Endomyocardial fibrosis

Context.-Drowning is a frequent cause of accidental death in childhood, but the association of myocarditis and drowning has only rarely been reported.

Objective.-To report 5 cases of drowning in children with coexistent myocarditis.

Design.-A retrospective review of autopsy records of patients O years to 18 years of age was performed during a 6-year period (1998-2003, total cases reviewed = 1431).

Results.-Twenty-two drownings were identified, in 14 male and 8 female children. Five patients (23%), 3 female and 2 male children, had coexistent myocarditis. The 5 patients ranged in age from 23 months to 13 years (mean, 7 years 2 months). None of the patients had antecedent symptomatology suggestive of myocarditis. In all patients, the myocarditis was focal mild or moderate, and the inflammatory infiltrate comprised lymphocytes with smaller numbers of neutrophils. All 5 patients had foci of myocyte necrosis. One patient had histologic evidence of myocardial hypertrophy but no evidence of a cardiomyopathy. Microbiologic studies, including culture, immunohistochemistry, polymerase chain reaction, and reverse transcriptase polymerase chain reaction, revealed Mycoplasma pneumoniae DNA in 1 case.

Conclusions.-The finding of myocarditis in a significant proportion of drowning victims in this series highlights the importance of a thorough autopsy examination in apparently straightforward cases and has clinicopathologic significance.

(Arch Pathol Lab Med. 2005;129:205-209)

Drowning is the second leading cause of accidental death in the pediatric population.1-4 At autopsy, findings are often nonspecific, and the diagnosis rests on a combination of circumstances and clinicopathologic correlation.5,6 Some authors consider the diagnosis to be one of exclusion.5,7 Occasionally, after careful review of the history and analysis of the laboratory studies, additional findings are uncovered that suggest a more complex series of events resulting in death. Usually, such findings are related to central nervous system or cardiovascular pathology,7,8 but the coexistence of myocarditis is rare.9,10 This study describes the occurrence of myocarditis in 5 children whose deaths were thought to be straightforward cases of drowning.

MATERIALS AND METHODS

Autopsy Review

Autopsy records for the period January 1, 1998, to November 30, 2003, were examined for cases of drowning (total number 1431: 739 forensic cases, 692 hospital cases).

Histologic Analysis

Routine sections of heart taken at autopsy included right ventricular inflow and outflow tracts, left ventricular anterior and posterior papillary muscles, and interventricular septum. Sections were fixed in 10% buffered formalin overnight and processed for routine histologic analysis. Sections were cut at 4 µm, mounted on glass slides, and stained with hematoxylin-eosin using routine methods.

Sections of myocardium were reviewed, and the myocarditis was classified using the Dallas criteria.11 However, these criteria were designed for endomyocardial biopsies. In addition, the definitions of focal, diffuse, mild, moderate, and severe myocarditis are not clearly defined by the Dallas criteria. Therefore, for the purposes of this study, an arbitrary working classification of myocarditis severity was designed. Mild myocarditis was defined as fewer than 4 clusters of interstitial lymphocytes per ×40 field; moderate myocarditis was defined as between 4 and 7 clusters of lymphocytes per ×40 field; and severe myocarditis was defined as 8 or more clusters of lymphocytes per ×40 field. All cases required the presence of myocyte necrosis, and the myocarditis was classified on the most severely affected field. For the myocarditis to be classified as diffuse, all sections needed to be involved, and some degree of confluence of the infiltrate was required.

Immunohistochemistry and In Situ Hybridization

Immunohistochemistry was performed on 3-µm sections of formalin-fixed, paraffin-embedded tissue placed onto silane-coated glass slides. Antibodies against parvovirus B19 (mouse monoclonal antibody, Novocastra Laboratories Ltd, Newcastle upon Tyne, United Kingdom, 1:25), adenovirus (mouse monoclonal antibody, Chemicon International, Temecula, Calif, 1:400), measles virus (mouse monoclonal antibody, Chemicon International, 1: 600), and cytomegalovirus early antigen (mouse monoclonal antibody, Chemicon International, 1:500) were used. The Ventana DAB kit (Ventana Medical Systems, Tucson, Ariz) was then used according to manufacturer's instructions, and the final color reaction was carried out by using diaminobenzidine as a chromogen. In situ hybridization was performed using the Epstein-Barr virus-encoded RNA probe (oligonucleotide, Novocastra, no dilution) according to previously published procedures.12

Polymerase Chain Reaction for Mycoplasma pneumoniae

DNA was extracted from ten 20-µm sections of formalin-fixed, paraffin-embedded tissue after xylene deparaffinization and alcohol rehydration (100%, 90%, 75%, 50%). An extended method of the standard proteinase K digestion was used, followed by organic purification with phenol/chloroform. The DNA was precipitated, dissolved in Tris-EDTA buffer, and used directly for polymerase chain reaction (PCR). Polymerase chain reaction for M pneumoniae DNA was done using primers specific for the Pl adhesin gene of M pneumoniae13 and was performed as described elsewhere.14 This method is specific for M pneumoniae and does not amplify DNA from other Mycoplasma species.15

Reverse Transcriptase PCR for Enteroviruses

RNA was extracted from ten 20-µm sections of formalin-fixed, paraffin-embedded tissue using a previously published method.16 Reverse transcriptase PCR (RT-PCR) for enteroviruses was done using the primers EV1b and EV2b,17 which target a segment in the 5'NC region of the enterovirus genome that is highly conserved among all enteroviruses, including coxsackie viruses B.17,18 Briefly, the reverse transcription step was carried out using the primer EV1b and avian myeloblastosis virus reverse transcriptase (Promega, Madison, Wis), essentially as described.19 For the PCR, 10 µL of the RT reaction mix was transferred to a tube with 40 µL of a PCR master mix with primers EV1b and EV2B and AmpliTaq Gold (Roche Molecular Systems, Pleasanton, Calif), overlaid with 50 µL of mineral oil. The reaction was incubated at 95°C for 10 minutes, followed by 40 cycles consisting of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and elongation at 72°C for 1 minute; a final incubation at 72°C for 3 minutes was then done. The PCR products were analyzed by electrophoresis on agarose gels containing ethidium bromide and were photographed under ultraviolet transillumination.

RESULTS

Autopsy Review

During the 6-year period, 1431 autopsies were performed at our institute in infants and children O years to 18 years of age. Of these, 739 were forensic autopsies, which constituted 25% of all forensic autopsies performed in children younger than 18 years in the province of Ontario. Twenty-two drownings were identified in 14 male and 8 female children whose ages ranged from 6 months to 17 years (mean, 4 years 3 months). Five patients (23%) had coexistent myocarditis; 3 of these were female and 2 were male children. The 5 patients ranged in age from 23 months to 13 years (mean, 7 years 2 months). None of the 5 patients had antecedent symptomatology suggestive of myocarditis.

Clinical Details

Patient 1.-A previously well girl, 12 years 5 months old, was swimming in shallow lake water (approximately 90 cm deep) with friends. There was no adult supervision, and she was described as a good swimmer. She complained of feeling "dizzy" and then floated on her back for a short period of time. Approximately 5 minutes later, she was found face down and unresponsive. Her friends pulled her out of the water. Upon arrival of emergency personnel, she had no vital signs and was pronounced deceased after 1 hour of resuscitative efforts at a nearby hospital.

Patient 2.-A previously well boy, 4 years 7 months old, was swimming in a stream with a moderate flow of water at a public park. He was with his 11-year-old sibling and under adult supervision. He was swept under tree branches and carried further downstream. The supervising adult was unable to find him in the stream, and his body was recovered approximately 2 km downstream, 1 hour after having disappeared. He was pronounced deceased after 30 minutes of resuscitative efforts.

Patient 3.-A previously well girl, 4 years 2 months old, was left on the dock by a cottage while her older siblings took a boat out onto the lake. Approximately 1 hour later she was found head down in the water. Resuscitative attempts by family at the scene and by emergency personnel were unsuccessful, and she was pronounced deceased 2 hours later.

Patient 4.-A previously well 23-month-old girl was found face down and unresponsive in a steep-banked ditch. The ditch was filled with approximately 40 cm of water. Resuscitative attempts were unsuccessful.

Patient 5.-A previously well 13-year-old boy was attempting to swim to a raft on a lake. He was described as not a strong swimmer. He began to struggle, and a bystander (approximately 15 years old) attempted to come to his aid but had to kick him away to avoid being pulled underwater. His body was recovered a few hours later, and he was pronounced deceased at the scene.

Autopsy Findings

The cardiac anatomy and gross appearance of the myocardium was unremarkable in all patients. The heart weights of the 2 oldest patients (patients 1 and 5) exceeded the standard for body length by 14% and 34%, respectively (Table).20 In both patients, the weights of the systemic organs (liver, kidneys, pancreas) were also proportionately increased above the standards for body length (results not shown). Furthermore, there was no gross evidence of a cardiomyopathy or a second disease process.

Histologically, all 5 patients had focal myocarditis. Two patients were classified as mild (patients 1 and 5) and 3 as moderate (patients 2, 3, and 4) using the definitions outlined in the "Materials and Methods" section. All patients had an infiltrate comprising lymphocytes and occasional neutrophils, as well as foci of myocyte necrosis (Figure 1). Microscopic foci of myocardial hypertrophy and interstitial fibrosis were present in patient 1; however, neither patient 1 nor patient 5, with the proportionately heavier hearts, had evidence of myofiber disarray, endocardial fibroelastosis, or diffuse fibrosis and alternate fiber hypertrophy and atrophy suggestive of a cardiomyopathy. No viral inclusions, infective organisms, or evidence of a vasculitic process were present in any of the sections examined. Significant unexpected extracardiac pathology was not seen.

Immunohistochemistry, PCR, and Enteroviral RT-PCR

No staining for adenovirus, parvovirus B19, measles virus, or cytomegalovirus early antigen was present in any of the myocardial samples. DNA was successfully extracted from all cases, 1 of which (patient 2) was positive for M pneumoniae DNA (Figure 2). RNA was successfully extracted from all cases, none of which was positive for enterovirus transcripts (Table).

COMMENT

Drowning is the second most common cause of accidental death in children in the developed world.1-4 Post-mortem examination of drowning victims may reveal positive findings, such as a white frothy exudate from the mouth and nose and bulky edematous lungs. Although characteristic, these findings are not specific for drowning,5,6 and an accurate account of the events leading up to death are necessary for the diagnosis to be made with any certainty.5,7 Occasionally, review of the history and post-mortem examination reveals additional factors that may have contributed to death. In 2 published series of childhood drownings, 9 (14%) of 65 patients had other significant factors: 6 had epilepsy, 1 had an intellectual handicap, 1 suffered a subarachnoid hemorrhage while in the water, and 1 was found to have a severely hypoplastic right coronary artery.7,8

In the present series, 23% of patients who drowned were found to have coexistent myocarditis. An infectious cause was established in patient 2, where PCR revealed M pneitmonine DNA in cardiac tissue. In the other 4 patients, no infectious cause was established by immunohistochemistry, PCR, or RT-PCR. There was no unexpected significant extracardiac pathology. Cardiac output was not reestablished in any of the patients, and all patients were pronounced deceased within a few hours of the drowning episode. Interestingly, 2 patients had heart weights above the expected weight based on body length. Detailed gross and microscopic examination of the heavier hearts did not reveal evidence of a cardiomyopathy or a second disease process, and no cause for the increased heart weight could be established. However, the weights of the systemic organs (liver, kidneys, pancreas) were also proportionately increased above the standard for body length in both patients.

Subclinical myocarditis is a well-described cause of sudden unexpected death in children and adults.21 Because many patients are asymptomatic, clues as to its true prevalence have come from autopsy studies. Such studies have identified myocardial inflammation in 1% to 9% of otherwise routine autopsies.21-24 However, many of these studies were performed prior to the introduction of the strict Dallas criteria11 for the diagnosis of myocarditis; thus, comparison with present-day studies is difficult. Etiologies of myocarditis include viral infection, particularly enteroviruses such as coxsackie virus; bacterial infections, including M pnenmoniae (as in patient 2); drugs and toxins; and autoimmune diseases, especially systemic lupus erythematosus, scleroderma, and sarcoidosis.21 Mycoplasma-associated myocarditis is an uncommon complication of a common pathogen. In a recent review of 21 cases of Mycoplasma-associated myocarditis, the clinical features ranged from an absence of symptoms to rapid-onset cardiac failure and death.25 Most patients were adults, but the youngest patient was 4 years of age (as in the present patient).

The finding of incidental myocarditis in accidental and traumatic deaths has been reported previously in both adults26-28 and children.29-30 For example, myocarditis has been described in motor vehicle and building site accident victims, homicide victims, and pilots involved in fatal aircraft accidents.26-28 However, the co-occurrence of myocarditis and drowning is rare. Two cases of myocarditis have been reported in the German-language literature, both occurring in adult women while swimming.10 A similar case has been published in the English-language literature, that of a previously well 7-year-old boy who died while immersed in a whirlpool.9 Autopsy showed no positive features of drowning. Histologic analysis revealed isolated foci of lymphocytes within the myocardium, and RT-PCR for coxsackie B3 virus on RNA extracted from formalin-fixed cardiac tissue was positive.9

Myocarditis is a well-known cause of sudden death.5,21 However, when other potential causes of death are also present, the significance of myocarditis is more difficult to determine. This is not an unusual phenomenon in autopsy examination; when more than 1 lesion known to cause death is present, determining the precise role of each in the fatal episode may be problematic.30,31 Nevertheless, given that both myocarditis21 and swimming32 increase the risk of arrhythmias, the combination of both may predispose the individual to sudden cardiac death. Furthermore, in a mouse model of coxsackie myocarditis, mice that were forced to swim after infection showed increased levels of virus during the acute stages of the infection and had significantly higher mortality rates compared with mice that were rested.33,34 The circumstances surrounding the death of patient 1 seem to suggest a role for myocarditis in causing death: a good swimmer, in shallow water, complaining of feeling "dizzy." However, in the other 4 cases, there was insufficient eyewitness information to draw firm conclusions as to the role of myocarditis in the fatal episode.

The present study indicates that cardiac pathology sufficient to raise a competing hypothesis for the cause of death may be seen in apparently straightforward cases of drowning. This has obvious implications for the families involved and for death certification and highlights the need for thorough autopsy examination and clinicopathologic correlation.

We thank Michael Ho, MLT, and Wilson Chan, MSc, for expert help with the immunohistochemistry; Carlos Pereira, BSc, and Grant Johnson, BSc, for help with the molecular microbiologic analyses; Louis Litsas, BSc, for help with archival searches; and Sandra Viero, MD, for many helpful discussions regarding the manuscript. Gratitude is extended to the chief coroner, Barry McLellan, MD, for permission to publish these cases.

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Gino R. Somers, MD, PhD; Charles R. Smith, MD; Gregory J. Wilson, MD; Maria Zielenska, PhD; Raymond Tellier, MD; Glenn P. Taylor, MD

Accepted for publication September 29, 2004.

From the Division of Pathology (Drs Somers, Smith, Wilson, Zielenska, and Taylor) and the Division of Microbiology (Dr Tellier), Department of Pediatric Laboratory Medicine, Hospital for Sick Children, Toronto, Ontario; and the Office of the Chief Coroner, Toronto, Ontario (Drs Smith, Wilson, and Taylor).

Presented in part at the meeting of the Society for Pediatric Pathology, Vancouver, British Columbia, March 2004.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Gino R. Somers, MD, PhD, Division of Pathology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8 (e-mail: gino.somers@sickkids.ca).

Copyright College of American Pathologists Feb 2005
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