Download Cardiomyopathy Using Gene Amplification

8
Clinical Investigation
Detection of Enterovirus RNA in Myocardial
Biopsies From Patients With Myocarditis and
Cardiomyopathy Using Gene Amplification
by Polymerase Chain Reaction
Ou Jin, MD, Michael J. Sole, MD, Jagdish W. Butany, MD, Wah-Kiam Chia, MSc,
Peter R. McLaughlin, MD, Peter Liu, MD, and Choong-Chin Liew, PhD
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Recent molecular studies have suggested that viral myocarditis frequently underlies human
congestive cardiomyopathy; however, only moderately sensitive and specific techniques were
used. Polymerase chain reaction (PCR) gene amplification is a sensitive, specific technique
ideally suited for the diagnosis of viral disease in small tissue samples where low copy numbers
of the viral genome may be present. Using PCR and high stringency condition, we screened
biopsies taken from 48 patients with clinically suspected myocarditis or dilated cardiomyopathy. Five patients demonstrated positive enteroviral signals by PCR; two of them had
myocarditis by pathology, whereas the other three had changes consistent with cardiomyopathy.
Four other patients had myocarditis diagnosed by pathology from 3 months to 1 year earlier but
were now negative by both PCR and pathology. Both pathology and PCR were negative for
active myocarditis in all other patients. Ventricular samples taken from left ventricular
myectomy in four additional patients with hypertrophic cardiomyopathy, normal human
ventricle samples, and uninfected monkey kidney cells were also negative by PCR. This study
supports a link between viral infection and dilated cardiomyopathy in some patients. PCR gene
amplification provides a new diagnostic approach to patients with suspected myocarditis.
(Circulation 1990;82:8-16)
E-
nteroviruses, particularly the coxsackievi-
believed to be the most common
viral agents for the pathogenesis of human
myocarditis.' Although serological studies have indicated an association of coxsackievirus B viral infection with myocarditis,2-5 viral cultures of myocardial
tissue obtained by endomyocardial biopsies from
patients with biopsy-proven myocarditis or dilated
cardiomyopathy are almost always negative, even
when the clinical history or serological studies indicate recent viral infection.67 In addition, immunocytochemical studies of myocardial tissue have
ruses, are
From the Laboratory of Molecular Cardiology, Departments of
Medicine, Clinical Biochemistry, Pathology, and Microbiology,
The Centre for Cardiovascular Research, The Toronto Hospital,
University of Toronto, Ontario, Canada.
Supported by grants from the Medical Research Council of
Canada and the Heart and Stroke Foundation of Ontario. M.J.S.
is a Distinguished Research Professor of the Heart and Stroke
Foundation of Ontario. P.L. is a Research Scholar of the Heart
and Stroke Foundation of Ontario.
Address for correspondence: Dr. Michael J. Sole, Room 13-208
EN, Toronto General Hospital, 200 Elizabeth Street, Toronto,
Ontario M5G 2C4, Canada.
Received October 4, 1989; revision accepted February 27, 1990.
repeatedly failed to demonstrate the presence of viral
antigens in patients with myocarditis and chronic
dilated cardiomyopathy.8
Recombinant DNA technology holds great promise for the study of virus-associated myocarditis by
detecting the presence of the viral genome in endomyocardial biopsy tissue. The molecular genetics of
the enteroviruses, particularly coxsackievirus, and
poliovirus, are well documented. The sequences of
coxsackievirus Bi, B3, and B4 (CVB1, CVB2, and
See p 294
CVB3) and poliovirus 1, 2, and 3 (PV1, PV2, and PV3)
have been reported.9 12 Using coxsackievirus B complementary DNA (cDNA) probes, two groups have
reported the presence of CVB2 and coxsackieviru's B4
(CVB4) signals in myocardial biopsies from patients
with myocarditis or dilated cardiomyopathy using slotblot and in situ hybridization techniques.13'14 In these
studies, nonspecific hybridization was a concern.
Polymerase chain reaction (PCR) gene amplification is a technique that allows rapid and substantive
amplification of specific DNA sequences. This sensi-
Jin et al Enterovirus RNA in Myocardium
tive and specific technique is ideally suited to the
diagnosis of viral disease where low copy numbers of
the viral genome may be present. In this study, we
report the application of PCR gene amplification to
the diagnosis of viral myocarditis using cardiac biopsies taken from 48 patients with clinically suspected
myocarditis or dilated cardiomyopathy.
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Methods
Preparation of Coxsackievirus B RNA
African Green monkey kidney cell culture in a
concentration of 106 cells/ml and suspended in
medium 199 containing 10% fetal calf serum and
antibiotics was supplied by Connaught Laboratories,
Toronto, Ontario. This cell suspension was seeded in
25-cm2 tissue culture flasks and incubated at 370 C
for 24 hours. The medium was then replaced with
minimum essential medium (MEM) supplemented
with 10% fetal calf serum, penicillin (100 units/ml),
streptomycin'(100 ,gg/ml), and fungizon (2.5 ,ug/ml).
The cell culture flasks were further incubated at
370 C until a confluent monolayer of cells was
observed before viral inoculation.
Cell cultures were inoculated with CVB3 at a total
titer of' 106 particles or 50% tissue culture infection
dose (106 TCID50). After a 1-hour incubation at room
temperature, the infected cell culture was washed
twice with MEM and then incubated in 5.0 ml MEM
containing antibiotics without fetal calf serum at
370 C. The infected cell culture monolayer was examined daily for 25-50% cytopathic effect. The cells
were then suspended in 0.5 ml of 8 M guanidine-HCl
(pH 5.0), passed twice through a 25-gauge needle,
and precipitated with one-half volume of ethanol at
-20° C overnight. The precipitate was dissolved in
autoclaved H2O, and the RNA was quantified by
ultraviolet spectrophotometry.
Myocardial Biopsies and Histopathology
Right ventricular endomyocardial biopsy samples
were obtained from 48 patients with a Stanford
bioptome by the internal jugular approach.15 Five
samples were usually taken from each patient, and
four were fixed in 10% formalin, embedded in paraffin, and cut into 4-5-gm sections; one section
(usually less than 1 mg) was instantly frozen in liquid
nitrogen for molecular diagnosis.
Multiple sections of the biopsy were stained with
hematoxylin and eosin and examined by light microscopy. Each section was studied in detail, and the
diagnosis of myocarditis was based on the Dallas
criteria.16
Myocardial biopsy samples for study by PCR were
homogenized with 0.5 ml of 8 M guanidine-HCl (pH
5.0) in a small homogenizer and spun down to
remove tissue debris. Total nucleic acids were then
precipitated with ethanol, dissolved in 15 gul of autoclaved H20, and prepared for further assay.
9
Blood DNA Isolation
The procedure for rapid DNA isolation from blood
was a modification of the method described by Lindblom et al.17 Blood samples (5 ml) were lysed with
cold sucrose buffer (10 ml, pH 7.5, 1% Triton X-100
in 320 mM sucrose, 1 mM Tris-HCI, and 5 mM
MgCl2). After centrifugation (2,000 g at 40 C for 20
minutes), the pellet was resuspended in the same
buffer and incubated in proteinase K (500 ,ug/ml,
fresh stock solution: 1 mg/ml in 1% sodium dodecyl
sulfate [SDS] 2 mM EDTA [pH 8.0]). Proteins were
extracted by the addition of the same volume of
Tris-EDTA-saturated phenol: chloroform: isoamyl
alcohol (25:24: 1). After centrifugation (10,000 g at
40 C for 10 minutes), the upper aqueous phase was
transferred to a new tube, and DNA was precipitated
by adding 2 vol of absolute ethanol (200 C for 2 hours
or longer). The DNA was dissolved in sterilized H O
and quantitated by ultraviolet spectrophotometry.
Oligonucleotide Synthesis and Labeling
Oligonucleotides (20-60 base pairs) were prepared by a solid-phase phosphotriester method18 and
labeled at the 5' end with phosphorus-32-r-ATP
(specific activity, 3,000 Ci/mmol, New England
Nuclear, Boston, Mass.). Oligonucleotide (0.6-1 jug)
was incubated in standard kinase buffer'6 plus 150
,uCi "2P-r-ATP and 10 units polynucleotide kinase
(Boehringer Mannheim Biochemicals, Indianapolis,
Ind.) in 50 ,lI total volume for 1 hour at 370 C.
Labeled oligonucleotide was separated from unincorporated ATP by passage through a G-50 column
(Pharmacia, Sweden) in 10 mM Tris-HCl (pH 7.5)
and 0.1 mM EDTA. The specific activity of the
probes was 7-8 x 107 cpm/,g.
Viral cDNA Synthesis and PCR Gene Amplification
First strand cDNA synthesis. We synthesized the
first strand cDNA using a virus-specific primer (see
Figure 1). Four microliters (0.5 ,tg) of kidney cell
RNA and 15 ,l of biopsy RNA samples (RNA
heated to 700 C for 5 minutes before synthesis) were
added to 40 ,ul of 500 mM Tris-HCl (pH 8.3), 500
mM KCl, 100 mM MgCl2, 10 mM 1,4-dithiothreitol
(DTT), 10 mM EDTA, 100 gg/ml bovine serum
albumin (BSA), 80 mM sodium pyrophosphate, 10
mM spermidine-HCI, 10 mM deoxynucleotide triphosphates (dNTP), and 100 ,ug/ml oligo d(T),"2-18 1
,uM of each primer, and 1,000 units/ml avian murine
virus (AMV) reverse transcriptase. These mixtures
were incubated at 420 C for 50 minutes and then at
650 C for 10 minutes to denature the AMV reverse
transcriptase. The samples were then ready for PCR
gene amplification.
PCR gene amplification. Two microliters (0.025 ,ug)
of both uninfected and infected cell samples and 20
,u1 of biopsy samples containing the first strand
cDNA as described above were added to 40 gl of 50
mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCI,,
0.01% (wt/vol) gelatin, 200 ,uM each deoxynucleo-
Circulation Vol 82, No 1, July 1990
10
Probe 1
0
5'
Probe 2
'S
3'
200
cDNA
600
400
I
Primer A
5'
Primer B
i 3r
Primer C
3P
*
(-) strand
B
750
|
3' 3'
5'
Viral RNA
3'
3'
Denature
Denature
-
Primer D
Anneal to
Anneal
primer A
Probe 1 3'--GCGATCGTGAGACCATTAGTGCCATGGAAACACGCGGACAMAA
TATGGGGGAGGGGGTTGA--5'
Primer A: 5'--AGCCTGTGGGTTGATCCCAC--3'
Primer B: 3'--CTAGTTGTCAGTCGCACCGT--5'
METHOD 2
Probe 2: 3'--CAGCATTGCCCGTTGAGACGTCGCC1TGGCTGATGAAACCCA
CAGGCACAAAGTAATA--5'
Primer C: 5'--TCCTCCGGCCCCTGAATGCG--3'
Primer D: 3'--ACCGACGAATACCACTGTTA--5'
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FIGURE 1. Sequences of synthetic oligonucleotide primers
and probes and their relation to the target enterovirus genomic
RNA (positive strand) region. Primers A and C are complementary to the negative strand, and primers B and D are
complementary to the positive strand. Probes 1 and 2 are
complementary to the coxsackievirus genomic RNA.
tide triphosphates, and 1 /LM of each primer
(Perkin-Elmer-Cetus protocol). After 10 minutes at
940 C, 5 units of Taq polymerase (Stratagene, La
Jolla, Calif.) and 80 gl mineral oil were added, and
PCR cycles were started (denaturation: 1 minute at
900 C; annealing: 2 minutes at 520 C; elongation: 3
minutes at 720 C).
Blot Hybrdization
After PCR gene amplification, the products (40 ,tl
each) were added to 120 ,l of a solution of 6.15 M
formaldehyde and lOx SSC (same as cDNA control:
2 ,ul of uninfected cell, and infected cell and 20 gl of
biopsy cDNA, respectively, with dH2O added to 40
,ul). These mixtures were incubated for 15 minutes at
650 C. The denatured samples were loaded onto a
Schleicher and Schuell Minifold IL Slot Blot apparatus (containing a nitrocellulose filter that had been
soaked in water) with suction. Each sample well was
washed with 200 ,ul of lOx SSC. The nitrocellulose
sheets were vacuum-dried for 2 hours at 800 C. Filters
were prehybridized in 6 x SSC, 5 x Denhardt's solution (0.1% Ficoll, 0.1% BSA, 0.1% polyvinylpyrrolidone,19 0.5% SDS, and 100 ,gg/ml sheared salmon
sperm DNA) at 550 C for at least 2 hours. After
prehybridization, a second hybridization was performed with the prehybridization solution except that
approximately 1 x 107 cpm of radiolabeled probe was
added. After 8-16 hours at 55° C, filters were rinsed
in 6x SSC and 0.1% SDS at room temperature,
washed in 3x SSC and 0.1% SDS for 15 minutes at
550 C twice and 65° C for 5-10 minutes, and autoradiographed at -70° C for various times with intensifying screens.
primer C
Extension
Extension
METHOD 1
to
p
R
(-
strand
|B
Denature
Annealing (Primer A and B)
Extension
C
()
strand
|
Denature
Annealing (Primer C and D)
Extension
Repeat Cycles
Schematic representation of the PCR protocol
FIGURE 2. Schematic representation of the PCR protocol
demonstrating virus-specific complementary (cDNA) synthesis
from enteroviral RNA by reverse transcriptase and PCR
gene-amplification procedure (see "Methods").
Results
Enteroviruses exhibit areas of extensive nucleotide
sequence homology even between disparate members such as the coxsackievirus and polioviruses.
Other regions are virus specific. Using virus-specific
primers (see Figure 1) to synthesize specific regions
of cDNA, we could select primer pairs that would
amplify sequences of a broad range of enteroviruses.
The regions between these pairs could then be
identified by probes exhibiting homologies to specific
viruses or to the broad family. The sequence of
synthetic oligonucleotide primers and probes and
their relation to the target enterovirus region is
shown in Figure 1. Comparison of probes 1 and 2 to
CVB1, CVB3, CVB4, PV1, PV2, and PV3 revealed
that probe 1 is most homologous with CVB3. Primers
A and B are most homologous to CVB3, whereas
primers C and D have 100% sequence homology with
six viruses (CVB1, CVB3, CVB4, PV1, PV2, and
PV3). Probe 2 exhibits more than 96% homology
with CVBl, CVB3, and CV4 and more than 90%
with PV1, PV2, and PV3. Therefore, probe 2 is more
group specific for the enteroviruses than probe 1.
Overall, both primers and probes should allow detection of the presence of either coxsackievirus and
poliovirus families. Figure 2 briefly describes the
method used to synthesize cDNA from enteroviral
RNA derived from either infected culture kidney
cells or biopsies by reverse transcriptase and PCR
gene amplification.
Jin et al Enterovirus RNA in Myocardium
.a
b
2
3
X~~~~~~~~~~~~~~~~~~~....
i..
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4
...
8
_
11
c
FIGURE 3. Slot-blot hybridization
of probe 1 (coxsackievirus B3
[CVB3]) to different samples: 1) cells
infected with CVB3 (1.5 gg), 2) cells
infected with CVB6 (1.5 gg), 3) uninfected cells (1.5 gg), 4) biopsy A, 5)
biopsy B, 6) biopsy C, 7) biopsy D, 8)
normal blood DNA (1.5 ag), and 9)
normal total human ventricular RNA
(1.5 gg). Isolation method of biopsies
B, C, and D is the same as biopsy A
and 48 other patients except cell
debris was not removed. Amount of
RNA could not be quantitated due to
minute size of the biopsy samples.
This filter was hybridized with CVB3specific oligonucleotide probe 1 at
550 C and washed with 3 x SSC and
0.1% sodium dodecyl sulfate buffer
at 1) room temperature for 5 minutes
and 550 C for 15 minutes, 2) 550 C
for 15 minutes and 65° C for 10
minutes, and 3) 650 Cfor 60 minutes.
9
_
EvL
initial experiments, we did not use PCR
amplification but instead used a conventional
method in which synthetic oligonueleotides derived
from coxsackievirus sequences served as diagnostic
probes.13 Figure 3 shows the results of slot-blot
analysis of four biopsy samples by the conventional
method. Biopsy sample A was from a patient whose
clinical diagnosis indicated myocarditis with severe
left ventricle dysfunction and who had myocarditis
and pericarditis by pathology. Biopsy B was from a
patient who had ventricular tachyarrhythmias after
myocardial infarction a few weeks earlier but showed
no evidence of coronary disease by coronary angiography and pathology. Biopsies C and D were from
two patients whose clinical diagnoses indicated
dilated cardiomyopathy and whose pathology indicated changes compatible with congestive cardiomyopathy but showed no evidence of myocarditis. The
cell debris was not removed from biopsies B, C, and
D after guanidine-HCI homogenation, whereas the
cell debris from sample A was removed. After RNA
precipitation, all four samples were dissolved in 30 gl
autoclaved H20 and subjected to slot-blot analysis.
From these experiments, it is clear that crosshybridization exists between viral RNA and human
DNA and RNA, leading to false-positive results
under low stringency conditions and negative results
In
gene
our
under higher stringency conditions. To improve both
the sensitivity and specificity, we used PCR geneamplification methodology in subsequent studies.
Figure 4 shows the sensitivity and specificity of
probe 1, primer A, and primer B after 30 PCR cycles.
It indicates that virus-specific primer was better than
oligo d(T) for synthesizing specific eDNA in 5'
region. Because the CVB3 genome is 7.5 kb in length,
it will be difficult to synthesize a full-length eDNA.
Figure 5 shows the results of agarose gel electrophoresis and oligonucleotide hybridization analysis of
PCR-amplified DNA samples from monkey kidney
cells. A single band is present, migrating at 184 bp in
DNA from infected cells but not from uninfected
cells. This result also demonstrates the sensitivity and
specificity of the PCR gene-amplification method.
Figure 6 shows a typical PCR analysis of Enterovirus RNA from endomyocardial biopsy samples using
probe 2, primer C, and primer D. Like other patients
whose data are presented in Table 1, the cell debris
was removed before slot-blot analysis. Negative and
positive controls were used for each experiment.
Unlike conventional slot-blot analysis, PCR analysis
does not reveal a continuum of hybridization signal
intensity. Signals were either positive or absent.
Among 48 patients screened by PCR gene amplification, probe 1, primer A, and primer B were used
Circulation Vol 82, No 1, July 1990
12
AftXer.
Before
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FIGURE 4. Assessment of the sensitivity and specificity
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of probe 1, primer A, and primer B after 30 polymerase
chain reaction (PCR) cycles: 1) uninfected cells + oligo
d(T)12 18 primer, 2) cells infected with coxsackievirus B3
4:ii< (CVB3) + oligo d(T)12 18primer, 3) uninfected cells +
CVB3 specific primers A and B, and 4) cells infected with
CVB3 + CVB3-specific primers A and B. For infected
cells, the total RNA was isolated when 25% of cells
showed cytopathic effect by infection of CVB3: 5) uninfected cells + oligo d(T)12 18primer, 6) cells infected with
6 CVB3 + oligo d(T)f2l8 primer, 7) uninfected cells +
CVB3-specificprimersA anzd B, and 8) cells infected with
7 CVB3 + CVI33-specific primers A and B. For infected
cells, the total RNA was isolated when 50% of cells
showed cytopathic effect by infection of CVB3. cDNA,
8 complementwy DNA.
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in patient samples 1-7, lla, 14, iSa, and 16. Probe 2
and primers C and D were used in all other patients,
including llb, 15b, and 15c. Five patients (14-16, 29,
and 42) contained enterovirus RNA in myocardial
tissue as shown by PCR gene amplification. A biopsy
from one of these patients (patient 15) 3 months later
continued to be positive. However, a third biopsy 4
months later was negative. The results derived from
PCR analysis were correlated to the pathological
results (see Table 1). In two patients (15 and 42), both
PCR analysis and pathology indicated the presence of
myocarditis. Three patients (14, 16, and 29) with congestive cardiomyopathy but not active myocarditis as
indicated by pathology exhibited definite viral signals by
PCR. Four patients (1, 8, 11, and 19) with myocarditis
by pathology 3 months to 1 year earlier had a negative
PCR result and no evidence of myocarditis by pathology when examined during this study. PCR analysis was
performed using blood from one of these patients
(15a), but no viral RNA was indicated. Samples were
taken from the left ventricle in four additional patients
with hypertrophic obstructive cardiomyopathy, from
normal human ventricles, and from uninfected monkey
kidney cells (not shown). No viral RNA was indicated
by PCR analysis in these tissues.
Discussion
The genomic sequence relation of the enteroviruses
such as CVB1, CVB3, CVB4, PV1, PV2, and PV39-12
allow the introduction of methods using synthetic oli-
Jin et al Enterovirus RNA in Myocardium
E ..:
I
:.
2
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FIGURE 5. Agarose gel electrophoresis 1) of amplified coxsackievirus B3 (CVB3) sequence fragment by polymerase chain reaction
(PCR) after 40 cycles with probe 1,
primer A, and primer B. Lane 1:
Uninfected cells. Lane 2: Infected
cells (by CVB3). These samples
were run on 3% Nusieve/1% agarose in 1 xtris-HCI acetate EDTA
buffer. Southern blot hybridization
analysis 2) of PCR-amplified fragments of CVB3 genome from
infected cells.
-:184 bp
(1)
gonucleotides, which are CVB- and Enterovirus-specific
for the detection and analysis of viral genomic RNA
sequence. These probes, used in conjunction with PCR
technology, are particularly well suited to the diagnosis
of viral infections of heart tissue because myocardial
biopsies are of limited size and the viral RNA copy
number in cardiac cells is probably very low.
PCR is a unique in vitro gene-amplification method
that can produce a greater than 105-fold increase in
the amount of target sequence, permitting analysis of
as little as 1 ng of genomic DNA.211 The enteroviral
genome is a single-stranded, positive-sense RNA
molecule.21 Available sequence data allowed us to
choose a viral region that would exhibit homology to a
broad range of enteroviruses in a sensitive and specific
manner. We screened 48 patients with clinically sus-
13
(2)
pected myocarditis and cardiomyopathy with this
method; only five patients exhibited an enteroviral
genome in their myocardium.
One of our patients presented with a postpartum
cardiomyopathy; however, her endomyocardial biopsy
revealed active myocarditis by pathological examination. It is of interest to note that when this patient was
examined 3 months later, pathology was negative for
myocarditis, but the PCR result was still positive. Four
months later, both pathology and PCR produced
negative results. This case demonstrates that viral
myocarditis can be the underlying etiology of some
cases of postpartum cardiomyopathy.
Three other patients had biopsies that were positive by PCR analysis. These patients had clinically
manifested cardiomyopathy and exhibited negative
14
Circulation Vol 82, No 1, July 1990
Before
PCR
(cDNA)
After
PCR
26
27
28
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29
30
31
32
33
34
35
15c
Uninf Cell
lnf Cell (CVB3)
FIGURE 6. Examples of polymerase chain reaction (PCR)
analysis of Enterovirus RNA in
endomyocardial biopsy samples.
cDNA, complementary DNA;
CVB3, coxsackievirus B3.
(50 cycles)
pathology by the Dallas criteria but had histories
suggestive of myocarditis. Although the pathology
indicated no evidence of active myocarditis, there
was a significant amount of fibrosis and myocyte
hypertrophy present, suggesting considerable myocardial damage due possibly to myocarditis. In contrast to the PCR gene-amplification results, conventional slot-blot hybridization failed to exhibit a viral
signal with coxsackievirus probes under stringent
conditions in each of these four patients. Thus, in
these patients, the level of viral RNA was less than
the limit of conventional detection or the viral
genome was incomplete, possibly accounting for the
failure to isolate infectious progeny virus or to demonstrate the presence of virus-specific antigens from
biopsy samples in previous studies.
These results suggest that residual viral genome
may be present in myocardial cells in the absence of
acute, pathologically manifest myocarditis and may
predispose such individuals to ensuing dilated cardiomyopathy. It is also possible that the ditference
between pathology and PCR evaluation is a manifestation of sampling heterogeneity and that the biopsy
samples analyzed by hybridization may have exhibited pathologically evident myocarditis had they been
subjected to microscopy. One of our patients was an
18-year-old woman who had symptoms of myocarditis and a positive pathological diagnosis on her first
biopsy that showed evidence of an inflammatory
infiltrate and scattered muscle fiber necrosis. Unfortunately, her initial biopsy sample was not available
for PCR. Six months later when a tissue sample was
Jin et al Enterovirus RNA in Myocardium
15
TABLE 1. Detection of Enteroviral RNA Sequences in Myocarditic Human Heart Biopsies by Polymerase Chain Reaction Technique
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Patient
1
3
5
8
9
10
lla
llb
12
14
15a
15b
15c
16
19
29
Clinical diagnosis
Myocarditis
Myocarditis
Myocarditis
Myocarditis
Myocarditis
Myocarditis fibrosis
Myocarditis, CMP
Myocarditis, CMP
CMP
Myocarditis, CMP
Postpartum CMP
Postpartum CMP
Postpartum CMP
Myocarditis, CMP
Myocarditis
Myocarditis
30
31
32
39
40
41
42
43
46
47
48
17, 20,
21, 22, I
23, 26,
27, 34,
35
J
Myocarditis
Myocarditis
Myocarditis
Myocarditis
Myocarditis
2,4,6,
7,13,
18, 24,
25, 28,
33, 36,
37, 38, J
Myocarditis
Myocarditis
Myocarditis
Myocarditis
Myocarditis
Myocarditis
Pathology result
Dilated CMP (myocarditis by biopsy 1 year earlier)
Interstitial vessels show luminal stenosis
Mild interstitial fibrosis
Mild, focal interstitial fibrosis (myocarditis by biopsy 4 months earlier)
Right ventricular dysplasia
Mild interstitial
Congestive CMP (myocarditis by biopsy 4 months earlier)
Myocarditis
Pompe's disease
Congestive CMP
Active myocarditis
No evidence of active myocarditis
No evidence of active myocarditis
Congestive CMP
Mild, focal interstitial fibrosis (myocarditis by biopsy 1 year earlier)
Dilated CMP (moderately severe interstitial fibrosis; focal mild
increase in interstitial mononuclear cells)
Increased adipose tissue, possible right ventricular dysplasia
Active myocarditis (1 week after onset of herpes zoster)
Hypertrophic CMP (Becter's muscular dystrophy)
Mild subendocardial fibrosis
Patchy mild interstitial fibrosis
Patchy muscle fiber hypertrophy
Focal myocarditis
Presence of amyloid (on special stains)
Tissue inadequate for diagnosis
Patchy endocardial and interstitial fibrcOSiS
Significant iron overload
Myocarditis
Normal
Myocarditis
Dilated CMP
PCR result
+
+
44, 45
PCR, polymerase chain reaction; CMP, cardiomyopathy; -, negative; +, positive.
available for analysis by PCR, it was negative.
Although pathology revealed no active myocarditis,
there was myocyte destruction and fibrosis consistent
with clinical congestive cardiomyopathy. This patient
went for a heart transplant in October 1988 due to
severe dilated congestive cardiomyopathy but subsequently died. Her myopathic heart was available for
detailed hybridization study with different enteroviral primers and probes, but although pathology
exhibited areas of myocarditis, PCR analysis was
consistently negative. One possible explanation was
that the free viral RNA in the myocardium had
degraded despite the relatively rapid RNA isolation
because the heart was not immediately frozen.
Another patient, a 50-year-old woman, who had a
sudden onset of pulmonary edema after the onset of
herpes zoster infection 1 week earlier, showed active
myocarditis by pathology but no enteroviral signal by
PCR. Myocarditis in this case may have been due to
infection by herpes virus rather than an enterovirus.
Three other patients had a definite history and biopsyderived evidence of myocarditis; however, when
biopsied 3 months to 1 year later, both pathology and
PCR analyses were negative. The negative PCR and
pathological data in these cases could be due to
sampling heterogeneity. Furthermore, although we
used primers and probes designed to capture most
enteroviruses, there are other viral families that can
cause viral myocarditis.
16
Circulation Vol 82, No 1, July 1990
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We also took blood samples from 14 patients to
determine the usefulness of PCR analysis during
theoretical viremia. All blood studies were negative
for viral signals, including a sample from one patient
(15) whose heart biopsies were positive by both PCR
and pathology.
This study supports a link between Enterovirus
infection and congestive cardiomyopathy in some
patients. However, the frequency of Enterovirus-like
RNA sequences in clinically suspected myocarditis
and cardiomyopathy samples is not as high as
reported by others using slot-blot hybridization without PCR or in situ hybridization.1314 Sampling has
been a major impediment to the pathological diagnosis of myocarditis from biopsy analysis. It is estimated that 17.2 samples are required per patient for
79% diagnostic sensitivity; such sample numbers are
clinically unattainable.22 Although sampling heterogeneity may account for differences between PCR
studies and those using standard hybridization techniques, it is unlikely to account for a difference of this
magnitude. It would seem more likely that homologies between human DNA and RNA and viral
sequences led to false-positive results under conditions that were not highly stringent. In conclusion,
this study supports a link between viral infection and
dilated cardiomyopathy in some patients. It appears
that the number of viral RNA copies present in
myocardial cells from human myocarditis hearts is
very low; however, the PCR gene-amplification
method can provide a very sensitive and specific
means for the study of such tissue.
6. Daly K, Richardson PJ, Olsen EGJ, Capner PM, Mcsorley C,
Jackson G, Jewitt DE: Acute myocarditis: Role of histological
and virological examination in the diagnosis and assessment of
immunosuppressive treatment. Br Heart J 1984;51:30
7. Parrillo JE, Aretz HT, Palacios I, Fallon JT, Block PC: The
results of transvenous endomyocardial biopsy can frequently
be used to diagnose myocardial diseases in patients with
idiopathic heart failure: Endomyocardial biopsies in 100 consecutive patients revealed a substantial incidence of myocarditis. Circulation 1984;69:93
8. Rose AG, Beck W: Dilated (congestive) cardiomyopathy: A
syndrome of severe cardiac dysfunction with remarkably few
morphological features of myocardial damage. Histopathology
1985;9:367
9. lizuka N, Kuge S, Nomoto A: Complete nucleotide sequence
of the genome of coxsackievirus B,. Virology 1987;156:64-73
10. Lindberg AM, Stalhandske POK, Petterson U: Genome of
coxsackievirus B3. Virology 1987;156:50-63
11. Jenkins 0, Booth JD, Minor PD, Almond JW: The complete
nucleotide sequence of coxsackievirus B4 and its comparison to
other members of picornaviridae. J Gen Virol 1987;
68:1835-1848
12. Toyoda H, Kohara M, Kataoka Y, Suganuma T, Omata T:
Complete nucleotide sequences of all three poliovirus serotype genomes. J Mol Biol 1984;174:561-585
13. Bowles NE, Richardson PJ, Olsen EGJ, Archard LC: Detection of coxsackie B virus specific RNA sequences in myocardial biopsy samples from patients with myocarditis and dilated
cardiomyopathy. Lancet 1986;1 :1120
14. Kandolf R: The impact of recombinant DNA technology on
the study of enterovirus heart disease, in Benclinelli M,
Friedman H (eds): Coxsackievirus: A General Update. Plenum
Publishing, 1988, pp 303-305
15. Richardson PJ: Endomyocardial biopsy technique and evaluation of a new disposable forceps and catheter sheath system,
in Bolte HD (ed): Viral Heart Disease. Berlin, Springer-Verlag,
1984, pp 173-176
16. Aretz HT, Billingham ME, Edwards WD, Factor SM, Fallon
JT, Fenolio JJ Jr, Olsen EG, Schoen FJ: Myocarditis, a
histopathologic definition and classification. Am J Cardiovasc
Acknowledgments
We are grateful to Mr. Jack Liew for synthetic
oligonucleotides and Dr. Samuel Mok for technical
assistance. We thank Sian Liem and Douglas Anderson for secretarial assistance.
17. Lindblom B, Holmlund G: Rapid DNA purification for restric-
Pathol 1987;1:3-14
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KEY WORDS *
dilated cardiomyopathy * coxsackievirus
myocarditis * polymerase chain reaction * complementary DNA
.
Detection of enterovirus RNA in myocardial biopsies from patients with myocarditis and
cardiomyopathy using gene amplification by polymerase chain reaction.
O Jin, M J Sole, J W Butany, W K Chia, P R McLaughlin, P Liu and C C Liew
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Circulation. 1990;82:8-16
doi: 10.1161/01.CIR.82.1.8
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