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10]

Lab Sciences

Polymerase Chain Reaction as a Diagnostic Tool in Human Viral

Myocarditis
Nivedita Pathak, Bimal Kumar Das1
Departments of Pediatrics, Microbiology, All India Institute of Medical Sciences, NewDelhi, India
1

Abstract
Viral myocarditis is now acknowledged as a leading cause of morbidity as well as mortality in cardiovascular diseases. Its treatment is
highly dependent on its proper diagnosis as its clinical features overlap with or mimic many other cardiovascular conditions. Histology by
endomyocardial biopsy(EMB) confirms its diagnosis but given its own limitations and complications, the noninvasive imaging methods
such as echocardiogram and magnetic resonance imaging as well as the molecular techniques like polymerase chain reaction(PCR) have
redefined the entire scenario. Of these, PCR can detect the viral epitopes in peripheral blood samples, heart biopsy tissues samples, or
urine/stool sample. Moreover, the best use of PCR is exemplified in the EMB samples where scarcity of the sample is not a limiting factor
unlike histopathological examination. Detecting the subclinical infections, identifying different strains, and detecting pathogens which are
otherwise difficult to grow gives PCR an edge. As it is said time is money, thus rapid detection of specific nucleic acid sequences from
minute samples, and the overall costeffectiveness makes PCR a technique of choice in the diagnostic armamentarium.

Key words: DNA/RNA, endomyocardial biopsy, polymerase chain reaction, primers, viral myocarditis

Introduction reactions in cardiac tissues,[4] however, as early as in 1806

In the last few decades, there has been significant progress
in the prevention and treatment of cardiovascular disease,
but still the incidence and prevalence of chronic heart failure
has risen continuously. Few decades back, regardless of
the underlying etiology, a mortality of up to 50% has been
recorded from heart failure within 1year of diagnosis.[1] The
underlying etiology in more than 40% of patients undergoing
heart transplantation in the Western world was myocarditis
which still remains a common and important cause of dilated
cardiomyopathy.[2]
The diagnosis of acute myocarditis is still a complex and
challenging task in cardiology. [3] Histologically, Dallas
criteria define cardiomyopathy as an inflammation of the
myocardium which is associated with necrosis and an
absence of ischemia.[48] Poor sensitivity and specificity
remains the major factor using Dallas criteria in the diagnosis
of myocarditis because of sampling errors, nonhomogenous
distribution of lesions, and variability in interpretations.[9,10]
Moreover, one more limitation of Dallas classification is that
it does not focus on the viral infections and immunological
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Corvisart described a cardiac inflammatory disorder that
resulted in progressive abnormalities of cardiac function
after disappearance of all the evidences of infective
agent, suggesting of a relation between the infection and
chronic heart disease.[3] Viral infection is one of the major
causes of myocarditis in patients.[11] Apart from histology,
additional evaluation techniques such as polymerase chain
reaction (PCR) are nowadays touchstone for diagnosing
myocarditis.[11] The PCR has been used as the new gold
standard for detecting a wide variety of templates including
viral genome. PCR is an extremely sensitive method which
allows the detection of even low copies of the viral genome
to establish a diagnosis of viral myocarditis from very less
endomyocardial biopsy samples.[1222]
Address for correspondence: Dr.Nivedita Pathak,
Department of Pediatrics, All India Institute of Medical Sciences,
NewDelhi110029, India.
EMail:nivisaiims@gmail.com
This is an open access article distributed under the terms of the Creative Commons
AttributionNonCommercialShareAlike 3.0 License, which allows others to remix,
tweak, and build upon the work noncommercially, as long as the author is credited and
the new creations are licensed under the identical terms.
For reprints contact: reprints@medknow.com

DOI: How to cite this article: Pathak N, Das BK. Polymerase chain reaction
10.4103/2395-5414.166338 as a diagnostic tool in human viral myocarditis. J Pract Cardiovasc Sci
2015;1:168-75.

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Pathak and Das: PCR in Viral Myocarditis

Here, in this review, we will focus on the use of PCR in the transplant recipients, with the involvement of multiple organs
detection of pathogenic viruses in the endomyocardial tissue also.[37,38] In transplanted patients treated with ganciclovir for
of the patients of myocarditis. a previous CMV infection, the features of cells are altered and
they often neither show the characteristic basophilic inclusions
Etiology nor cytomegaly in the biopsy sample.[3941] Parvovirus B19,
the causative agent of erythema infectiosum, also known as
A number of agents including viruses are came out to be the
fifth disease, has been reported as rare but severe cause of
most common causative agents of myocarditis. With the current
myocarditis in infants and children,[4244] also accounted for
techniques and recent approaches, our understanding of viral
the conditions such as hydrops fetalis and fetal death.[45,46]
myocarditis has increased. Viruses known to cause myocarditis
The B19 receptor(erythrocyte Pantigen) has been found on
are listed in Table1.
fetal myocardial cells, suggesting intrauterine myocarditis
Coxsackie B viruses are most commonly associated with viral be the reason for the development of fetal hydrops.[42] This
myocarditis. A member of the picornavirus family and the cardiotropic virus infects most adults at some time during a
enterovirus genus, it is related closely to other enteroviruses lifetime. In the pathogenesis of endocardial fibroelastosis,
such as poliovirus, rhinovirus, and echovirus. The prevalence mumpsinduced myocarditis has been demonstrated to be
of the enteroviruses have been reported in many clinical the first step. The incidence of the disease, in recent years
studies; being an infective agent in viral myocarditis.[2328] which was previously considered a significant cause of infant
In a series of experiment around 86% of the healthy adult mortality, has phenomenally declined due to vaccination.[47,48]
patients being tested and neutralizing antibody against
two serotypes of Coxsackie B were detected in the sera.[8] Pathogenesis
Numerous respiratory tract viruses namely EpsteinBarr
A clear understanding of the pathophysiology of progression
viruses, influenza viruses, adenoviruses, etc., are associated
of heart muscle damage and the development of dilated
with viral myocarditis.[2934] Both in childhood [25,26] and
cardiomyopathy is important in the management of this often
adulthood cases of myocarditis and dilated cardiomyopathy,
fluctuating disease. The pathogenesis of myocarditis is evident
adenoviruses are found to be an important causative agent.[35]
by experiments demonstrating virus induced myocarditis in
Cytomegalovirus (CMV) is fairly uncommon in otherwise
mice models.[18]
healthy people and belongs to herpes group of viruses, and
is an acknowledged cause of acute infectious myocarditis.[36] Progression of myocarditis comprises of three distinct phases:
In some patients presenting with acute myopericarditis, the The viral stage is defined as the period of time when the
CMVspecific genome was detected in the biopsy and active replication of the live virus is occurring within
myocytes.[37] CMV infection might be considered a more the myocardium. In this stage, the virus gains access
frequent cause of myocarditis than what was previously to the cardiomyocytes and induce the innate immune
established. CMV infection is a particular viral disease in response comprising macrophages, natural killer cells,
and various chemical messengers. [19,20] The innate
immune response clears the viral load leaving behind
Table1: Infective agents of viral myocarditis injured cardiomyocytes which result in nonsymptomatic
Types of virus causing Nucleic acid (ss)/(ds) myocarditis. [21] Viruses that successfully avoid the
human myocarditis elimination by the innate immune system begin to
Adenovirus DNA ds replicate, producing viral proteins that can cause direct
Coxsackievirus RNA ss myocardial injury
EBV DNA ds The second phase, the immune response is generated, and
Cytomegalovirus DNA ds the viral antigens are detected, processed, and presented
Hepatitis C virus RNA ss by the antigen presenting cells and then killed by the
Hepatitis B virus DNA Partially ds major histocompatibility complex restricted lymphocytes.
Human herpesvirus 6 DNA ds This destruction of viral antigens can also leave behind
HIV 1, 2 RNA ss the injured cardiomyocytes. In addition, some of the host
Mumps virus RNA ss myocardial tissue share the epitope similarity with the
Parvovirus B19 DNA ss viral epitopes called as molecular mimicry can also lead to
Poliomyelitis virus RNA ss the destruction of cardiomyocytes while clearing the viral
Rabies virus RNA ss
particles[22]
Rubella virus RNA ss
The third phase, dilated cardiomyopathy which is a result
Vaccinia virus DNA ds
of viral and autoimmune injury of the cardiomyocytes, with
Varicella virus DNA ds
the disappearance of the pathological signs of myocarditis
Variola virus DNA ds
EBV: EpsteinBarr virus, DNA: Deoxyribonucleic acid, RNA: Ribonucleic
and an increase in tissue fibrosis.[19] In most cases, it may
acid, EBV: Epstein-Barr virus, HIV: Human immunodeficiency virus, not even be possible to determine whether the inciting event
ss:Singlestranded, ds: Doublestranded was a viral infection.

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Pathak and Das: PCR in Viral Myocarditis
This highlights the importance of specific and sensitive
diagnostic methods for viral myocarditis that can be used at
early stages of viral infection.
Diagnosis of Viral Infections of the Myocardium
and Their Bottlenecks
A case of myocarditis has to be confirmed by histological
examination, but one has to remain the cautious about observer
dependent variables and patchy inflammatory infiltrates
causing sampling errors which are limitations to the utility of
endomyocardial biopsy (EMB). Moreover, the risks of EMB
should be always taken into the consideration which could be
acute or delayed. The perforation of heart leading to the pericardial
tamponade, ventricular or supraventricular arrhythmias, heart
block, pneumothorax, puncture of the central arteries, pulmonary
embolization, nerve paresis, venous hematoma, tricuspid valve
damage, and creation of arterial venous fistula within the heart
could be the immediate risks of biopsy. The risks of EMB
are much variable depending upon: Patients clinical status,
operators experience, presence or absence of left bundlebranch
block, the site of access, and bioptome too. Access site bleeding,
tricuspid valve damage, pericardial tamponade, and deep venous
thrombosis comprise the delayed complications. The precise
frequency of these complications is not known as these have
been only known through case reports.[49,50]
Diagnosing myocarditis now is based upon noninvasive cardiac
imaging surmounting the low sensitivity of the Dallas criteria in
histologically diagnosing myocarditis. Diagnostic noninvasive
cardiac imaging techniques to detect the myocarditis include:
Echocardiography, nuclear imaging, and cardiac magnetic
resonance imaging(MRI).[51]
To ascertain the left ventricular function, echocardiography is a
very prime component of the diagnostic workup and to rule out
other causes of heart failure as well.[52,53] Tissue characterization
by sonogram may prove to be more useful despite the anatomic
features on echocardiography (i.e., chamber dimensions,
ejection fraction, and wall motion abnormalities) being not
sufficient to differentiate myocarditis from the other forms
of cardiomyopathy. Features suggestive of myocarditis by
echocardiogram are often nonspecific yet can be useful in
identifying a fulminant course, if any. To determine its clinical
utility, additional validation studies are needed usually.[5457]
Cardiac MRI is very promising in diagnosing the myocarditis
showing an evolution of contrast enhancement from the focal
to disseminated disease and could be useful to diagnose the
myocarditis associated with edema, hyperemia, or fibrosis
sensitive sequences.[58-60] To identify the patients who should
undergo a biopsy, MRI is useful, and could be used as a guided
approach to the abnormal region of the myocardium. Such a
focused approach could improve the sensitivity of EMB to
establish a correct histological diagnosis. The ability of MRI
to differentiate the viral myocarditis from other causes of acute
dilated cardiomyopathy is still, unfortunately, unclear.[61]
170 Journal of the Practice of Cardiovascular Sciences MayAugust 2015 Volume 1 Issue 2
With the emergence of recent molecular biology techniques,
the diagnosis of viral myocarditis, specifically during acute
viral infection, is based on the detection of the viral epitopes
in peripheral blood samples, heart biopsy tissues samples,
or urine/stool sample. The viral genome can be amplified by
the molecular biology techniques as PCR from EMB samples
[Figure 1 (i) and (ii)]. There could be few problems performing
the PCR from EMB samples:(1) After the second phase of
viral infection, it is difficult to get viral genome from EMB
sample to make out that resultant dilated cardiomyopathy
is due to viral infection,(2) if the sample is not rapidly and
properly transported from the procedure room to the laboratory
bench, PCR analysis for viral genomes can yield false results.
Pathogenfree biopsy devices and storage vials are generally
used to prevent the sample degradation and contamination.
There are certain commercially available fixatives such
as RNAlater which allow PCR and a reverse transcription
PCR(RTPCR) to be performed on the samples transported on
dry ice at room temperature without loss of sensitivity when
compared with frozen tissue that is transported on ice.[49]
Polymerase Chain Reaction: Principle and Procedure
Astute observations, dedicated researchers, years of diligence,
perseverance, simple organic molecules to most complex
DNA/RNA genomes all have contributed to reach the
Eureka moment of PCR discovery.
PCR employs a pair of synthetic oligonucleotides or
primers each hybridizes to one strand of a doublestranded
DNA (dsDNA) target, with the pair crossing a region that
will be exponentially reproduced. The primer hybridizes to
the target gene of interest and acts as a substrate for a DNA
polymerase (most commonly derived from the thermophilic
bacterium Thermus aquaticus and called as Taq) to bind, which
finally creates a complementary strand via sequential addition
of deoxynucleotides. Deoxynucleotide triphosphates(dNTPs,
sometimes called deoxynucleotide triphosphates; nucleotides
containing triphosphate groups), are the buildingblocks, which
help the DNA polymerase to synthesize a new DNA strand.[62,63]
For setting PCR buffer solution is required, which provides
a suitable chemical environment for optimum activity and
stability of the DNA polymerase.
Along with some bivalent cautions such as magnesium or
manganese ions; generally Mg2+is needed for amplification
process[Figure2].[64]
PCR mostly consists of a series of 2040 repeated temperature
changes, called cycles, with each cycle commonly consisting
of 23 discrete temperature variations.
The process can be summarized in three steps:(i) Denaturation:
dsDNA separates at temperatures>90C, the high temperature
is required to break the hydrogen bonds that connect the two
DNA strands. Before starting the first cycle, the DNA and the
primers both need to get denatured and completely separated in
to single strands. The time required for this primary denaturation
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Pathak and Das: PCR in Viral Myocarditis

Figure1: (i) Course and pathophysiology of viral myocarditis(invivo) which leads to the damage and injury of cardiomyocytes.(ii) In vitro isolation
of cells from the biopsy sample and the extraction of DNA and RNA for downstream polymerase chain reaction reactions.

Annealing of primers at wrong temperature during this step can

Figure2: Reaction mixture for setting up a polymerase chain reaction
typically consists of viral nucleotide(DNA), target gene specific primers,
deoxynucleotide triphosphates, and Thermus aquaticus polymerase
enzyme mixed in buffer solution.
is up to 5min. Taqpolymerase is also activated at this higher
temperature(ii) annealing: After denaturing the DNA strands,
a somewhat lower temperature is required to let the primers
hybridize themselves to the singlestranded DNA on which,
the gene of interest is lying. This step is called annealing. The
temperature of this stage usually depends upon the sequence of
the primers(usually 5065C) and can be calculated for each
set of primers. The optimum annealing temperature should be
5C below the melting temperature of the primer sequence.
result in mispriming. (iii) Extension: In the final step, the DNA
polymerase synthesizes a new DNA strand complementary to
the original DNA template strand by adding dNTPs in 5 to
3 direction. The 5phosphate group of the dNTPs condenses
with the 3hydroxyl group at the end of the nascent(extending)
DNA strand. The extension time depends both on the DNA
polymerase used and on the length of the DNA fragment to
be amplified, both. As a rule of thumb, 1min/1 kilo base pair
is usually required [Figure 3]. The temperature of this step
depends upon the type of polymerase used in the reaction.
The optimal extension is at 7278C for Taq polymerase.
The rate of temperature change or ramp rate, the length of the
incubation at each temperature and the number of times each
set of temperatures(or cycle) is repeated are controlled by a
programmable thermal cycler[Figure4ac].[6567]
Stages of Polymerase Chain Reaction
The whole PCR process can be divided into various stages:
Exponential amplification
Assuming 100% reaction efficiency, virtually the amount
of product is amplified exponentially in every cycle. The
sensitivity of the reaction is so high that even few copies of
the target sequence can be amplified.
Leveling off
For the continuous synthesis of new strands, primers and
dNTPs are required. After a certain point of time as the reaction
goes on; primers and dNTPs are used up which results in

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Pathak and Das: PCR in Viral Myocarditis

Figure3: Schematic representation of the first cycle of polymerase chain reaction showing denaturation of template DNA, annealing of primers to the
target sequence and during extension, and synthesis of the complementary strand.

slowing down of the reaction. DNA polymerase also loses its
activity after a certain number of cycles.
Plateau phase
As the reagents and enzyme are used up and exhausted, no
more product forms and piles up.
PCR has displaced some of the established gold standards as
cell culture and various serological assays.[68] The existing
combinations of PCR and detection assays have been used to
obtain the quantitative data with promising results.
Polymerase Chain Reaction/Reverse
Transcription Polymerase Chain Reaction in
Viral Genome Detection
During PCR, a fragment of the viral genome is amplified
using specific primers as mentioned above. For RNA viruses,
synthesis of complementary DNA strand (cDNA) via RT
(viral RNA to cDNA) is necessary prior to the PCR. Such PCR
is called as RTPCR.[69]
Reverse Transcription Polymerase Chain
Reaction
During RT, cDNA from RNA is synthesized using a primer
which helps the reverse transcriptase(RNAdependent DNA
polymerase) to bind and make the first strand cDNA. Three
types of primers are commonly used: Random hexamer
primers, polythymine/oligo dT primers, and gene specific
primers:
Random hexamer primers are the short singlestranded
DNA fragments with all possible combinations of bases.
They are short, nonspecific primers, and will produce pieces
of cDNA scattered all over the messenger RNA(mRNA)
molecule. The RT reaction will nonspecifically produce
cDNAs from all the mRNA present in the reaction mixture,
but cDNA would not be of full length[70,71-73]
Polythymine(T16) primers/Oligo dT primers are usually
1216 baselong thymine primers that will hybridize with
the polyadenine tail at the 3 end of mRNA. For efficiency
of the reaction, it is necessary that mRNA should have
polyadenine tail. If the mRNA is degraded then using these
primers would be of less use[7274]
Gene specific primers are used when only a subset of cDNA
from total mRNA is required. Gene specific primers will
bind only to the targeted region of the mRNA and transcribe
only the required sequence[Figure5].[75]
The RT step is not necessary for viruses whose genome is
composed of DNA.
For the detection of each particular virus, specific sets of primers
need to be designed. Sequences of conserved regions or genes
found in the viral genome are used for designing of primer sets
which can hybridize with a number of different members from
a particular viral family. This way, with the use of a limited
number of primers, many viruses could be screened at a time.

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Pathak and Das: PCR in Viral Myocarditis
a
b
c
Figure4:(a) Setting up a polymerase chain reaction,(b) Temperature and
time needed per cycle in a typical polymerase chain reaction(temperature
and time varies as per the specific requirement of a reaction), and
(c) Exponential amplification of target gene; process is repeated during
cycles and gene of interest is amplified after a specified number of cycles
in a polymerase chain reaction run.
For example, in adenoviruses a conserved region codes for the
capsid hexon. Primers specific to this region could be used in the
detection of different subtypes (2, 40, and 41) of adenoviruses
possessing same conserved region.[76] Sometimes, the conserved
5 noncoding region of the enterovirus genome is also used
for designing the primers for the detection of poliovirus,
Coxsackievirus, and echovirus which belong to the same
family.[70] Other variable regions in the genome of the virus are
useful for typing viral isolates in the epidemiological studies.[73]
The final, PCR product is analyzed by agarose gel
electrophoresis, which determines the correct size of the
Journal of the Practice of Cardiovascular Sciences MayAugust 2015 Volume 1 Issue 2
Figure5: Different types of primers used to set up a reverse transcription
polymerase chain reaction. The choice of the primers depends upon the
target sequence, which is to be amplified.
PCR product. However, the confirmation of the PCR product
is recommended by sequencing or other assays.
Realtime quantitative PCR(qPCR) quantitatively determines
the amount of viral genome present in the sample. [77,78]
During a qPCR assay, in each cycle, the produced product
is quantified in two ways: By using(1) SYBR Green which
binds nonspecifically to dsDNA, or(2) a fluorescent internal
probe which binds specifically to the DNA, containing probe
sequence.[61] In both cases, the fluorescence is measured during
each cycle. The sample is considered positive and when the
amount of fluorescence exceeds the certain threshold level.
The number of cycles needed to reach the threshold level,
commonly referred to as the cycle threshold value, correlates
with the amount of target in the sample prior to amplification.
Realtime PCR is less time consuming mainly because of
the precise and stringent thermal cycling/ramp rates, the use
of fluorescence dyes and probes to sensitively detect their
emissions and removal of postPCR detection procedures such
as gel electrophoresis. Various diagnostic methods and their
advantages/disadvantages are discussed in Table2.
Conclusion
Todays, the molecular techniques such as PCR, realtime PCR,
and gene sequencing are the techniques of choice for rapid,
specific, and sensitive identification of infective agents and
can be applied on the same EMB specimen, so the scarcity of
sample is no more a limiting factor. The numerous advantages
of PCR analysis for definitive diagnosis of viral myocarditis
are:(i) Rapid detection of specific viral nucleic acid sequences
in minute quantities,(ii) detection of infective agents that are
difficult to grow or cannot be grown on a media,(iii) detecting
strains of the pathogens, and(iv) detection of the subclinical
and manifested infections.
Advancement in the molecular techniques will allow us to get
more information about the epidemiology, risk stratification,
and newer treatment approaches.
Financial support and sponsorship
Nil.
173
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Pathak and Das: PCR in Viral Myocarditis

Table2: Different methods for diagnosis of viral myocarditis

Methods Time Advantage Disadvantage
Single target PCR Hours Sensitive, viral quantitation, and less costly Can recognize only targeted viruses
Multiplex PCR Hours Sensitive, viral quantitation, and more cost Limited sensitivity as compared to single target PCR
effective than single target PCR
Cell culture Days to week Low reagent cost, diagnosis of unknown viruses Poor sensitivity without pathognomonic relevance
Direct fluorescent assay 2days Improved sensitivity, costly Limited viruses, over all less sensitive
Serology Hours More specific, less costly Limited sensitivity
In situ hybridization 1-2days Identifies specific infected cell, more costly Less sensitive than PCR
PCR: Polymerase chain reaction

Conflicts of interest Experimental CVB3induced chronic myocarditis in two murine strains:

There are no conflicts of interest.
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