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| LAB SCIENCES |
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| Year : 2015 | Volume
: 1
| Issue : 2 | Page : 168-175 |
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Polymerase chain reaction as a diagnostic tool in human viral myocarditis
Nivedita Pathak1, Bimal Kumar Das2
1 Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India
2 Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India
| Date of Web Publication | 30-Sep-2015 |
Correspondence Address:
Nivedita Pathak
Department of Pediatrics, All India Institute of Medical Sciences, New Delhi - 110 029
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/2395-5414.166338
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 cost-effectiveness makes PCR a technique of choice in the diagnostic armamentarium. Keywords: DNA/RNA, endomyocardial biopsy, polymerase chain reaction, primers, viral myocarditis
How to cite this article:
Pathak N, Das BK. Polymerase chain reaction as a diagnostic tool in human viral myocarditis. J Pract Cardiovasc Sci 2015;1:168-75 |
How to cite this URL:
Pathak N, Das BK. Polymerase chain reaction as a diagnostic tool in human viral myocarditis. J Pract Cardiovasc Sci [serial online] 2015 [cited 2022 Jan 23];1:168-75. Available from: https://www.j-pcs.org/text.asp?2015/1/2/168/166338 |
| Introduction | |  |
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 1-year 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.[4],[5],[6],[7],[8] 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 reactions in cardiac tissues,[4] however, as early as in 1806 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.[12],[13],[14],[15],[16],[17],[18],[19],[20],[21],[22]
Here, in this review, we will focus on the use of PCR in the detection of pathogenic viruses in the endomyocardial tissue of the patients of myocarditis.
| Etiology | | |
A number of agents including viruses are came out to be the most common causative agents of myocarditis. With the current techniques and recent approaches, our understanding of viral myocarditis has increased. Viruses known to cause myocarditis are listed in [Table 1].
Coxsackie B viruses are most commonly associated with viral myocarditis. A member of the picornavirus family and the enterovirus genus, it is related closely to other enteroviruses such as poliovirus, rhinovirus, and echovirus. The prevalence of the enteroviruses have been reported in many clinical studies; being an infective agent in viral myocarditis.[23],[24],[25],[26],[27],[28] In a series of experiment around 86% of the healthy adult patients being tested and neutralizing antibody against two serotypes of Coxsackie B were detected in the sera.[8] Numerous respiratory tract viruses namely Epstein–Barr viruses, influenza viruses, adenoviruses, etc., are associated with viral myocarditis.[29],[30],[31],[32],[33],[34] Both in childhood [25],[26] and adulthood cases of myocarditis and dilated cardiomyopathy, adenoviruses are found to be an important causative agent.[35]Cytomegalovirus (CMV) is fairly uncommon in otherwise healthy people and belongs to herpes group of viruses, and is an acknowledged cause of acute infectious myocarditis.[36] In some patients presenting with acute myopericarditis, the CMV-specific genome was detected in the biopsy and myocytes.[37] CMV infection might be considered a more frequent cause of myocarditis than what was previously established. CMV infection is a particular viral disease in transplant recipients, with the involvement of multiple organs also.[37],[38] In transplanted patients treated with ganciclovir for a previous CMV infection, the features of cells are altered and they often neither show the characteristic basophilic inclusions nor cytomegaly in the biopsy sample.[39],[40],[41] Parvovirus B19, the causative agent of erythema infectiosum, also known as fifth disease, has been reported as rare but severe cause of myocarditis in infants and children,[42],[43],[44] also accounted for the conditions such as hydrops fetalis and fetal death.[45],[46] The B19 receptor (erythrocyte P antigen) has been found on fetal myocardial cells, suggesting intrauterine myocarditis be the reason for the development of fetal hydrops.[42] This cardiotropic virus infects most adults at some time during a lifetime. In the pathogenesis of endocardial fibroelastosis, mumps-induced myocarditis has been demonstrated to be the first step. The incidence of the disease, in recent years which was previously considered a significant cause of infant mortality, has phenomenally declined due to vaccination.[47],[48]
| Pathogenesis | | |
A clear understanding of the pathophysiology of progression of heart muscle damage and the development of dilated cardiomyopathy is important in the management of this often fluctuating disease. The pathogenesis of myocarditis is evident by experiments demonstrating virus induced myocarditis in mice models.[18]
Progression of myocarditis comprises of three distinct phases:
- The viral stage is defined as the period of time when the active replication of the live virus is occurring within the myocardium. In this stage, the virus gains access to the cardiomyocytes and induce the innate immune response comprising macrophages, natural killer cells, and various chemical messengers.[19],[20] The innate immune response clears the viral load leaving behind injured cardiomyocytes which result in nonsymptomatic myocarditis.[21] Viruses that successfully avoid the elimination by the innate immune system begin to replicate, producing viral proteins that can cause direct myocardial injury
- The second phase, the immune response is generated, and the viral antigens are detected, processed, and presented by the antigen presenting cells and then killed by the major histocompatibility complex restricted lymphocytes. This destruction of viral antigens can also leave behind the injured cardiomyocytes. In addition, some of the host myocardial tissue share the epitope similarity with the viral epitopes called as molecular mimicry can also lead to the destruction of cardiomyocytes while clearing the viral particles [22]
- The third phase, dilated cardiomyopathy which is a result of viral and autoimmune injury of the cardiomyocytes, with the disappearance of the pathological signs of myocarditis and an increase in tissue fibrosis.[19] In most cases, it may not even be possible to determine whether the inciting event was a viral infection.
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: Patient's clinical status, operator's experience, presence or absence of left bundle-branch 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 work-up 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.[54],[55],[56],[57]
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],[59],[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]
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. Pathogen-free 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] | Figure 1: (i) Course and pathophysiology of viral myocarditis (in vivo) 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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| 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 double-stranded 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 building-blocks, 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 Mg 2 + is needed for amplification process [Figure 2].[64] | Figure 2: 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.
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PCR mostly consists of a series of 20–40 repeated temperature changes, called cycles, with each cycle commonly consisting of 2–3 discrete temperature variations.
The process can be summarized in three steps: (i) Denaturation: dsDNA separates at temperatures > 90°C, 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 is up to 5 min. Taq-polymerase 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 single-stranded 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 50–65°C) and can be calculated for each set of primers. The optimum annealing temperature should be 5°C below the melting temperature of the primer sequence. Annealing of primers at wrong temperature during this step can 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 5'-phosphate group of the dNTPs condenses with the 3'-hydroxyl 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, 1-min/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 72–78°C 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 [Figure 4]a,[Figure 4]b,[Figure 4]c.[65],[66],[67] | Figure 3: 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.
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 | Figure 4: (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.
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| 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 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 (RNA-dependent 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 single-stranded 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],[72],[73]
- Polythymine (T16) primers/Oligo dT primers are usually 12–16 base-long 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 [72],[73],[74]
- 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 [Figure 5].[75]
 | Figure 5: 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.
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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. 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 PCR product. However, the confirmation of the PCR product is recommended by sequencing or other assays.
Real-time 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. Real-time 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 post-PCR detection procedures such as gel electrophoresis. Various diagnostic methods and their advantages/disadvantages are discussed in [Table 2].
| Conclusion | | |
Today's, the molecular techniques such as PCR, real-time 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.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]
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