Journal of Clinical Virology 42 (2008) 207–210

Case report

Detection of enteroviral RNA on Guthrie card dried blood of

a neonate with fatal Coxsackie B3 myocarditis on day 17
Koenraad Smets a,∗ , Annelies Keymeulen b , Elke Wollants c , Katrien Lagrou d ,
Marc Van Ranst c , Elizaveta Padalko e
aDepartment of Neonatology, Ghent University Hospital, De Pintelaan 185, B-9000 Gent, Belgium
b
Department of Pediatrics, Ghent University Hospital, De Pintelaan 185, B-9000 Gent, Belgium
c Department of Clinical and Epidemiological Virology, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium
d Department of Experimental Laboratory Medicine, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium
e Department of Virology, Ghent University Hospital, De Pintelaan 185, B-9000 Gent, Belgium

Received 20 September 2007; received in revised form 19 December 2007; accepted 7 January 2008

Abstract
A fatal case of Coxsackie B3 myocarditis in a neonate is described. The clinical features became evident in the 3rd week of life,
but enteroviral RNA was detected in the dried blood spot of the baby collected on day 4 after birth. This is the first report on the
detection of enteroviral RNA in Guthrie card dried blood using reverse-transcriptase PCR. Materials and methods used are described in
detail.
© 2008 Elsevier B.V. All rights reserved.

Keywords: Neonatal; Coxsackie virus B3; Myocarditis; PCR; Dried blood spot

1. Introduction disease (Bendig et al., 2003; Verboon-Maciolek et al., 2003).

Enterovirus infection is a common cause of acute viral
illness worldwide. In the perinatal period enterovirus
infections may vary in clinical presentation from mild
non-specific symptoms to severe multisystem disease,
including meningoencephalitis, myocarditis, hepatitis and
serious generalized sepsis (Abzug, 2001; Bauer et al., 2002;
Bendig et al., 2003; Bryant et al., 2004; Sawyer, 1999).
Perinatal enterovirus infection can be associated with high
morbidity and mortality. Peak incidence is during summer
and autumn. Coxsackie virus B3 (CVB3) has been identified
as a major causative agent of acute and chronic myocarditis
(Jeonghyun et al., 2005).
Most frequently neonates are infected directly from
mother to child at delivery. This may result in more serious
Abbreviations: CVB3, Coxsackie virus B3; RT-PCR,
reverse-transcriptase-polymerase chain reaction.
∗ Corresponding author. Tel.: +32 9 3323535; fax: +32 9 3326105.
E-mail address: koenraad.smets@ugent.be (K. Smets).
The infection can also be acquired postnatally from sources
other than the mother. When neonates are symptomatic at
birth, in utero transmission is suggested (Bendig et al., 2003).
The incubation period is 3–5 days (Bauer et al., 2002).
Diagnosis can be made by serological testing and viral
cultures. However, the presence of passively acquired mater-
nal antibodies, the need for specific assays and the delay in
the diagnosis render these methods not very useful. Reverse-
transcriptase-polymerase chain reaction (RT-PCR) increases
the sensitivity and speed of enterovirus diagnosis, and is
therefore the method of choice (Bendig et al., 2003; Bryant
et al., 2004; Sawyer, 1999).
We describe a neonate with disseminated intravascular
coagulopathy and severe cardiomyopathy on day 17 after
birth with fatal outcome due to CVB3 infection. In an attempt
to determine the time of transmission, RT-PCR on a Guthrie
card dried blood spot, collected on day 4 after birth, was used
to detect enteroviral RNA. To our knowledge this is the first
report on detection of enteroviral RNA in dried blood spots
using the Guthrie card.

1386-6532/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jcv.2008.01.004
208 K. Smets et al. / Journal of Clinical Virology 42 (2008) 207–210
2. Case report
A neonate, born at 39 weeks gestation, was admitted
shortly after delivery to a neonatal department because of per-
sistent peripheral cyanosis and cold hands and feet. After a
few hours in a warm environment the boy recovered quickly.
On day 3 after birth he regained weight. On day 4 he was
discharged from the hospital in good health.
On day 17 after birth the child presented in the emer-
gency room with grunting, poor feeding, hypothermia and
cardiovascular collaps. While being prepared for intubation
he required aggressive resuscitation, and was transferred to
our neonatal intensive care unit requiring assisted ventilation
and extensive haemodynamic support.
On admission the peripheral white blood cell count was
normal, but within 6 h had increased with a shift to the
left. Intravenous antibiotics were given. Arterial blood gases
showed mixed metabolic and respiratory acidosis. Lactate
serum level was 273 mg/dl. Over the next 6 h there was a
significant raise in levels of liver enzymes; clotting time was
prolonged, but there was no thrombocytopenia.
On echocardiography a severe cardiomyopathy was found
with poor heart function and areas of very thin myocardium,
suggesting ischaemia or myocarditis. Despite aggressive
medical treatment, the boy died within 6 h after admission.
Bacterial blood cultures remained negative.
Autopsy showed pericardial effusion and ascites, both
suggestive for infection. Bacterial cultures of these samples
were negative. Kidneys and brain were normal at autopsy.
Microscopy of the myocardial tissue showed dispersed
foci of disturbed architecture of muscle fibers: fibers were
thinned and inflammatory cells were clearly present. These
findings were interpreted by the pathologist as compatible
with myocarditis.
Neutralization antibody titers to CVB3 in serum were 1/16
for IgM and negative for IgG. The mother and other family
members had not been symptomatic in the several weeks
before and after delivery. However, in the second week of his
life the baby had been in close contact with a family friend
who shortly thereafter developed Guillain-Barré syndrome.
In order to confirm the type and determine the timing of
infection, paraffin sections of myocardium and frozen myoca-
rdial tissue, as well as Guthrie card dried blood from the 4th
day of life, were analyzed for the presence of enteroviral
RNA. Recovered blood tubes obtained before the baby died
(sodium fluoride (NaF) and citrate tubes) were also sent for
PCR. Unfortunately, viral cultures were not performed pre-
or post-mortem.
3. Materials and methods
3.1. Real-time RT-PCR for the detection of enterovirus
RNA
RNA was extracted from NaF-containing and citrate-
containing plasma by using the QIAamp® Viral RNA Kit
(Qiagen Benelux B.V., The Netherlands). Both samples were
obtained while the patient was alive in the neonatal inten-
sive care unit. RNA extraction from paraffin sections of
myocardium and frozen myocardial tissue was performed
using Rneasy Mini Kit (Qiagen Benelux B.V., The Nether-
lands). RNA from the Guthrie card was extracted using
Rneasy Mini kit after Proteinase K digest (200 ␮l PBS buffer
(Fluka, Buchs, Germany), 20 ␮l Proteinase K (Sigma, Stein-
heim, Germany) and 180 ␮l ATL-buffer (Qiagen, Benelux))
for 3 h at 56 ◦ C. Real-time RT-PCR for the detection of
enterovirus RNA was performed as previously published
(Verstrepen et al., 2001; Verstrepen et al., 2002) with primers
and probe directed to the conserved sequences in the 5 -UTR
of the enterovirus genome. Tests included low and high pos-
itive controls as well as negative controls (including paper
from this specific Guthrie card and paper used for its prepa-
ration). Each sample was tested for inhibition and was tested
in duplicate.
3.2. Diagnostic 5 NCR RT-PCR
To confirm that the extracted RNA contained enterovirus
RNA, a 231 bp gene fragment in the second half of the
5 -noncoding region was amplified with a one-step RT-
PCR (Qiagen OneStep RT-PCR Kit, Qiagen Benelux B.V.,
The Netherlands) using 10 ␮l viral RNA sample and two
5 NCR-specific primers E4KB-F and E1-R (Thoelen et al.,
2003). The RT-PCR was undertaken containing 10 ␮l 5×
Qiagen OneStep RT-PCR buffer, 400 ␮M of each dNTP,
1.5 ␮l enzyme mix (an optimized combination of Omniscript
and Sensiscript reverse transcriptase and a HotStarTaq DNA
Polymerase; Qiagen), 0.6 ␮M of each primer and RNase-
free water to 50 ␮l. The reaction was carried out with an
initial reverse transcription step at 50 ◦ C for 30 min, fol-
lowed by PCR activation at 95 ◦ C for 15 min, 40 cycles
of amplification (94 ◦ C, 30 s; 55 ◦ C, 30 s; 72 ◦ C, 1 min),
and a final extension of 10 min at 72 ◦ C in a GeneAmp
PCR System 9700 thermal cycler (Perkin-Elmer, Foster
City, CA). PCR-products were run on a polyacrylamide gel,
stained with ethidium bromide and visualized under UV-
light.
3.3. Amplification and sequencing of the amino-terminal
part of VP1
A VP1 one-step RT-PCR (Qiagen OneStep RT-PCR Kit)
was carried out on plasma, frozen heart tissue and Guthrie
card dried blood, using 10 ␮ RNA sample, 10 ␮l 5× Qia-
gen OneStep RT-PCR buffer, 400 ␮M of each dNTP, 1.5 ␮l
Qiagen OneStep RT-PCR enzyme mix, 1.2 ␮M of the degen-
erative primers ENTNES-F and ENTNES-R and RNase-free
water to 50 ␮l (Thoelen et al., 2003). The amplification pro-
file involved a reverse transcription step at 50 ◦ C for 30 min,
followed by PCR activation at 95 ◦ C for 15 min, 40 cycles of
amplification (94 ◦ C, 30 s; 55 ◦ C, 30 s; 72 ◦ C, 1 min), and a
final extension of 10 min at 72 ◦ C in a GeneAmp PCR System
 K. Smets et al. / Journal of Clinical Virology 42 (2008) 207–210
9700 thermal cycler (Perkin-Elmer). The 346 bp PCR ampli-
cons were subjected to polyacrylamide gel electrophoresis,
stained with ethidium bromide and visualized under UV-
light. The amplicons were purified using the Invitek MSB
SpinPCRapace (Westburg, The Netherlands), and sequenced
in forward and reverse directions with the respective PCR
primers using the ABI PRISM BigDye Terminator Cycle
Sequencing Reaction Kit (version 3.0) on a ABI PRISM
3100 DNA sequencer (Applied Biosystems), according to
the manufacturer’s instructions.
3.4. Sequence analysis and phylogenetic analysis of the
VP1 amplicons
Chromatogram sequencing files were inspected with
Chromas 2.3 (Technelysium, Helensvale, Australia), and
coatings were prepared using SeqMan II (DNASTAR, Madi-
son, WI). The obtained VP1 consensus sequences of 346 bp
length were compared to the corresponding enterovirus
sequences available in GenBank using BLAST (Basic Local
Alignment Search Tool) in order to identify the enterovirus
type (Altschul et al., 1990). A VP1 nucleotide sequence
identity of more than 75% to a certain reference strain in
GenBank, indicates that the patient sample is of the homolo-
gous type, provided that the second-highest identity score
(next closest genotype) is less than 70% (Oberste et al.,
2000). Multiple sequence alignments were prepared using
CLUSTALX and manually edited in the GeneDoc alignment
editor (Nicholas et al., 1997; Thompson et al., 1997).
4. Results
4.1. Real-time RT-PCR for the detection of enterovirus
RNA
Both paraffin section of myocardium and frozen myocar-
dial tissue contained enteroviral RNA. Enteroviral RNA was
also detected in Guthrie card dried blood and NaF-containing
plasma samples (citrated plasma tested negative).
4.2. Diagnostic 5 NCR RT-PCR and amplification of the
amino-terminal part of the VP1 segment of enteroviral
RNA from plasma, frozen heart tissue, and Guthrie card
dried blood
Fragments of 231 bp were seen on the polyacrylamide
gel of the 5 NCR RT-PCR for NaF-containing and citrated
plasma and frozen myocardial tissue. The degenerative VP1
primers ENTNESS-F/R successfully amplified enterovirus
RNA from these samples. The nucleotide sequence of
the virus was submitted to GenBank (accession number
EF599487). BLAST analyses were carried out and a sequence
similarity of 94% with CVB3 was found. Although the
Guthrie card dried blood showed a positive signal after
amplification with the enteroviral-specific primers, there was
209
insufficient product to further amplify for additional genotyp-
ing.
5. Discussion
Enteroviruses cause a wide spectrum of severe manifes-
tations in neonates, including severe, fulminant, sometimes
fatal disease with myocarditis. Coxsackie B viruses often
infect the heart and in particular CVB3 causes irreversible
damage to the cardiomyocytes and is considered a major
cause of myocarditis, thereby causing irreversible damage
to the cardiomyocytes (Arbustini et al., 2000; Bauer et al.,
2002; Dettmeyer et al., 2002; Jeonghyun et al., 2005; Sawyer,
1999).
The evolution of a severe enteroviral infection with
myocarditis may be biphasic, with a period of one to 7
days of apparent well-being interspersed between the initial
symptoms and the appearance of more serious manifesta-
tions (Abzug, 2004; Chanzy and Msélati, 2005; Rigourd et
al., 2002).
There is a correlation between timing of transmission and
morbidity. It is known that pre- and perinatal transmission
causes more severe disease than postnatal transmission from
the mother or other family members to the baby (Abzug,
2004; Bryant et al., 2004; Rigourd et al., 2002). Presence
of maternal illness in the 2 weeks prior to delivery suggests
that the mother is a likely source of infection. In perinatally
infected children symptoms may appear within the first week
of life and may be more severe. If the symptoms become
evident 14 days after birth, it is more likely that the infection
was transmitted postnatally (Bendig et al., 2003; Bryant et
al., 2004; Verboon-Maciolek et al., 2003).
In this case diagnosis of CVB3 infection was confirmed
post-mortem by RT-PCR on myocardium tissue and by sero-
logical tests. IgM serum neutralizing antibody for CVB3
virus was positive, whereas anti-CVB3 IgG antibody was not
detected. This suggests that there was no transfer of maternal
antibodies transplacentally and therefore prenatal infection
was less likely, although infection shortly before delivery,
prior to maternal development of IgG or transplacental pas-
sage of antibody, is still possible. The clinical signs did not
become evident until the third week of life, suggesting hori-
zontal transmission. However, the symptoms were so severe
with fatal outcome, that perinatal infection was more likely
(Bryant et al., 2004; Verboon-Maciolek et al., 2003). The lack
of maternal antibodies may partially explain the severity of
illness in this child (Abzug, 2004).
We noted some discrepancies between the results of the
NaF and citrate plasma samples. Citrate plasma is a suit-
able sample for molecular diagnostical procedures. It is
possible that the citrate plasma was negative in real-time
RT-PCR due to the low viral load of the sample: the posi-
tivity of NaF plasma was seen after cycle 40, which indicates
a very weak signal, and could indicate low viral titer in
this patient. Variable results in diagnostic RT-PCR can be
210 K. Smets et al. / Journal of Clinical Virology 42 (2008) 207–210
due to differences in procedures involved (extraction, choice
of primers, amplification protocol, etc.) between both PCR
methods.
In order to try to refine the time of onset of infection,
RT-PCR on a dried blood spot Guthrie card was performed
and enteroviral RNA was found. The detection of virus
on the Guthrie card is consistent with prenatal infection,
perinatal acquisition, or even early horizontal transmission
after delivery. However, a rather strong positive PCR sig-
nal is required for highly qualitative sequencing analysis.
That is why, despite the positivity in real-time RT-PCR and
diagnostic 5 NCR RT-PCR, sequencing could not be per-
formed. Therefore we cannot prove that the virus detected
on the card was the same as the virus causing illness on
day 17.
PCR on dried blood spots is well known and widely used
for retrospective diagnosis of congenital cytomegalovirus
infection (Barbi et al., 2006; Binda et al., 2004). To our knowl-
edge, this is the first report of detection of enteroviral RNA in
a dried blood spot from a Guthrie card. Our case emphasizes
the potential value of Guthrie card dried blood for diagnosis
and approximate timing of perinatal infections by molecular
methods and the ability to extract enteroviral RNA from dried
blood spots.
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