VIRUMILK

Biosensor for CMV Detection in Breast Milk from Lactating Women of Preterm

Infants Less than 33 Weeks

S. Py

, B. Wacogne

1,2

, L. Pazart

A. Coaquette

, W. Boireau

G. Herbein

3,4

and G. Thiriez

INSERM-CIC 1431, Besançon University Hospital, Besançon, France

Institute FEMTO-ST, University of Franche-Comté, Besançon, France

Laboratory of Virology, Besançon University Hospital, Besançon, France

UPRES EA4266, SFR FED 4234, Pathogens and Inflammation Laboratory, Department of Virology,

University of Franche-Comté, Besançon, France

Department of Neonatal Medicine, Besançon University Hospital, Besançon, France

Keywords: Cytomegalovirus, Screening, Biochips, Preterm Infants, Breastfeeding.

Abstract: Cytomegalovirus (CMV) is the leading cause of neonatal viral infection and can have a significant impact

on the neurosensory development of newborns and especially preterm infants. While congenital CMV

infection affects about 2-5% of very preterm infants, the risk of postnatal infection, particularly through

breast milk, is much higher in this population (20%). However, infection could be considerably reduced by

an early and fast screening of breast milk. Indeed, a treatment (freezing or pasteurization) of contaminated

breast milk only could eliminate the virus. The idea of this position paper is that breast milk screening

would help defining an appropriate and personalized feeding strategy. We explain how to develop a CMV

biosensor to detect the virus in milk. It employs specific CMV antibodies grafted on a biochip surface to

capture viral material and additional detection antibodies in a “sandwich assay” type system. Detection is

based on optical absorption. It will be tested with a device developed previously. However, preliminary

results obtained in ELISA technique with breast milk and homemade antibodies are presented in this

position paper. The ulterior motive of this work is the fabrication of an autonomous and automated device

that will be experimented in subsequent diagnosis strategy trial.

1 CONTEXT

Cytomegalovirus (CMV), member of the sub-family

of β-herpesvirus, is only present in humans and 40 to

90% of the world population is infected. This virus,

rarely dangerous for immune-competent person, is a

real threat for immune-depressed people, as for

example, organ transplanted or pregnant women.

Following a primo-infection, CMV diffuses in the

whole body and alternates latency and re-activation

periods. CMV is the most frequent etiologic agent of

congenital and postnatal infection of newborns and

can have a significant impact on the neurosensory

development of newborns and especially preterm

infants (Hayashi et al. 2011). Postnatal transmission

of CMV can occur during blood transfusions, while

absorbing infected biological liquids like mother

cervical secretions during delivery or during

breastfeeding. CMV excretion in breast milk is the

main source of postnatal infection.

1.1 Postnatal CMV Infection via Breast

Milk

Breastfeeding is now clearly recognized as being

superior to artificial feeding for the future of

newborns and, more particularly of preterm infants.

Indeed, preterm newborns are more vulnerable to

digestive and neurological problems. Breast milk is

better accepted and limits the risk of feared

complications like necrotising enterocolitis. Its

incomparable time-varying composition offers the

best chances of cognitive evolution on the long term.

However, breastfeeding plays a major role in the

epidemiology of transmission and postnatal CMV

infection. It is now well established that CMV is

excreted in milk from seropositive lactating mothers,

the majority of whom are asymptomatic, especially

due to a reactivation of the virus. Excretion can start

since the first post-partum week with a low viral

115

Py S., Wacogne B., Pazart L., Coaquette A., Boireau W., Herbein G. and Thiriez G..

VIRUMILK - Biosensor for CMV Detection in Breast Milk from Lactating Women of Preterm Infants Less than 33 Weeks .

DOI: 10.5220/0005251801150120

In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2015), pages 115-120

ISBN: 978-989-758-071-0

 2015 SCITEPRESS (Science and Technology Publications, Lda.)

charge and reaches a maximum value 4 to 8 weeks

after birth and declines steadily thereafter. Mother-

to-child transmission generally occurs during the

period where the virus level (DNA or viral particles)

in milk is about its maximum (Hamprecht et al.

2003; Hamprecht et al. 2008).

A review paper (Kurath et al. 2010) related to

CMV transmission by breastfeeding in preterm

infants shows that 87% (median value) of mothers

whose CMV serology is positive for IgG (CMV

mothers) excrete CMV in their milk. Among the

children, about 20% (median value) are CMV

positive by PCR or ELISA technique for IgM and

IgG (CMV

newborns) and the contamination risk

increases with lactating duration (figure 1). Usually,

for term babies, symptomatic infection does not

exist because mothers start to transmit their

antibodies during the 29th pregnancy week.

Conversely, for preterm infants, the weak

transmission of mother antibodies and the non-

mature immune system increases the risk of

symptomatic CMV infection. A small birth weight

(<1.5 kg) and an early postnatal transmission

constitute risk factors of symptomatic infection

(Lombardi et al. 2012). Recent studies showed that

postnatal CMV infection in preterm infants can lead

to serious clinical consequences like respiratory

distress, neutropenia, thrombocytopenia,

hepatomegaly and septic syndromes, and can lead to

death in rare cases (Lanzieri et al. 2013; Hamele et

al. 2010; Hamprecht et al. 2008). According to

Kurath et al., 3.7% (median value) of the positive

children develop CMV related symptomatic clinical

complication with, for 0.7% (median value) of them,

appearance of a severe septic syndrome (Kurath et

al. 2010). Although the long-term follow-up of the

neurosensory development of congenitally infected

preterm infants is well documented, very few studies

concern postnatal infected preterm infants and

results obtained are generally controversial, in

particular because of the reduce number of infants in

the cohorts

(Kurath et al. 2010; Bevot et al. 2012;

Goelz et al. 2013).

Up to now, there is no consensus between

learned societies of pediatrics concerning the

attitude to be adopted and actions to be started to

prevent CMV infection via breast milk in preterm

infants less than 33 weeks.

Today, almost no national recommendations on

the manipulation of the breast milk of CMV positive

mothers are proposed. However, methods exist to

treat breast milk and consist in heating or freezing

the milk (Forsgren 2004; Hamprecht et al. 2004).

Systematic pasteurization of human milk is not done

because it alters the immune components of milk

which are particularly precious for very preterm

infants (Chang et al. 2013). Freezing at -20 °C does

not completely destroy the virus, but better preserves

biological properties of human milk (Buxmann et al.

2009).

Figure 1: Simplified epidemiology of CMV infection in

breastfed preterm newborns (Kurath, 2010).

For these reasons, neonatologists are squeezed in

their clinical practice between the potential risk to

transmit infection when breast milk is picked up to

be given to the infant, and the risk to favor digestive

complications or not to give the better chances of

neurologic development if the breast milk is not

used. In order to address this issue, the ideal solution

would be to differentiate “at risk” and “non at risk”

situations. In fact, treating the milk of only the “at

risk” population of CMV contamination via the

breast milk would be extremely more satisfactory

than a systematic attitude.

1.2 CMV Screening Tools in Breast Milk

Most of the time in France, the, presence of CMV in

lactating mother’s milk is not screened and milk

does not normally undergo specific pre-treatments in

a breast milk bank. However, an early detection of

CMV in breast milk is feasible. Studies concerning

CMV transmission via breast milk are based on

detecting the viral DNA and/or the infectious virus.

Qualitative or quantitative techniques used to detect

viral DNA are PCR, RT-PCR or nested PCR

(Hamprecht et al. 2008). These techniques are

expensive, time consuming and require the milk to

be previously prepared in 2 or 3 fractions (lipidic,

whey and cellular fractions) using various

centrifugations. Conventional cell culture gives a

result only several days after sampling and often

fails because of the native mother milk cytotoxicity.

This greatly reduces the detection sensitivity of the

infectious virus and further requires fractionation of

mother milk. Therefore, PCR and cell culture are not

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adapted to rapid and early CMV detection in breast

milk.

In this position paper, we show early results

concerning CMV detection in breast milk and we

propose the development of a CMV screening

biosensor. The idea presented in this position paper

is to prevent a postnatal CMV contamination for a

majority of preterm newborns by using an adapted

milk treatment. To do this, it is necessary to detect

CMV in this liquid with a simple, fast and low-cost

system.

VIRUMILK project clearly consists of bridging a

clinical problem to an innovative technological

solution that opens up perspectives of medical

advances. It relies on previous biotechnological

research studies realized during the MEDICALIP

project (Mangeat et al. 2011) whose main scientific

achievements are:

• Homemade production of anti-CMV antibodies,

called PAbH (polyclonal human antibodies) able to

trap viral material at the surface of a biosensor.

• Grafting of these antibodies on the gold surface

of the biosensor while keeping their CMV capture

properties. Indeed, the use of commercially available

antibodies was disappointing because they lost

(partially or completely) their capture properties

when grafted onto the biochip. Grafting methods as

well as chemical environment can, in some cases,

reduce their capture efficiency. Success in producing

homemade and efficient antibodies in the virology

unit of Besançon University Hospital allowed

continuing the project.

• Set-up of methods and opto-fluidic devices for

reactive and sample flow control as well as the

optical detection of the test results.

2 THE BIOSENSOR

2.1 General Concept

The technique we proposed to detect CMV is based

on antigen/antibody recognition. The biosensor

consists of a gold coated biochip grafted with human

polyclonal anti-CMV antibodies. CMV potentially

present in the breast milk sample is captured by

these antibodies. CMV detection uses a specific

secondary antibody coupled to horseradish

peroxidase (HRP) enzyme which subsequently

recognizes the captured virus. After addition of a

substrate of this enzyme, a colorimetric reaction

occurs and allows transforming the substrate to a

blue product. When reaction is stopped, the blue

product turns into yellow. Then, the optical reading

relies on an absorbance measurement around 450

nm. A schematic representation of the biosensor is

given in figure 2.

Figure 2: The CMV biosensor.

2.2 Immunosensor Engineering

Design and production of homemade chips

compatible with Surface Plasmon Resonance

imaging (SPRi) have been performed as previously

described with the help of the MIMENTO

technological platform, Besançon, France (Remy-

Martin et al. 2012). Gold coated biochips are made

using magnetron sputtering. Gold present the

advantage of enhancing the sensitivity, reducing the

detection time and improving the specificity of the

interaction under study.

A strategy of functionalization and grafting of

homemade polyclonal anti-CMV antibodies was

optimized to guarantee an optimum anti-CMV

antibodies surface density and also to reduce non-

specific interactions onto the biochip surface.

This

strategy is based on thiol chemistry and is well

managed by the CLIPP platform (Clinical and

Innovation Proteomic Platform), Besançon, France

(Bruchard et al. 2013).

Chips are incubated in a

solution of 11-mercapto-1-undecanol/16-

mercaptohexadecanoic acid (97/3 by mole) (Sigma-

Aldrich) overnight at room temperature (RT).

Surfaces were rinsed by ethanol and ultra-pure

water. Then, 200 µl of 1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide (EDC) at 200

mM/N-hydroxysulfosuccinimide (sulfo-NHS) at 50

mM (Amine Coupling Kit from Biacore AB,

Uppsala, Sweden) are added on each surface and

incubated during 30 min at RT. This step is

necessary to activate C11/C16 layer. Surfaces were

rinsed by ultra-pure water and different batches of

PAbH directed against viral proteins from various

strains (AD169 and 3 clinical strains) were spotted

(0.27 µl/spot) on the chips during 30 min at RT

under sonication in a humid chamber. To ensure

optimal grafting of the PAbH, antibodies are diluted

in an acetate buffer at 100 µg/mL, pH 5. Then

surfaces were rinsed by ultra-pure water and a

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blocking agent (Rat Serum Albumine 40 µg/mL, pH

5.2) was used to passivate the surface by incubation

at RT for 30 min. Surfaces are finally rinsed with

water and C11/C16 layer is deactivated using 200 µl

Ethanolamine-HCl (1 M pH 8.5) during 30 min à

RT. After a last rinsing by water, biochips were

ready for use in SPRi experiments.

2.3 CMV Capture and Detection

Control of the grafting of homemade antibodies onto

the chip surface and CMV capture were performed

in a SPRi-PlexII imager (Horiba Scientific, France)

equipped with an 810 nm wavelength LED and a

CCD camera. Experiments were carried out at 25°C,

in phosphate buffered saline (PBS) 1X. The flow

rate in the chamber was 20 µl/min. CMV was

commercial antigen from AD169 strain (ETI-

CYTOK-M reverse plus, Diasorin). After injection

(volume of 200 µl) the biochip surface was rinsed

for 1 min with detergent (n-Octyl-beta-D-

glucopyranoside, 40 mM)

to remove unbound

ligands.

Figure 3: Sensorgram and contrast plasmon image of spots

obtained with the SPRi-PlexII after injection of

commercial CMV on grafted PAbH (4 spots and curves

correspond to different batches of PAbH).

As seen in figure 3, a signal is observed on the

four spots grafted with different batches of PAbH

whereas no significant signal is observed on the non-

grafted spots. The interaction is therefore specific

and shows the absence of undesired non-specific

binding and/or adsorption on the surface. These

results are consistent with those obtained by ELISA

(Enzyme-Like Immunosorbent Assays) experiments

using the same batches of PAbH and the same viral

material (data not shown).

In order to test whether or not similar results can

be obtained with CMV positive breast milk samples,

a direct sandwich ELISA experiment was

performed. One CMV positive breast milk sample

(volume of 5 mL) was centrifuged at 400 g during

10 min at RT in order to separate milk into 3

fractions: the cell fraction, whey and the lipidic

portion which was discarded. The CMV positivity

was assessed by PCR analysis and cell culture. In

the meantime, remaining volumes (slightly less than

1 ml) of cell and whey fractions were stored at -

80°C. When positivity is confirmed, an ELISA

experiment was conducted as follows. Fetal Calf

Serum (FCS) at a concentration of 1 µg/µL or two

batches of PAbH (produce from two different

clinical strains) at a concentration of 3.75 µg/mL,

7.5 µg/mL or 15 µg/mL in 100 µL of

carbonate/bicarbonate buffer were coated overnight

at 4°C in 96 wells microplates. The day after, a

rinsing of wells with PBS 1X followed by a

saturation step of the surface with Bovine Serum

Albumin (BSA) 3% (200 µl/well) during 1 h at RT

was performed. A mixture containing commercial

antigen or the cell fraction (diluted at 2.5%, 7%,

22%, 50% or 66% in PBS 1X) or whey (diluted at

2%, 7%, 20%, 50% or 61% in PBS 1X) and an anti-

CMV antibody conjugated to HRP diluted at 1/70

(ETI-CYTOK-M reverse plus, Diasorin) was added

(100 µL/well) and incubated during 1 h at RT. Five

washing (200 µL/well) were realised with a wash

solution composed of PBS-Tween (ETI-CYTOK-M

reverse plus, Diasorin) and the HRP substrate

(hydrogen peroxide and tetramethylbenzidine) was

incubated (100 µL/well) during 30 min at obscurity

and at RT. The reaction was stopped with 0.2 N

sulphuric acid. Absorbance of the yellow solution

obtained was immediately measured around 450/630

nm. Results are presented in the histograms in figure

Figure 4: Absorbance values obtained by ELISA

experiment using two batches of PAbH and different

CMV sources (commercial antigen, breast milk cell

fraction and milk whey).

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A positive control is represented by the incubation

with commercial antigen in which high absorbance

values are obtained. Decay is observed with the

increasing dilution of capture antibodies.

Concerning the cell fraction and whey, values are

lower overall and decay is observed with the

increasing dilution of capture antibodies but also

with the increasing dilution of milk fraction. As

expected, incubation of CMV with FCS shows a

very low signal.

SPRi and ELISA results show the capacity to

capture CMV with homemade antibodies on

different surfaces. Moreover, viral particles

contained in a breast milk sample can also be

captured and detected in ELISA experiment.

3 A POSSIBLE DEVICE

In the frame of another research program called

SmarTTransfuser (Charrière et al. 2012), a

laboratory prototype had been fabricated (figure 5).

It consists of a fluidic system containing a biochip

inserted into a cartridge. Syringes contain reagents

which are driven on biochips surfaces by fluidics

arrangements. The system allows controlling the

fluid flows and interaction durations.

Immunocapture and immunodetection of CMV are

performed on a biochip. In parallel, a second biochip

is used as negative control.

Preliminary results were obtained with an

experimental protocol approaching as much as

possible the conditions used in ELISA experiments

(concentrations and incubation times of reagents are

the same). Homemade functionalised biochips were

designed and produced as described above and

PAbH were grafted following the same protocol

used for SPRi experiments. BSA was first

introduced manually to the biochip followed by the

mixture of commercial antigen and detection

antibody conjugated to HRP at a flow rate of 100

µL/min during 2 min. A washing was realised by

500 µL of wash solution at a flow rate of 250

µL/min. Finally, substrate was added at a flow rate

of 100 µL/min and incubated at obscurity. The

excess of solution contained at the outlet of the

cartridge was discarded and 100 µL of blue product

was recovered in a tube and directly turned in yellow

by addition of the stop solution. Negative control

consists of a biochip incubated with all the reagents

except the mixture antigen/antibody which is replace

by only antibody diluted at 1/70 in PBS 1X. Optical

reading is performed with a spectrophotometer by

measurement about 450/630 nm.

Figure 5: General concept of the CMV detection in an

already available device (SmartTransfuser project). A

fluidic system ensures the flow of different reagents (BSA,

antigen, antibody detection coupled to HRP, wash solution

and HRP substrate) on the biochip of interest.

Absorbance values obtained in first experiments

were lower in comparison with ELISA results but a

difference in positive and negative biochips was

indeed present (factor of 5). Additional experiments

are necessary to really prove the possibility to detect

CMV in such a laboratory model and particularly

with breast milk samples.

However, this preliminary

result shows that laboratory SPR and SPRi technique

can be transposed to a more bulk device and that

fluid flow condition in this fluidic cartridge allows

specific detection of CMV.

When finalized, the biosensor validated with

breast milk will be integrated into a built-in system

which will be of simple use (presence or not of the

virus indicated with red/green LEDs). It will allow

controlling fluid flows by means of an automated

management of micro-fluidic and also timing of

different biochemical interactions in a similar way to

what we already presented before (Mangeat et al.

2011). It will include the optical reading system of

the test result

which is based on an absorbance

measurement. Finally, it will include a

human/machine

interface to use the device by a non-

expert user.

The finalized biodevice will then allow

rapid and simplified CMV detection in breast milk

of lactating mothers of preterm infants. Contact with

a company was established to develop the device,

but for reasons of intellectual property it is still too

early to assess the cost of producing the proposed

biosensor in a larger scale.

4 CONCLUSIONS

Although the risk of CMV congenital infection is

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relatively low (prevalence of about 1%), the risk of

postnatal contamination, in particular via breast

milk, can be dramatic for preterm infants. Currently,

the question is: should we favour a better

development and take the risk of using contaminated

breast milk, or should we use treated milk, even

when the CMV infection is low enough to be

considered safe?

To address this problem, and in the current

context of breastfeeding promotion, we propose to

develop a CMV biosensor based on sandwich

ELISA principle. First SPRi, ELISA and assays in a

laboratory model lead us to assume that a biochip

CMV capture and detection is possible. However,

more tests, especially with positive and negative

breast milk samples, are required to validate our

biosensor.

This position paper presents studies that have

just started, but we think it is possible to set-up an

easy to use and rapid "point-of-care" device to detect

CMV in milk of lactating mothers of preterm

infants. Therefore, a third answer can be proposed to

the above mentioned question. The idea is to screen

CMV on a routine basis and to define a personalized

feeding strategy for “at risk” population only.

Without such a rapid CMV test, this third solution

may never exist.

ACKNOWLEDGEMENTS

The authors would like to thank the French Agence

Nationale de la Recherche, INSERM and the DGOS.

This work is developed in the frame of the Biom'@x

transversal axis at FEMTO-ST.

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