**6. Diagnosis of viral causes of myocarditis**

Many common human viruses can be the etiological cause of acute or chronic myocarditis in children or young adult patients (Andréoletti et al., 2009). Because of the large number and serotypes of human viruses potentially responsible for cardiac infection the clinical interest

**Dilated** 

(PrvB19) <1 - 37% 51% Kühl et al. 2005a, 2005b

(HHV6) 11% 6-22% Kühl et al. 2005a, 2005b,

Adenovirus 8.1 - 23% 2 - 12% Kühl et al. 2005a, 2005b,

(CMVH) 3% 0.8% Kühl et al. 2005a, 2005b

(EBV) <1% 2% Kühl et al. 2005a, 2005b

(HSV) <1% - Kühl et al. 2005a, 2005b

Influenza viruses <1 - 2% - Kühl et al. 2005a, 2005b

Hepatitis C - - - HIV - - -

Table. 1. Prevalence of viruses detected by molecular biology-based techniques in cardiac tissue samples taken from patients with acute myocarditis or dilated cardiomyopathy.

2005a). Moreover in these patients, the absence of associated myocardial inflammation suggests that viral persistence can be responsible for a modulation of the immune response, which would be decreased from the beginning of the chronic phase of myocarditis (Kawai,

Many common human viruses can be the etiological cause of acute or chronic myocarditis in children or young adult patients (Andréoletti et al., 2009). Because of the large number and serotypes of human viruses potentially responsible for cardiac infection the clinical interest

*(-) Not determined; \* Prevalence determined in cases of solid organ transplantation.* 

**Cardiomyopathy References** 

12% 27% Kühl et al. 2005a, 2005b

Li et al. 2000; Andréoletti et al. 2000, Kühl et al. 2005a, 2005b

Mahrhodt et al.2004

Bowles et al. 2003

**Acute myocarditis** 

Human enteroviruses 14 - 33 % 8 - 35%

**Human cardiotropic Viruses** 

Parvovirus B19

Human Herpes Virus 6

Multiple infections (60% of cases = PrvB19+HHV6)

 Rare viral causes in immunocompetent patients\* (non restrictive list)

Cytomegalovirus

Epstein Barr Virus

Herpes Simplex Virus

1999; Yajima & Knowlton, 2009).

**6. Diagnosis of viral causes of myocarditis** 

of viral serological assay remains of a limited interest in clinical practice (Mhafoud et al., 2011). A recent European study demonstrated that virus serology has no relevance for the diagnosis of myocardial infection in young adults; this study indicated that comparatively to the molecular analysis of endomyocardial biopsy tissues, the positive predictive value was 25% and that the negative predictive value was 49% (Mhafoud et al., 2011).Therefore the etiological diagnosis of viral myocarditis is based on the detection of the viruses or viral components (proteins or genomes) in peripheral blood samples at the time of viremia phase (clinically characterized by fever), but also at the entry and the excretion sites (throat, urine and stool samples), and in heart tissue samples that corresponds to the organ site of viral replication. Therefore, endomyocardial biopsy remains the gold standard for unequivocally establishing the histopathological and virological diagnosis of unexplained cardiomyopathies as acute or chronic myocarditis (Li et al., 2000; Mahrholdt et al., 2004). Its clinical impact on prognosis and treatment largely depends on establishing a rapid and standardized set of diagnostic methods including histopathological and virological analyses of endomyocardial tissue taken by endomyocardial biopsy (Aretz et al., 1987; Mahrholdt et al., 2004).

#### **6.1 Endomyocardial biopsy (EMB)**

As recommended by the Heart Failure Society of America and the Heart Failure Association of the European Society of Cardiology, the implementation of a right or left ventricular EMB is indicated in the case of acute symptoms of heart failure refractory to standard management, a substantial worsening of ejection fraction despite optimized pharmacological therapy, the development of hemodynamically significant arrhythmias, an heart failure with concurrent rash, fever, or peripheral eosinophilia, an history of collagen vascular disease such as systemic lupus, erythematosus, scleroderma, or polyarteritis nodosum, and a new-onset cardiomyopathy in the presence of known amyloidosis, sarcoidosis, or hemochromatosis when no obvious cause, in particular ischemic, could be established (Mahrholdt et al., 2004). EMB remains the gold standard for unequivocally establishing the diagnosis of unexplained cardiomyopathy Li et al., 2000). However, its sensitivity and its specificity are limited by the often-focal distribution of the specific histological lesions (Baughman, 2006).

Cardiac biopsy samples should be obtained in more than one area of the right ventricular septum and the number of samples should range from 5 to 10 of a volume from 1 to 2 mm3. Five of these samples should be fixed in neutralized 10% formalin or 10% PFA; five should be flash-frozen or placed immediately conserved at –80°C to perform further classical or molecular virological techniques (Mahrholdt et al., 2004). Interestingly, it has been recently demonstrated that there were no differences in the number of positive left ventricular-EMB, right ventricular-EMB, or left ventricular- and right ventricular-EMB findings when related to the site of cardiovascular magnetic resonance– based late gadolinium enhancement. Preferential biopsy in regions showing late gadolinium enhancement on cardiovascular magnetic resonance does not increase the number of positive diagnoses of myocarditis. (Yilmaz et al., 2010).

#### **6.2 Histopathological evaluation of cardiac biopsy samples**

Histological evaluation of cardiac biopsies from patients with clinically suspected myocarditis is routinely done according to the Dallas criteria (Cooper, 2009). Since

Viral Myocarditis: Physiopathology and Diagnosis 97

clinical threshold related to a significant endomyocardial inflammation (Bock et al., 2010) Only the strategy of associated classical RT-PCR and PCR assays or PCR-microarrays for the detection of all the most common cardiotropic viruses can provide an accurate diagnosis of viral myocarditis. However, sampling error in this focal cardiac disease and the frequent late timing of EMB can also hamper the clinical application of these molecular assays after disease onset. Finally, a positive RT-PCR or PCR result can provide an etiological diagnosis and have to be evaluated into the clinical context, whereas negative PCR results do not exclude a viral-related cardiac disease. Moreover, the only use of the molecular techniques cannot distinguish an active from a persistent viral cardiac infection. Therefore during the clinical course of myocarditis, the immunohistochemical detection of *enterovirus*, *adenovirus or parvovirus B19* capsid proteins or *herpesviruses* late proteins is necessary in order to differentiate a viral cardiac infection with replication activities from a persistent or latent cardiac infection (Matsumori et al, 2006; Sudano et al., 2006). In the present time, the combination of molecular and immunohistochemical assays on cardiac tissue samples provides a reliable diagnostic strategy for a complete diagnosis of a potential viral-induced acute or latent/persistent cardiac infections. The direct detection of viral genomes in association with the immunohistochemical detection of *viral* capsid or late proteins in cardiac tissues is crucial to characterize the phase of the viral infection (acute or persistent/chronic) and therefore to specifically adapt the therapeutic strategies (Andréoletti

et al., 2009; Badorff et al., 1999; Gravanis & Sternby, 1991; Sudano et al., 2006).

**patients with myocarditis or dilated cardiomyopathy** 

biopsies.

**6.4 Use of new techniques for the virological analyses of heart samples taken from** 

In the present time, classical real time PCR techniques allow a reliable qualitative detection of viral genomes in cardiac tissues of patients in cases of medical diagnosis of hypokinetic cardiomyopathies (Cooper, 2009) or in series of post mortem or explanted heart tissues (Andréoletti et al., 2000, Dennert et al., 2008; Cooper, 2009). The two critical points remain the number of endomyocardial biopsies (5 to 10) their anatomic origin as well the extraction phase that determine the quality of and the sensitivity of detection of the DNA or RNA viral genomic sequences (Cooper, 2009). The standardization of the extraction phase is now possible by the use of semi-automated or totally automated use of standardized procedure using certified available systems (Renois et al.,2010). A pool of several EMBs taken, from different heart tissue areas have to be extracted together in order to optimize the efficiency of viral nucleic acid recovery rates (Copper, 2009; Dennert et al., 2008). Combination of such automated extraction procedures with monoplex or multiplex classical real time RT PCR techniques can allow an international standardization of the molecular qualitative or quantitative detection of DNA or RNA cardiotropic viruses in human endomyocardial

It is now possible to use new multiplex amplification assays followed by a microarray hybridization system allowing a simultaneous of 9 to 12 cardiotropic viruses (herpesviruses or human enteroviruses) in a single analysis. We experienced this system in our laboratory and we observed that multiplex PCR-microarray assay provided a consistently robust qualitative detection of at least 9 viruses in on reaction tube (HSV1, HSV2, CMVH, VZV, HHV6, HHV7, HHV8, enteroviruses species A & B) (unpublished data). We showed that there was a 100% concordance for positive virus detection results and that the theoretical limits of detection were as low as 10 genome copies for CVB3 Nancy per microgram of total RNA. Because the system used intergrated a internal extraction and ampliciation control, it

myocarditis is a focal disease, 4 to 5 biopsy samples obtained in more than one area of the right ventricular septum should be analysed for light microscopic examination and immunohistochemical assays (Mahrholdt et al., 2004). For routine light microscopy examination, EMB tissue is embedded in paraffin and serial sections are obtained and stained with hematoxylin, eosin. Masson trichrome or Sirius red can be useful for a better evaluation of the fibrosis (Cooper, 2009). Additional slides have to be performed for subsequent immunohistochemical assays (Aretz et al., 1987; Mahrholdt et al., 2004).

To improve the histological diagnosis, additional immunological evaluation of cardiac tissues is required with immunohistochemical techniques allowing quantification, identification, and differentiation of inflammatory cells (Aretz et al., 1987). Criteria for immunohistological diagnosis in the EMB of inflammatory cardiomyopathy is specified quantitatively as >14 infiltrating leukocytes/mm², preferably T-lymphocytes or activated Tcells. If foci of T-lymphocytes are present, active myocarditis can be diagnosed due to the nature of the infiltrate even when the critical level of 14 leukocytes/mm² is not reached (Aretz et al., 1987). Subsequent biopsies will allow reliable follow-up of the myocarditis with a semi-quantitative evaluation of myocardial inflammation, necrosis and healing. If the levels of inflammation appear unchanged from the most recent previous cardiac biopsies, the term ongoing (or persistent) myocarditis will be used (Cooper, 2009).

Finally in cases of clinically suspected viral myocarditis or unexplained cardiomyopathy, Dallas criteria have to be associated with classical immunohistological assays for the identification of inflammatory cells and also for the detection of viral capsid or early or late antigens in EMBs (Andréoletti et al., 2009).

#### **6.3 Classical virological analyses of samples taken from patients with myocarditis**

The etiological diagnosis of viral myocarditis, specifically during the acute phase corresponding to the acute viral infection, is based on the detection of the viruses or viral components (proteins or genomes) in heart biopsy tissue samples and additionally to detection of viral genomes in peripheral biological samples as whole blood (viremia) , throat (classical entry site of viruses) and urine and stool samples. The direct virological techniques are now based on molecular biology (PCR, RT-PCR) and immunohistochemical assays for EMBs. By comparison, the contribution of viral serological assays to the clinical diagnosis of acute or chronic myocarditis is relatively poor and the serological assays are of interest only for a late and retrospective diagnosis of viral myocarditis (Dennert et al., 2009). However, Hepatitis C and B and HIV serologies should be systematic in a patients suffering from acute or chronic myocarditis (Aretz et al., 1987).

Using classical molecular techniques, the genome of enteroviruses, adenoviruses, human hepatitis viruses or *herpesviruses* can be detected in 40 to 70 % of the cardiac tissues of patients suffering from an histological–proven acute myocarditis (Table 1) (Badorff et al., 1999; Gravanis & Sternby, 1991). These molecular techniques must be performed on a pool of several EMBs taken, from different heart tissue areas that should be extracted together in order to optimize the efficiency of viral nucleic acid recovery rates (Mahrholdt et al., 2004). Some specialized laboratories use real-time PCR assays that allow a quantitative approach to estimate viral loads of the majority of cardiotropic viruses. However, no published data exist on the clinical value of real-time PCR viral loads and the determination of clinical thresholds that could be capable to differentiate a viral cardiac replicative infection from a persistent or latent viral endomyocardial infection. Only one published study reported a mean value of 500 copies of PVB19 genome per one microgram of extracted DNA as the

myocarditis is a focal disease, 4 to 5 biopsy samples obtained in more than one area of the right ventricular septum should be analysed for light microscopic examination and immunohistochemical assays (Mahrholdt et al., 2004). For routine light microscopy examination, EMB tissue is embedded in paraffin and serial sections are obtained and stained with hematoxylin, eosin. Masson trichrome or Sirius red can be useful for a better evaluation of the fibrosis (Cooper, 2009). Additional slides have to be performed for

To improve the histological diagnosis, additional immunological evaluation of cardiac tissues is required with immunohistochemical techniques allowing quantification, identification, and differentiation of inflammatory cells (Aretz et al., 1987). Criteria for immunohistological diagnosis in the EMB of inflammatory cardiomyopathy is specified quantitatively as >14 infiltrating leukocytes/mm², preferably T-lymphocytes or activated Tcells. If foci of T-lymphocytes are present, active myocarditis can be diagnosed due to the nature of the infiltrate even when the critical level of 14 leukocytes/mm² is not reached (Aretz et al., 1987). Subsequent biopsies will allow reliable follow-up of the myocarditis with a semi-quantitative evaluation of myocardial inflammation, necrosis and healing. If the levels of inflammation appear unchanged from the most recent previous cardiac biopsies,

Finally in cases of clinically suspected viral myocarditis or unexplained cardiomyopathy, Dallas criteria have to be associated with classical immunohistological assays for the identification of inflammatory cells and also for the detection of viral capsid or early or late

**6.3 Classical virological analyses of samples taken from patients with myocarditis**  The etiological diagnosis of viral myocarditis, specifically during the acute phase corresponding to the acute viral infection, is based on the detection of the viruses or viral components (proteins or genomes) in heart biopsy tissue samples and additionally to detection of viral genomes in peripheral biological samples as whole blood (viremia) , throat (classical entry site of viruses) and urine and stool samples. The direct virological techniques are now based on molecular biology (PCR, RT-PCR) and immunohistochemical assays for EMBs. By comparison, the contribution of viral serological assays to the clinical diagnosis of acute or chronic myocarditis is relatively poor and the serological assays are of interest only for a late and retrospective diagnosis of viral myocarditis (Dennert et al., 2009). However, Hepatitis C and B and HIV serologies should be systematic in a patients suffering from

Using classical molecular techniques, the genome of enteroviruses, adenoviruses, human hepatitis viruses or *herpesviruses* can be detected in 40 to 70 % of the cardiac tissues of patients suffering from an histological–proven acute myocarditis (Table 1) (Badorff et al., 1999; Gravanis & Sternby, 1991). These molecular techniques must be performed on a pool of several EMBs taken, from different heart tissue areas that should be extracted together in order to optimize the efficiency of viral nucleic acid recovery rates (Mahrholdt et al., 2004). Some specialized laboratories use real-time PCR assays that allow a quantitative approach to estimate viral loads of the majority of cardiotropic viruses. However, no published data exist on the clinical value of real-time PCR viral loads and the determination of clinical thresholds that could be capable to differentiate a viral cardiac replicative infection from a persistent or latent viral endomyocardial infection. Only one published study reported a mean value of 500 copies of PVB19 genome per one microgram of extracted DNA as the

subsequent immunohistochemical assays (Aretz et al., 1987; Mahrholdt et al., 2004).

the term ongoing (or persistent) myocarditis will be used (Cooper, 2009).

antigens in EMBs (Andréoletti et al., 2009).

acute or chronic myocarditis (Aretz et al., 1987).

clinical threshold related to a significant endomyocardial inflammation (Bock et al., 2010) Only the strategy of associated classical RT-PCR and PCR assays or PCR-microarrays for the detection of all the most common cardiotropic viruses can provide an accurate diagnosis of viral myocarditis. However, sampling error in this focal cardiac disease and the frequent late timing of EMB can also hamper the clinical application of these molecular assays after disease onset. Finally, a positive RT-PCR or PCR result can provide an etiological diagnosis and have to be evaluated into the clinical context, whereas negative PCR results do not exclude a viral-related cardiac disease. Moreover, the only use of the molecular techniques cannot distinguish an active from a persistent viral cardiac infection. Therefore during the clinical course of myocarditis, the immunohistochemical detection of *enterovirus*, *adenovirus or parvovirus B19* capsid proteins or *herpesviruses* late proteins is necessary in order to differentiate a viral cardiac infection with replication activities from a persistent or latent cardiac infection (Matsumori et al, 2006; Sudano et al., 2006). In the present time, the combination of molecular and immunohistochemical assays on cardiac tissue samples provides a reliable diagnostic strategy for a complete diagnosis of a potential viral-induced acute or latent/persistent cardiac infections. The direct detection of viral genomes in association with the immunohistochemical detection of *viral* capsid or late proteins in cardiac tissues is crucial to characterize the phase of the viral infection (acute or persistent/chronic) and therefore to specifically adapt the therapeutic strategies (Andréoletti et al., 2009; Badorff et al., 1999; Gravanis & Sternby, 1991; Sudano et al., 2006).

#### **6.4 Use of new techniques for the virological analyses of heart samples taken from patients with myocarditis or dilated cardiomyopathy**

In the present time, classical real time PCR techniques allow a reliable qualitative detection of viral genomes in cardiac tissues of patients in cases of medical diagnosis of hypokinetic cardiomyopathies (Cooper, 2009) or in series of post mortem or explanted heart tissues (Andréoletti et al., 2000, Dennert et al., 2008; Cooper, 2009). The two critical points remain the number of endomyocardial biopsies (5 to 10) their anatomic origin as well the extraction phase that determine the quality of and the sensitivity of detection of the DNA or RNA viral genomic sequences (Cooper, 2009). The standardization of the extraction phase is now possible by the use of semi-automated or totally automated use of standardized procedure using certified available systems (Renois et al.,2010). A pool of several EMBs taken, from different heart tissue areas have to be extracted together in order to optimize the efficiency of viral nucleic acid recovery rates (Copper, 2009; Dennert et al., 2008). Combination of such automated extraction procedures with monoplex or multiplex classical real time RT PCR techniques can allow an international standardization of the molecular qualitative or quantitative detection of DNA or RNA cardiotropic viruses in human endomyocardial biopsies.

It is now possible to use new multiplex amplification assays followed by a microarray hybridization system allowing a simultaneous of 9 to 12 cardiotropic viruses (herpesviruses or human enteroviruses) in a single analysis. We experienced this system in our laboratory and we observed that multiplex PCR-microarray assay provided a consistently robust qualitative detection of at least 9 viruses in on reaction tube (HSV1, HSV2, CMVH, VZV, HHV6, HHV7, HHV8, enteroviruses species A & B) (unpublished data). We showed that there was a 100% concordance for positive virus detection results and that the theoretical limits of detection were as low as 10 genome copies for CVB3 Nancy per microgram of total RNA. Because the system used intergrated a internal extraction and ampliciation control, it

Viral Myocarditis: Physiopathology and Diagnosis 99

patients, our findings indicated that EV (12.5%), PVB19 (12.5%) and dual EV-PVB19 (25%) infections were identified as major potential etiological causes of idiopathic DCM (personal data; not shown). Moreover this new system allowed a rapid semi-quantitative detection of EV associated with a genotyping identification of the EV strains (CVB3 or CVB5; personal data not shown) (Figure 3) .Finally this system allows a rapid and valuable quantitative detection and genotyping identification of common viruses in heart tissues and can be used

**2000**

**1000**

**amplitude (molecules per Da)**

Fig. 3. Detection of parvovirus B19 (panel A) or coxsackievirus B3 (Panel B) in cardiac tissues of two patients (A and B) with idiopathic dilated cardiomyopathy using broadrange PCR amplification followed by ionisation and mass spectrometry analysis (personnal

To improve histological diagnosis of viral myocarditis, additional virological evaluation of cardiac tissues is required with immunohistochemical and polymerase chain reaction (PCR) techniques allowing identification and quantification of viral infection markers. The diagnostic gold standard is endomyocardial biopsy (EMB) with the histological Dallas criteria, in association with new immunohistochemical and PCR analyses of cardiac tissues. These new viral diagnostic approaches can lead to better identification of the aetiology of myocarditis and can improve the clinical or therapeutic monitoring of viral causes of human myocarditis. Therapeutic strategies adapted specifically to the phase of the disease are currently under evaluation and may improve prognosis and clinical outcomes significantly. It might be more efficient to use positive immunomodulators (interleukins, interferon alpha, interferon gamma) alone or in combination with specific antiviral components such as ribavirin in the initial phase of the disease when viral replication activity can be detected in the cardiomyocytes (Külh et al., 2003). By contrast, immunosuppressive drugs would be more appropriate in the chronic phase of myocarditis, when no or low viral replication

Further specific strategies could consist to specifically block the entry of the virus in target cardiac cells, by preventing interaction of viruses with their cellular receptor and their

activities are detectable in cardiac tissues by immunohistochemistry assays.

**0**

**Internal Calibrant (A24 G28 C39 T26)**

**35.9 37.1 38.3**

**molecular mass (kDa)**

**Human Coxsackievirus B3 (A25 G32 C40 T25)**

in clinical practice for an accurate diagnostic of viral cardiac infection.

**A B**

**26.7 27.7 28.7 molecular mass (kDa)**

**7. Conclusions and therapeutic perspectives** 

**Human Parvovirus B19 (A33 G22 C16 T21)**

**Internal Calibrant (A29 G22 C16 T20)**

**1500**

**750**

**amplitude (molecules per Da)**

**0**

data 2011).

should be assumed that negative results indicate the absence of targeted viruses (Figure 2). The use of such systems could help to standardize the diagnostic detection of cardiotropic viruses in endomyocardial tissues and can be routinely used for a rapid viral detection and screening; however this system is only qualitative and cannot provide semi-quantitative or quantitative viral load values for each detected virus. Therefore in cases of a positive viral detection in cardiac tissues we currently use a second quantitative approach using standardized classical real time viral quantitative detection assays. The obtained results are standardized in viral genome copies per one ug of extracted DNA (Andréoletti et al., 2009, Bock et al., 2010) (Figure 2).

Fig. 2. Detection of a dual Human Enterovirus and HHV6 cardiac infection using a PCR-DNA microarray system in an adult patient with an idiopathic dilated cardiomyopathy. **A.** *PCR-DNA microarray system (CLART® Entherpex,Genomica, Madrid, Spain) allowing the detection and typing of 8 Human Herpes viruses and of the Human Enterovirus (HEVs) group. The study patient is co-infected by HHV-6 and Human Enterovirus (Poliovirus, Echovirus or Coxsackievirus).***B.** *Quantitative detection of HEVs in biopsy heart tissues from the same idiopathic DCM patient using classical real time PCR assay and confirming the HEV cardiac infection Amplification curves obtained for positive and negative controls and one HEV positive DCM patient X. Positive control corresponded to the CVB3 Nancy strain; Negative control corresponded to healthy heart tissue; CT, cycle threshold.* 

A new strategy for the molecular detection of cardiac viral infections couples broad-range PCR amplification to electrospray ionization/ mass spectrometry analysis (PCR/ESI-MS). Previous versions of this method were known commercially as the Ibis T5000 and the current commercial hardware platform that conducts the MS analysis is now known as the Abbott PLEX-ID (Ecker et al., 2008). This technique uses primers designed to genomic regions highly conserved regions across viral domains of life. The method was initially developed for the identification of viruses, including previously unknown or unculturable viral agents in samples where multiple microbes may be present, primarily for biodefense applications (Ecker et al., 2008). It is now being developed for diagnosis of human cardiac infections by our team (Figure 3). Preliminary data obtain from 52 heart biopsy samples taken from 24 patients with idiopathic DCM showed a Kappa test correlation of 0.7 +/- 0.22 between PleX-ID detection system and 8 monoplex Q rt-PCR assays. Among the 24 study

should be assumed that negative results indicate the absence of targeted viruses (Figure 2). The use of such systems could help to standardize the diagnostic detection of cardiotropic viruses in endomyocardial tissues and can be routinely used for a rapid viral detection and screening; however this system is only qualitative and cannot provide semi-quantitative or quantitative viral load values for each detected virus. Therefore in cases of a positive viral detection in cardiac tissues we currently use a second quantitative approach using standardized classical real time viral quantitative detection assays. The obtained results are standardized in viral genome copies per one ug of extracted DNA (Andréoletti et al., 2009,

Fig. 2. Detection of a dual Human Enterovirus and HHV6 cardiac infection using a PCR-DNA microarray system in an adult patient with an idiopathic dilated cardiomyopathy. **A.** *PCR-DNA microarray system (CLART® Entherpex,Genomica, Madrid, Spain) allowing the detection and typing of 8 Human Herpes viruses and of the Human Enterovirus (HEVs) group. The* 

**A B. CT = 37** 

Human ENTEROVIRUS

Positive Control

Negative Patie

*Coxsackievirus).***B.** *Quantitative detection of HEVs in biopsy heart tissues from the same idiopathic DCM patient using classical real time PCR assay and confirming the HEV cardiac infection* 

*Amplification curves obtained for positive and negative controls and one HEV positive DCM patient X. Positive control corresponded to the CVB3 Nancy strain; Negative control corresponded to* 

A new strategy for the molecular detection of cardiac viral infections couples broad-range PCR amplification to electrospray ionization/ mass spectrometry analysis (PCR/ESI-MS). Previous versions of this method were known commercially as the Ibis T5000 and the current commercial hardware platform that conducts the MS analysis is now known as the Abbott PLEX-ID (Ecker et al., 2008). This technique uses primers designed to genomic regions highly conserved regions across viral domains of life. The method was initially developed for the identification of viruses, including previously unknown or unculturable viral agents in samples where multiple microbes may be present, primarily for biodefense applications (Ecker et al., 2008). It is now being developed for diagnosis of human cardiac infections by our team (Figure 3). Preliminary data obtain from 52 heart biopsy samples taken from 24 patients with idiopathic DCM showed a Kappa test correlation of 0.7 +/- 0.22 between PleX-ID detection system and 8 monoplex Q rt-PCR assays. Among the 24 study

*study patient is co-infected by HHV-6 and Human Enterovirus (Poliovirus, Echovirus or* 

Bock et al., 2010) (Figure 2).

HHV-6

Aligment Markers

Amplification Control

*healthy heart tissue; CT, cycle threshold.* 

patients, our findings indicated that EV (12.5%), PVB19 (12.5%) and dual EV-PVB19 (25%) infections were identified as major potential etiological causes of idiopathic DCM (personal data; not shown). Moreover this new system allowed a rapid semi-quantitative detection of EV associated with a genotyping identification of the EV strains (CVB3 or CVB5; personal data not shown) (Figure 3) .Finally this system allows a rapid and valuable quantitative detection and genotyping identification of common viruses in heart tissues and can be used in clinical practice for an accurate diagnostic of viral cardiac infection.

Fig. 3. Detection of parvovirus B19 (panel A) or coxsackievirus B3 (Panel B) in cardiac tissues of two patients (A and B) with idiopathic dilated cardiomyopathy using broadrange PCR amplification followed by ionisation and mass spectrometry analysis (personnal data 2011).
