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(Circulation. 1997;96:3549-3554.)
© 1997 American Heart Association, Inc.

Articles

Association of Parvovirus B19 Genome in Children With Myocarditis and Cardiac Allograft Rejection

Diagnosis Using the Polymerase Chain Reaction

Kenneth O. Schowengerdt, MD; Jiyuan Ni, MD; Susan W. Denfield, MD; Robert J. Gajarski, MD; Neil E. Bowles, PhD; Geoffrey Rosenthal, MD, PhD; Debra L. Kearney, MD; Julia K. Price, RN; Beverly B. Rogers, MD; Gail M. Schauer, MD; Richard E. Chinnock, MD; ; Jeffrey A. Towbin, MD

From the Departments of Pediatrics (K.O.S., J.N., S.W.D., R.J.G., N.E.B., G.R., D.L.K., J.K.P., J.A.T.), Pathology (D.L.K.), and Molecular and Human Genetics (J.A.T.), Texas Children's Hospital and Baylor College of Medicine, Houston, Tex; Department of Pathology (B.B.R.), University of Texas, Dallas, Tex; Departments of Laboratory Medicine (G.M.S.), Children's Hospital, and Pathology (G.M.S.), The Ohio State University, Columbus, Ohio; and Department of Pediatrics (R.E.C.), Loma Linda University Children's Hospital, Loma Linda, Calif. Dr. Schowengerdt is now at the University of Florida School of Medicine, Gainesville.

Correspondence to Jeffrey A. Towbin, MD, Pediatric Cardiology, Baylor College of Medicine, One Baylor Plaza, Room 333E, Houston, TX 77030. E-mail jtowbin{at}bcm.tmc.edu

	Abstract

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Background Inflammatory diseases of the heart, including myocarditis and cardiac transplant rejection, are important causes of morbidity and mortality in children. Although viral infection may be suspected in either of these clinical conditions, the definitive etiology is often difficult to ascertain. Furthermore, the histology is identical for both disorders. Coxsackievirus has long been considered the most common cause of viral myocarditis; however, we previously demonstrated by polymerase chain reaction (PCR) analysis that many different, and sometimes unexpected, viruses may be responsible for myocarditis and cardiac rejection. In this study, we describe the association of parvovirus genome identified through PCR analysis of cardiac tissue in the clinical setting of myocarditis and cardiac allograft rejection.

Methods and Results Myocardial tissue from endomyocardial biopsy, explant, or autopsy was analyzed for parvovirus B19 using primers designed to amplify a 699–base pair PCR product from the VP1 gene region. Samples tested included those obtained from patients with suspected myocarditis (n=360) or transplant rejection (n=200) or control subjects (n=250). Parvoviral genome was identified through PCR in 9 patients (3 myocarditis; 6 transplant) and no control patients. Of the 3 patients with myocarditis, 1 presented with cardiac arrest leading to death, 1 developed dilated cardiomyopathy, and the other gradually improved. Four of the 6 transplant patients had evidence of significant rejection on the basis of endomyocardial biopsy histology. All transplant patients survived the infection.

Conclusions Parvovirus is associated with myocarditis in a small percentage of children and may be a potential contributor to cardiac transplant rejection. PCR may provide a rapid and sensitive method of diagnosis.

Key Words: parvovirus • polymerase chain reaction • myocarditis • rejection

	Introduction

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The family Parvoviridae is a group of DNA viruses that are known to infect both animals and humans. Members of the genus Parvovirus include the common animal pathogens feline leukopenia virus and canine parvovirus. Parvovirus B19 is the only member of the recently created genus Erythrovirus and is the only known human pathogen in this family of viruses. The parvovirus B19 genome consists of a linear single-stranded DNA molecule of 5.5 kb.¹ Unique features of the parvoviral genome include the use of a single promoter, multiple polyadenylation signals, and the presence of several large introns.² The viral particle itself is icosahedral, and structural proteins include a major structural protein (VP2) and the minor capsid protein (VP1). The cellular receptor for parvovirus B19 has been identified as blood group P antigen (or globoside, a tetrahexose ceramide),³ and persons with the rare phenotype of absence of P antigen have been shown to be resistant to B19 infection.⁴ The identification of this receptor serves to explain the tropism of this virus for proliferating red cell precursors; however, the antigen has also been found on endothelial cells⁵ and fetal myocardial cells.^6,7

Parvovirus B19 infection is relatively common in humans, with 50% of the population having detectable IgG antibodies by age 15 years, increasing to 90% in the elderly.⁸ Human parvoviral infection most commonly causes asymptomatic infection or erythema infectiosum ("Fifth disease") accompanied by a characteristic facial rash.^9–12 Less commonly, human parvoviral infection has been described as a cause of transient aplastic crisis in patients with underlying hemolytic disorders,¹³ as well as polyarthropathy syndrome,^11,12 nonimmune fetal hydrops,^14–16 and transient erythroblastopenia of the newborn.¹⁷ Parvoviral infections are more common during late winter through early summer, and the infection is generally spread via the respiratory route, although the virus has also been reported to have been transmitted via administration of blood products.¹⁸

Canine parvovirus has been known for some time to be a cause of fulminant myocarditis seen in young dogs.¹⁹ In addition, as noted above, human parvovirus B19 infection has been previously described as a known cause of hydrops fetalis with associated cardiac compromise. The cardiac insufficiency seen in this setting, however, has been attributed to profound fetal anemia with resultant high-output failure. Reports of parvovirus B19 infection as a cause of pediatric or adult myocarditis are extremely rare.^20–23

The definitive diagnosis of viral infections in general, particularly those involving the heart, is often difficult. This is especially true in the postoperative cardiac transplant patient, in whom inflammatory changes within the myocardium secondary to viral infection are identical histologically to those of acute cellular rejection. Serological studies, peripheral viral cultures, and histopathology have traditionally been used as aids in the diagnosis of viral myocarditis; these methods, however, tend to be time consuming and lack sensitivity and specificity.^24,25 Certain viruses, such as parvovirus B19, cannot be grown in standard cell culture systems. Because these routine methods appear to definitively identify only a portion of patients with suspected cardiac infection, additional methods have been sought to improve the diagnostic sensitivity in cases of suspected myocarditis. Therefore, molecular genetic techniques, including PCR, have been used to detect the presence of viral genome in several tissue types and body fluids, including cardiac tissue.^26–48 PCR can rapidly and efficiently amplify up to 1 billion–fold desired nucleic acid sequences (ie, viral sequence) of low copy number present in very small amounts of tissue. This method has been successfully used in other clinical settings in which the detection of parvoviral genome was sought.^49–52

In the present study, PCR was used to identify parvoviral genome in the myocardium of previously normal children with clinical findings consistent with myocarditis and in postoperative pediatric cardiac transplant patients presenting with findings of acute, chronic, or late unexplained rejection. The results obtained provide new information regarding the epidemiology of parvovirus B19 infection in these patient groups.

	Methods

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Patient Selection
Patients were selected for analysis based on the clinical findings of heart failure and echocardiographic evidence of ventricular dysfunction. Children evaluated included those with suspected myocarditis (myocarditis group) and patients status-post cardiac transplantation (transplant group). Tissue was also analyzed from patients in whom there was no history of inflammatory heart disease, recent infection, or history of prior heart transplantation (control group). In all patients and control subjects, endomyocardial biopsy, explants at time of transplant, or autopsy tissue samples were available for histological evaluation and PCR analysis.

Study Design and Patient Population
PCR analysis was performed (in duplicate) on RVEMB or right ventricular autopsy samples obtained from patients with suspected myocarditis (n=360 patients; 720 sample determinations) or from postoperative cardiac transplant patients undergoing cardiac catheterization and RVEMB for routine rejection surveillance or suspected acute rejection (n=200 patients; 400 sample determinations). Myocarditis patient ages ranged from 1 day to 21 years (mean, 7.4 years), and transplant patient ages ranged from 6 months to 21 years (mean, 8.1 years). All biopsy samples were either fresh frozen in liquid nitrogen (432 of 720 myocarditis samples; 220 of 400 transplant samples) or formalin fixed (288 of 720 myocarditis samples; 180 of 400 transplant samples). PCR analysis of fixed tissue has been successfully performed in our laboratory, with results comparable to those with the use of frozen tissue.³⁹ For the transplant group, histopathological diagnosis of rejection grade was performed using the ISHLT rejection scale,⁵³ whereas the diagnosis of myocarditis was based on the Dallas Criteria.⁵⁴ Tissue samples from pediatric patients with no known evidence of myocarditis, dilated cardiomyopathy, or other ongoing inflammatory processes were used as negative controls (n=250); these included autopsy samples (164 of 250) or tissue from explanted hearts obtained at time of transplantation (86 of 250) from patients with complex congenital heart disease (n=116), hypertrophic cardiomyopathy (n=18), trauma (n=25), or other disease-related deaths in children (n=91) who had no evidence of myocardial infection and no history of recent viral illness. All samples were analyzed by PCR for the presence of parvovirus nucleic acid (VP1 gene region) using presynthesized primers based on the published viral sequence of parvovirus, f-5'-ATAAATCCATATACTCATT-3', nucleotides 2936 to 2954; and parvovirus, r-5'-CTAAAGTATCCTGACCTTG-3'; nucleotides 3617 to 3635.⁵⁰ PCR analysis was performed blinded to clinical, serological, culture, and histopathological data. Parvoviral serological results were analyzed when available. PCR findings were subsequently correlated with the patient's clinical course, routine histology (diagnosis of myocarditis based on Dallas Criteria⁵⁴ or the ISHLT rejection grade in the transplant group), and serology data. Positive serology was defined as a "high" titer for an acute serology (ie, >2 SDs above the mean) or a fourfold titer rise when paired serologies were obtained.

Primer Design and Synthesis
PCR primers were synthesized with an Applied Biosystems model 380 oligonucleotide synthesizer using published viral sequences. The parvoviral primer pair used produces a 699-bp amplimer from the VP1 gene region of parvovirus B19.⁵⁰ To confirm nucleic acid extraction from all samples, a primer pair was designed to amplify a 135-bp sequence from the 12th codon region of K-ras, a single-copy proto-oncogene present in all cells,⁵⁵ and has been used previously for this purpose (K-ras, f-5'-TATTATAAGGCCTGCTGAAAATGACTGAAT-3', nucleotides 180 to 202; and K-ras, r-5'-TTACCTCTATTGTTGGATCATATTCGTCCA-3', nucleotides 278 to 304).

Template Preparation and PCR
Frozen and formalin-fixed tissue (1 to 2 mm³; 1.5 to 2 mg) obtained on endomyocardial biopsy with a 1.8-mm bioptome or a sterile blade to cut similar-sized specimens from explant or autopsy tissue was homogenized using a Brinkmann Polytron homogenizer. Genomic DNA, viral DNA (if present), and total RNA were extracted simultaneously from patient specimens using a modification⁵⁶ of the RNAzol method originally described by Chomczynski and Sacchi.⁵⁷ Suitability of extraction was evaluated by gel electrophoresis without nucleic acid quantification. Contamination was controlled between homogenization of samples as follows: the homogenizer was washed with DEPC-treated deionized H₂O for 1 minute, washed with 1% SDS for 10 minutes, and then placed in 100% ethanol for 10 minutes. The homogenizer was autoclaved for 20 minutes and cooled before reuse. Water homogenate negative controls were tested for contamination between uses.

Parvoviral target DNA sequences were amplified in a total reaction volume of 100 µL containing a 100 µmol/L concentration of each dNTP, a 1 µmol/L concentration of each primer, 10 µL of 10x PCR buffer, and a 3-µL sample. Taq polymerase, 2.5 U (Perkin-Elmer Cetus), was added after an initial incubation at 94°C for 3 minutes. Thirty-three rounds of amplification were carried out at the following conditions: 94°C for 2 minutes, 42°C for 2 minutes, and 72°C for 3 minutes (72°C x 7 minutes extension) with an automated thermocycler (MJ Research, Inc, or Biometra). Parvovirus B19 DNA was used as the positive viral control. Positive and negative controls were run with all samples to control for contamination and PCR fidelity.

K-ras primers (2 mmol/L each) were combined with 5 µL of 10x PCR buffer, 8 µL of 1.25 mmol/L dNTP concentrations, 5 µL of sample, and deionized H₂O to achieve a total volume of 50 µL. This mixture was boiled at 100°C for 6 minutes. Taq polymerase (2.5 U) was added, and 40 rounds of amplification were performed at the following conditions: 94°C for 2 minutes, 60°C for 90 seconds, and 72°C for 60 seconds.⁵⁵ The extension time was 60 seconds for the first cycle; this was extended by 10 seconds for each additional cycle.

For each reaction, 10 µL was analyzed on a 2% agarose gel (FMC BioProducts) containing 0.5 mg/ml ethidium bromide (Sigma Chemical Co.). The gels were then placed under UV light for visualization of the amplified products.

All samples were run with a simultaneous positive control (parvovirus B19 DNA) and negative control (ie, reaction mixture minus template nucleic acid). If a band was visualized in the negative control lane, the PCR sample was considered contaminated, and the sample was reanalyzed or reextracted and analyzed. All samples were analyzed without prior knowledge of clinical or culture/serological data for each patient, and all PCR-positive samples were reanalyzed to confirm the positive result. Control PCR amplification to verify the presence of nucleic acid extracted from each sample was performed using primers designed to amplify K-ras.⁵⁵ If the K-ras primers failed to amplify the appropriate 135-bp amplimer, the sample was reextracted or excluded. Southern blotting and hybridization, as well as direct sequencing of the PCR product,⁵⁸ were used to confirm positive results. Radioactive labeling of internal probes and hybridization of PCR products were carried out as previously described (parvovirus probe, 5'-CTAACTCTGTAACTTGTAC-3', nucleotides 3222 to 3240; and K-ras probe, 5'-CCTACGCCACCAGCTC CAAC-3', nucleotides 217 to 236).^50,55

Serological Analysis
Serological results for parvovirus (when available) are described in the following section. When performed, serological analysis for anti–parvovirus B19 IgM was carried out using ELISA methods.

Statistical Analysis
The two-tailed Fisher's exact test was used for all comparisons between groups.⁵⁹ The proportions with positive PCR findings among transplant and myocarditis patients were each compared with that among controls. In addition, the proportion of transplant patients with positive PCR results was compared with the proportion among myocarditis patients. The Bonferroni correction to minimize the risk of false rejection of any of the null hypotheses of no difference in proportions with PCR positive results is applicable.⁶⁰

	Results

Top Abstract Introduction Methods Results Discussion References

 
PCR Analysis
PCR amplification of parvoviral genome occurred in cardiac tissue specimens from 3 of 360 patients (0.8%) undergoing evaluation for suspected myocarditis and in 6 of 200 transplant patients (3%) undergoing acute rejection surveillance. None of the 250 "negative control" patients analyzed amplified parvoviral genome (Table 1). All samples included in the analysis tested positively for the presence of nucleic acid using K-ras primers. Because this template represents a single-copy gene from the human genome and should be representative of other normal human gene sequences that are present in all cells,⁵⁵ amplification of the 135-bp sequence provided evidence of adequate nucleic acid extraction from the sample, indicating that the parvovirus PCR-negative samples were true negative results. Representative gel electrophoresis results for the presence of parvoviral genome in a myocarditis patient is shown in the Figure (below). Direct sequencing of the PCR product or Southern blot hybridization (Figure) confirmed positive PCR results.

View this table: [in this window] [in a new window]   Table 1. Summary of Results

View larger version (51K): [in this window] [in a new window]   Figure 1. PCR analysis of RVEMB sample obtained from a patient (patient 3, Table 2) with suspected myocarditis (left). The patient sample lane (RVEMB) demonstrates the parvoviral 699-bp PCR amplimer. Lane 2, Parvoviral positive control for comparison. The negative control lane is devoid of PCR products. Right, Autoradiogram after blotting and membrane hybridization with parvovirus probe confirms the identity of the PCR products.

Results of Statistical Analysis
The difference between the proportion of control subjects and patients with myocarditis having positive PCR results for parvoviral genome did not achieve statistical significance (P=.273). No important differences in the proportion with positive PCR was observed between those with myocarditis and those with acute transplant rejection (P=.076). However, the difference between the proportion of control subjects and patients with acute transplant rejection having a positive parvoviral PCR result was statistically significant (P=.007).

Correlation Between Histopathology and PCR Results in the Myocarditis Group
Table 2 outlines patient ages, clinical findings, histopathology results, and serological findings (when available) in the 3 patients evaluated for suspected myocarditis who had biopsy samples positive for the presence of parvoviral genome by PCR. All 3 patients in this group demonstrated histological evidence of lymphocytic myocarditis. In no cases were parvoviral inclusions seen; in the one patient in which serology had been obtained (patient 2, Table 2), parvovirus infection was confirmed.

View this table: [in this window] [in a new window]   Table 2. Myocarditis Group With Positive Parvovirus PCR

Correlation Between Transplant Rejection Grade and PCR Results
Table 3 outlines patient ages, clinical findings, ISHLT rejection grades, and serological findings (when available) in the 6 cardiac transplant patients who had biopsy samples positive for the presence of parvoviral genome by PCR. Four of the 6 PCR positive patients in this group (patients 4 through 7; Table 3) manifested concurrent biopsy scores of 3A (multifocal and diffuse moderate-to-severe rejection). Two of these patients (patients 4 and 5, Table 3) had a history of chronic rejection that was difficult to control with the usual antirejection therapeutic regimens. In the 2 patients in whom serology was subsequently obtained, parvoviral infection was confirmed (patients 5 and 7, Table 3).

View this table: [in this window] [in a new window]   Table 3. Transplant Group With Positive Parvovirus PCR

Correlation Between Parvoviral Infection and Prognosis
All patients survived who had transplant rejection associated with parvovirus B19 genome identification. Two of these 6 patients (patients 4 and 5, Table 3), however, had persistent rejection despite aggressive antirejection therapy. The clinical course of patients with myocarditis was no different than that of other children with myocarditis matched for age and sex. In 1 of 3 patients (patient 1, Table 2), death occurred; in the 2 survivors, 1 child developed dilated cardiomyopathy (patient 2, Table 2), and the other had normalization of ventricular function (patient 3, Table 2).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The etiological diagnosis of myocarditis is often difficult. Although coxsackievirus has long been considered the most common cause of viral myocarditis, we previously demonstrated that several other viruses (most notably, adenovirus) may also cause this disorder in children.^61,62 In addition, the histological picture of cardiac transplant cellular rejection mimics the findings of myocarditis; for this reason, it has been speculated that viral infection of the heart may contribute to some cases of rejection. In this setting, the diagnostic algorithm has classically included serological studies and peripheral viral cultures for selected viruses.⁶³ These methods are time consuming and may lack specificity and sensitivity. More recently, PCR has become a useful adjunct to the diagnostic armamentarium for the identification of viral genome in both patient groups.

As previously outlined, canine parvovirus (genus Parvovirus) is known to be a common cause of canine myocarditis.¹⁹ Parvovirus B19 (genus Erythrovirus) has also been demonstrated to cause significant disease in humans, such as erythema infectiosum (Fifth disease),^9–12 hydrops fetalis and fetal death,^14–16,64 transient aplastic crisis in patients with various forms of anemia,^12,13 and chronic anemia in immunocompromised patients.⁶⁵ Janner et al⁶⁶ reported a case of severe pneumonia caused by parvovirus B19 that occurred in a 5-year-old boy after cardiac transplantation. In addition, a recent report⁶⁷ described the occurrence of fetal parvovirus B19 infection and liver disease in a patient noted to have Ebstein's anomaly postnatally. It is of potential relevance that the B19 receptor (erythrocyte P antigen) has been identified on fetal myocardial cells,^6,7 suggesting that intrauterine myocarditis contributes to the development of fetal hydrops after parvovirus B19 infection. As previously mentioned, rare, isolated cases of parvovirus as a cause of human myocarditis have been reported based on clinical presentation, in combination with immunocytochemical techniques or serological data.^20–22 In this report, we demonstrated parvoviral genome to be present within the myocardium in the setting of human myocarditis both in previously normal patients presenting with signs and symptoms of cardiac insufficiency and inflammation and in postoperative cardiac transplant patients with acute or chronic rejection. It is of note that 2 of the 3 PCR-positive patients in the myocarditis group (patients 2 and 3; Table 2) manifested no other clinical evidence of parvoviral infection, underscoring the importance of PCR in the identification of an etiological agent in cases of myocarditis that have often been only presumptively ascribed to be due to viral infection. Similarly, in the transplant group, 4 of the 6 PCR-positive patients (patients 4, 5, 8, and 9; Table 3) had no clinical evidence of a recent viral illness. Interestingly, 2 of these patients (patients 4 and 5) had a history of chronic unexplained rejection. We believe the results described here demonstrate the usefulness of PCR as a sensitive method for the detection of parvoviral genome in cardiac biopsy samples obtained in these settings. Although the overall incidence of myocarditis associated with PCR detection of parvovirus genome in our study is admittedly low (3 of 360 myocarditis patients and 6 of 200 transplant patient; total of 9 of 560 of patients [1.6%] positive for parvoviral genome), we believe these findings are clinically significant in light of the paucity of current literature reports (three cases) of presumed parvoviral myocarditis.^20–22 Although not statistically significant, the findings of parvoviral genome within the myocardium of children with clinical myocarditis should be considered a clinically relevant, although uncommon, association with this severe disease. It is possible that host factors are responsible for the ability of certain children to become infected with this virus; credence to this hypothesis is supported by the findings in the immunocompromised post-transplant group in whom a statistically significant association of parvovirus and the myocarditis-like process of rejection is shown here. Furthermore, the fact that no control children were found with parvoviral genome supports the potential cause-and-effect association of parvoviral genome and inflammatory heart disease.

One of the questions raised through this study and others concerns the likelihood of viral persistence in tissue from previous infection. Studies in animals and humans suggest that, at least in the case of certain viruses, viral persistence may occur.^{26–29,40,68} The mechanism that allows viral persistence, if it occurs, is unclear at this time but may involve complex immunological characteristics of both the virus and its host.⁶⁹ In the cases in our series in which repeated follow-up biopsies were performed (transplant patients), parvoviral genome has not been detected on subsequent biopsies. In addition, we performed PCR of blood concurrently with that of endomyocardial biopsy specimens, and the blood PCR for parvoviral genome was negative in the face of a positive cardiac PCR. This supports the conclusion that virus exists within the cardiac tissue itself and that our results are not simply due to amplification of viral genome present in blood elements within the tissue sample (ie, during acute viremia).

In summary, PCR offers a rapid and sensitive way to detect the presence of parvoviral nucleic acid from the heart itself. When used in conjunction with the standard clinical evaluation and routine histopathology, it appears to improve the accuracy and rapidity of the diagnosis of parvoviral infection of the heart in patients with suspected myocarditis as well as in immunosuppressed postoperative cardiac transplant patients. It appears that parvoviral myocarditis in humans, although rare, may be more common than previously identified and reported. It is likely that PCR will prove to be a useful adjunct to the care of these patients, providing a rapid and specific assessment technique in children suspected of having myocarditis or transplant rejection due to unknown etiological agents. Further study of these conditions in conjunction with the availability of a rapid diagnostic method of viral identification may ultimately lead to improved treatment regimens (ie, intravenous immunoglobulin therapy) that may serve to limit inflammatory damage to the heart.

	Selected Abbreviations and Acronyms

 

ISHLT = International Society of Heart and Lung Transplantation PCR = polymerase chain reaction RVEMB = right ventricular endomyocardial biopsy

	Acknowledgments

 
This work was supported in part by NIH grant 1-KO8-HL-03254 (K.O.S.) and the Pediatric Cardiomyopathy Registry (grant NIH-1-RO1-HL-53392; J.A.T.). This work was performed in the Phoebe Willingham Muzzy Pediatric Molecular Cardiology Laboratory, Baylor College of Medicine, and we thank Valerie R. Price for her expert secretarial support.

	Footnotes

 
Guest editor for this article was Jeffrey M. Leiden, MD, PhD, University of Chicago (Ill).

Received February 6, 1997; revision received July 11, 1997; accepted August 1, 1997.

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