The present study reports 102 patients (89 males and 13 females) clinically selected for presentation with inflammatory heart diseases, and aged between 17 and 46 years (mean age 30.8 years). Myocardium and/or pericardium inflammation was presumed to be due to a typical history of viral infection at the time of cardiac disease onset. A number of clinical signs of viral impregnation, including rhinorrhea, coughing, myalgia, muscle soreness, high fever as well as cardiac symptoms, such as chest pain, breathlessness, cardiac arrhythmia, easy fatigability, electrocardiographic changes and reduced exercise, were observed. In terms of susceptibility and history, the patients are most commonly adolescents and young adults. Further biological tests were performed. In fact, a complete blood count marked by a hyperleukosytosis with a lymphocyte predominance, marginally raised C-reactive protein and procalcitonin, supported the suspicion of a viral heart infection. The samples were obtained from patients within 48 h following admission to the Cardiology Departments at Fattouma Bourguiba and Sahloul hospitals. The sampling was performed at three university hospitals located on the coast of Tunisia: Fattouma Bourguiba (Monastir), Farhat Hached (Sousse) and Sahloul (Sousse) between November 2006 and November 2011. A total of 100 adult subjects presenting with other cardiac pathologies than infectious ones were used as the controls. The study was approved by the ethics committees of the University Hospitals Fattouma Bourguiba (Monastir, Tunisia), Farhat Hached and Sahloul (Sousse, Tunisia). Each Ethic Committee is designed by the hospital where the sampling was performed. Written informed consent forms were signed by patients and the controls participating in this study.

The viral RNA was extracted from blood, pericardial fluid samples using the TRIzol^® Plus RNA Purification kit (Invitrogen Life Technologies) according to the manufacturer’s instructions. DNase treatment during RNA purification was adopted using the PureLink™ DNase (Invitrogen Life Technologies) in order to obtain DNA-free total RNA. One-step RT-PCR for the detection of enterovirus RNA was performed with primers directed to the conserved sequences in the 5′-UTR of the enterovirus genome. A fragment of 155 bp of the extracted RNA was amplified by one-step RT-PCR (Invitrogen SuperScript™ One-Step RT-PCR with Platinum^® Taq) using 006 and 007 primers (36). The RT-PCR was performed on a mixture containing 25 μl 2X reaction mix (a buffer containing 0.4 mm of each dNTP; 2.4 mm MgSO₄); 0.2 μM each of sense and anti-sense primers, 1 μl of enzyme mix (RT/Platinum^® Taq; Invitrogen Life Technologies) and RNase free water to 50 μl. The reaction was conducted with an initial reverse transcription step at 42°C for 30 min, followed by PCR activation at 94°C for 5 min, 30 amplification cycles (94°C, 30 sec; 42°C, 1 min; 72°C, 2 min) and a final 10-min extension at 72°C in an Eppendorf Mastercycler Thermal Cycler. The PCR products were run on a 2% agarose gel stained with ethidium bromide and visualized under UV light.

First, the PCR amplicons were purified using the ExoSAP-IT-PCR Clean-Up Reagent (USB^® Products from Affymetrix, Inc) which constitutes a one-step enzymatic cleanup of PCR products. Next, they were sequenced in forward and reverse directions with the respective PCR primers. The chromatogram sequencing files were inspected with Finch TV (version 1.4.0). The obtained enterovirus sequences were compared with the corresponding ones available in the GenBank using Basic Local Alignment Search Tool (BLAST) in order to identify the enterovirus type (37,38).

Heart biopsies obtained from the patients and controls were fixed in formalin (neutral-buffered formaldehyde, 30% diluted to 1/10) for 24 h and embedded in paraffin. The sections (5 μm) were cut from the paraffin-embedded tissues with a microtome. All of the sections were stained with hematoxylin-eosin staining (Invitrogen Life Technologies) and the slides were investigated for the presence of infectious markers (39–42).

In all the heart biopsies, immunohistochemical investigations were performed on the enteroviral capsid protein VP1 Mab 5-D8/1 (DAKO, Carpenteria, VT, USA). Tris-buffered NaCl solution with Tween-20, the target retrieval solution, serum-free protein block, antibody diluent, mayer’s hematoxylin, EnVision⁺ system-HRP (AEC) and Glycergel^® Mounting Medium (aqueous) were all purchased from DAKO. The immunohistochemical procedures included antigen exposure, blocking, incubation with primary antibody, incubation with the secondary antibody in the En-Vision detection system and appropriate wash between steps using Tris-buffered saline with Tween-20. All the incubations were performed at room temperature (5,43–45). Briefly, paraffin-embedded tissue sections (5 μm) were dewaxed with xylene and rehydrated with graded ethanol. Antigen exposure was achieved by heat in a water bath (95–99°C) mediated by target retrieval. The endogenous peroxidase activity was blocked with a peroxidase-blocking reagent for 15 min. The tissue sections were blocked with the protein block for 10 min and then incubated with primary antibody (appropriately diluted to 1:100–1:500 in antibody diluent) for 30 min and washed. The secondary antibody in the En-Vision detection system was the goat anti-mouse Ig conjugated with dextran polymer, on which numerous peroxidase molecules were labeled. The sections were incubated with this reagent for 30 min, washed and then reacted with substrate chromogen for 5–10 min. The slides were immersed in aqueous hematoxylin for counterstaining. The color reaction was stopped by a wash in distilled water. Finally, the mounted sections were examined and confirmed under a Nikon Eclipse 50i microscope (Nikon, Tokyo, Japan).

Virus isolation was performed on KB cells (human squamous carcinoma cell line). The cells were grown in 24-well microplates in RPMI-1640 medium (Invitrogen Life Technologies), washed twice with phosphate-buffered saline (PBS) and then inoculated with 50 μl of the patients’ samples (blood and pericardial fluid). The samples were allowed to adsorb for 1 h at 37°C with gentle swirling every 15 min. Eagle’s minimal essential medium with 5% fetal calf serum, 100 U/ml penicillin, 200 μg/ml streptomycin and 1 mg/ml l-glutamine (Gibco-BRL, Invitrogen Life Technologies) were then added to the infected cells. The cells were monitored daily for characteristic cytopathic effect (CPE) (6).

To detect viral internalization, the cells were fixed in cold acetone (15 min) and subjected to an indirect immunofluorescence assay intended to detect the VP1 capsid protein. Briefly, the slides were incubated with enterovirus group specific monoclonal antibody (mAb) 5-D8/1 (diluted at 1/40 in PBS; Dako, Trappes, France) for 30 min at room temperature. Following three washes in PBS, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody (Jackson ImmnuoResearch Laboratories, Inc., West Grove, PA, USA) was applied at a dilution of 1:25 in blue Evans for a further 30 min at room temperature. Subsequently, the slides were washed again with PBS, dried, mounted with glycerol medium and examined using a fluorescence microscope (Olympus, Rungis, France) (5).

To determine the risk groups, a statistical analysis (chi-squared test) with a 95% significance level was performed using Epi Info version 6.04 (CDC, Atlanta, GA, USA).

Of the 102 evaluated affected individuals, 51 patients were clinically diagnosed with myocarditis, 31 with pericarditis, 12 with myopericarditis and 8 with dilated cardiomyopathy. The patients were divided into groups according to age: adolescents (13–18 years old; n=23), young adults (19–29 years old; n=63) and adults (aged >29 years; n=16). The samples were tested for the presence of genomic RNA by RT-PCR using primers located in the 5′-UTR of the enterovirus genome. The RT-PCR amplified the enteroviral genome in 9 of 51 samples obtained from myocarditis patients, in 15 of 31 samples obtained from pericarditis patients, in 2 of 12 samples obtained from myopericarditis patients and in 2 of 8 samples obtained from dilated cardiomyopathy patients. Pericarditis infection was then more frequent than myocarditis (P<0.05) or myopericarditis (P=0.05). Of the 28 positive RT-PCR samples, 17 were simultaneously positive in blood and pericardial fluid samples. Fig. 1 shows an example of detection by agarose gel electrophoresis after RT-PCR. However, of the 100 control samples, none were RT-PCR positive.

When analyzed according to age group, the differences in outcome were notable between groups (Fig. 2). Five out of 23 adolescents, 19 out of 63 young adults and 4 out of 16 adults tested positive for enteroviral RNA by RT-PCR. Infection was higher in young adults than in adults (P<0.5) or adolescents (P<0.5). When analyzed by seasonal variation, the differences in outcome were also significant. Fig. 3 demonstrates the following distribution: enteroviral RT-PCR was positive in 9 of 32 in autumn, 16 of 49 in winter and 3 of 19 in spring. The analysis of the enteroviral RT-PCR positivity according to gender shows the following distribution: 25 of 89 males and 3 of 13 females were positive. Thus, males seem more susceptible (P<0.5; Fig. 4).

The sequence analysis of enterovirus amplimers showed that 27 strains were identical to the CV-B3 sequence in 9 myocarditis cases, 15 pericarditis cases, 1 myopericarditis case and 2 dilated cardiomyopathy cases. Only one strain was identical to the CV-B1 sequence in 1 myopericarditis case.

Following investigation of the paraffin tissue blocks from patients, diffuse inflammation was marked (Fig. 5A and B). The slides from the paraffin tissue blocks from the controls demonstrated no significant pathological findings (Fig. 5C).

To determine whether the enterovirus cardiac infection was associated with a viral protein synthesis activity, all of the heart biopsies obtained from hospitalized patients and controls were examined by immunohistochemical assays specific for the enteroviral capsid protein VP1. The VP1 capsid protein in the heart biopsies was detected in enterovirus-positive cases revealed by means of RT-PCR (Fig. 6A and B). The slides from the control subjects demonstrated a negative result with no significant pathological findings (Fig. 6C).

Enterovirus was cultured from 1 of 43 pericardial fluid samples whereas no positive viral cultures were found in all of the blood samples (n=102). Within the control group, enteroviral cell culture was negative. The sample demonstrating a positive cytopathic effect in cell culture was also positive for VP1 antigen detection by immunostaining assay (Fig. 7).