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PCR in Viral Myocarditis PDF
PCR in Viral Myocarditis PDF
10]
Lab Sciences
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
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.
168 2015 Journal of the Practice of Cardiovascular Sciences | Published by Wolters Kluwer - Medknow
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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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This highlights the importance of specific and sensitive With the emergence of recent molecular biology techniques,
diagnostic methods for viral myocarditis that can be used at the diagnosis of viral myocarditis, specifically during acute
early stages of viral infection. viral infection, is based on the detection of the viral epitopes
in peripheral blood samples, heart biopsy tissues samples,
Diagnosis of Viral Infections of the Myocardium or urine/stool sample. The viral genome can be amplified by
the molecular biology techniques as PCR from EMB samples
and Their Bottlenecks [Figure 1 (i) and (ii)]. There could be few problems performing
A case of myocarditis has to be confirmed by histological the PCR from EMB samples:(1) After the second phase of
examination, but one has to remain the cautious about observer viral infection, it is difficult to get viral genome from EMB
dependent variables and patchy inflammatory infiltrates sample to make out that resultant dilated cardiomyopathy
causing sampling errors which are limitations to the utility of is due to viral infection,(2) if the sample is not rapidly and
endomyocardial biopsy (EMB). Moreover, the risks of EMB properly transported from the procedure room to the laboratory
should be always taken into the consideration which could be bench, PCR analysis for viral genomes can yield false results.
acute or delayed. The perforation of heart leading to the pericardial Pathogenfree biopsy devices and storage vials are generally
tamponade, ventricular or supraventricular arrhythmias, heart used to prevent the sample degradation and contamination.
block, pneumothorax, puncture of the central arteries, pulmonary There are certain commercially available fixatives such
embolization, nerve paresis, venous hematoma, tricuspid valve as RNAlater which allow PCR and a reverse transcription
damage, and creation of arterial venous fistula within the heart PCR(RTPCR) to be performed on the samples transported on
could be the immediate risks of biopsy. The risks of EMB dry ice at room temperature without loss of sensitivity when
are much variable depending upon: Patients clinical status, compared with frozen tissue that is transported on ice.[49]
operators experience, presence or absence of left bundlebranch
block, the site of access, and bioptome too. Access site bleeding, Polymerase Chain Reaction: Principle and Procedure
tricuspid valve damage, pericardial tamponade, and deep venous Astute observations, dedicated researchers, years of diligence,
thrombosis comprise the delayed complications. The precise perseverance, simple organic molecules to most complex
frequency of these complications is not known as these have DNA/RNA genomes all have contributed to reach the
been only known through case reports.[49,50] Eureka moment of PCR discovery.
Diagnosing myocarditis now is based upon noninvasive cardiac PCR employs a pair of synthetic oligonucleotides or
imaging surmounting the low sensitivity of the Dallas criteria in primers each hybridizes to one strand of a doublestranded
histologically diagnosing myocarditis. Diagnostic noninvasive DNA (dsDNA) target, with the pair crossing a region that
cardiac imaging techniques to detect the myocarditis include: will be exponentially reproduced. The primer hybridizes to
Echocardiography, nuclear imaging, and cardiac magnetic the target gene of interest and acts as a substrate for a DNA
resonance imaging(MRI).[51] polymerase (most commonly derived from the thermophilic
To ascertain the left ventricular function, echocardiography is a bacterium Thermus aquaticus and called as Taq) to bind, which
very prime component of the diagnostic workup and to rule out finally creates a complementary strand via sequential addition
other causes of heart failure as well.[52,53] Tissue characterization of deoxynucleotides. Deoxynucleotide triphosphates(dNTPs,
by sonogram may prove to be more useful despite the anatomic sometimes called deoxynucleotide triphosphates; nucleotides
features on echocardiography (i.e., chamber dimensions, containing triphosphate groups), are the buildingblocks, which
ejection fraction, and wall motion abnormalities) being not help the DNA polymerase to synthesize a new DNA strand.[62,63]
sufficient to differentiate myocarditis from the other forms For setting PCR buffer solution is required, which provides
of cardiomyopathy. Features suggestive of myocarditis by a suitable chemical environment for optimum activity and
echocardiogram are often nonspecific yet can be useful in stability of the DNA polymerase.
identifying a fulminant course, if any. To determine its clinical
utility, additional validation studies are needed usually.[5457] Along with some bivalent cautions such as magnesium or
manganese ions; generally Mg2+is needed for amplification
Cardiac MRI is very promising in diagnosing the myocarditis process[Figure2].[64]
showing an evolution of contrast enhancement from the focal
to disseminated disease and could be useful to diagnose the PCR mostly consists of a series of 2040 repeated temperature
myocarditis associated with edema, hyperemia, or fibrosis changes, called cycles, with each cycle commonly consisting
sensitive sequences.[58-60] To identify the patients who should of 23 discrete temperature variations.
undergo a biopsy, MRI is useful, and could be used as a guided The process can be summarized in three steps:(i) Denaturation:
approach to the abnormal region of the myocardium. Such a dsDNA separates at temperatures>90C, the high temperature
focused approach could improve the sensitivity of EMB to is required to break the hydrogen bonds that connect the two
establish a correct histological diagnosis. The ability of MRI DNA strands. Before starting the first cycle, the DNA and the
to differentiate the viral myocarditis from other causes of acute primers both need to get denatured and completely separated in
dilated cardiomyopathy is still, unfortunately, unclear.[61] to single strands. The time required for this primary denaturation
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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.
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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 primers, polythymine/oligo dT primers, and gene specific
activity after a certain number of cycles. primers:
Random hexamer primers are the short singlestranded
Plateau phase
DNA fragments with all possible combinations of bases.
As the reagents and enzyme are used up and exhausted, no
They are short, nonspecific primers, and will produce pieces
more product forms and piles up.
of cDNA scattered all over the messenger RNA(mRNA)
PCR has displaced some of the established gold standards as molecule. The RT reaction will nonspecifically produce
cell culture and various serological assays.[68] The existing cDNAs from all the mRNA present in the reaction mixture,
combinations of PCR and detection assays have been used to but cDNA would not be of full length[70,71-73]
obtain the quantitative data with promising results. Polythymine(T16) primers/Oligo dT primers are usually
1216 baselong thymine primers that will hybridize with
Polymerase Chain Reaction/Reverse the polyadenine tail at the 3 end of mRNA. For efficiency
of the reaction, it is necessary that mRNA should have
Transcription Polymerase Chain Reaction in polyadenine tail. If the mRNA is degraded then using these
Viral Genome Detection primers would be of less use[7274]
During PCR, a fragment of the viral genome is amplified Gene specific primers are used when only a subset of cDNA
using specific primers as mentioned above. For RNA viruses, from total mRNA is required. Gene specific primers will
synthesis of complementary DNA strand (cDNA) via RT bind only to the targeted region of the mRNA and transcribe
(viral RNA to cDNA) is necessary prior to the PCR. Such PCR only the required sequence[Figure5].[75]
is called as RTPCR.[69] The RT step is not necessary for viruses whose genome is
composed of DNA.
Reverse Transcription Polymerase Chain For the detection of each particular virus, specific sets of primers
Reaction need to be designed. Sequences of conserved regions or genes
During RT, cDNA from RNA is synthesized using a primer found in the viral genome are used for designing of primer sets
which helps the reverse transcriptase(RNAdependent DNA which can hybridize with a number of different members from
polymerase) to bind and make the first strand cDNA. Three a particular viral family. This way, with the use of a limited
types of primers are commonly used: Random hexamer number of primers, many viruses could be screened at a time.
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