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9PREVALENCE OF DNA PARVOVIRUS B19 AND HUMAN 6 TYPE-HERPES-VIRUS IN THE HEART TISSUE AND SERUM OF THE PA-TIENTS WITH DILATED
CARDIOMYOPATHY
Vaikhanskaya Tatiyana G.
leading researcher of the medical Information Technology department, PhD, MD, Republican Scientific and Practical Center of Cardiology, Minsk, Belarus
Shumavets Vladimir V.
heart surgeon of the cardiac surgery department N 2, PhD, MD, assistant professor Republican Scientific and Practical Center of Cardiology, Minsk, Belarus
Kurushka Tatiyana V. functional diagnostics doctor, Republican Scientific and Practical Center of Cardiology, Minsk, Belarus
Bohush Zoya F.
researcher of the Laboratory with natural reservoir infections, Research and Practical Centre of Epidemiology and Microbiology, Minsk, Belarus
ABSTRACT
This study intend to narrow the gap in our current knowledge regarding the diagnostic workup of dilated phenotype heart disease, particularly as related to viral cardiac infections. The article presents the range of occurrence cardiac viruses (enteroviruses, Epstein-Barr virus, varicella-zoster virus, parvovirus B19, adenoviruses, herpes simplex virus, human herpesvirus 6, cytomegalovirus) in patients with dilated cardiomyopathy (DCM) and influence these pathogens on clinical phenotype of the disease. The blood serum level of detection markers of the viral agents was identified 14,1% in patients with DCM disorder. The nucleic acids of viral pathogens were determined in heart tissue of 74,4 % of samples. Among the wide range of viruses most often were detected parvovirus B19 and human herpes virus type 6. The objective of this study was to assess virus-related heart diseases as a precipitating factor for heart failure in patients with dilated cardiomyopathy (DCM).
Methods and results: 135 patients with DCM and 30 controls were evaluated. Samples (109) of heart tissue taken from 39 patients who underwent open-heart surgery and serum of 135 patients were collected. Cardiotropic DNA viruses like as enteroviruses (EV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), parvovirus B19 (PVB19), adenoviruses (AdV), herpes simplex virus (HSV), human herpesvirus 6 (HHV6), cytomegalovirus (CMV) were determined by quantitative polymerase chain reaction (PCR) test. Total DNA detection markers of viral agents were 35,6% (48/135), in particular nucleic acids of viral pathogens determined in heart tissue were 74,4% (29/39) of the EMB samples: for monovirus 51,7%, for mixtviruses 48,3%. Among the wide range of viruses, the most common were PVB19 (37%) and HHV6 (20%); subsequently in the descending order: EBV (12%), AdV (11%), EV (9%), CMV (6%), VZV (3%), and HSV (2%). The patients showed signs of viremia B19 PV in 5,93% of cases, EBV in 2,96%, both CMV and ADV in 2,22%, HHV6 in 1,48% (versus absent in group serum control). Virus-positive patients had more severe signs of right ventricular (RV) negative cardiac remodeling: RV end-diastolic volume (p=0,005) and RV end-systolic volume (p=0,029), pulmonary artery pressure (p=0,041). The positive correlation between virus-positivity and right ventricle negative remodeling was detected; coefficients were: r = 0,66 (p = 0.006) for RV ES volume and r = 0,69 (p = 0.005) for RV ED volume.
Conclusion. Myocarditis and DCM are related etiopathogenically mainly by persistence of cardiotropic viral infection. Among Belarusian population with DCM persistence of PV B19 and HHV6 prevalence in the heart was common. Virus-positive patients had a more severe negative cardiac-right remodeling.
Key words: dilated cardiomyopathy (DCM), endomyocardial biopsy (EMB), cardiac viruses, nucleic acid (NA)
Dilated cardiomyopathy (DCM) is the most common most of the complications of viral etiology after the heart
myocardial disease observed among primary lesions transplantation are associated with it. To date, the range of
characterized by increased heart cavities and decreased systolic cardiotropic viruses is considerably extended and replenished
function. Among the etiological factors of DCM pathogenesis by enteroviruses, Epstein-Barr virus (EBV), herpes simplex
viral infections (cardiotropic), autoimmune disorders, and viruses of 1th and 2th types, varicella-zoster virus (VZV),
genetic abnormalities [1,2] can be determined. Viral and human herpes viruses (HHV) of the 6th and 8th types, virus of
postviral myocarditis are the major causes of acute and chronic hepatitis C and human immunodeficiency virus [9-12].
dilated phenotype of cardiomyopathy. THE AIM OF RESEARCH
Over the last decade, viral agents were defined in biological In view of the association established for some viral agents
tissues of patients with DCM has been dominating such as: with the development of cardiomyopathies, the aim of this
parvovirus B19 (PV B19), herpes simplex virus (HSV) and a study was to investigate the range of viral pathogens identified
human herpesviruses (HHV) of the 6 and 8 types [3-8]. Then in pts with DCM and analysis of virus-positive effects on the
one can add up enteroviruse infections associated with DCM. clinical phenotype of the disease.
It is noteworthy that cytomegalovirus (CMV) plays a MATERIALS AND METHODS
significant part among cardiotropic viruses, and its DNA is Patients with the diagnosis of DCM were enrolled in the
found in 15 % samples of DCM patients' endomyocardial research made according to International working group
biopsies [10]. CMV should be specifically underlined because criteria of cardiomyopathies [1,2]. Research was approved by
local ethical committee.
The patients with the diagnostic criteria confirming DCM were examined (135 pts; 101X74,8% male, NYHA functional class 2,8±0,43; aged 46,7±11,6 years). Controls were 30 healthy volunteers compared by gender and age to the main group of patients.
All patients underwent by examinations including physical examination, ECG-12 lead registration, selective coronary angiography, Holter ECG 24h monitoring, echocardiography, bicycle ergometric test, and virological screening of blood samples and endomyocardial biopsy samples using molecular and genetic methods.
Endomyocardial biopsy (EMB) was performed by means of standard technique including collection of myocardial material from right ventricular apex using biopsy forceps. In the settings of valve surgical corrective operation and orthotopic heart transplantation, biopsy was taken from RV and LV apexes, septal area and from atrial auricles.
Reverse transcription reaction was obtained using «RevertAid» return transcription kit by «Fermentas» (Lithuania) and «REVERTA-L» by «Amplisens», (Russia) to receive enteroviral DNA. Amplification of viral nucleic acids was carried out using commercial test systems by «Amplisens»(Russia).
Clinical materials (blood, endomyocardial biopsy samples) were studied using molecular genetics technologies to define genetic markers (DNA, RNA) of 8 viral infections causative agents associated with the development of DCM (HSV1-2, CMV, EBV, HHV 6, VZV, PV B19, ADV and an EV).
Genetic study of 109 myocardial samples taken from 39 patients was per-formed. Endomyocardial biopsies (EMB) were taken during surgical valvular correction operation in 20 patients, during orthotopic heart transplantation from 17 explanted hearts and using standard percutaneous catheter endocardial procedure EMB were taken in 2 patients. Our
To diagnose EV, VZV and PV B19, PCR test systems were used considering real-time results. AdV, HSV, CMV, HHV6t and EBV were detected using PCR test systems and electrophoretic result analysis.
Statistical analysis of test results was performed based on biostatistical methods using Statistica-7 and SPSS (version 20.0) computer programs for Windows. Quantitative parameters were presented as arithmetic average (M) and mean-square deviation of an average (sd). Paired Student's coefficient and correlation analysis were used for statistical analysis of the figures. For qualitative indices, the analysis of frequency reliability was performed using x2 criterion and the exact Fisher's method. In case of appreciable deviations of distributions examined from the normal, Whitney's-Mann criterion was used. A P-value of < 0.05 was considered statistically significant.
RESULTS
135 patients with DCM aged between 17 and 59 years old underwent Integrated examination (males 101X74,8 %, NYHA 3,02±0,3; sinus rhythm was defined in 109X80,7%; atrial fibrillation in 26X19,26%; ventricular tachyarrhythmias in 43X31,8 %; complete left bundle branch block was defined in 37X27,4 %; average duration of QRS complex was 124±25 ms). Clinical characteristics of patients that were observed are presented in Table 1.
Table 1.
findings showed increased viral persistence in cardiomyocytes of patients with DCM: in 74,4 % EMB (in 29 of 39) genetic material of one or more viral pathogens were present. Positive results were seen in respect of 7 out of 8 pathogens detected among dominating PV B19 (43,6 %) and HHV6t (35,9 % ). The next more important of virus agents represent EV which nucleic acids were present in cardiac tissues of 5 patients of the studied group and it was 12,8 %. Then followed in the descending order, respectively VEB and ADV (10,3 % each), VZV and CMV(on 2,56 %). The genetic material of HSV 1, 2 types was not found in EMB samples studied (Table 2).
Clinical characteristics of 135 DCM patients enrolled in the study.
Parameter Value, M±sd Number of patients with an estimation, n
Age, years 46,7±11,6 135
NYHA, class 3,02±0,3 135
Left ventricular end-systolic volume, ml 224,5 ±86,5 135
left ventricular end-diastolic volume, ml 297±97,4 135
Left ventricular ejection fraction,% 26,3±9,61 135
Right ventricular end-systolic volume, ml 62,7 ±35,4 135
Right ventricular end-diastolic volume, ml 99,4 ±47,7 135
Right ventricular ejection fraction,% 39,3±11,5 135
Viral screening: -serum blood PCR positive, n (%) -endomyocardial biopsy PCR positive, n (%) 19 (14,1) 29 (74,4) 135 39
Table 2.
Genetic material (nucleic acids) of viruses in cardiac tissue (EMB) of DCM patients
Material: heart tissue (EMB) n = 39 Number of DNA-positive results, n/ percentage detection, n/ % Total DNApositive patients, n/%
HSV VZV CMV EBV HHV6 PV B19 ADV EV
0 1/2,56 1/2,56 4/10,3 14/35,9 17/43,6 4/10,3 5/12,8 29 /74,4
Signs of mono-infection were identified in 51,7 % the EMB-positive patients (15 of 29), and the mixed infection of heart tissues with two or more viral pathogens was detected in 48,3 % (14 of 29).
Genetic study revealed the presence of all examined cardiotropic viral nucleic acids in blood serum, and cumulative
level of detected viral genetic markers was 14,1%. PV B19 (5,93 %) was dominating among viral pathogens detected then followed by VEB (2,96 %), ADV and CMV genomes at the similar rate (2,22 %) and nucleic acid HSV, HHV6, EV and VZV (on 0,74 %) were detected in one case (Table 3).
Table 3.
Genetic markers of the viral agents in blood serum of DCM patients and Controls
Material-blood serum Number of DNA-positive results, n / % (percentage detection) Total DNApositive patients, n/%
HSV VZV CMV EBV HHV6 PV B19 ADV EV
DCM, n=135 1/0,74 1/0,74 3/2,22 4/2,96 1/0,74 8/5,93 3/2,22 1/0,74 19/14,1
Controls, n=30 0 0 0 0 0 0 0 1 0
Controls did not show any genetic markers of viral infections. Differences in a genetic viral research of blood serum of patients with the clinical diagnosis of DCM (n = 135, of them positive - 19) and Controls (n = 30, of them positive - 0) were statistically evident by the adjusted x2 criterion (p = 0,029) and by the exact unilateral Fisher's method (p = 0,031).
DNA detection markers of the viral agents were identified in 35,6 % (48/135) pts, nucleic acids of viral pathogens were determined in the samples of heart tissue in 74,4% (29/39) of cases: monovirus in 51,7 %, mixed viruses in 48,3 %. Results of the distribution of viral DNA localization are presented in Figure 1. tissue.
Figure 1. Distribution of viral DNA (mono/mixed) in the heart tissue and serum blood samples
The total results obtained from a multifaceted virology screening are follows as: 48 (35,6 %) virus-positive patients with DCM by genetic diagnosis of blood serum samples (n=19) and genetic diagnosis of EMB of the samples (n=29). Absolute superiority in virus PCR diagnosis of serum blood and EMB samples belongs to PV B19 (37 % from all identified
genomes), followed by HHV6 (20 %) and EBV (12 %) and then in the descending order: ADV (11 %), EV (9 %), CMV (6 %), VZV (3 %), HSV (2 %). Figure 2 shows dominant presence of parvovirus B19 among 135 patients with DCM and viral genome detection rate.
Figure 2. Serum blood and EMB viral genomes detection rate structure in patients with DCM.
Some peculiarities of viral genomes detection levels in a nearly similar rate as blood and EMB, but nucleic acids HHV6 biological samples were noted: parvovirus B19 was detected at were predominantly seen in myocardial samples (Figure 3).
Figure 3. Comparative characteristics of the identified viral DNA in serum and the heart tissue.
Comparative analysis of data obtained from virus-positive and virus-negative patients showed no evident differences between anamnesis (previous viral infection, p=0,104), cardiac rhythm disturbances or conduction (atrial fibrillation/flutter, p=0,77; ventricular tachyarrhythmias, p=0,62; left bundle branch block, p=0,75) and NYHA heart failure (p=0,23). Only disease duration periods were significantly different: virus-positive patients had a shorter duration of the disease (mean 19,2±9,39 months) vs 38,2±12,9 months from the onset of DCM in virus-negative patients (p=0,001). Viruspositive patients had a more severe signs of negative cardiac right ventricle remodeling: RV end-diastolic volume (p=0,005) and RV end-systolic volume (p=0,029), systolic pulmonary artery pressure (PAPsyst), p=0,041. The positive correlation between two signs (virus-positive and right ventricle negative remodeling) was detected, and Spearman coefficients for RV end-systolic volume r = 0.66 (p = 0,006), for RV end-diastolic volume r = 0.51 (p=0,01) were determinated respectively.
DISCUSSION
The results of our study were similar to those of German scientists, obtained by PCR analysis of endomyocardial biopsy specimens of 245 patients with idiopathic dilated cardiomyopathy (Charite Hospital, Berlin), the genetic material of a wide range of viruses was detected in 67.4% of pts. Patients with multiple infections were detected in 27.3% of cases.
As for the spectrum of viral DNA detected in EMB, nucleic acids HHV6 and PV B19 were dominated [11]. In another large study in Germany (University Hospital of Tübingen) parvo-virus B19 genomes were significantly more likely to be detected in the endomyocardial biopsy specimens of patients with myocarditis (in 322 of 498 patients/64.7 %) and dilated cardiomyopathy (in176 of 498 patients /35.3%) than in non-inflamed control hearts (in 7 of 91 patients/7.7 %), the level of significant differences was p<0.01 [12].
However, the stunning results obtained by another group of scientists (University of Dresden) for the study of PV B19 parvovirus and species - human Bocavirus (HBoV ) in atrial biopsies from patients not suffering from myocarditis and dilated cardiomyopathy can not be neglected. In nosology study group, a coronary pathology was domineering in 65% (biopsies were collected during surgery coronary artery bypass grafting) and in 35% it was valve pathology (biopsies were performed during surgical valvular correction). Of 100 patients, 85% showed PV B19 DNA detected in the atrial tissues, and 5% of patients showed DNA HBoV in atrial biopsies. Symptoms of parvovirus B19 viremia (presence of DNA PV B19 in the blood) were observed in 4% of cases [13].
So what is the role of PV B19? Is parvovirus B19 a major factor etiopathogenetic DCM or a «witness» of the patient's disease? Whether to pursue an active antiviral therapy, trying
to eliminate parvovirus? It is known that not all patients with viral myocarditis develop dilated cardiomyopathy in the future [14]. An appropriate example of this may serve the results of long-term monitoring of patients who experienced viral myocarditis. The development of cardiomyopathy occurs in 3-3.5 % of patients with myocarditis [14,15]. Then crucial question is: why the prognosis of myocarditis is so different? The most logical explanation given in the literature is the presence of genetically determined disorders of the immune response. Damage produced by is only a trigger complex of the whole chain of pathological processes leading to cardiomyopathy. Viral infection can initiate the synthesis of antibodies to the cells depressor, improve the activity of T- and B- cell production of antibodies and cytokines. After the heart damage, the virus may disappear having induced the autoimmune process that leads to the development of dilated cardiomyopathy [15].
CONCLUSION
Myocarditis and DCM are linked the etiology and pathogenesis mainly by persistence of cardiac viral infection. In Belarusian population with DCM persistence of PV B19 and HHV type 6 was predominant in the heart tissue, whereas PV B19 viremia and EBV viremia were prevailing in serum. Virally positive patients had a promote development of a more severe right cardiac negative remodeling. And our findings presented above demonstrate the high rate of detection of viral genetic markers in patients with dilated cardiomyopathy. The presence of viral genomes in endomyocardial biopsies in 74.4% of cases and the detection of viremia in 14.1% of patients with dilated cardiomyopathy is (in our view) a good evidence base in favor of the alleged role of cardiotropic viruses as potential etiopathogenetic factors contributing to the development of DCM. This is confirmed by the results obtained from correlation analysis of clinical and hemodynamic parameters of virus-positive and virus-negative patients with dilated cardiomyopathy. Significantly more pronounced signs of adverse cardiac remodeling (increased right ventricular volume parameters, increased PAPsyst ) were identified in the group of virus-positive patients. The correlation coefficients between virus-positivity and negative right ventricular remodeling were so: r = 0,66 (p = 0.006) for RV EDV and r = 0,69 (p = 0.005 ) for RV ESV, re-spectively. The high prevalence of parvovirus B19 genomes in 37 % heart tissues of patients with dilated cardiomyopathy and greater right cardiac remodeling strongly suggests that chronic cardiac disease develops from previous parvovirus B19-associated myocarditis.
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