M yocarditis and inflammatory cardiomyopathy: microbiological and

Cardiovascular Research 60 (2003) 11–25www.elsevier.com/ locate/cardiores

Review

M yocarditis and inflammatory cardiomyopathy: microbiological andmolecular biological aspects

*Fiorella Calabrese , Gaetano ThieneDepartment of Pathology, University of Padua Medical School, Via Gabelli, 61, 35121Padua, Italy

Received 5 February 2003; received in revised form 20 May 2003; accepted 5 June 2003

Abstract

Myocarditis is an inflammatory disease of the myocardium associated with cardiac dysfunction. The natural history of myocarditis isfrequently characterised by the evolution in dilated cardiomyopathy. Due to its variable clinical manifestation from latent to very severeclinical forms, such as acute congestive heart failure and sudden death, its prevalence is still unknown and probably underestimated. Inspite of the development of various diagnostic modalities, early and definite diagnosis of myocarditis still depends on the detection ofinflammatory infiltrates in endomyocardial biopsy specimens according to the Dallas criteria. Routine application of immuno-histochemistry, used for identification and characterisation of inflammatory cell populations, has now significantly increased thesensistivity of the diagnosis of inflammatory cardiomyopathy. Various molecular techniques, such as PCR, gene sequencing and real-timePCR, often applied on the same endomyocardial specimen, have become an essential part of the diagnostic armamentarium for rapid,specific and sensitive identification of infective agents. The correct application of molecular techniques will allow increasingly moreinformation to be obtained: new epidemiology, new patient risk stratification and overall more appropriate medical treatment. 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Keywords: Myocarditis; Cardiomyopathy; Inflammation; Inflammation/ infection

1 . Introduction: role of endomyocardial biopsy sis, is a frequent observation. This subtle substrate, to-gether with the possible inflammatory involvement of the

According to the current WHO classification of car- conduction system, seems highly arrhythmogenic and maydiomyopathies, myocarditis is an inflammatory heart mus- account for unexpected arrhythmic cardiac arrest[3].cle disease associated with cardiac dysfunction, and dilated Heterogeneity of symptoms, including subclinical orcardiomyopathy (DC) may represent the chronic phase of asymptomatic forms, could be the reason why the preval-the disease[1]. Clinical features of the disease are variable ence of myocarditis is still unknown and probably under-including unexplained congestive heart failure, chest pain estimated. The large spectrum of clinical forms—frommimicking myocardial infarction[2], arrhythmias, syncope subclinical to severe—depends on several factors such asand sudden death. Sudden death may occur both in the genetic determinants of infective agents, the genetics, ageactive or healed phases as a consequence of life-threaten- and gender of the host, and host immunocompetence.ing ventricular arrhythmias that develop mostly in the In spite of the development of various diagnosticsetting of an unstable vulnerable myocardial substrate, modalities, early and definite diagnosis of myocarditis stillnamely inflammatory infiltrates, interstitial edema, myocar- depends on the detection of inflammatory infiltrates indial necrosis and fibrosis. Patchy inflammatory infiltrate, in endomyocardial biopsy (EMB) specimens according to thethe form of a starry sky-like feature (,14 leucocytes/ Dallas criteria[4]. The major limitation of EMB remains

2mm ), and not necessarily associated with myocyte necro- the low sensitivity due to sampling error, particularly inthe presence of focal disease. The size and number of theheart biopsy specimens as well as the processing of*Corresponding author. Tel.:139-049-827-2268; fax:139-049-827-

2269.E-mail address: [email protected](F. Calabrese). Time for primary review 21 days.

0008-6363/03/$ – see front matter 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.doi:10.1016/S0008-6363(03)00475-9

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12 F. Calabrese, G. Thiene / Cardiovascular Research 60 (2003) 11–25

myocarditis of|50% using four to five biopsy samples.When 17 biopsy specimens per case were considered, thesensitivity reached 79%[7]. It has also been shown thatserial sectioning and multiple level examination of EMBsincrease the sensitivity of EMB in the evaluation ofmyocarditis[9,10], thus facilitating the diagnosis of focalmyocarditis with a rate of sensitivity similar to that indiffuse myocarditis[10].

Although some authors have demonstrated that biven-tricular EMB (obtaining several samples from multipleareas of the left and right ventricles) may improve thesensitivity in the detection of myocarditis[11], it should beemphasised that this aggressive approach increases the riskof complications. However most experts in the field agreethat an actual increase in the sensitivity of EMB has nowbeen reached using immunohistochemistry together withroutine histology. A large panel of monoclonal and poly-clonal antibodies is now mandatory to identify and char-acterize the inflammatory cell population as well as theactivated immunological processes[12–15].

Even though sampling and processing techniques havesignificantly increased the sensitivity of EMB in theevaluation of myocarditis, morphological analysis today isstill greatly limited in the detection of infective pathogens,particularly viral agents, the commonest cause of inflam-matory cardiomyopathy (IC). Viral myocarditides usually

Fig. 1. Adenoviral (AV) myocarditis in a young adult man (37 years old). lack specific cytopathic effects (Fig. 1a,b), especially those(a) Histology of EMB showing lymphomonocyte inflammatory cell caused by RNA viruses. With the exception of some rareinfiltrates associated with myocyte injury. No viral inclusion bodies are

forms of cytomegaloviral myocarditis, these effects, whenevident. Hematoxylin and eosin3100. (b) PCR analysis for adenovirus:observed, neither necessarily imply the presence nor arelane 1: DNA marker; lane 2: AV positive control; lane 3: EMB AV PCR

positive; lane 4: beta-globin (house-keeping gene); lane 5: negative useful in detecting the type of virus, since they maycontrol (reagents without template). represent degenerative changes or myocyte nuclear hy-

perplasia.

specimens are known to influence the sensitivity of EMBin the detection of myocarditis. 2 . Etiology

The greater sensitivity of the Stanford-Caves and Cordisbioptomes over other small bioptomes has been proven by IC has multiple causes, both non-infectious and infecti-previous reports[5,6]. In particular, the number of biopsy ous, and viruses account for the disease in the majority ofsamples also directly increases the likelihood of detecting paediatric cases (Table 1).foci of myocarditis. Chow et al.[7] and Hauck et al.[8] Different cardiotropic viruses have been implicated inindependently reported a sensitivity for detection of more than 50% of paediatric cases with IC[16,17]. The

T able 1Myocarditis: infective agents

Viruses RNA Picornavirus (entero, rhino); orthomyxovirus (influenza A,B); paramyxovirus (rubeola, mumps);retrovirus (HIV-1); hepatitis C virus (HCV)

Viruses DNA Adenovirus, herpesvirus (cytomegalo, Epstein–Barr virus, varicella-zoster), Parvovirus BigBacteria Staphylococcus, Streptococcus, Pneumococcus, Meningococcus, Gonococcus,

Salmonella, Corynebacterium diphtheriae, Haemophilus influenzae,Mycoplasma pneumoniae, Brucella species

Mycobacteria Tuberculosis, Avium intracellulare,LepraeFungi Aspergillus, Candida, Actinomyces, Blastomyces, Cryptococcus, HistoplasmaProtozoa Toxoplasma gondii, Trypanosoma cruziRickettsiae Coxiella burnetti (Q fever),Rickettsia rickettsii, Rickettsia tsutsugamuschiChlamydiae Trachomatis, PsittaciParasitic Trichinella spiralis

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F. Calabrese, G. Thiene / Cardiovascular Research 60 (2003) 11–25 13

remaining forms may be triggered by other environmental the characteristic cytomegaly, and the intranuclear inclu-factors such as toxins, alcohol, cytotoxic chemotherapy, sions may not be basophilic[34–36].metabolic abnormalities or more frequently sustained by During or following mumps, mild or subclinicalimmunologic mechanisms. These latter may characterize myocardial infection may be present with 15% of casesthe autoimmune myocarditis ab initio or more often are the showing ST-T wave changes. Mumps-induced myocarditisconsequence of viral myocarditis itself[18]. has been demonstrated to be the first step in the patho-

Among cardiotropic viral agents, coxsackie viruses are genesis of endocardial fibroelastosis[17,37]. In recentthought to be most frequently implicated[19,20]. Group B years the incidence of the disease, which was previouslycoxsackie viruses consist of six serotypes within the considered a significant cause of infant mortality, hasenterovirus genusPicornaviridae, a large family of single- dramatically declined, probably due to mumps vaccine.stranded positive-sense RNA viruses. In the WHO record Parvovirus B19, the causative agent of erythema infec-during the 10-year period from 1975 through 1985, the tiosum, also called fifth disease, has been reported to be acoxsackie B viruses represent the most frequent inflamma- rare but severe cause of myocarditis in infants and childrentory agents in cardiovascular disease (34.6 per 1000), [38–40]. The virus has also been considered as beingfollowed by influenza B virus (17.4 per 1000), influenza A responsible for hydrops fetalis and foetal death[41,42].(11.7 per 1000), coxsackie A (9.1 per 1000) and cyto- The B19 receptor (erythrocyte P antigen) has been recog-megalovirus (CMV) (8.0 per 1000)[21]. nized on foetal myocardial cells[38,42] suggesting that

The prevalence of the enteroviruses as infective agents intrauterine myocarditis contributes to the development ofassociated with myocarditis has been reported in several fetal hydrops after parvovirus B19 infection.clinical studies[19–22]. Patients with IC had significantly The importance of hepatitis C virus (HCV) in patientshigher serological titres when compared with controls with myocarditis and DC has recently been stressed[43–[23–25]. In contrast, a coxsackie virus infection could be 45]. Myocyte tropism has also been demonstrated byproven in only 2% of consecutively studied myocarditis Takeda et al. using in situ hybridisation[46] and recentlypatients in a 5-year study at a military hospital in Finland, by our group using immunohistochemistry with TORDJI-where traditional enteroviral diagnostic methods were used 22 monoclonal antibody[45].[26]. Variable aetiology was found and was mainly repre- IC caused by other non-viral infective agents is nowa-sented by vaccine inoculation, followed by other infective days rare. It usually occurs in immunosuppressed patientsagents such as adenovirus, Epstein–Barr virus,Myco- with secondary involvement of the myocardium.plasma pneumoniae and Chlamydia. Myocarditis is the most common cause of death in

Using molecular techniques, the frequency of myocar- diphtheria by the action of exotoxin produced bydial enteroviral infection is now estimated in|30–50% of Corynebacterium diphtheriae. Although still a cause ofcases with infectious etiology[16,17,27,28].Various respi- morbidity and mortality in developing countries[47],ratory tract viruses—adenoviruses, Epstein–Barr viruses diphtheria is now rare in the West, most probably because(EBV), influenza viruses, etc.—may cause myocarditis at of vaccination. It re-appeared in Scandinavia in the mid-variable frequencies[20,21,26,29]. In particular, adeno- 1980s, mostly in alcoholics[48].viruses have been shown to be an important cause of Non-rheumatic myocarditis may be a complication ofmyocarditis and dilated cardiomyopathy both in childhood beta-haemolytic streptococci (Streptococcus pyogenes),[16,17] and adulthood[30]. due to bacterial exotoxins[49]. Rheumatic carditis with

It is noteworthy that rhinovirus-associated myocarditis involvement of both the valvular apparatus andhas been rarely reported in the literature[31], although perimyocardium accounts for the majority of pediatricseveral cases have been registered with the U.S. Centers hospitalizations and cardiac deaths world-wide, particu-for Disease Control[21]. larly in developing countries. Immune responses to group

Cytomegalovirus (CMV) is a recognized cause of acute A streptococcal antigens during pharyngitis, resulting ininfectious myocarditis in the herpes group of viruses, antibodies and immunocompetent cells that cross-reactalthough CMV is considered to be uncommon in previous- with myocardial antigens, are thought to be central to thely healthy people[32]. However some authors detected a pathogenesis of rheumatic carditis. The recent finding ofCMV-specific genome in the myocytes of EMBs in up to enteroviral RNA replication and protein synthesis in15% of patients with acute myopericarditis[33]. Therefore valvular tissue from patients with chronic rheumatic heartCMV infection may be considered a more frequent cause disease may provide intriguing evidence for a viral etiolo-of myocarditis than was previously thought. CMV in- gy or cofactor, at least in some cases[50]. Bacterialfection is a peculiar viral disease in transplant recipients, endotoxin is also considered to play a role in meningococ-with multiorgan involvement[32–34], since specific cyto- cal myocarditis. Myocarditis has been found in up to 78%pathic features are only exceptionally observed. In trans- of cases of meningococcal septicaemia with marked endo-planted patients, treated with ganciclovir for a previous toxinaemia[51].CMV infection, the biopsy features of CMV myocarditis Even though rarely, myocarditis has been found inappear modified since the infected cells often do not show Salmonella septicaemia caused bySalmonella typhi or

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paratyphi. Yersiniosis myocarditis, probably more frequent the increasing number of immunocompromised patients.than is commonly thought, is in most cases a quite mild The major risk factors for severe fungal disease includeform and considered to be immune in the pathogenesis administration of broad-spectrum antibiotics, corticoste-[52]. Myocarditis is a well documented complication (1– roids and cytotoxic agents, invasive medical procedures8% of cases) inBorrelia burgdorrferi infection. In- and HIV. In this latter condition the association of fungalvolvement of the specialized myocardium may account the and other infective agents may be quite frequent[67].onset of atrioventricular block[53]. Mycoplasma pneumo- Pre-mortem diagnosis of fungal myocarditis is difficultniae infection is commonly accompanied by mild myocar- since clinical findings of myocardial involvement are oftenditis with up to 33% of cases exhibiting ECG changes[54]. absent or ambiguous, blood cultures are often negative and

Chlamydia psittaci infection is complicated by sub- other laboratory tests are not effective.clinical or asymptomatic myocarditis in 5–15% of cases Among parasitic infections,Trichinella spiralis is most[55], but cardiac insufficiency may be marked[56]. In prone to cause myocarditis, however other elmints canChlamydia trachomatis infection myocarditis is rare. A infect the myocardium usually in the setting of multi-organfew published cases have been reported in the literature, involvement (Fig. 2a–d).even fatal, with the majority of these involving smallchildren [55]. Chlamydia pneumoniae infection combinedwith myocarditis has been also described as a mild form 3 . Pathogenesis[57], although one case of sudden death in a young athlete[58]. Acute myocarditis, more frequently viral forms, can be

Cardiac involvement is often found in the setting of resolved without sequela, however progression in therickettsial infection with vasculitis as a prominent feature, chronic form, DC, is not a rare event. Two differentbecause the rickettsiae have special tropism for endothelial theories have been recognized to explain myocyte damagecells. Even though endocarditis is considered more com- and the progression from acute myocarditis to chronicmon, myocarditis may also present in Q fever caused by forms/DC: autoimmunity and direct cytotoxicity due toCoxiella burnetii [59]. persistent viral infection.

Tripanozoma cruzi (Chagas’ disease) is a well-recog-nized cause of myocarditis and cardiomyopathy in both 3 .1. Autoimmunityurban and rural areas of South America[60]. Also theAfrican trypanosomes,Tripanozoma gambiense and Tri- In 1957 Witebsky et al., modelling on Koch’s post-panozoma rhodesiense, occasionally cause myocarditis and ulates, proposed rationales to explain the autoimmuneinflammatory cardiomyopathy[61]. basis of clinical disease: an autoimmune response, as either

Toxoplasma gondii poses significant problems among humoral or cell-mediated, is recognized when a specificrecipients of cardiac transplants. A study reported that 57% antigen is identified and when an analogous autoimmuneof transplanted patients lacking antibodies to that agent response is induced in an experimental animal[68].developed toxoplasma myocarditis[62]. However, tox- Aberrant cellular and humoral immune responses haveoplasmosis may become reactivated in antibody-positive been proposed to explain the progression of viral myocar-transplant recipients, and myocarditis has been reported in ditis.4–53% of transplant cases. This great variation rate isprobably due to differences in antibody testing methods 3 .1.1. Aberrant cellular immune response[63]. After the introduction of pyrimethamine prophylaxis, The original discovery that T-cells play a major role inthis complication has decreased substantially. the pathogenesis of coxsackie virus B infection was

Myocarditis seems to be quite frequent in AIDS pa- reported over two decades ago. Mice depleted of T-cells bytients, with a rate of 45–52% of cases[64]. Cardiac pre-treatment with antithymocyte serum and then infecteddysfunction in AIDS may, however, have various forms of with coxsackie virus B3 exhibit a marked reduction inpathogenesis. The aetiology of myocarditis in AIDS is also myocardial disease. The degree of cardiac inflammationvariable and may be difficult to establish, especially in and necrosis was much less than that found in immuno-cases with multiple opportunistic infections. Viruses are logically intact mice or in mice reconstituted with bothconsidered to be the most common cause, such as the bone marrow and thymus cells[69].human immunodeficiency virus (HIV) itself and coxsackie Inflammation of the heart muscle can be transferred intoviruses. In one autopsy study,Toxoplasma gondii infection non-immunized recipient mice by purified T-cells fromof the myocardium was found in 12% of cases[65]. mice with active myocarditis[70].

Fungal myocarditis frequently occurs in the setting ofdisseminated disease. The major fungal pathogen respon-3 .1.2. Aberrant humoral immune responsesible for myocardial infection isAspergillus fumigatus The hypothesis that humoral immunity plays a role in[66]. The incidence of invasive fungal disease has dramati- the development of post-infectious myocarditis is derivedcally increased over the past few decades corresponding to from the finding that immunization with cardiac myosin in

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Fig. 2. EMB in multiorganToxocara canis infection. Toxocara larva in myocardium (hematoxylin and eosin3100) (a) with birefringence of the larva atpolarized light (b) and giant cell granulomatous reaction hematoxylin and eosin360 (c). Toxocara larva in liver biopsy (cross-section), hematoxylin andeosin330 (d). Courtesy of Dr F. Bomio, Lugano, Switzerland.

susceptible strains of mice induces myocarditis[71]. This deduced amino acid sequence of viral precursor polypro-result was further supported by subsequent studies: mice tein with the sequence of murine cardiac myosin showedimmunized with cardiac C protein[72] or streptococcal M however a very low degree of genetic homology[80].protein peptide[73] developed T-cell dependent myocar- It is very likely that the development of autoimmuneditis, or adoptive transfer of myosin-reactive cells or myocarditis following viral myocarditis is sustained by asplenocytes after myocardial infarction[74] or transplanta- complex interaction between cellular and humoral immunetion of a normal heart into a virus-treated host after response as proposed by Rose and Hill[80].documentation of viral clearance[75] led to the develop- Abnormal human leucocyte antigen (HLA) expressionment of T-cell dependent myocarditis. Even though cel- on endomyocardial biopsy[12,13,81] increased levels oflular necrosis may explain the presentation of cardiac circulating cytokines and cardiac autoantibodies[82,83]released myosin to the immune system thus triggering the are similarly also found in human myocarditis /DC, highlyautoimmune disease, recent studies suggest that molecular suggestive of an immune pathogenetic mechanisms of viralmimicry may also be involved in the pathogenesis of myocarditis in the clinical setting.autoimmune myocarditis.

The finding of cross-reactive epitopes between cardiac 3 .2. Persistent viral infectionmyosin and infectious agents confirms this concept[76–78]. Molecular mimicry has been proposed as an explana- Continuous virus replication or persistent viral infectiontion for coxsackie virus B3-induced autoimmune myocar- in the heart could play a role in the maintenance and/orditis. The hypothesis is supported by data from studies of progression of disease.monoclonal antibodies directed against coxsackie virus Molecular investigations have shown coxsackie virus B3group B that recognizes epitopes on murine cardiac myosin to persist in mouse myocardium in different models for up[79]. Comparison of the nucleotide sequence and the to 56 days[84,85] and in hamsters for up to 180 days[85].

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Viral RNA was found to persist from 80 days to 12 months above), have various limits because of low sensitivity andin other non-productive picornavirus myocardium infec- specificity.tions in mice[86]. Molecular studies in various experimen-tal models show that enterovirus can persist in the chronic 4 .1. Culturestages of myocarditis[87]. The mechanisms of enteroviruspersistence so far is not well known. A defective virus, It is important to recognize that the traditional methodsmainly resulting from the altered function of the viral RNA of pathogen identification by culture isolation, phenotypicpolymerase[88], is now considered to be one of the expression via biochemical reactions and determination ofpossible mechanisms of persisting enteroviral infection. antibiotic sensitivity remain the gold standard for|90% ofTherefore acute myocytolytic inflammatory disease infectious disease pathogens. However many infectiveevolves to persistent infection with a defective virus, agents, particularly viruses, can not be cultivated as theyunable to induce cytolysis or elicit the host cellular are very difficult to isolate.immune response. For many viruses, such as enteroviruses, isolation

Tam and Messner recently demonstrated that viral RNA procedures are poorly standardized and virus isolation dataperseveres in a double-stranded form in a murine model of may vary considerably among laboratories. The type andcoxsackie virus B1 infection of skeletal muscle. It is quality of the specimen, the timing of specimen collection,possible that double-stranded RNA is resistant to ribonu- and storage before arrival in the laboratory can highlyclease activity and is connected to reduced viral antigen influence the sensitivity[96]. The choice of cell types usedexpression, which in turn minimizes immune-mediated for virus isolation is also important. For example no singlekilling of persistently infected cells[89]. Therefore, if cell line supports the growth of all known enteroviruscoxsackie virus B3 persists in a double-stranded form, it serotypes[97] and many serotypes of adenovirus do notmay be responsible for the long half-life of viral RNA replicate efficiently in conventional cell cultures (Hep-2during the chronic phase of infection. and A549 cells)[98].

Recently some authors have demonstrated that coxsackie Virus isolation from blood components is useful forvirus B3 protease 2A can directly cleave dystrophin in providing evidence of systemic infection but not formyocytes, these data support the idea of a mechanism of providing information on specific organ disease.direct viral destruction during the pathogenesis of chronic Detection of enterovirus in the alimentary tract (throatmyocarditis /DC[90]. swabs, rectal swabs, or stool specimens) allows only

The activation of mitogen-activated protein kinase circumstantial evidence of etiology, since viral shedding at(MAPK) as a consequence of the P21 guanosine tri- these sites may occur in asymptomatic subjects, especiallyphosphatase-activating protein (RasGAP) cleavage, has in infants and during epidemic seasons[99].been more recently suggested to also play a role in the Isolation of a virus, particularly of enterovirus fromdevelopment of chronic myocarditis. The MAPK pathway affected tissues of patients with severe inflammatoryis a mechanism employed by different viruses for control myocardial diseases, has seldom been successful[100–of cell proliferation and survival[91]. During enteroviral 103].replication, activation of MAPKs may participate in themobilization of intracellular calcium and may thereby 4 .2. Serologycontribute to morphological and physiological destructionof infected myocytes[92]. Following the isolation of a virus, the identification and

Several studies have also documented viral persistence overall serotypic characterization can be determined byin the myocardium of patients with DC until end-stage neutralization of infectivity with serotype-specific antisera.disease requiring cardiac transplantation[28,93–95]. Serologic diagnosis requires demonstration of IgM

antibody or a fourfold increase in IgG antibody titre whenpaired serologies are obtained and is therefore retrospec-

4 . Diagnostic tools for infective myocarditis tive.Every viral family includes, however, a large number of

Different diagnostic tools are known to detect infective serotypes (among the family Picornaviridae, 66 serotypesagents responsible for IC. Non-viral infective agents are have been identified for the genus enterovirus) and typingoften morphologically distinctive and identifiable by direct by neutralization, with reference antisera for all serotypesmicroscopic examination with routine and special stains individually, is surely not practical. In order to overcome(Gram’s, Gomori’s, periodic acid-Schiff, Ziehl-Neelsen, this problem for the enterovirus family, equine type-spe-etc.). For these forms microbiologic culture and other cific hyperimmune sera have been mixed to providelaboratory tests are considered important for definitive intersecting pools containing different combinations ofidentification of causative infective agents. individual antisera[96].

For viruses, the main pathogens of IC, traditional Serotypic identification of an enterovirus isolate general-diagnostic tools, including morphological analysis (see ly takes 1–2 weeks and usually has little impact on patient

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care. Consequently, in the clinical setting a generic diag- target sequence (i.e. the pathogen in question) from verynosis of ‘enterovirus infection’ it is often put forward low numbers, as RNA viral genomes usually are, towithout further specifying the serotype[97]. literally millions of copies without cloning technology.

Another important limit of serological analysis is in the The decision to develop and apply PCR for routinehigh background prevalence of these responses. This likely diagnosis of infective myocarditis must be considered inreflects the ubiquity of many viruses (enterovirus, adeno- relation to the speed, sensitivity and specificity of morevirus, etc.), which circulate seasonally among the general conventional culture and/or serological methods[16,17].population. Moreover immunoglobulin M responses may However it should be stressed that the presence offrequently be detectable for 6 months or longer after acute bacterial or viral nucleotide sequences does not auto-infection (such as for enterovirus)[104], may decline very matically imply a direct role in the pathogenesis ofrapidly resulting in negative values after only a few weeks myocarditis. Indeed an infective agent detected by molecu-(such as parvovirus B19)[105] or can result as a false lar techniques, especially with PCR/nested-PCR, may notnegative in immunodeficient persons or children younger be a cofactor but just an innocent bystander. Therefore it isthan 4 years (such as for EBV)[106]. extremely important to use a molecular technique as a

Direct detection of different antigens in peripheral blood diagnostic tool ancillary to other mandatory investiga-leukocytes by immunocytochemical staining (antigenemia) tions—clinical and morphological—and apply it withis now used efficiently for the diagnosis of different skilled expertise.infective agents, particularly for viruses. Direct detection Enterovirus, particularly coxsackie virus B, was the firstof the lower matrix phosphoprotein (pp65) in peripheral virus investigated in myocarditis / IC based on coxsackieblood leukocytes has now been demonstrated to be a very virus B3 experimental studies that provided inferential datasensitive tool for the diagnosis and monitoring of CMV to implicate enteroviruses as etiologic agents in humaninfection. However the tool is almost laborious and shows myocarditis[20,108,109]. Several reports have investi-quite low sensitivity for detecting the very early or chronic gated the presence of enteroviral genome using PCR,phase of the disease[107]. overcoming the limits of the non-specificity of slot-blot

and the low sensitivity of in situ hybridization and4 .3. Molecular biology technical problems particularly when RNA probes are used

[110,111].4 .3.1. Etiological diagnosis of inflammatory cardio- However enteroviral RNA amplification studies, per-myopathy formed either on EMB specimens or on fragments from

The development of molecular biological techniques, whole heart, have produced controversial data[94,112–particularly amplification methods such as polymerase 125].chain reaction (PCR) or nested-PCR, allows the detection InTable 2the principal studies that investigated only theof low copy of viral genomes even from an extremely presence of enterovirus are reported: the rate of viralsmall amount of tissue such as endomyocardial samples. amplification ranged from 0 to 80% for myocarditis andPCR is an enzymatic amplification technique whereby very from 0 to 57.9% for DC. The wide discrepancy in resultsfew copies of RNA or DNA sequences can be amplified could have different explanations. It may be due to themore than a million-fold. This allows transformation of a different detection procedures adopted by the investigators

T able 2Enteroviral PCR proven myocarditis /dilated cardiomyopathy

Authors, year Material Myocarditis DC

Weiss et al.[112], 1992 EMB – 5/11 (45%)Grasso et al.[113], 1992 NH – 0/21(0%)Katsuragi et al.[114], 1993 NH 0/1 (0%) 3/11 (27%)Schwaiger et al.[115], 1993 EMB – 6/19 (31.6%)Giacca et al.[116], 1994 EMB 1/3 (33%) 4/53 (7%)Khan et al.[117], 1994 EMB – 16/8 (50%)Nicholson et al.[118], 1995 NH 5/6 (83%) –Ueno et al.[119], 1995 EMB 4/5 (80%) 7/42 (17%)Andreoletti et al.[120], 1996 EMB – 11/19 (57.9%)Muir et al. [121], 1996 NH 5/75 (6.6%)Fujioka et al.[122], 1996 EMB 6/31 (19%) 5/28(18%)Archard et al.[123], 1998 EMB – 9/21 (42.9%)Pauschinger et al.[30], 1999 EMB 18/45 (40%) –Calabrese et al.[94], 1999 NH 1/2 (50%) 1/27 (3%)Li et al. [124], 2000 EMB 6/11 (54.5%) 3/8 (37.5%)Crespo-Leiro et al.[125], 2000 NH – 0/68 (0%)

DC, dilated cardiomyopathy; EMB, endomyocardial biopsy; F, female; M, male; NH, native heart.

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as well as to the prevalence of enteroviruses in local Patients were not, however, evaluated for the presence ofpopulations and the stage of the disease. This discrepancy microorganisms, and thus many of the patients treated withemphasizes the need for a standardization of protocols immunosuppressive therapy may have harboured viral(RNA extraction, reverse transcriptase and PCR reaction) material, not detected by routine morphology. It is wellto achieve the same level of sensitivity as the PCR known that, when there is active viral replication, immuno-technique used in different molecular laboratories. More suppression may be harmful. Our retrospective studyrecently other cardiotropic viruses have been investigated confirms the inefficacy of immunosuppressive therapy inusing molecular techniques and it has been shown that patients with per proven viral myocarditis[83]. Immuno-enterovirus is not the sole viral cause of IC. Adenovirus, suppressive therapy has been demonstrated to have benefi-especially in pediatric myocarditis, is another key etiologi- cial effects only when the autoimmune reaction is associ-cal agent that should be investigated when the etiology is ated with IC[85,132].sought: adenovirus were found to be more prevalent than Different molecular strategies have now been developedenterovirus in the case series of Martin et al.[16] and as and applied to improve identification of an infective agentcommon as enterovirus in our recent study[17] performed thus achieving a more complete etiological diagnosis.on children where myocarditis was the most frequent cause Positive PCR results obtained on EMB should be alwaysof heart failure. investigated on a blood sample collected at the time of

EMB may be technically difficult to carry out in biopsy. A type of algorithm might be developed forchildren for a diagnosis of myocarditis, which frequently conclusive molecular diagnosis of myocarditis. The ab-complicates infectious respiratory disorders. sence of a viral genome in the blood sample rules out the

Recently Towbin’s group have demonstrated that trache- possibility of passive blood contamination while viralal aspirates may be a useful substrate for identification of blood positivity requires additional investigation usingcausative agents by PCR analysis in young patients with quantitative PCR analysis. The presence of viral genomicmyocarditis and presumed pneumonitis. In this study all copies, significantly less in the EMB than in blood, doesPCR performed on EMB specimens demonstrated results not necessarily mean that the heart is infected due toidentical to those obtained by tracheal aspirate PCR[126]. viremia, a situation that has recently been reported in a

Even though enterovirus and adenovirus are the princi- case of fatal parvovirus myocarditis[40].pal infective agents detected in myocardial specimens from On more than 600 EMBs (from 1992 to 2001) frompatients with IC, other cardiotropic viruses are now seen as patients with IC investigated by PCR in our molecularimportant pathogens[43,127,128].Thus a specimen from laboratory, concomitant viral detection, both in blood andpatients with clinical suspicion of IC should always be EMB, was observed in only one EBV positive case. Theinvestigated for at least for eight cardiotropic viruses: frequent occurrence of negative PCR blood finding hasenterovirus, adenovirus, CMV, EBV, parvovirus b19, HCV been reported by other authors[16,17]. This is in keepingand influenza virus A and B. An exact etiological diag- with the hypothesis that the viruses are in the blood onlynosis, providing new prognostic information as well as for a brief period followed by rapid clearance from thespecific therapeutic indications, is now extremely impor- bloodstream, whereas false negatives may be due to poortant for appropriate management of patients. handling of blood samples.

Viral infection represents an independent unfavourable Among molecular biological techniques used to dif-prognostic factor in patients[27], particularly in the ferentiate viral genomes[24,116,133,134],gene sequenc-paediatric population, affected by IC either as acute ing is the most informative allowing not only exactmyocarditis or DC[16,17]. In our experience all patients characterization of the infective agent but also elucidationwith viral PCR proven DC underwent heart transplantation of the molecular basis of cardiotropism as well as car-or died within 1 year of diagnosis[17]. diovirulence.

The importance of specific viral treatment in cases with Genomic sites determining the virulence phenotypes areproven molecular diagnosis has been recently stressed by now considered the principal factors responsible for theseveral authors[17,28,82,129]. severity of the disease. Different reports have documented

Apart from the ongoing European Study of Epidemiolo- that the quantity of virus recovered from the myocardiumgy and Treatment of Cardiac Inflammatory Disease did not correlate with the extent of myocardial lesions.(ESETCID) trial [130], the Myocarditis Treatment Trial is This is particularly true for RNA viruses, such as en-the only large multicenter randomized study to have ever teroviruses. Most of the efforts of the experts in the field,been accomplished[131]. Patients in this trial, classified using strains engineered in the laboratory, have beenwith active myocarditis based on the Dallas criteria, principally addressed to the study of the coxsackie virus Breceived immunosuppressive therapy (either azathioprine genome[135,136].A recent work, which used two pheno-and prednisone or cyclosporine and prednisone vs. typically and genotypically distinct clinical CVB3 strainsplacebo) in addition to conventional therapy. The results for constructing six intratypic chimeric viruses, has iden-were inconclusive without any hemodynamic or prognostic tified cardiovirulence determinants in the coxsackie virusbenefit after 6 months of immunosuppressive treatment. B3 59 non-translated region (59NTR) [137].

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Identification of cardiovirulent strains could provide notonly important prognostic indications on the clinicalprogression of the disease but also useful therapeuticsuggestions on responsiveness to specific medical treat-ment. Moreover, in the future the knowledge of genomicsites which determine the cardiovirulence could lead to thedevelopment of vaccines for patients with a major risk oflethal complications[138].

It is now equally important to have more preciseetiological characterization in order to exactly define theinfective status of viruses. The detection of viral replica-tion has been demonstrated to be quite useful not only forthose DNA viruses known to be in the latent form, but alsofor RNA viruses. Different authors have shown that thedetection of CMV mRNA transcripts (early and late gene)may be considered diagnostic markers of the disease withexpression of early gene preceding the antigenemia de-tection in most cases[139]. Both qualitative and quantita-tive determination of human CMV immediate-early andlate (pp67) transcripts are now used for monitoring ofCMV infections in the post transplantation period[140].

During viral replication of the enterovirus genome, asingle-strand monocistronic RNA of positive polarity(plus-strand) acts as a template for the transcription of areplicate minus-strand RNA intermediate through the

polRNA-dependent RNA polymerase (3D ). This minus-polstrand RNA is then used as a template again for the 3D

to generate multiple copies of viral plus-strand genomesthat are translated into enteroviral structural proteins andultimately packaged into new virions. The identification ofminus-strand enteroviral RNA is an indicator of activeviral replication in enteroviral positive cases (positivestrands). Some authors have emphasised the importance ofdistinguishing between active viral replication and thelatent persistence of enteroviral genomes not only forprognosis with different clinical course but also for a more

Fig. 3. AV myocarditis in a female child (2 years old). (a) Histology ofappropriate treatment regime[17,28,141]. A high fre-EMB showing focus of lymphocytes associated with myocyte necrosis,

quency of active enteroviral RNA replication was detected hematoxylin and eosin3100; (b) PCR analysis for AV: lane 1: DNAnot only in patients with myocarditis or clinically sus- marker; lane 2: beta-globin (house-keeping gene); lane 3: AV positivepected myocarditis but also in patients with end stage control; lane 4: EMB AV PCR positive; lane 5: negative control (reagents

without template); (c) histology of EMB, 6 months after therapy withDCM [17,28], where the active viral replication wasspecific immunoglobulin; note the complete absence of inflammatory cellassociated with a poor prognosis.infiltration, hematoxylin and eosin360; (d) PCR analysis for adenovirus:

Similar data are reported in HCV myocarditis where lane 1: DNA marker; lane 2: beta-globin (house-keeping gene); lane 3:both positive and negative strands of HCV RNA were AV positive control; lane 4 EMB (after therapy) AV PCR negative; lane 4:detected in cardiac tissue from patients with acute and uninfected cell line; lane 5: negative control (reagents without template).

chronic forms[43,44].

4 .3.2. Monitoring of inflammatory cardiomyopathy three out of eight viral myocarditis cases followed by EMBPCR analysis (including detection of viral replicative merged into DC with PCR evidence of persistent viral

status and viral load) should be applied on follow-up genome and two of them died[17]. In these cases thebiopsies of patients with viral myocarditis and may progression into DC provides further evidence of the linkrepresent the best way to verify the efficacy of specific between viral myocarditis and DC.antiviral therapy (Fig. 3a–d) [142]. The diagnosis of viral myocarditis, which ‘per se’ is

Furthermore viral persistence has been also detected by difficult particularly when based only on morphologicalseveral studies that investigated the viral genome on features, becomes quite challenging in the monitoring ofsubsequent biopsies[17,112,143]. In our recent study, post-transplant endomyocardial biopsies where inflamma-

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tory infiltrates within the myocardium, as a result of viralinfection, are difficult to differentiate histologically fromthose of acute cellular rejection (Fig. 4a–c).

CMV is generally regarded as the most importantinfectious agent affecting organ transplant recipients, andlatently infected allografts constitute a major source of thevirus. The disease may result from either primary infectionor reactivation of the virus in the immunosuppressedpatient. More recently Towbin’s group in a large series ofcases (553 consecutive biopsy samples from 149 transplantrecipients) has demonstrated that different types of cardiot-ropic viruses are frequently implicated[144]. In thisinvestigation the authors stressed the crucial role of theviruses to adversely influence graft survival especiallyfavouring chronic vascular rejection. Previous studies haveshown an association between CMV infection and vascularrejection and coronary artery disease[145,146].Differenthypotheses have been postulated for the pathogenesis ofallograft substrates of vasculopathy. It has been speculatedFig. 3. (continued)that viruses may transform infected cells and thereby alter

Fig. 4. Post-transplant cytomegaloviral (CMV) myocarditis: (a) histology of EMB showing interstitial edema and polymorphous inflammatory cellinfiltration. No cytomegaly and/or viral inclusion bodies are seen; hematoxylin and eosin350; (b) histology of EMB (3 months after therapy withganciclovir; note the absence of inflammatory cell infiltration), hematoxylin and eosin350; (c) RT-PCR analysis for CMV: lane 1: DNA marker; lane 2:CMV positive control; lane 3: EMB (a: before treatment): CMV positive; lane 4: negative control (reagents without template); lane 5: skipped slot; lane 6:DNA marker; lane 7: CMV positive control; lane 8: EMB (b: after ganciclovir therapy): CMV PCR negative; lane 9: negative control (reagents withouttemplate).

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T able 3aViral PCR1 myocarditis /dilated cardiomyopathy: recurrence of viral infection

Sex/age Clinical presentation Histology Viral PCR Viral recurrence

M/15 CHF AM EV 1

M/17 Dyspnea DC EV –M/16 Dyspnea DC EV –F/13 Fatigue DC AV/EV –F/1 CHF DC EV –M/6 Acute CHF AM HSV 1

M/1.5 Fatigue DC EV –F/18 CHF DC EV

AM, active myocarditis; AV, adenovirus; CHF, congestive heart failure; DC, dilated cardiomyopathy; EBV, Epstein–Barr virus; EV, enterovirus; HSV,herpes simplex virus.

a From Calabrese et al.[17].

release of growth factors leading to cellular proliferation in minute quantities; (ii) detection of agents that are[147]. In addition, viruses may act directly to cause difficult to cultivate or which cannot be cultivated; (iii)vascular endothelial damage within the heart, leading to detection of latent and active infections; (iv) strain typing;increased expression of cell adhesion molecules and other and (v) detection of virulence and antimicrobial resistanceinflammatory mediators that then promote leukocyte re- determinants.cruitment to the site. Viral particles, attached to the blood Correct application of molecular techniques will allowvessel wall, may also serve to activate the immune system, increasingly more information to be obtained: new epi-with resultant damage to the vascular endothelial surface, demiology, current patient risk stratification and overalland viral particles may serve to alter alloantigenicity more appropriate medical treatment.resulting in rejection[148]. The high cost of biopsy processing including molecular

De novo viral infection is not surprising in heart investigation with an apparently unfavourable cost:benefittransplant recipients, given the immunocompromised ratio, is counterbalanced by the current high morbidity andstatus, however the possibility of recurrence of viral mortality (56% of patients die within 4.3 years of diag-myocarditis, favoured also by the immunosuppression nosis) calling for precise etiological diagnosis with inher-regimen, should be considered as well (Table 3) [17]. ent therapeutic implications.

The demonstration of viral disease recurrence in all William Osler, acutely ahead of his time, stated: ‘‘Therecardiac grafts after cardiac transplantation stresses the are three phases to treatment: diagnosis, diagnosis andimportance of etiologic characterization of myocarditis at diagnosis’’, stressing that the best patient managementthe time of transplantation. Strict surveillance of these resides fundamentally in the most accurate diagnosispatients using molecular techniques after transplantation is provided by expertise and experience[149].mandatory for early detection of the recurrent myocarditisand consequently administration of more specific treat-ment, including antiviral therapy. R eferences

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M yocarditis and inflammatory cardiomyopathy: microbiological and

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