbott, Wiesbaden, Germany).
Colostrum samples were obtained from these same mothers
between 1 and 4 days after delivery. Ten women had more than
one collected milk sample (ve women gave two samples; two gave
three samples; and three gave four samples).
Breast milk samples were collected by manual expression or
with a mechanical suction pump (Medela Lactina Select (Lactaset),
Medela Inc., McHenry, IL, USA), placed in sterile asks, and stored
frozen (20 C) until processing. Bleeding was not apparent during
collection. Milk samples were centrifuged at 2000 rpm for 15 min
to separate the fatty phase from the intermediate phase (serum) as
previously described.14 This milk serum fraction was concentrated
using the Centricon YM-10 kit (Microcon Centrifugal Filter Devices,
Bedford, MA, USA) according to the manufacturers instructions.
The mean concentration factor obtained was 3.5 times. For testing
after pasteurization, 10 ml of the serum fraction of raw milk was
heated to 62.5 C for 30 min and then cooled to 7 C (Holder pasteurization process) and 2 ml of pasteurized milk was also concentrated
for the laboratory assays. Before and after pasteurization, whole
milk aliquots, and concentrated milk serum fractions were stored
at 20 C until testing. Milk samples were tested for HBsAg before
and after milk concentration, and before and after milk processing
by Holder pasteurization.

Mean maternal age was 26.5 years (range: 1742). Gestational


age ranged from 33 to 41 weeks (median = 39 weeks).
Anti-HBc antibodies were detected in the peripheral blood of all
24 HBsAg-positive subjects, and HBe antigen was detected in 4 of
them (17%); the remaining 20 participants were anti-HBe positive.
HBV-DNA was found in 17 of 20 (85%) blood samples tested (four
samples could not be tested due to technical problems). All four
HBeAg-positive carriers, and 13/16 (81.2%) HBeAg-negative women
were positive for HBV-DNA in blood.
HBsAg was detected in 36% (15 of 42) of non-concentrated milk
samples. The frequency of detection increased to 55% (23/42) after
concentration (p < 0.001). Considering this nding, all the results
concerning HBV markers in milk were obtained by testing concentrated samples.
Table 1 shows the frequency of detection of HBV markers in milk
samples before and after pasteurization. At least one viral marker
(HBsAg and/or HBV-DNA) was detected in most samples, whether
considering the rst samples obtained from the 24 participants or
all samples analyzed. Detection of HBsAg before and after pasteurization was 83% concordant, while the detection of HBV-DNA was
71% concordant. For both markers, most discordance was due to a
decrease in positive tests occurring after pasteurization.
Table 1
Detection of HBsAg and HBV-DNA in milk of HBsAg carriers, before and after Holder
pasteurization.
HBV markers

Before pasteurization

After pasteurization

Colostrum
HBsAg
HBV-DNA
At least one marker

14/24 (58%) [3778]


18/24 (75%) [5390]
20/24 (83%) [6396]

8/18a (44%) [2169]


12/18 (67%) [4187]
15/18 (83%) [5996]

0.37
0.55
0.67

All milk samples


HBsAg
HBV-DNA
At least one marker

23/42 (55%) [3970]


29/42 (66%) [5382]
32/42 (76%) [6188]

12/35b (34%) [1952]


20/35 (57%) [3974]
27/35 (77%) [6090]

0.32
0.39
0.92

3.3. DNA extraction and PCR assays


Concentrated milk samples and serum samples were submitted to DNA extraction and HBV-DNA PCR amplication as
previously described.15,16 Briey, viral DNA was extracted using
phenol/chloroform after treatment of 250 l of serum or concentrated milk samples with 0.5 mg/ml of proteinase K in the
presence of 0.2 M NaCl, 0.25% SDS, for 4 h at 37 C. After
precipitation with ethanol, the pellet was dried and resuspended in 30 l of distilled water. The pre-S region was
amplied by a double step (semi-nested) PCR assay. The rst

Positive/total (%) [95%CI]

a
b

Six samples were not tested.


Seven samples were not tested.
p = 0.26, 2 -test for comparison between proportions of HBsAg and HBV-DNA.

P.R. de Oliveira et al. / Journal of Clinical Virology 45 (2009) 281284

283

Table 2
HBsAg and HBV-DNA in serial samples of breast milk, tested before and after pasteurization.
Mother number

Samples (median time after birth)


1st (1 day)

1
2
3
4
5
6
7
8
9
10
a

2nd (4 days)

3rd (9 days)

4th (14.5 days)

HBsAga

HBV-DNAa

HBsAg

HBV-DNA

HBsAg

HBV-DNA

HBsAg

HBV-DNA

/
/
+/+
/+
+/+
/
/
+/
+/+
+/+

+/+
+/
+/
/
+/+
+/+
+/+
+/+
/
+/

/
/
+/
/
+/+
+/
/
+/
+/
+/+

+/
+/+
+/
/
/+
+/
+/+
+/+
+/
+/+

/
/
+/+

/+
/+
/

+/+

+/+

+/

+/+

+/

+/+

Before/after Holder pasteurization.

With respect to the relationship between detection of viral


markers in blood and milk, both HBsAg and HBV-DNA were identied in the colostrum of the four mothers who were HBeAg-positive
in blood. HBsAg was detected in 10 (50%) and HBV-DNA in 13 (65%)
rst milk samples from the remaining 20 participants who were
not carriers of HBeAg in blood, with no association between the
presence of at least one viral marker in milk and the detection of
the e antigen in blood (100% vs. 80%, in positive and negative samples, respectively). HBsAg and/or HBV-DNA were identied in the
colostrum of 12/17 (71%) mothers who were carriers of HBV-DNA in
blood. Also, in all three women in whom HBV-DNA was not detected
in blood, at least one viral marker was detected in milk.
Table 2 shows the data for 10 women from whom more than
1 milk sample was obtained. Although HBV markers were intermittently detected over time in most mothers, once detected
subsequent samples tended to be positive, independently of milk
pasteurization.
5. Discussion
Markers of HBV infection were detected with high frequency
in milk from HBsAg-positive lactating subjects in a population
with low HBV endemicity. Markers in milk were obtained in both
HBe-positive and HBeAg-negative mothers. Although a decrease
in positive tests occurred after pasteurization, we could not
demonstrate that Holder pasteurization signicantly affected the
probability of detection of HBsAg or HBV-DNA.
Our nding of detection of HBV markers in milk is comparable to observations made in carrier mothers from highly endemic
regions, either for HBsAg or viral DNA, which have ranged from
56% to 71%,2,4,7 and 43% to 73%,69 respectively. Differently from
studies on Asian populations,8,9 only 4 (17%) women studied by
us were HBe carriers, while the presence of anti-HBe antibodies
in the remaining 20 women indicates that most of these women
had low or very low viral replication. The nding of HBV markers
in most milk samples from these lactating mothers may be a consequence of the very sensitive DNA amplication technique used
by us (as low as 100 copies of HBV-DNA/ml16 ), which could detect
viral HBV-DNA in the blood of 81% HBeAg-negative women. However, the presence of pre-core mutations in these HBeAg-negative
mothers17 may be another possible explanation. It is interesting
to note that we found the HBV-viral markers in milk of 3 mothers without HBV-DNA in their blood who were anti-HBe positive
in blood. These results might suggest that HBV replicates in mammary glands which have not been demonstrated so far. However,
although laboratory tests were performed with the recommended
precautions to avoid contamination, we cannot completely discard
this possibility. Ideally, the likelihood of HBV shedding by mammary glands in the absence of HBV-DNA in blood should be tested

with a larger sample size employing quantication of HBV-DNA in


blood and milk.
The biological implications of our ndings remain to be determined. Since HBV cannot be cultured in vitro, the demonstration
of viral viability in women milk will require experiments on
chimpanzee18 or duck HBV models.19 Although the study methodology was criticized,3 a study performed before the neonatal
immunoprophylaxis availability did not demonstrate that breastfeeding increased the risk of vertical transmission of HBV in term
babies.20 Even though the uncertain clinical signicance of our
study must be recognized, our ndings are worrisome. HBV is a
highly contagious virus.21 Heat pasteurization at 60 C for 10 h22 followed by solvent/detergent treatment 23 is required for maximum
HBV viral inactivation in human plasma products as demonstrated
by biological experiments. Considering that most recipients of
banked human milk are preterm infants, mainly those with very
low birth weight who receive banked human milk for long periods
of time, the possibility of exposure to viable viral HBV inoculum is a
concern. The overall immaturity of local host defenses of the oral24
and gastrointestinal mucosa25 of preterm infants could predispose
these infants to infection.
In the absence of donor screening and elimination of milk
donated by HBV-infected women, and if Holder pasteurization fails
to eliminate infectious HBV from human milk, early routine vaccination of newborn infants would protect them against a possible
exposure by this route. However, even for term neonates the protective efcacy of vaccination alone is inferior to that obtained
when vaccination is combined with hyperimmune immunoglobulin against HBV.26 Additionally, the lower immunogenicity of the
HBV vaccine in preterm infants,27 the main users of human milk
banks, may further increase their vulnerability to this infection.
In conclusion, the present study demonstrated the presence of
HBV markers in the milk of HBsAg carrier mothers, including those
who were HBeAg-negative, independently of milk processing by
Holder pasteurization. These data support the recommendation for
serologic screening for HBV of human milk donors at human milk
banks.
Acknowledgements
This work was partially supported by CNPq, Brazil. Funding: No
authors have any competing interests to declare. Ethical approval:
This study was approved by the Research Ethics Committee of the
Hospital (Process # 9032/2001). Written informed consent was
obtained from all subjects.
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