Tissue tropism, pathology and pathogenesis of enterovirus infection
Atis Muehlenbachs, Julu Bhatnagar, and Sherif R. Zaki.
Infectious Diseases Pathology Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, GA, 30030.
Key words: Enterovirus, autopsy, immunohistochemistry, PCR, immunohistochemistry, meningoencephalitis & neonatal and congenital infections.
Corresponding author: Atis Muehlenbachs Infectious Diseases Pathology Branch, Division of High-Consequence Pathogens and Pathology NCEZID, CDC 1600 Clifton Rd NE, MS G32 Atlanta, GA 30329-4018 Tel: 404-639-4649 Cell: 404-692-8682 Fax: 404-639-3043 Email: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/path.4438
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ABSTRACT Enteroviruses are very common and cause infections with a diverse array of clinical features. Enteroviruses are most frequently considered by practicing pathologists in cases of aseptic meningitis, encephalitis, myocarditis, and disseminated infections in neonates and infants. Congenital infections have been reported, and transplacental transmission is thought to occur. Although skin biopsies during hand foot and mouth disease are infrequently obtained, characteristic dermatopathological findings can be seen. Enteroviruses have been implicated in lower respiratory tract infections. This review will highlight histopathological features of enterovirus infection and discuss diagnostic modalities for formalin-fixed paraffin-embedded tissues and their associated pitfalls. Immunohistochemistry can detect enterovirus antigen within cells of affected tissues, however assays can be non-specific and detect other viruses. Molecular methods are increasingly relied upon, but due to the high frequency of asymptomatic enteroviral infections, clinical-pathological correlation is needed to determine significance. Of note, diagnostic assays on central nervous system or cardiac tissues from immunocompetent patients with prolonged disease courses are most often negative. Histopathological, immunohistochemical and molecular studies performed on clinical specimens also provide insight into enteroviral tissue tropism and pathogenesis.
INTRODUCTION The genus Enterovirus is within the family Picornaviridae and consists of non-enveloped, icosahedral, single-stranded RNA viruses. Based on molecular and serological characteristics, the following species are defined: enterovirus (EV)-A (which contains EV-71 and several Coxsackievirus group A (CVA) viruses), EV-B (Coxsackievirus group B (CVB)
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viruses and echoviruses), EV-C (polioviruses 1-3 and several CVA viruses), EV-D (EV-68 and 70), and the rhinoviruses [1]. Natural recombination occurs within species [2, 3].
Enterovirus infections occur most frequently in children under the age of 10 (reviewed in [1]). In temperate regions, infections are most common in summer and fall whereas, in tropical regions, infections occur year round. Age has a strong association with clinical presentation: central nervous system (CNS) disease occurs most frequently in 5-15 year olds, myocarditis in 20-40 year olds, severe infections (including myocarditis, CNS disease and sepsis-like illness) in neonates and infants, and hand foot and mouth disease in children less than 5 years of age. A risk factor for infection is low socioeconomic status, most likely associated with poor sanitation and close living quarters. Enterovirus transmission is generally by faecal-oral contamination or by respiratory droplets. Further, enteroviruses are relatively resistant to various solvents and detergents at ambient temperatures.
Infections with enteroviruses are very common and most are frequently asymptomatic, but they can be severe and life-threatening [1]. The enteroviruses of greatest public health concern are the polioviruses, which have been eliminated from much of the world, but are currently circulating in parts of sub-Saharan Africa, the horn of Africa, Pakistan, Afghanistan and Syria despite global eradication efforts. Other enteroviruses, notably EV-71 and several CVA viruses can cause neurological disease similar to poliomyelitis. Enteroviruses are associated with a wide array of clinical presentations including aseptic meningitis and meningoencephalitis (CVA viruses), myocarditis and pericarditis (CVB viruses and echoviruses), systemic neonatal infections (CVB viruses and echoviruses), hand foot and mouth disease (EV-71 and CVA viruses), pleurodynia (CVB viruses and echoviruses),
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pancreatitis (CVB viruses), hepatitis, respiratory disease (rhinoviruses and EV-68) and undifferentiated febrile illness. Some enteroviruses have rather unique disease associations, such as CVA-24 and EV-70 with acute haemorrhagic conjunctivitis, and EV-71 with neurogenic pulmonary oedema.
Following exposure, the site of primary replication is thought to be the gastrointestinal and respiratory epithelium [4, 5]. Viraemia can occur [6, 7] and can even be detectable in asymptomatic cases of poliovirus infection [8]. Certain enteroviruses have characteristic tissue tropisms. Enteroviruses can infect a variety of target cells within tissues, including neurons, cardiomyocytes and epithelial cells. Detection of viral antigens in human tissues is generally associated with cellular injury and the host inflammatory response, which will be discussed in the sections below. Prior exposure to an enterovirus serotype does not limit reinfection. However, during re-infection, viral replication is thought to be limited to the site of primary replication without haematogenous dissemination or secondary tissue infection.
Immunity Immunity is believed to be primarily humoral, and patients with X-linked agammaglobulinaemia are notable for severe and prolonged courses of infection [9]. Severe enterovirus infections have also been reported in patients undergoing rituximab therapy [10] and haematopoietic stem cell transplantation [11, 12]. Persistent viral shedding of a vaccine poliovirus strain has been reported in patients with common variable immune deficiency for up to two decades [13], but it is thought to be a rare occurrence [14]. Enteroviral persistence in immunocompetent individuals has been postulated to play a role in the pathogenesis of post-poliomyelitis progressive muscular atrophy and dilated
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cardiomyopathy; evidence for this has been supported by some studies [15-17], but not others [18, 19]. Autoimmune or post-inflammatory mechanisms may have a role in chronic sequelae; CVB viruses can cause pancreatitis [20], and have been implicated in the pathogenesis of human insulin-dependent diabetes [21, 22], although the issue remains controversial [1].
DIAGNOSIS The conventional methods for detection and serotyping of enterovirus infections are virus isolation and immunofluorescence assays (IFA), which are time consuming and require specialized laboratory facilities [23, 24]. Serological diagnosis (reviewed in [1]) can be made by comparing titres in paired acute and convalescent sera. IgM assays are commercially available and have been used to infer recent infection. However the high prevalence of enteroviruses and serological cross-reactivity limits clinical and epidemiological utility. Several studies in the last decade have described that molecular diagnosis based on the polymerase chain reaction (PCR) is a more rapid, specific and sensitive approach for detection and typing of enterovirus infections than virus isolation [25-29]. Thus, more recently, clinical laboratory diagnosis is generally accomplished by direct molecular testing of fresh clinical specimens to detect enteroviruses [30-33]. Often appropriate clinical specimens for routine enteroviral diagnosis are not collected at the time of a biopsy or an autopsy. Diagnosis of enterovirus infection can be made by using formalin-fixed paraffin embedded (FFPE) tissues, but requires specialized diagnostic modalities discussed below.
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Immunohistochemistry has the advantage of identifying viral antigens and their localization within tissues. Several antibodies against enteroviruses are commercially available for immunohistochemistry, and a commercially available antibody is routinely used at the Infectious Diseases Pathology Branch, CDC for primary immunohistochemical diagnosis. The major pitfall of this antibody is that it cross-reacts with a wide variety of other viruses in our experience, including flaviviruses, adenoviruses, eastern equine encephalitis virus, astrovirus, hepatitis A virus and others. Serotype-specific immunohistochemistry assays may be available at reference laboratories; however, these often tend to be less sensitive than assays using the broadly cross-reactive antibody. During infection, enterovirus antigens are localized to host cell cytoplasm and have a fine granular appearance. To distinguish true from non-specific staining, expertise and negative controls are essential. Challenging cases are frequently encountered where only a few foci of immunostaining are present; in these cases, enteroviral antigen localization is often within foci of inflammation. Because of the non-specificity of immunohistochemistry, PCR is increasingly relied upon for a definitive diagnosis.
Based on studies performed in our laboratory [34-36] and other published reports [37, 38], molecular methods have high sensitivity to detect viral RNA, including enterovirus RNA, in FFPE tissue. PCR-based assays for the detection of human enteroviruses in clinical specimens generally target highly conserved areas in the 5′ untranslated region (5′UTR) [3942]. The 5’ UTR cannot accurately identify various serotypes and is not usually used for enterovirus typing because it is quite conserved among enteroviruses, and some recent studies detected recombination in the 5’ UTR between enteroviruses [43, 44]. For the
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typing of enterovirus, the VP1 capsid gene is considered to be an ideal target because of variability of the region [45, 46]. In addition, phylogenetic analysis of VP1 gene sequences also correlates well with serotyping using serum neutralization assays [1].
Nonetheless, molecular detection and identification of enteroviruses from FFPE tissues has some limitations, including low viral load in the specimens, poor quality fragmented RNA due to formalin fixation (difficult to amplify more than 300-bp segment of the target sequence), and false negative results if no good quality control assay is used simultaneously. On the other hand, because enterovirus infections have high prevalence, PCR can be positive in respiratory swabs when enteroviral infection is not the underlying cause of illness. Thus, to understand the clinical significance of molecular results, it is important to correlate clinical, epidemiological, histopathological and immunohistochemical findings. According to the studies performed by our group (unpublished data), enterovirus PCR is frequently positive in cases that are negative by immunohistochemistry but show histopathological features of enterovirus infection.
CENTRAL NERVOUS SYSTEM DISEASE Aseptic meningitis is the most commonly reported CNS infection, and enteroviruses are the leading pathogens identified (reviewed in [1, 47]). Enteroviral aseptic meningitis and meningoencephalitis occur most frequently in 5 to 15 year olds. Enteroviral aseptic meningitis is typically associated with fever and rash and is self-limited. Enteroviral encephalitis and encephalomyelitis are less common, but more often have severe manifestations including long term debilitation and death. Polioviruses may cause spinal or bulbar paralysis resulting in respiratory failure. Recovery from poliovirus infection can be
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prolonged and incomplete such that extremities may remain permanently weak and develop atrophy. Large EV-71 outbreaks have occurred in Asia, and sporadic cases occur in North America. EV-71 is associated with hand foot and mouth disease and herpangina, and may result in severe brain stem encephalitis and neurogenic pulmonary oedema (figure 1A). CVA viruses can also cause neurological disease. Chronic meningoencephalitis is reported in individuals with agammaglobulinaemia [9] and with rituximab therapy [10]. In infants and neonates, disseminated infections with CVB and echoviruses frequently involve the CNS.
The histopathology of aseptic meningitis typically shows leptomeningitis with a varying degree of oedema and lymphohistiocytic infiltration. Within the parenchyma, perivascular lymphocytic cuffing and neutrophils may be present. In encephalitis or encephalomyelitis, gliosis, microglial nodules, neuronophagia, neutrophilic infiltrates and necrosis may be seen. Following poliovirus infection, the anterior and posterior horns of the spinal cord can show marked neuronal dropout [48]. During EV71 infection, inflammation in the brain stem nuclei and spinal cord is often more severe than in the cortex or cerebellum [49, 50], and neutrophils can be prominent (figure 1B). By immunohistochemistry in cases of fatal EV71 infection, brainstem neurons typically show intense antigen positivity (figure 1C). with immunostaining also seen in neuronal processes and inflammatory foci [49, 51, 52]. Neurons and their processes can also show immunostaining in encephalitis caused by other enteroviruses including CVA viruses. With increasing duration of illness, immunostaining is often limited to granules within microglial nodules (figure 2). Of note, immunohistochemistry is frequently negative in immunocompetent patients who have undergone a lengthy disease course. In infants, disseminated CVB and echoviruses infections can range from aseptic meningitis to necrotizing meningoencephalitis (figure 3).
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The differential diagnosis includes infections with flaviviruses, herpesviruses, eastern equine encephalitis virus, other viruses, and agents of bacterial meningitis in addition to autoimmune causes of encephalitis.
Pathogenesis The poliovirus receptor (PVR, also known as CD155) is an immunoglobulin-like nectin-like protein [53] which is involved in cell-cell contact and signaling. PVR is expressed in the developing CNS, particularly in structures giving rise to the anterior horn motor neurons [54]. Two receptors for EV-71 have been identified: scavenger receptor class B, member 2 (SCARB2, also known as CD36L2) [55] and P-selectin glycoprotein ligand (SELPLG, also known as PSGL-1 and CD162) [56]. SCARB2 is also a receptor for CVA16, but not poliovirus [55]. It is a ubiquitously expressed and integral membrane glycoprotein; gene mutations have been identified in action myoclonus-renal failure syndrome [57], an autosomal recessive progressive epilepsy associated with accumulation of storage material within the brain. SELPLG is a high-affinity P-selectin ligand expressed by myeloid cells and Tlymphocytes; of interest, it is also the cellular receptor for Anaplasma phagocytophilum (the bacterial agent of anaplasmosis, formerly known as human granulocytic ehrlichiosis) to infect granulocytes [58]. SCARB2 has been localized to neurons [5, 59], tonsillar crypt squamous epithelium [5] and intestinal epithelium [60], suggesting a role in the determination of EV-71 cellular tropism, whereas SELPLG has been localized to macrophages/microglia, neutrophils, and vascular endothelium [5].
MYOCARDITIS
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Acute enteroviral myocarditis is most prevalent in adults aged 20 to 40 years. It is frequently self-limited but can cause sudden cardiac death. In autopsy series of patients with myocarditis, enteroviruses have been identified in 12.5-18.5% of cases [36, 61]. The classic presentation is of a biphasic illness, with cardiac symptoms following 2-3 weeks after a viral-like illness. Episodes of enteroviral myocarditis have been implicated in the pathogenesis of dilated cardiomyopathy [62]. CVB and echoviruses are the most frequently identified viruses. Neonatal and infant infections often involve the heart, and can present as myocarditis [63].
Histopathological evaluation of the heart typically shows oedema, interstitial lymphohistiocytic inflammatory infiltrates, a varying degree of neutrophil infiltration, and cardiomyocyte necrosis (figure 4a). Fibrosis may be present depending on length of disease course. The inflammatory infiltrates can be subendocardial, multifocal, patchy or diffuse. By immunohistochemistry, enteroviral antigens are localized to the cytoplasm of cardiomyocytes (figure 4B) [36, 61]. In neonatal and infant cases, immunohistochemistry is much more likely to be positive than in adult cases (figure 5), which are more often negative in the setting of positive PCR. In tissue from individuals with prolonged disease courses, both diagnostic immunohistochemistry and PCR are frequently negative. The differential diagnosis for acute myocarditis is broad, and includes other pathogens such as human parvovirus B19, parechoviruses, Borrelia burgdorferi and Neisseria meningitides among many others. Myocarditis is also thought to result from post-infectious or other inflammatory reactions, and may also be autoimmune.
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Pathogenesis Decay acceleration factor (DAF) is involved in the attachment of CVB to the cell surface [64], and the Coxsackie virus and adenovirus receptor (CAR) has been identified as the cellular receptor for CVB viruses [65]. CAR is highly expressed in developing brain and heart, is an integral membrane protein localized to tight junctions [66], is involved in cardiac cell-cell communication and conduction [67], and is upregulated in dilated cardiomyopathy [68]. A role for CVB interaction with CAR has been postulated in the development of fatal cardiac conduction disturbances and cardiac injury.
NEONATAL , INFANT AND CONGENITAL INFECTIONS Although the majority of enterovirus infections in neonates are asymptomatic [69], neonates and infants are at risk for disseminated CVB and echoviral, and to a lesser extent CVA, infections [70]. The majority of cases occur between the 3rd and 5th days of life, suggesting that acquisition is in the immediate perinatal period. Risk factors include acute maternal illness within a week of delivery [71], and absent neonatal serum neutralizing antibodies [72].
Nosocomial transmission has been well documented [71, 73]; these
infections tend to occur later (10 to 14 days postpartum) and are associated with milder disease [71]. Clinical presentation can involve a sepsis-like condition, hepatitis, meningoencephalitis, myocarditis, pancreatitis, dermatitis and diffuse intravascular coagulopathy.
Neonatal and infant infections typically involve multiple organs, including the CNS and heart as detailed above. Liver histopathological findings can include portal lymphohistiocytic inflammatory infiltrates and multifocal to extensive hepatocellular necrosis and
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haemorrhage (figure 6A). Inflammatory foci can also be present in the adrenal glands, pancreas, spleen, skeletal muscle and other tissues [70]. By immunohistochemistry, enterovirus antigens can be localized to a variety of cell types including neurons, cardiomyocytes, hepatocytes (figure 6B), renal tubular cells (figure 7A), pancreatic islet cells (figure 7B), cells of the adrenal gland (figure 8), pulmonary interstitial cells (figure 9), smooth muscle cells, and brown adipocytes. The differential diagnosis for neonatal and infant infections includes bacterial sepsis and herpesvirus infection, including cytomegalovirus.
Placental villitis and necrosis can be seen in cases that are positive for enterovirus infection at delivery (figure 10) [74]. One study identified coxsackievirus RNA in 6 of 7 placentas by PCR from infants with severe respiratory failure and subsequent neurodevelopmental abnormalities [75]; by immunohistochemistry, enteroviral antigen was reported to be localized to the syncytiotrophoblast. Congenital infections acquired earlier in pregnancy have been documented. In one case, echovirus 11 was detected in amniotic fluid at 22 weeks gestational age from a woman who had a co-twin demise at 14 weeks gestational age [76]; abnormal lung development was noted, and the child died after spontaneous delivery at 38 weeks gestational age with primary pulmonary hypoplasia detected at autopsy. In another woman, EV-71 was isolated from amniotic fluid at 17 weeks gestational age; the fetus developed hepatosplenomegaly and hydrocephalus and stillbirth occurred at 26 weeks gestational age [77]. Cord blood was positive for EV-71 by PCR, and immunohistochemistry was positive in fetal midbrain and liver. In a case of spontaneous abortion at 17 weeks gestational age, echovirus 33 was isolated from maternal stool and placenta; placental villitis and fetal hepatic necrosis were present [78, 79]. Reports such as these are
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infrequent, and the relationship between maternal enterovirus infection and congenital anomalies is unclear.
Pathogenesis It is likely that the increased susceptibility of neonates and infants to severe infection is due to the nature of the developing immune system, particularly in the absence of maternal neutralizing antibodies. As detailed above, DAF and CAR are the attachment factor and cellular receptor for CVB, respectively. Murine neonatal neural stem cells express high levels of CAR and experimentally support CVB infection [80], such that a role for increased susceptibility and neurotropism has been hypothesized. CVB tropism for pancreatic islet cells has been observed in infants who died of CVB infection (Figure 7B) [81-83]; this tropism has been postulated to play a role in the development of insulin-dependent diabetes. DAF is expressed on polarized epithelial cells [84], including the placental syncytiotrophoblast [85]. One study demonstrated that primary human trophoblasts and a choriocarcinoma cell line can be infected with CVB in vitro [86]; infection was dependent upon both DAF and CAR and required lipid rafts, suggesting a mechanism for transplacental infection.
HAND FOOT AND MOUTH DISEASE Hand foot and mouth disease is a viral exanthem, and the majority of cases occur in children aged under 5 years. It is a transient vesicular maculopapular rash involving anterior hands, feet and mouth and typically associated with clinical symptoms of a mild viral illness. Biopsies are generally not obtained in children with the classic clinical presentation, but can be taken from older individuals or those with an atypical disease course. CVA-16 is the most
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frequently implicated virus in children, and there is also a strong association with EV-71. More recently, CVA-6 has been identified as a cause of atypical hand foot mouth disease [87, 88] characterized by a vesiculobullous rash of the trunk and extremities, with differential diagnosis including toxic epidermal necrolysis; nail shedding has been reported during recovery.
On skin biopsy of typical cases, the epidermis shows keratinocyte necrosis that is most prominent in the upper layers. Intraepidermal edema, vesicle formation, and neutrophilic infiltrates can be seen (figure 11A). The papillary dermis can show oedema and a degree of perivascular lymphohistiocytic infiltration. By immunohistochemistry, enteroviral antigens are seen predominantly in the upper half of the epidermis, are localized to the cytoplasm of keratinocytes and are associated with areas of epidermal necrosis (figure 11B). The differential diagnosis primarily includes herpesvirus infections, but no multinucleated giant cells or viral inclusions can be seen.
Pathogenesis As detailed above, SCARB2 is a receptor for EV-71 and CVA-16. At the time of this review, no reports of expression of SCARB2 by keratinocytes can be identified, although SCARB2 has been reported in squamous cells of the tonsillar crypts [5]. Enteroviral tropism for keratinocytes of the anterior hands and feet (in addition to squamous cells of the oral mucosa) suggests targeting of anatomical sites that most facilitate transmission between individuals through direct contact or contamination of surfaces. Determination of the molecular details of this tropism would be of particular interest.
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RESPIRATORY INFECTIONS Enteroviruses are associated with both upper and lower respiratory tract disease. EV-68 has been reported in outbreaks of lower respiratory tract illness [89, 90]. Rhinovirus infections are generally considered to be clinically limited to the upper respiratory tract, although viraemia has been documented [6]. Rhinoviruses are increasingly detected in cases of severe bronchiolitis [91, 92], raising the possibility that symptomatic lower respiratory tract infection may occur. The high prevalence of rhinoviruses in asymptomatic individuals [93] makes it challenging to interpret a causative role in severe disease. Of note, experimental evidence in human volunteers suggests that bronchiolar epithelium can be infected by rhinoviruses [94].
Pathogenesis Intracellular adhesion molecule-1 (ICAM-1) is the cellular receptor for the majority of rhinoviruses [95, 96], and is reported to be expressed by bronchial epithelium [97] and by pneumocytes [98], with upregulation during experimental murine pneumonia [99]. Of interest, a case of sudden infant death was recently submitted to the CDC for evaluation (figure 12); bronchiolitis was present, abundant immunostaining was seen by enterovirus immunohistochemistry in cells of the lower respiratory tract, and rhinovirus was detected by PCR from FFPE lung tissue; no other viruses were detected. Systematic autopsy studies that localize rhinoviral antigens and provide molecular data in cases of fatal bronchiolitis would likely provide insight into the pathogenesis of severe rhinovirus infection or coinfection.
SUMMARY
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Enteroviral infections are common, are caused by a wide variety of different enteroviruses, and have diverse clinical presentations. Characteristic clinical and pathological findings can be associated with certain types of enteroviruses. Clinical and histopathological correlation with immunohistochemical and molecular test results can provide insight into tissue tropism and the pathogenesis of enteroviral infections.
ETHICAL STATEMENT: Tissues were obtained at biopsy or autopsy from patients and were submitted to the CDC for laboratory evaluation; this activity is not considered to be research that required review by an institutional review board or informed consent from the patients.
AUTHOR CONTRIBUTIONS: All authors wrote, read and approved the final manuscript.
DISCLAIMER: The findings and conclusions herein are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
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FIGURE LEGENDS Figure 1. Fatal EV-71 infection in a young child during an outbreak in Asia. A) Neurogenic pulmonary oedema (H&E). B) Brainstem encephalitis (H&E). C) Enterovirus antigen (red) localized to brainstem neurons and processes by immunohistochemistry (alkaline fast red).
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Figure 2. EV-A encephalitis in a 15 year old child. A) Brain biopsy showing encephalitis (H&E). B) Focal enterovirus antigen (red) localized to a glial nodule by immunohistochemistry (alkaline fast red). The patient was on rituximab for an autoimmune disorder, had a several month long disease course and ultimately died.
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Figure 3. Necrotizing encephalitis in a 7-day old infant caused by CVB1 (H&E). The mother was febrile at delivery at 34 weeks gestational age.
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Figure 4. Enteroviral myocarditis in a 26-year old adult . A) Dense myocardial infiltrates and cardiomyocyte necrosis (H&E). B) Rare cardiomyocytes showing enterovirus antigen (red) by immunohistochemistry (alkaline fast red). The patient was hospitalized for respiratory distress, pleuritic chest pain and died of cardiac arrhythmia and seizure.
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Figure 5. CVB3 myocarditis in a 6-month old infant. A) Mocardium with diffuse myocardial infiltrates and contraction band necrosis (H&E). B) Diffuse enterovirus antigen localization to cardiomyocytes by immunohistochemistry (alkaline fast red).
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Figure 6. CVB3 hepatitis in a 6 month old infant. A) Portal inflammatory infiltrates and single cell hepatocyte necrosis (H&E). Enterovirus antigen localized to degenerating hepatocytes (red) by immunohistochemistry (alkaline fast red).
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Figure 7. Kidney and pancreas in paediatric CVB infections. Enterovirus antigen (red) localized to A) renal tubular epithelium and B) pancreatic islet cells by immunohistochemistry (alkaline fast red).
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Figure 8. Adrenal gland in a neonatal CVB1 infection. A) Adrenal necrosis (H&E). B) Enterovirus antigen localization to necrotic cells (red) by immunohistochemistry (alkaline fast red).
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Figure 9. Lung in paediatric CVB3 infection. Interstitial pneumonitis (H&E). Focal enterovirus antgen localized to interstitium (red) by immunohistochemistry (alkaline fast red).
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Figure 10. Necrotizing villitis in a placenta from a stillbirth complicated by CVB infection at 32 weeks gestational age (H&E). This case was positive by PCR but negative by immunohistochemistry.
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Figure 11. Hand foot mouth disease caused by CVA-6 in a 13 year old child. A) Epidermal necrosis with vesicle formation (H&E). B) Enterovirus antigen localized to keratinocyte cytoplasm (red) by immunohistochemistry (alkaline fast red).
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Figure 12. Sudden death in an 8 month old infant associated with rhinovirus infection. A) Bronchiolitis (H&E). B) Extensive immunostaining seen by Enterovirus immunohistochemistry in cells of the lower respiratory tract. PCR performed on FFPE lung tissue was positive for rhinovirus, and negative for respiratory syncytial virus, influenza and parainfluenza viruses, metapneumovirus and Mycoplasma species.
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Atis Muehlenbachs, Julu Bhatnagar, and Sherif R. Zaki.
Infectious Diseases Pathology Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, GA, 30030.
Key words: Enterovirus, autopsy, immunohistochemistry, PCR, immunohistochemistry, meningoencephalitis & neonatal and congenital infections.
Corresponding author: Atis Muehlenbachs Infectious Diseases Pathology Branch, Division of High-Consequence Pathogens and Pathology NCEZID, CDC 1600 Clifton Rd NE, MS G32 Atlanta, GA 30329-4018 Tel: 404-639-4649 Cell: 404-692-8682 Fax: 404-639-3043 Email: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/path.4438
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ABSTRACT Enteroviruses are very common and cause infections with a diverse array of clinical features. Enteroviruses are most frequently considered by practicing pathologists in cases of aseptic meningitis, encephalitis, myocarditis, and disseminated infections in neonates and infants. Congenital infections have been reported, and transplacental transmission is thought to occur. Although skin biopsies during hand foot and mouth disease are infrequently obtained, characteristic dermatopathological findings can be seen. Enteroviruses have been implicated in lower respiratory tract infections. This review will highlight histopathological features of enterovirus infection and discuss diagnostic modalities for formalin-fixed paraffin-embedded tissues and their associated pitfalls. Immunohistochemistry can detect enterovirus antigen within cells of affected tissues, however assays can be non-specific and detect other viruses. Molecular methods are increasingly relied upon, but due to the high frequency of asymptomatic enteroviral infections, clinical-pathological correlation is needed to determine significance. Of note, diagnostic assays on central nervous system or cardiac tissues from immunocompetent patients with prolonged disease courses are most often negative. Histopathological, immunohistochemical and molecular studies performed on clinical specimens also provide insight into enteroviral tissue tropism and pathogenesis.
INTRODUCTION The genus Enterovirus is within the family Picornaviridae and consists of non-enveloped, icosahedral, single-stranded RNA viruses. Based on molecular and serological characteristics, the following species are defined: enterovirus (EV)-A (which contains EV-71 and several Coxsackievirus group A (CVA) viruses), EV-B (Coxsackievirus group B (CVB)
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viruses and echoviruses), EV-C (polioviruses 1-3 and several CVA viruses), EV-D (EV-68 and 70), and the rhinoviruses [1]. Natural recombination occurs within species [2, 3].
Enterovirus infections occur most frequently in children under the age of 10 (reviewed in [1]). In temperate regions, infections are most common in summer and fall whereas, in tropical regions, infections occur year round. Age has a strong association with clinical presentation: central nervous system (CNS) disease occurs most frequently in 5-15 year olds, myocarditis in 20-40 year olds, severe infections (including myocarditis, CNS disease and sepsis-like illness) in neonates and infants, and hand foot and mouth disease in children less than 5 years of age. A risk factor for infection is low socioeconomic status, most likely associated with poor sanitation and close living quarters. Enterovirus transmission is generally by faecal-oral contamination or by respiratory droplets. Further, enteroviruses are relatively resistant to various solvents and detergents at ambient temperatures.
Infections with enteroviruses are very common and most are frequently asymptomatic, but they can be severe and life-threatening [1]. The enteroviruses of greatest public health concern are the polioviruses, which have been eliminated from much of the world, but are currently circulating in parts of sub-Saharan Africa, the horn of Africa, Pakistan, Afghanistan and Syria despite global eradication efforts. Other enteroviruses, notably EV-71 and several CVA viruses can cause neurological disease similar to poliomyelitis. Enteroviruses are associated with a wide array of clinical presentations including aseptic meningitis and meningoencephalitis (CVA viruses), myocarditis and pericarditis (CVB viruses and echoviruses), systemic neonatal infections (CVB viruses and echoviruses), hand foot and mouth disease (EV-71 and CVA viruses), pleurodynia (CVB viruses and echoviruses),
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pancreatitis (CVB viruses), hepatitis, respiratory disease (rhinoviruses and EV-68) and undifferentiated febrile illness. Some enteroviruses have rather unique disease associations, such as CVA-24 and EV-70 with acute haemorrhagic conjunctivitis, and EV-71 with neurogenic pulmonary oedema.
Following exposure, the site of primary replication is thought to be the gastrointestinal and respiratory epithelium [4, 5]. Viraemia can occur [6, 7] and can even be detectable in asymptomatic cases of poliovirus infection [8]. Certain enteroviruses have characteristic tissue tropisms. Enteroviruses can infect a variety of target cells within tissues, including neurons, cardiomyocytes and epithelial cells. Detection of viral antigens in human tissues is generally associated with cellular injury and the host inflammatory response, which will be discussed in the sections below. Prior exposure to an enterovirus serotype does not limit reinfection. However, during re-infection, viral replication is thought to be limited to the site of primary replication without haematogenous dissemination or secondary tissue infection.
Immunity Immunity is believed to be primarily humoral, and patients with X-linked agammaglobulinaemia are notable for severe and prolonged courses of infection [9]. Severe enterovirus infections have also been reported in patients undergoing rituximab therapy [10] and haematopoietic stem cell transplantation [11, 12]. Persistent viral shedding of a vaccine poliovirus strain has been reported in patients with common variable immune deficiency for up to two decades [13], but it is thought to be a rare occurrence [14]. Enteroviral persistence in immunocompetent individuals has been postulated to play a role in the pathogenesis of post-poliomyelitis progressive muscular atrophy and dilated
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cardiomyopathy; evidence for this has been supported by some studies [15-17], but not others [18, 19]. Autoimmune or post-inflammatory mechanisms may have a role in chronic sequelae; CVB viruses can cause pancreatitis [20], and have been implicated in the pathogenesis of human insulin-dependent diabetes [21, 22], although the issue remains controversial [1].
DIAGNOSIS The conventional methods for detection and serotyping of enterovirus infections are virus isolation and immunofluorescence assays (IFA), which are time consuming and require specialized laboratory facilities [23, 24]. Serological diagnosis (reviewed in [1]) can be made by comparing titres in paired acute and convalescent sera. IgM assays are commercially available and have been used to infer recent infection. However the high prevalence of enteroviruses and serological cross-reactivity limits clinical and epidemiological utility. Several studies in the last decade have described that molecular diagnosis based on the polymerase chain reaction (PCR) is a more rapid, specific and sensitive approach for detection and typing of enterovirus infections than virus isolation [25-29]. Thus, more recently, clinical laboratory diagnosis is generally accomplished by direct molecular testing of fresh clinical specimens to detect enteroviruses [30-33]. Often appropriate clinical specimens for routine enteroviral diagnosis are not collected at the time of a biopsy or an autopsy. Diagnosis of enterovirus infection can be made by using formalin-fixed paraffin embedded (FFPE) tissues, but requires specialized diagnostic modalities discussed below.
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Immunohistochemistry has the advantage of identifying viral antigens and their localization within tissues. Several antibodies against enteroviruses are commercially available for immunohistochemistry, and a commercially available antibody is routinely used at the Infectious Diseases Pathology Branch, CDC for primary immunohistochemical diagnosis. The major pitfall of this antibody is that it cross-reacts with a wide variety of other viruses in our experience, including flaviviruses, adenoviruses, eastern equine encephalitis virus, astrovirus, hepatitis A virus and others. Serotype-specific immunohistochemistry assays may be available at reference laboratories; however, these often tend to be less sensitive than assays using the broadly cross-reactive antibody. During infection, enterovirus antigens are localized to host cell cytoplasm and have a fine granular appearance. To distinguish true from non-specific staining, expertise and negative controls are essential. Challenging cases are frequently encountered where only a few foci of immunostaining are present; in these cases, enteroviral antigen localization is often within foci of inflammation. Because of the non-specificity of immunohistochemistry, PCR is increasingly relied upon for a definitive diagnosis.
Based on studies performed in our laboratory [34-36] and other published reports [37, 38], molecular methods have high sensitivity to detect viral RNA, including enterovirus RNA, in FFPE tissue. PCR-based assays for the detection of human enteroviruses in clinical specimens generally target highly conserved areas in the 5′ untranslated region (5′UTR) [3942]. The 5’ UTR cannot accurately identify various serotypes and is not usually used for enterovirus typing because it is quite conserved among enteroviruses, and some recent studies detected recombination in the 5’ UTR between enteroviruses [43, 44]. For the
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typing of enterovirus, the VP1 capsid gene is considered to be an ideal target because of variability of the region [45, 46]. In addition, phylogenetic analysis of VP1 gene sequences also correlates well with serotyping using serum neutralization assays [1].
Nonetheless, molecular detection and identification of enteroviruses from FFPE tissues has some limitations, including low viral load in the specimens, poor quality fragmented RNA due to formalin fixation (difficult to amplify more than 300-bp segment of the target sequence), and false negative results if no good quality control assay is used simultaneously. On the other hand, because enterovirus infections have high prevalence, PCR can be positive in respiratory swabs when enteroviral infection is not the underlying cause of illness. Thus, to understand the clinical significance of molecular results, it is important to correlate clinical, epidemiological, histopathological and immunohistochemical findings. According to the studies performed by our group (unpublished data), enterovirus PCR is frequently positive in cases that are negative by immunohistochemistry but show histopathological features of enterovirus infection.
CENTRAL NERVOUS SYSTEM DISEASE Aseptic meningitis is the most commonly reported CNS infection, and enteroviruses are the leading pathogens identified (reviewed in [1, 47]). Enteroviral aseptic meningitis and meningoencephalitis occur most frequently in 5 to 15 year olds. Enteroviral aseptic meningitis is typically associated with fever and rash and is self-limited. Enteroviral encephalitis and encephalomyelitis are less common, but more often have severe manifestations including long term debilitation and death. Polioviruses may cause spinal or bulbar paralysis resulting in respiratory failure. Recovery from poliovirus infection can be
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prolonged and incomplete such that extremities may remain permanently weak and develop atrophy. Large EV-71 outbreaks have occurred in Asia, and sporadic cases occur in North America. EV-71 is associated with hand foot and mouth disease and herpangina, and may result in severe brain stem encephalitis and neurogenic pulmonary oedema (figure 1A). CVA viruses can also cause neurological disease. Chronic meningoencephalitis is reported in individuals with agammaglobulinaemia [9] and with rituximab therapy [10]. In infants and neonates, disseminated infections with CVB and echoviruses frequently involve the CNS.
The histopathology of aseptic meningitis typically shows leptomeningitis with a varying degree of oedema and lymphohistiocytic infiltration. Within the parenchyma, perivascular lymphocytic cuffing and neutrophils may be present. In encephalitis or encephalomyelitis, gliosis, microglial nodules, neuronophagia, neutrophilic infiltrates and necrosis may be seen. Following poliovirus infection, the anterior and posterior horns of the spinal cord can show marked neuronal dropout [48]. During EV71 infection, inflammation in the brain stem nuclei and spinal cord is often more severe than in the cortex or cerebellum [49, 50], and neutrophils can be prominent (figure 1B). By immunohistochemistry in cases of fatal EV71 infection, brainstem neurons typically show intense antigen positivity (figure 1C). with immunostaining also seen in neuronal processes and inflammatory foci [49, 51, 52]. Neurons and their processes can also show immunostaining in encephalitis caused by other enteroviruses including CVA viruses. With increasing duration of illness, immunostaining is often limited to granules within microglial nodules (figure 2). Of note, immunohistochemistry is frequently negative in immunocompetent patients who have undergone a lengthy disease course. In infants, disseminated CVB and echoviruses infections can range from aseptic meningitis to necrotizing meningoencephalitis (figure 3).
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The differential diagnosis includes infections with flaviviruses, herpesviruses, eastern equine encephalitis virus, other viruses, and agents of bacterial meningitis in addition to autoimmune causes of encephalitis.
Pathogenesis The poliovirus receptor (PVR, also known as CD155) is an immunoglobulin-like nectin-like protein [53] which is involved in cell-cell contact and signaling. PVR is expressed in the developing CNS, particularly in structures giving rise to the anterior horn motor neurons [54]. Two receptors for EV-71 have been identified: scavenger receptor class B, member 2 (SCARB2, also known as CD36L2) [55] and P-selectin glycoprotein ligand (SELPLG, also known as PSGL-1 and CD162) [56]. SCARB2 is also a receptor for CVA16, but not poliovirus [55]. It is a ubiquitously expressed and integral membrane glycoprotein; gene mutations have been identified in action myoclonus-renal failure syndrome [57], an autosomal recessive progressive epilepsy associated with accumulation of storage material within the brain. SELPLG is a high-affinity P-selectin ligand expressed by myeloid cells and Tlymphocytes; of interest, it is also the cellular receptor for Anaplasma phagocytophilum (the bacterial agent of anaplasmosis, formerly known as human granulocytic ehrlichiosis) to infect granulocytes [58]. SCARB2 has been localized to neurons [5, 59], tonsillar crypt squamous epithelium [5] and intestinal epithelium [60], suggesting a role in the determination of EV-71 cellular tropism, whereas SELPLG has been localized to macrophages/microglia, neutrophils, and vascular endothelium [5].
MYOCARDITIS
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Acute enteroviral myocarditis is most prevalent in adults aged 20 to 40 years. It is frequently self-limited but can cause sudden cardiac death. In autopsy series of patients with myocarditis, enteroviruses have been identified in 12.5-18.5% of cases [36, 61]. The classic presentation is of a biphasic illness, with cardiac symptoms following 2-3 weeks after a viral-like illness. Episodes of enteroviral myocarditis have been implicated in the pathogenesis of dilated cardiomyopathy [62]. CVB and echoviruses are the most frequently identified viruses. Neonatal and infant infections often involve the heart, and can present as myocarditis [63].
Histopathological evaluation of the heart typically shows oedema, interstitial lymphohistiocytic inflammatory infiltrates, a varying degree of neutrophil infiltration, and cardiomyocyte necrosis (figure 4a). Fibrosis may be present depending on length of disease course. The inflammatory infiltrates can be subendocardial, multifocal, patchy or diffuse. By immunohistochemistry, enteroviral antigens are localized to the cytoplasm of cardiomyocytes (figure 4B) [36, 61]. In neonatal and infant cases, immunohistochemistry is much more likely to be positive than in adult cases (figure 5), which are more often negative in the setting of positive PCR. In tissue from individuals with prolonged disease courses, both diagnostic immunohistochemistry and PCR are frequently negative. The differential diagnosis for acute myocarditis is broad, and includes other pathogens such as human parvovirus B19, parechoviruses, Borrelia burgdorferi and Neisseria meningitides among many others. Myocarditis is also thought to result from post-infectious or other inflammatory reactions, and may also be autoimmune.
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Pathogenesis Decay acceleration factor (DAF) is involved in the attachment of CVB to the cell surface [64], and the Coxsackie virus and adenovirus receptor (CAR) has been identified as the cellular receptor for CVB viruses [65]. CAR is highly expressed in developing brain and heart, is an integral membrane protein localized to tight junctions [66], is involved in cardiac cell-cell communication and conduction [67], and is upregulated in dilated cardiomyopathy [68]. A role for CVB interaction with CAR has been postulated in the development of fatal cardiac conduction disturbances and cardiac injury.
NEONATAL , INFANT AND CONGENITAL INFECTIONS Although the majority of enterovirus infections in neonates are asymptomatic [69], neonates and infants are at risk for disseminated CVB and echoviral, and to a lesser extent CVA, infections [70]. The majority of cases occur between the 3rd and 5th days of life, suggesting that acquisition is in the immediate perinatal period. Risk factors include acute maternal illness within a week of delivery [71], and absent neonatal serum neutralizing antibodies [72].
Nosocomial transmission has been well documented [71, 73]; these
infections tend to occur later (10 to 14 days postpartum) and are associated with milder disease [71]. Clinical presentation can involve a sepsis-like condition, hepatitis, meningoencephalitis, myocarditis, pancreatitis, dermatitis and diffuse intravascular coagulopathy.
Neonatal and infant infections typically involve multiple organs, including the CNS and heart as detailed above. Liver histopathological findings can include portal lymphohistiocytic inflammatory infiltrates and multifocal to extensive hepatocellular necrosis and
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haemorrhage (figure 6A). Inflammatory foci can also be present in the adrenal glands, pancreas, spleen, skeletal muscle and other tissues [70]. By immunohistochemistry, enterovirus antigens can be localized to a variety of cell types including neurons, cardiomyocytes, hepatocytes (figure 6B), renal tubular cells (figure 7A), pancreatic islet cells (figure 7B), cells of the adrenal gland (figure 8), pulmonary interstitial cells (figure 9), smooth muscle cells, and brown adipocytes. The differential diagnosis for neonatal and infant infections includes bacterial sepsis and herpesvirus infection, including cytomegalovirus.
Placental villitis and necrosis can be seen in cases that are positive for enterovirus infection at delivery (figure 10) [74]. One study identified coxsackievirus RNA in 6 of 7 placentas by PCR from infants with severe respiratory failure and subsequent neurodevelopmental abnormalities [75]; by immunohistochemistry, enteroviral antigen was reported to be localized to the syncytiotrophoblast. Congenital infections acquired earlier in pregnancy have been documented. In one case, echovirus 11 was detected in amniotic fluid at 22 weeks gestational age from a woman who had a co-twin demise at 14 weeks gestational age [76]; abnormal lung development was noted, and the child died after spontaneous delivery at 38 weeks gestational age with primary pulmonary hypoplasia detected at autopsy. In another woman, EV-71 was isolated from amniotic fluid at 17 weeks gestational age; the fetus developed hepatosplenomegaly and hydrocephalus and stillbirth occurred at 26 weeks gestational age [77]. Cord blood was positive for EV-71 by PCR, and immunohistochemistry was positive in fetal midbrain and liver. In a case of spontaneous abortion at 17 weeks gestational age, echovirus 33 was isolated from maternal stool and placenta; placental villitis and fetal hepatic necrosis were present [78, 79]. Reports such as these are
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infrequent, and the relationship between maternal enterovirus infection and congenital anomalies is unclear.
Pathogenesis It is likely that the increased susceptibility of neonates and infants to severe infection is due to the nature of the developing immune system, particularly in the absence of maternal neutralizing antibodies. As detailed above, DAF and CAR are the attachment factor and cellular receptor for CVB, respectively. Murine neonatal neural stem cells express high levels of CAR and experimentally support CVB infection [80], such that a role for increased susceptibility and neurotropism has been hypothesized. CVB tropism for pancreatic islet cells has been observed in infants who died of CVB infection (Figure 7B) [81-83]; this tropism has been postulated to play a role in the development of insulin-dependent diabetes. DAF is expressed on polarized epithelial cells [84], including the placental syncytiotrophoblast [85]. One study demonstrated that primary human trophoblasts and a choriocarcinoma cell line can be infected with CVB in vitro [86]; infection was dependent upon both DAF and CAR and required lipid rafts, suggesting a mechanism for transplacental infection.
HAND FOOT AND MOUTH DISEASE Hand foot and mouth disease is a viral exanthem, and the majority of cases occur in children aged under 5 years. It is a transient vesicular maculopapular rash involving anterior hands, feet and mouth and typically associated with clinical symptoms of a mild viral illness. Biopsies are generally not obtained in children with the classic clinical presentation, but can be taken from older individuals or those with an atypical disease course. CVA-16 is the most
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frequently implicated virus in children, and there is also a strong association with EV-71. More recently, CVA-6 has been identified as a cause of atypical hand foot mouth disease [87, 88] characterized by a vesiculobullous rash of the trunk and extremities, with differential diagnosis including toxic epidermal necrolysis; nail shedding has been reported during recovery.
On skin biopsy of typical cases, the epidermis shows keratinocyte necrosis that is most prominent in the upper layers. Intraepidermal edema, vesicle formation, and neutrophilic infiltrates can be seen (figure 11A). The papillary dermis can show oedema and a degree of perivascular lymphohistiocytic infiltration. By immunohistochemistry, enteroviral antigens are seen predominantly in the upper half of the epidermis, are localized to the cytoplasm of keratinocytes and are associated with areas of epidermal necrosis (figure 11B). The differential diagnosis primarily includes herpesvirus infections, but no multinucleated giant cells or viral inclusions can be seen.
Pathogenesis As detailed above, SCARB2 is a receptor for EV-71 and CVA-16. At the time of this review, no reports of expression of SCARB2 by keratinocytes can be identified, although SCARB2 has been reported in squamous cells of the tonsillar crypts [5]. Enteroviral tropism for keratinocytes of the anterior hands and feet (in addition to squamous cells of the oral mucosa) suggests targeting of anatomical sites that most facilitate transmission between individuals through direct contact or contamination of surfaces. Determination of the molecular details of this tropism would be of particular interest.
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RESPIRATORY INFECTIONS Enteroviruses are associated with both upper and lower respiratory tract disease. EV-68 has been reported in outbreaks of lower respiratory tract illness [89, 90]. Rhinovirus infections are generally considered to be clinically limited to the upper respiratory tract, although viraemia has been documented [6]. Rhinoviruses are increasingly detected in cases of severe bronchiolitis [91, 92], raising the possibility that symptomatic lower respiratory tract infection may occur. The high prevalence of rhinoviruses in asymptomatic individuals [93] makes it challenging to interpret a causative role in severe disease. Of note, experimental evidence in human volunteers suggests that bronchiolar epithelium can be infected by rhinoviruses [94].
Pathogenesis Intracellular adhesion molecule-1 (ICAM-1) is the cellular receptor for the majority of rhinoviruses [95, 96], and is reported to be expressed by bronchial epithelium [97] and by pneumocytes [98], with upregulation during experimental murine pneumonia [99]. Of interest, a case of sudden infant death was recently submitted to the CDC for evaluation (figure 12); bronchiolitis was present, abundant immunostaining was seen by enterovirus immunohistochemistry in cells of the lower respiratory tract, and rhinovirus was detected by PCR from FFPE lung tissue; no other viruses were detected. Systematic autopsy studies that localize rhinoviral antigens and provide molecular data in cases of fatal bronchiolitis would likely provide insight into the pathogenesis of severe rhinovirus infection or coinfection.
SUMMARY
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Enteroviral infections are common, are caused by a wide variety of different enteroviruses, and have diverse clinical presentations. Characteristic clinical and pathological findings can be associated with certain types of enteroviruses. Clinical and histopathological correlation with immunohistochemical and molecular test results can provide insight into tissue tropism and the pathogenesis of enteroviral infections.
ETHICAL STATEMENT: Tissues were obtained at biopsy or autopsy from patients and were submitted to the CDC for laboratory evaluation; this activity is not considered to be research that required review by an institutional review board or informed consent from the patients.
AUTHOR CONTRIBUTIONS: All authors wrote, read and approved the final manuscript.
DISCLAIMER: The findings and conclusions herein are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
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FIGURE LEGENDS Figure 1. Fatal EV-71 infection in a young child during an outbreak in Asia. A) Neurogenic pulmonary oedema (H&E). B) Brainstem encephalitis (H&E). C) Enterovirus antigen (red) localized to brainstem neurons and processes by immunohistochemistry (alkaline fast red).
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Figure 2. EV-A encephalitis in a 15 year old child. A) Brain biopsy showing encephalitis (H&E). B) Focal enterovirus antigen (red) localized to a glial nodule by immunohistochemistry (alkaline fast red). The patient was on rituximab for an autoimmune disorder, had a several month long disease course and ultimately died.
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Figure 3. Necrotizing encephalitis in a 7-day old infant caused by CVB1 (H&E). The mother was febrile at delivery at 34 weeks gestational age.
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Figure 4. Enteroviral myocarditis in a 26-year old adult . A) Dense myocardial infiltrates and cardiomyocyte necrosis (H&E). B) Rare cardiomyocytes showing enterovirus antigen (red) by immunohistochemistry (alkaline fast red). The patient was hospitalized for respiratory distress, pleuritic chest pain and died of cardiac arrhythmia and seizure.
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Figure 5. CVB3 myocarditis in a 6-month old infant. A) Mocardium with diffuse myocardial infiltrates and contraction band necrosis (H&E). B) Diffuse enterovirus antigen localization to cardiomyocytes by immunohistochemistry (alkaline fast red).
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Figure 6. CVB3 hepatitis in a 6 month old infant. A) Portal inflammatory infiltrates and single cell hepatocyte necrosis (H&E). Enterovirus antigen localized to degenerating hepatocytes (red) by immunohistochemistry (alkaline fast red).
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Figure 7. Kidney and pancreas in paediatric CVB infections. Enterovirus antigen (red) localized to A) renal tubular epithelium and B) pancreatic islet cells by immunohistochemistry (alkaline fast red).
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Figure 8. Adrenal gland in a neonatal CVB1 infection. A) Adrenal necrosis (H&E). B) Enterovirus antigen localization to necrotic cells (red) by immunohistochemistry (alkaline fast red).
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Figure 9. Lung in paediatric CVB3 infection. Interstitial pneumonitis (H&E). Focal enterovirus antgen localized to interstitium (red) by immunohistochemistry (alkaline fast red).
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Figure 10. Necrotizing villitis in a placenta from a stillbirth complicated by CVB infection at 32 weeks gestational age (H&E). This case was positive by PCR but negative by immunohistochemistry.
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Figure 11. Hand foot mouth disease caused by CVA-6 in a 13 year old child. A) Epidermal necrosis with vesicle formation (H&E). B) Enterovirus antigen localized to keratinocyte cytoplasm (red) by immunohistochemistry (alkaline fast red).
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Figure 12. Sudden death in an 8 month old infant associated with rhinovirus infection. A) Bronchiolitis (H&E). B) Extensive immunostaining seen by Enterovirus immunohistochemistry in cells of the lower respiratory tract. PCR performed on FFPE lung tissue was positive for rhinovirus, and negative for respiratory syncytial virus, influenza and parainfluenza viruses, metapneumovirus and Mycoplasma species.
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