Dengue is an arbovirosis caused by the Dengue Virus (DENV), an RNA-virus of the flaviviridae family, composed of four serotypes (called DENV-1 to DENV-4). It is transmitted to humans by mosquitoes, most commonly of the Aedes genus . It is an endemic disease in several parts of the world, especially in tropical and subtropical regions, where epidemics occur frequently . The incidence of dengue has grown around the world in recent decades. The number of dengue cases reported to the World Health Organization (WHO) increased over eight fold over the last two decades, from 505,430 cases in 2000, to over 2.4 million in 2010, and 5.2 million in 2019. Reported deaths between the year 2000 and 2015 increased from 960 to 4032. In 2021, still amidst the Coronavirus Disease (COVID-19) pandemic, endemic and mosquito-borne infections such as dengue are at potential risk of underreporting, underdiagnoses and reduced prevention measures, being a neglected disease that mainly affects underdeveloped countries. The combined impact of COVID-19 and dengue epidemics can potentially result in devastating consequences for the populations at risk .
According to WHO, in the revised and expanded edition of its comprehensive guidelines for prevention and control of dengue and dengue hemorrhagic fever, published in 2011, defines the different types of clinical presentation? The more nonspecific form, called undifferentiated fever, is common in infants, children and adults who have been infected with dengue virus, especially for the first time. The Dengue Fever (DF) is generally an acute febrile illness, and sometimes biphasic fever, which is generally self-limiting, and most common in older children, adolescents and adults. The more severe form is called severe dengue fever or Dengue Hemorrhagic Fever (DHF), more common in children less than 15 years of age in hyperendemic areas, and associated with repeated dengue infections. DHF can develop into a Dengue Shock Syndrome (DSS), which may be preceded by warning signs such as persistent vomiting, lethargy or restlessness, or irritability and oliguria .
The severity of clinical repercussions depends on multiple factors associated with the complex interaction between the host, especially its immune system, and the infectious agent [5-9]. Unusual or atypical manifestations have been designated Expanded Dengue Syndrome (EDS). These manifestations include the neurological impairment, such as seizures, lowered level of consciousness and motor disturbances (spasticity, transient paresis), and associated with multiple tissue changes (hemorrhages, edema, and inflammatory processes, among others) [4,10]. Unfortunately, in most of the non-fatal cases in well-known series, an autopsy was not performed, not allowing for an adequate histopathological analysis .
In this paper, aspects of dengue with neurological involvement are reviewed, as well as neuropathological findings described both in animal models and in human case reports and series, especially autopsies. Some aspects of the pathogenesis of these alterations are also discussed.
Neurological manifestations, in its clinical aspects, have been described in dengue since the 1960s [11-13] and, in some cases, the neurological presentation may be the first one related to the infection. Historically, such manifestations were considered secondary mainly to systemic alterations, especially vascular ones, and not to the presence of the virus, considered non-neurotropic . However, in recent decades, the neurotropism of DENV has been solidly demonstrated, with antigens and viral RNA being isolated both in tissues of the Central Nervous System (CNS) and Peripheral Nervous System (PNS), as well as in the Cerebrospinal Fluid (CSF) [15-24]. Serotypes 2 and 3 have been described as having the greatest potential to induce neurological disturbances, acting as neurovirulent elements similar to other flaviviruses [15,19,25-29]. Some clinical and laboratory parameters, like higher mean body temperature, low platelet count, elevated hematocrit, and liver dysfunction, have been described as risk factors for neurological dysfunction .
Neurological alterations associated with dengue are classified in different ways, with different criteria. Some authors prefer to use the mechanisms of pathogenesis as classifiers: Metabolic disturbances, resulting predominantly in encephalopathy; viral invasion, mainly associated with encephalitis, meningitis, myelitis and myositis; and exacerbated autoimmunity, including neuromyelitis optica, optic neuritis, Acute Disseminated Encephalomyelitis (ADEM) and Guillain-Barre syndrome [7,31,32]. Encephalopathy is strongly associated with DSS, due to the hydroelectrolytic, inflammatory and vascular alterations inherent to the shock condition, as well as commonly associated hepatic and renal injuries, and the possible presence of the virus in the tissue [21,33,34].
A characteristic finding of some cases is the Cerebellar syndrome mentioned by Weeratunga and colleagues, which described bilateral vertical and horizontal nystagmus, dysarthria, bilateral limb, and gait ataxia. A low-grade inflammatory process was the proposed mechanism . Solbrig and Perng, when proposing their classification, include cerebellitis in a broad category composed of inflammatory changes (encephalitis, meningitis, meningoencephalitis, myelitis, acute disseminated encephalomyelitis, and retinochoroiditis) and vascular disturbances (subdural hematoma, ischemic or hemorrhagic stroke, intracranial hemorrhage, cerebral vasculitis, retinal vasculopathy). Other categories proposed in this classification include encephalopathy, characterized by depressed sensitivity, cognitive disorders, convulsions, mood, personality and behavior disorders; peripheral nervous system syndromes, including Guillain-Barré, acute motor sensory axonal neuropathy, multiplex mononeuritis, brachial plexitis, hypokalemic quadriparesis or plegia, diaphragmatic paralysis, and myositis; and convalescent or post-dengue immune-mediated syndromes (ADEM, transverse myelitis, neuromyelitis optica, cranial neuropathies, arteritis, Miller-Fisher syndrome and arteritis) . Some excellent reviews were published on the neurological disorders associated with dengue in recent years [36,37].
The pathogenesis of neurological disorders is not fully understood. Various mechanisms that have been suggested include enhancing antibodies in secondary dengue, memory T?cell?mediated pathogenesis, immune complex disease, complement and its products, anti?NS1 antibodies that cross?react with vascular endothelium, cytokine “storm” and other soluble mediators, together with possible selection of virulent virus strains and host genetic polymorphism . The penetration of DENV into the CNS may occur through areas of hemorrhage, regions of plasma transudation, and/or damage to the blood-brain barrier by action mainly of inflammatory cells, but also of the virus itself [38,39]. DENV preferentially infects monocyte lineage cells [40,41]. Endothelial cells, neurons, glial cells and microglia are proven to be suitable receptor cells for the virus [39,42,43].
Neuropathological findings in humans and animal models
Morphological, both macroscopic and microscopic, descriptions associated with neurological signs and symptoms in dengue are limited in the literature, as most cases are non-fatal , biopsies are rarely needed in these individuals and, of those with a fatal evolution, only a variable percentage undergoes autopsy, the main method of obtaining tissue for analysis. Various techniques often performed in vivo for evaluating neuropathological alterations, such as Magnetic Resonance Imaging (MRI) and CSF laboratory analysis, allow us to obtain important information related to such alterations, without, however, replacing the histopathological evaluation.
Animal models are useful to demonstrate neuropathological changes in this context of limited amounts of material available. An and colleagues inoculated DENV-2 into the brain tissue of immunodeficient mice, and observed dissemination of the virions, to both brain and spinal cord neurons, and to myelinated and unmyelinated nerve fibers, suggesting spread through the CSF. On histopathological evaluation, they observed several pathological changes in the spinal cord, including edema and congestion, neuronal changes including decreased Nissl substance, vacuolization and neuronophagia. Astrocyte swelling was occasionally seen. With the virus replication, loss of the neurons was observed in the spinal cord anterior horn . Bordignon and colleagues performed intracerebral inoculation of neuroadapted DENV1 strains in mice, and described intense chronic lymphohistiocytic leptomeningitis and moderate encephalitis, with neuronal necrosis (red neurons), satellitosis of lymphocytes and microglia around dying neurons, and the presence of microglial nodules and perivascular lymphocytes. They compared the findings with strains considered to be non-neuroadapted, which showed much more mild inflammatory changes, predominantly in leptomeninges .
Amaral and colleagues inoculated DENV-3 intracranially in mice, and observed mild gliosis and multifocal areas of perivascular hemorrhages in brainstem, diffuse meningeal infiltration of neutrophils, mononuclear cells infiltration with associated vasculitis in brain and cerebellum, numerous rod-shaped microglial cells and intense neuronal destruction of the pyramidal layer (CA3 and CA4) of the hippocampus. They performed semi-quantitative analysis (0 to 4-point scale), and assessed the density of necrotic and apoptotic cells, thereby demonstrating progressive pathological changes in the meninges, cerebral cortex, cerebellum, hippocampus and brainstem .
Amorim and colleagues inoculated DENV2 in BALB/c mice by the intracerebral route, and several animals presented clinical manifestations related to neurological disorders and succumbed to the disease. They demonstrated several changes in brain tissue: Hemorrhages, microglial hyperplasia and hypertrophy, perivascular, meningeal and parenchymal inflammatory infiltrate (predominantly lymphocytic), and focal areas of reactive gliosis.They also performed a semiquantitative score (0 to 4-point scale) to measure histological changes, which demonstrated that the inflammatory infiltrates, as those found in the pia mater, showed a sustained increase until the end of the experiment, the same pattern being observed in relation to the perivascular infiltrates.In the cerebellar tissue, changes were similar, but milder than those observed in the brain, and morphological changes (retraction) in Purkinje neurons were observed. The authors demonstrated viral tropism and replication in resident cells of the brain and cerebellum, such as neurons, astrocytes, microglia and oligodendrocytes, and cited viral neuroadaptation as important in keeping the virus in the nervous tissue. The associated immune response was considered the main responsible for the observed histopathological alterations .
In reports and case series in humans, the main morphological changes in the CNS are associated with vascular alterations, especially plasma leakage and loss of wall integrity. Cerebral edema is a frequent alteration, both on MRI and gross evaluation, and it is mainly associated with the clinical aspects of encephalopathy . Edema is a process that can be routinely grossly evaluated, and Burke, in his pioneering work, described changes like an increase in brain weight, as well as meningeal congestion and edema, in all the 12 cases evaluated . Other authors described the changes associated with edema as an increase in brain mass and volume, obliteration of the sulci and flattening of the gyri, and meningeal congestion, or simply as brain edema [51-57]. Massive cerebral edema with consequent hemorrhage and bilateral tonsillar herniation leading to death was also described . By microscopic evaluation, Bhamarapravati and colleagues, in another pioneering work, described cerebral cortex, basal ganglia, pons, midbrain or medulla samples from a collection of 42 brains, and found microscopic edema-related changes (prominence of Virchow-Robin spaces, arachnoid layer edema) in “most cases” . Other relevant papers describe parenchymal and/or perineuronal vacuolation, meningeal edema, and associated vascular congestion [50-57].
Hemorrhagic events were also frequently described, both in fatal and non-fatal cases, mainly in MRI. These events take the many forms, such petechial, focal, multifocal, or even extensive hemorrhages, affecting different areas of the cerebral lobes, basal ganglia (sometimes with ventricular extension), cerebellum and pons, as well as meninges, in subarachnoid and subdural spaces, and epidural compartment [28,50-52,54,56,58-70]. Coagulation disorders, present in hemorrhagic forms of dengue, as well as the previously mentioned immune-mediated damage to endothelial cells and direct viral activity and injury to endothelial cells, were associated with bleeding events [27,39,40,58]. At the microscopic level, some authors showed diffuse thickening of white matter capillaries, sometimes with degeneration of elements of the vascular wall, and consequent weakening and ruptures [52,56]. Miagostovich and colleaguesdemonstrated, with immunohistochemical methods, virus-positive cells, mostly located within Virchow Robin space of medium size and small veins, infiltrating the white and grey matter, and often situated close to neurons displaying apparent cytopathic features. Furthermore, immunostaining for CD68 antigens demonstrated that most CD68-positive macrophages and dengue antigen-positive cells share similar morphology and localization, and suggested a unique identity for at least part of the cells, possibly responsible for the penetration of the virus into the CNS through the vessels . Ischemic alterations are reported in the literature, but with little anatomopathological substrate, as the cases are generally non-fatal, MRI being the main evaluation method [68,71,72]. Jois and colleagues described multiple microinfarcts throughout the midbrain, pons, medulla, cerebellum, cerebral cortex, and dura mater .
Regarding inflammatory processes, predominantly lymphomononuclear infiltrate affecting the perivascular compartment, without obvious encephalitic aspects, was described by some authors [52,56,63]. An encephalitic process is described by Jois and colleagues in three fatal cases, with a predominantly lymphomononuclear multifocal infiltrate, and scattered foci of neutrophils. The authors graded both inflammation and edema in semi quantitative scale (0 to 3-point), and found a significant association between the inflammation and edema in the medulla, pons, and midbrain, as wells as significant association between the frontal and temporal lobes, and the occipital and parietal lobes. They also suggested that inflammation in cases of dengue encephalitis is relatively more intense in the forebrain structures, and that the edema in the medulla, pons, and midbrain was probably a terminal event leading to death .
Pathological changes in brain parenchyma cells are also documented in the literature, like neuronal eosinophilic alterations associated with acute injury (red neuron), gliosis and microglial reactivity [56,58,73]. Salomão and colleagues showed intense neuronal degeneration, with some nuclei displaced to the periphery of the neuron, in the cortical area with no relevant alterations on the neuropil. The same authors proposed a quantitative analysis of glial cell numbers in cortex and white matter areas, compared with controls, and found an increment in both compartments in cases, compared to controls, demonstrating significant glial reactivity. In their important work, they also conducted a morphological analysis using immunohistochemical markers, showing hyperplasia of microglial cells. The astrocyte population also presented altered morphology - thicker and less numerous extensions either in the cortical area or in the white matter around blood vessels, compared to controls .
Descriptions of the neuropathological substrate of other neurological changes, especially related to inflammatory response, are scarce in the literature, although they are well documented by complementary techniques. Meningitis is a rare complication, usually determined by clinical signs and laboratory alterations (like lymphocytic pleocytosis) in the CSF, and not by tissue evaluation [74-77]. Transverse myelitis, or longitudinally extensive transverse myelitis, is alterations also commonly evaluated by CSF analysis, as well as MRI [67,70,78-86]. A specific form of involvement of the CNS is ADEM, and its assessment is predominantly through MRI [80,87-92]. Sometimes there is an association with hemorrhage . Few studies describe the ADEM alterations histologically as perivenous demyelination in the brain parenchyma [51,63].
Clinical features of myositis associated with dengue are very frequent, but there are few histopathological descriptions. Malheiros and colleagues studied fifteen muscular biopsies, and found perivascular mononuclear infiltrates (but no myositis), lipid accumulation, mild mitochondrial proliferation, few central nuclei, and rare foci of myonecrosis, but direct invasion of skeletal muscle by the dengue virus has not been demonstrated . Kalita and colleagues described a muscular biopsy containing necrotic fibers with evidence of regeneration, interstitial inflammatory reaction containing clusters of mononuclear and polymorphonuclear cells, and myophagocytosis, findings suggestive of myositis . Variation in fiber size, hyalinization and myophagocytois were described by Misra and colleagues in one muscular biopsy .
Being a disease widely spread across the globe, affecting a large number of people, the neurological alterations associated with dengue, clinically grouped in the so-called expanded dengue syndrome, is well known, and cause for concern, to the medical community. Complementary methods such as MRI and CSF laboratory analysis are very useful in the evaluation of non-fatal cases. However, despite a substantial number of deaths, descriptions of neuropathological alterations are scarce in the literature. Some animal models make it possible to document several neuropathological findings. As previously described, alterations are generally present in greater intensity and more diffusely than in humans. The mode of infection (intracerebral injection inoculation) by viral strains contributes to these findings, allowing for a higher viral load.
As we have seen, viral neurotropism is already well documented in the literature. The presence of the virus in the nervous system, associated with an exacerbated immune response, and vascular and metabolic disorders, results in several alterations, such as encephalopathy, encephalitis, meningitis, vasculitis, Guillain-Barre syndrome, myositis, various types of hemorrhagic phenomena, among others. These phenomena are more often evaluated by complementary methods, such as MRI and CSF laboratory analysis, in non-fatal cases.
In case reports and series, most commonly autopsies, the most frequent neuropathological findings are those related to disturbances of vascular function and integrity, such as edema and hemorrhage. There are descriptions of hemorrhagic phenomena of widely variable aspects and extension, affecting any compartment - parenchyma (brain, cerebellum, brainstem, and spinal cord), ventricular system, subarachnoid, subdural and epidural spaces. Ischemic phenomena, less frequent than hemorrhagic ones, are also described.
A few studies showed the presence of an inflammatory infiltrate, usually lymphomononuclear, in the parenchyma (encephalitis, myelitis) and around vessels (perivascular, sometimes vasculitis). In particular, the work of Jois and colleagues establishes degrees of parenchymal inflammation, and correlates them with changes associated with edema . Pathological responses in brain parenchyma cells like neuronal eosinophilic alterations associated with acute injury (“red neuron”), gliosis, and microglial reactivity, are also documented in some papers. Other neuropathological aspects that are described in the literature are perivenous demyelination in the brain parenchyma (in the context of ADEM) and myositis in a limited number of muscle biopsies.
It is interesting to note that some authors introduce semiquantitative rating scales for some histological parameters (such as edema, inflammation), both in animal and human models [47,48,57]. Others performed quantitative analysis of neuroglial cell numbers compared to controls using immunohistochemical methods . These types of approaches allow for a more objective, measurable assessment of morphologically observed phenomena, and could be further disseminated in future work.
From the above, it is clear that the presence of neuropathological alterations is well established in the literature, but more of these descriptions are needed for a better understanding of the pathogenesis of the important neurological alterations seen in expanded dengue syndrome. Animal models can also help achieve these goals. A more complete understanding of the effects of dengue on the nervous system, which includes an adequate and extensive tissue evaluation, in particular through autopsies, can result in more effective treatments and care protocols in an often neglected disease.