Journal of Clinical Virology 44 (2009) 215–218

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Journal of Clinical Virology

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Utility of newborn screening cards for detecting CMV infectionin cases of stillbirth

Jonathan Howarda,b, Beverley Hall a, Lyndall Eve Brennana, Susan Arbucklec,Maria E. Craiga,d, Nicole Grafc, William Rawlinsona,b,∗

a Virology Division, Department of Microbiology, SEALS, Prince of Wales Hospital, Randwick, 2031 Australiab School of Medical Sciences and School of Biotechnology and Biomolecular Sciences, University of New South Wales,Kensington, 2052 Australiac Department of Histopathology, The Children’s Hospital Westmead, Westmead, 2145 Australiad School of Women’s and Children’s Health, University of New South Wales, Kensington, 2052 Australia

a r t i c l e i n f o

Article history:Received 4 November 2008Received in revised form 17 December 2008Accepted 18 December 2008

Keywords:StillbirthCytomegalovirusNewborn screening cardsPCRCongenital infection

a b s t r a c t

Background: CMV infection may cause intrauterine deaths including stillbirths (intrauterine deaths at ≥20weeks gestation). In 2005, there were 1979 stillbirths in Australia, which is almost double the number ofdeaths reported for all children between 1 and 14 years age.Objectives: We evaluated the diagnostic utility of testing for the presence of CMV in newborn bloodscreening cards (NBSC) collected from stillborn babies, who had no known cause of death after post-mortem.Study design: Blood taken at post-mortem by cardiac puncture of 107 stillborn babies between July 2005and December 2006, was spotted onto NBSC. CMV infection was detected using nested PCR targeting theglycoprotein gene, gp58.Results: Of the 107 stillborn infants, 10 (9%) were CMV positive. The rate of CMV infection did not differbetween early stillbirths (8%) and late stillbirths (9%).

Conclusions: The use of NBSC is a convenient and accurate method for CMV detection in stillbirths. It iseasily collected, less laborious than viral culture, diagnostically useful and could be applied for epidemi-

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ological and retrospective

. Introduction

Despite improved obstetric and neonatal care, stillbirths com-rise the largest number of deaths between birth and 14 years ofge1, when all causes are examined. Stillbirths account for two-hirds of all perinatal deaths in Australia, and there has been littlehange in the frequency of stillbirths over the last 20 years.2 Knownauses of stillbirth such as congenital abnormalities, asphyxia,lacental abruption and umbilical cord accidents3 account for

pproximately half of all cases. Trauma, maternal obesity, low edu-ation, smoking, intrauterine growth restriction (IUGR), increasedaternal age, Rh disease and diabetes mellitus4 are associated with

tillbirth,3 although no prospective Australian or overseas study

Abbreviations: NBSC, newborn screening cards; CMV, cytomegalovirus; NAT,ucleic acid testing.∗ Corresponding author at: Virology Division, Department of Microbiology, South

astern Area Laboratory Services, Prince of Wales Hospital,andwick, 2031, Australia.

E-mail address: w.rawlinson@unsw.edu.au (W. Rawlinson).

386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.jcv.2008.12.013

stigation of the virus in the stillbirth population.© 2009 Elsevier B.V. All rights reserved.

has conclusively defined maternal characteristics as independentaetiological agents of stillbirth.

Several studies have shown that infectious agents includingcytomegalovirus (CMV) are able to cause stillbirth.5 Congeni-tal CMV infection, with a prevalence of 0.15–2%, is the leadingviral cause of congenital malformations in the developed worldincluding Australia.6,7 The role of CMV in stillbirth in Australiahas not been defined. There has been a wide range of studieson congenital CMV from a population,6 clinical,8 diagnostic,6,9,10

molecular,11,12 cellular,13 and pathogenetic14–18 viewpoint. Clearly,diagnostic studies are needed to determine the incidence of CMVinfection in the etiology of stillbirth.19

Determining rates of CMV infection in stillbirths requires aneasily obtainable clinical sample, high sensitivity, high specificityand preferably rapid detection method. During post-mortem exam-ination, organ tissues collected are usually formalin-fixed and

embedded in paraffin, making it difficult to extract good qualityDNA for nucleic acid testing (NAT). Routine methods of histologi-cal fixation and processing either destroy or damage RNA and DNA,and can cause extensive tissue cross-linking that renders the tissueunsuitable for further analysis.20

216 J. Howard et al. / Journal of Clinical

Table 1Prevalence of CMV DNA in the newborn screening cards tested.

Weeks gestational age Total no. CMV positive CMV negative

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3. Results

TC

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0–29 (early stillbirth) 36 3 (8%) 33 (92%)30 (late stillbirth) 71 7 (9%) 64 (90%)

Newborn blood screening cards (NBSC), also known as Guthrie’sards or dried blood spots are usually used to collect blood fromeonates with a heel prick. They have recently been used in

arge scale screening of livebirths for infectious agents includingMV,21–24 although, such specimens are not routinely collected

rom stillborn infants at post-mortem, where they could be usedo detect viral DNA and RNA.

The present study aimed to utilise NBSC collected during post-ortem examination of a retrospective case series of stillborn

nfants in New South Wales, Australia, to determine the prevalencef CMV in stillborn infants, and evaluate the utility of blood obtainedrom cardiac puncture at post-mortem of stillborn infants for CMVcreening.

. Materials and methods

.1. Newborn blood screening cards (NBSC)

NBSC were collected from 107 stillborn infants born in Newouth Wales, Australia between 2005 and 2006. Post-mortem waserformed in the Department of Histopathology, The Children’sospital at Westmead. Cardiac blood obtained during post-mortemas spotted onto NBSC and allowed to dry overnight. The sam-les were stored at room temperature prior to testing, in separatenvelopes. The NBSC were examined as part of the diagnosticorkup of stillbirths of unknown etiology undergoing post-mortem

xamination.

.2. CMV DNA extraction from NBSC

CMV DNA was extracted as previously reported21 with minorodifications. Three disks of 3 mm in diameter were prepared fromBSC using a Wallac 1296-071 DBS Puncher (PerkinElmer, Fin-

and). Carryover DNA contamination21,23 was excluded by punching0 disks from a clean, blank NBSC after punching each sam-le card. Three blank disks (as negative controls) were processeds test samples to detect any carryover DNA between cards.lood was eluted from the NBSC by incubating in 45 �l minimal

ssential media at 55 ◦C for 60 min prior to boiling at 100 ◦C formin. Samples were rapidly cooled, centrifuged at 10,000 × g formin and frozen at −80 ◦C for at least 1 h before further test-

ng.

able 2haracteristics of the 10 stillborn infants with CMV infection.

ex Weeks gestation Weight (g) Cause of death

ale 21 349 Not knownale 24 677 Not known

emale 26 1665 Not knownale 32 1862 Not knownale 33 1965 Not knownale 33 1752 Not knownale 37 2905 Not known but possible infection

emale 39 2815 Infectionemale 41 4625 Not known but possible infectionemale 41 1840 Not known

Virology 44 (2009) 215–218

2.3. Control preparation

To assess the sensitivity of the PCR reaction, plasmid constructof the target gene (gp58) was used as reaction control and tomeasure the limit of detection. First-round amplification product(150 base pair) was cloned using pGEM-T Easy Vector System II(Promega) and the construct extracted using the Wizard PCR PrepsDNA purification system (Promega). The sequences were verifiedusing the ABI PRISM Big Dye kit (PerkinElmer) and elucidatedusing NCBI BLAST. The concentration of the plasmid construct was1.01 × 1010 copies/�l. This was serially diluted (10 folds) to yielda final concentration of 1 copy/5 �l with CMV-negative blood and50 �l was spotted into the NBSC and dried overnight at roomtemperature. The cards were subjected to PCR amplification (asdescribe below) to determine the detection limit. Detection limitwas defined as the lowest dilution detected of a series of seriallydiluted (1:10) plasmid construct of the target sequence.

2.4. CMV PCR

A 5 �l sample of the extract was subjected to nested CMV DNAPCR amplification of gp58 target gene, using a published ampli-fication protocol with minor modifications.21 The first round PCRmixture contained 5× Taq polymerase buffer (Promega, Australia),1.5 mM MgCl2 (Promega, Australia), 0.2 mM of each deoxynucleo-side triphosphates dNTPs, (Promega, Australia), 0.25 �M of senseouter primer (gB1: 5′-GAGGACAACGAAATCCTGTTGGGCA-3′) andanti-sense outer primer (gB2: 5′-GTCGACGGTAGATACTGCTGAGG-3′), 1.5 U Taq polymerase (Promega, Australia) and was made to50 �l with sterile H2O. The first round of amplification involved aninitial denaturation step at 94 ◦C for 2 min followed by 35 cycles of94 ◦C for 30 s, 55 ◦C for 30 s and 72 ◦C for 30 s with a final extension at72 ◦C for 5 min and a 4 ◦C hold cycle. The second round of amplifica-tion included an initial denaturation at 94 ◦C for 2 min followed by30 cycles of 94 ◦C for 30 s, 53 ◦C for 30 s and 72 ◦C for 30 s with 5 minfinal extension at 72 ◦C and a 4 ◦C hold cycle on a MyCycler ThermalCycler (Bio-Rad, Australia). The second round PCR reaction mix-ture was identical to the first round mixture except for substitutionof 0.5 �M of sense inner (gB3: 5′-ACCACCGCACTGAGGAATGTCAG-3′) and anti-sense inner (gB4: 5′-TCAATCATGCGTTTGAAGAGGTA-3′)primers. Products were subjected to electrophoresis on 2% agarosegel containing ethidium bromide (0.5 �g/ml). CMV positive DNAand negative water controls were also included with every reac-tion. Positive results were confirmed by repeat punching of NBSC,extraction of DNA and PCR.

Results of CMV PCR of NBSC from a retrospective case series ofstillborn infants are shown in Table 1. Of the 107 stillbirths, CMVDNA was detected in 10 stillborn infants (9%). There were 36 still-

Organism detected by culture

Blood Tissue taken at post-mortem

Pseudomonas fluorescens NoneGroup B haemolytic Streptococcus NoneNone NoneNone NoneNone CMV inclusionsNone NoneNone Enterococcus on tissueEscherichia coli NoneNone Coagulase negative StaphylococcusNone Group B haemolytic Streptococcus

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J. Howard et al. / Journal of C

irths defined as early (between 20 and 29 weeks gestational age)nd 71 were late (≥30 weeks) and no differences in the rate of CMVnfection were observed among early (8%) vs late (9%) stillbirth.

The sensitivity of the amplification protocol was determined byerially diluting (1:10-fold) CMV gp58 plasmid construct with CMVegative blood from 108 to 1 copy/5 �l. Our limit of detection was0 copies in a 50 �l reaction.

The characteristics of the 10 stillborn infants positive for CMVre summarised in Table 2. The cause of death was unknownn eight cases and possibly due to bacterial sepsis in two cases.MV was not detected on routine post-mortem examination except

n one infant, where CMV inclusions were detected in the lungsnd placenta. The actual cause of death for this infant was notevealed by the pathologist, however, our PCR result confirmedhe CMV immunoperoxidase staining result obtained at post-

ortem. Other pathogens detected in fresh post-mortem tissuesncluded Pseudomonas fluorescens, Escherichia coli, coagulase-egative Staphylococcus, Enterococcus and Group B haemolytictreptococcus.

. Discussion

Several studies have documented the incidence of congeni-al CMV infection in Australia. The national seroprevalence ofMV in Australia for 1–59 years old in 2002 is 57%.7 Between993 and 2001, the average annual rate of CMV-related hospi-al admissions was 14.2 cases per 100,000.7 One study, based onotification data from Australian paediatricians as part of a nation-ide surveillance program, revealed that an average of 18 definite

ases of congenital CMV occurred yearly between 1999 and 2002,lthough this was considered a significant underestimation.25

n Australian study of CMV IgG prevalence and seroconver-ion in pregnant women showed 57% were CMV IgG positive atregnancy with 6% being also IgM positive.6 In this study, weid not have any details about the CMV status of the mothersor all the cases as these were retrospectively tested and de-dentified.

This study showed that the prevalence of congenital CMV infec-ion is 9% in stillborn infants undergoing autopsy. This value is highonsidering that the prevalence of active CMV infection in Australiand New Zealand is 0.03%.26 The high prevalence of CMV detectedn this current study is probably due to the biased in selecting still-orn infants as our study population. Nonetheless, our result wasxpected as CMV is the most common congenital viral infection27,28

nd has been associated with stillbirths in case reports.29 It is alsoossible that using sensitive methodologies (PCR and blood onBSC), we were able to detect more CMV infections. The extractionethod was simple, rapid, and NBSC has potential use in large epi-

emiological studies. Our detection limit of 10 copies per reactions approximately near the value previously obtained in a multiplexetting9 and when nested PCR was used to detect herpesvirusesn ocular and cerebrospinal fluid specimen,30,31 although otherseported 400 copies/ml as the lower limit of detection thresholdor their assay.22,32

There are several issues regarding the pathogenesis of CMV thatust be considered in assessing stillbirth using currently availableethods. Technical difficulties may prevent successful amplifica-

ion of DNA from paraffin-embedded tissues, making detectionf viral DNA in stillbirths a challenge.33 Detection of infectiousathogens in stillborn infants ideally requires an easily obtainablepecimen amenable to NAT, preferably not fixed in formalin, taken

oon after birth. In this study, testing of NBSC from a large propor-ion of stillbirths undergoing autopsy enabled us to investigate theossible role of CMV in stillbirth. This virus is an ideal candidateor an infectious cause of stillbirth, because it is relatively common,as a high rate of transplacental transmission and has been associ-

Virology 44 (2009) 215–218 217

ated with placental and fetal damage that is known to result in fetalmalformation and death.19,34

NBSC are a useful resource as they require minimal storageconditions, simple to collect, appear not prone to contaminationand can be retrospectively screened due to long term ana-lyte stability.35,36 NBSC were first used in neonatal testing forphenylketonuria, and are now widely used to test neonates formultiple metabolic disorders and genetic mutations37,38 and mark-ers for type 1 diabetes.39 The use of NBSC has been extended todetect viruses such as hepatitis C and HIV,40–48 congenital CMVinfections49–51 and in epidemiological investigations for CMV incountries including Argentina52 Italy49 and Japan.23

In investigating unexplained stillbirth for CMV, gestational agemay be of importance since the impact of CMV infection is usu-ally more severe early in pregnancy.28 Studies have demonstratedthe presence of CMV DNA in a variety of uterine and placental cellsespecially in the first and second trimester in pregnant women,indicating that vertical transmission of the virus occurs.17 It isknown that CMV is transmitted across the placenta, often withovert fetal damage.53 However, there is insufficient evidence forthe mechanism of fetal demise and such data are urgently needed.We did not find any association between gestational age and rateof CMV infection. Although CMV was detected in these stillbornbabies, it does not necessary meant that the virus caused the deathof these babies. In addition, several pathogens (including P. fluo-rescens, E. coli and group B Streptococcus) were also detected in theblood and tissues taken during post-mortem. It is possible that thepresence of these pathogens and CMV might contribute to the deathof the fetus. Nonetheless, NAT for CMV could be used as an adjunct inproviding a more accurate diagnostic result to determine the causeof the stillbirth.

The utility of NBSC PCR in screening newborns for congenitalCMV infection still requires further investigation. The determina-tion of DNA in blood by PCR at birth seems to be as sensitive andspecific as recovery from urine for diagnosis of congenital CMVinfection.54–56 However, in some cases, babies with congenital CMVinfection have undetectable CMV DNA in their blood.57 Anotherimportant consideration is albeit CMV was detected in these still-born babies, it might not be the causative agent for the death ofthe babies, however, CMV might be a contributing factor. There-fore, the value of a positive blood screening card for CMV remainsto be determined.

This study showed the successful extraction of DNA from NBSC,and the effectiveness of NBSC as a source material in screening forthe presence of CMV in stillbirth. This method could be appliedin investigating archival materials to offer counselling for fami-lies. Ultimately, this would provide useful information about theaetiology and pathogenesis of unexplained causes of stillbirth.

Conflict of interest

Authors declare no conflict of interest.

Acknowledgement

This work was supported by a charitable grant from SIDS andKids.

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