Clinical and Diagnostic Laboratory Immunology, July 2001, p. 818-821, Vol. 8, No. 4
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.4.818-821.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Viral Load in Breast Milk Correlates with
Transmission of Human Cytomegalovirus to Preterm Neonates, but
Lactoferrin Concentrations Do Not
B. W. A.
van der
Strate,1,*
M. C.
Harmsen,2,3
P.
Schäfer,4
P. J.
Swart,1,
T. H.
The,2
G.
Jahn,5
C. P.
Speer,6
D. K. F.
Meijer,1 and
K.
Hamprecht5
Department of Pharmacokinetics and Drug Delivery,
University Centre for Pharmacy, Groningen University Institute for
Drug Exploration, 9713 AV Groningen,1 and
Department of Clinical Immunology2 and
Department of Pathology/Laboratory
Medicine,3 Groningen University Institute for
Drug Exploration, University Hospital Groningen, 9713 GZ Groningen,
The Netherlands, and Institut für Medizinische
Mikrobiologie and Immunologie, Universitäts-Krankenhaus
Eppendorf, D-20246 Hamburg,4
Institute for Medical Virology and Epidemiology of Viral
Diseases, University Hospital Tübingen, D-72076
Tübingen,5 and Children's
Hospital, University of Würzburg, 97080 Würzburg,6 Germany
Received 27 December 2000/Returned for modification 7 February
2001/Accepted 11 April 2001
 |
ABSTRACT |
In vitro, lactoferrin (LF) strongly inhibits human cytomegalovirus
(HCMV), which led us to hypothesize that in vivo HCMV might also be
inhibited in secretions with high LF concentrations. In breast milk,
high viral loads observed as high viral DNA titers tended to coincide
with higher LF levels. However, the LF levels did not correlate to
virus transmission to preterm infants. The viral load in the
transmitting group was highest compared to the nontransmitting group.
We conclude that viral load in breast milk is an important factor for
transmission of the virus.
| |
TEXT |
Breast-feeding is a strong risk
factor for the postnatal transmission of human cytomegalovirus (HCMV)
(2, 21). The rate of transmission by consuming
HCMV-infected breast milk ranges from 58 to 76% (3, 24).
Although HCMV-infected cells have been isolated from breast milk
(1, 5) and cell-free virus has been detected in the whey
of HCMV-infected mothers (1, 5), the mechanism of virus transmission through breast milk has not been elucidated yet. In
contrast, HCMV is seldomly detected in colostrum (16).
Breast milk has a protective effect against microbial infections and one of the protective components is lactoferrin (LF).
LF, an 80-kDa iron-binding glycoprotein, is present in the secondary
vesicles of neutrophilic granulocytes (12). LF is also present in mucosal secretions (11, 13), where it is
produced by epithelial cells, e.g., by the mammary glands during
lactation (11, 13). At the mucosa, LF exerts its
antibacterial and fungicidal effect (10, 11, 13). In
vitro, LF exerts antiviral activities against a plethora of viruses,
including hanta, HIV and HCMV (6, 15, 18, 22).
Lactoferrin concentrations are highest in colostrum and tend to
decrease significantly within the first weeks of lactation (7,
14). We hypothesized that LF, among other defense proteins, would help to prevent the transmission HCMV to the newborn. In particular, for preterm newborns this nonspecific immunological defense
could be important.
We set out to determine the LF concentrations in breast milk
longitudinally to assess the relation between transmission of HCMV
and LF levels in vivo. The relation between LF concentrations and
the total amount of HCMV DNA in breast milk was studied in the same samples.
Study group.
Breast milk specimens were obtained from 23 breast-feeding mothers of preterm infants at the University Hospital of
Tübingen. These mothers were enrolled prospectively between July
1995 and June 1998 in a clinical study of postnatal mother-to-preterm
infant transmission of HCMV via breast milk (4). HCMV
screening of seronegative and seropositive mother-infant pairs was
performed by serology, virus culture, and PCR. Congenital and perinatal HCMV transmission were excluded. All mothers were informed of the aim
of the study, which was approved by the ethical comittee of the
University of Tübingen. All mothers were without clinical symptoms of HCMV invection and were classified into four groups. The
first group were seronegative controls (group 1, n = 4), i.e., without transmission, DNA-lactia, and virolactia. Groups
2 (n = 4), 3 (n = 8), and 4 (n = 7) all comprised seropositive mothers with
DNA-lactia. Transmission only occurred in group 4, for which the
mothers, as in group 3, had virolactia. Group 2 mothers had no virolactia.
Milk whey preparation.
Native expressed breast milk was
sampled longitudinally. Cell-free milk whey was prepared as described
previously (5) and stored as aliquots at 
20°C.
DNA extraction and qualitative nPCR from milk whey.
The
extraction of DNA and detection of HCMV DNA by nested PCR (nPCR) in
milk whey was performed as previously described (5). This
approach allowed detection of 200 genome equivalents (GE) per ml of
milk whey.
Determination of viral load by quantitative nPCR.
Extracted
DNA from breast milk samples were added to PCR reaction mixtures
containing 50 copies (high standard) or 10 copies (low standard) of a
cloned CMV standard (9, 17). Target sequences were
amplified with the external CMV-specific primers E1 and E2 (17). Then, 5 µl each of the external reaction was
reamplified in a second round of PCR with the internal CMV-specific
primers TGGE1B and TGGE2E. Standard and wild-type CMV PCR amplimers
were quantitated by hybridization analysis as described elsewhere
(17). For CMV DNA copies of 
20 in 2.5 µl, the data
from the high-standard reaction were used, and for CMV copies of <20,
data from the low-standard reaction were used. Results were expressed
as the number of CMV wild-type GE per ml of milk whey. Exact
quantification was possible at between 400 to 200,000 GE/ml.
Detection of virolactia and transmission.
HCMV was cultured
from milk whey by using human foreskin fibroblasts in the tube cell
culture system. Virus transmission to the preterm infant was documented
by positive viruria or DNA-uria not earlier than 3 weeks after
delivery. Viruria was detected by virus culture; DNA-uria was detected
by nPCR as outlined above.
Quantification of LF levels in breast milk.
LF concentrations
in breast milk were determined as described elsewhere
(23), with minor modifications. In brief, polyclonal antiserum against human LF (Jackson) was coated in 96-well plates (Hycult). Serial dilutions of breast milk were added to the wells. Human LF (Sigma) was used in the calibration curve. Bound antibody was
detected with horseradish peroxidase-labeled antibodies (Jackson). Color was developed with TMB (trimethyl benzidine; Sigma), and the
optical density (OD) at 450 nm was measured. These ODs were converted
to LF concentrations using a four-parameter curve-fitting algorithm.
Statistical analyses.
The courses of LF concentrations in
breast milk were calculated by using smoothing spline fits. Unpaired
t tests were performed to investigate differences in HCMV
DNA levels between the transmitting and nontransmitting groups.
In the milk whey of breastfeeding mothers of preterm infants, LF
concentrations were maximal in the colostrum, up to 7 mg/ml, and
decreased approximately sevenfold in 2 weeks (Fig.
1). This finding is consistent with
reported values found during term delivery (7, 14),
although for four mothers an increase in LF levels was observed.
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FIG. 1.
LF concentrations in breast milk decrease during
lactation. Using smoothing spline fitting, no significant differences
in decline or initial altitude of LF levels in mature milk were
observed.
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Present address: Yamanouchi Europe B.V., 2353 EW Leiderdorp,
The Netherlands.
) and nontransmitters (
). The HCMV DNA load in
milk whey of maternal transmitters is significantly increased compared
to the nontransmitters. *, significantly different compared to the
nontransmitters (unpaired t test, P = 0.024).