**8. Investigations and diagnosis**

In general, profound leucopenia is usually noted in patients with severe EV71 infections [56]. This is attributed to T-cell apoptosis as EV71 infection may increase FasL expression.

features and/or (iii) histochemical staining, (d) in situ hybridization assays or (e) polymerase chain reaction (PCR) to amplify viral nucleic acids [82, 83]. Since some enteroviruses such as coxsackievirus A [84] do not grow in standard cell cultures [85], the extensive use of PCR with its generally high specificity and sensitivity has greatly improved diagnosis for numer‐ ous pathogens. The overall sensitivity, specificity, positive and negative predictive values have been reported to be 85.7%, 93.9%, 61.7% and 98.3%,respectively, using viral culture as the gold standard [86]. However, the majority of clinically suspected viral encephalitis are still of unknown etiology. It is presumed as well that enterovirus will be detectable in the gastrointestinal (GI) tract, but in the case of chronic encephalitis, by the time the disease sur‐ faces, the virus may have cleared from the GI tract and be undetectable in the stool. Addi‐ tionally, due to low viral concentration, detection of viral RNA in the CSF early after

Viral Encephalitis with Focus on Human Enteroviruses

http://dx.doi.org/10.5772/52574

271

In EV71 encephalomyelitis, inflammation is stereotypical. Common areas of involvement are the spinal cord, brainstem, hypothalamus, cerebellar dentate nucleus, cerebral cortex and meninges. The anterior pons, corpus striatum, temporal lobe, hippocampus and cerebel‐ lar cortex are spared. These areas of involvement facilitate the differentiation of encephalitis due to EV71 from Japanese encephalitis virus [87]. The nature of the lesions, however, is non-specific, with inflammatory infiltration of perivascular and parenchymal tissue, edema,

The tests currently available have a low diagnostic yield, even in the case of PCR which has high specificity and high sensitivity. This is shown in a meta-analysis conducted in 2010 which reviewed 41 studies on the etiology of encephalitis [88]. In 26 of the studies, more than 50% of the cases were of unknown etiology [88]. Identifying the viral etiological agent

The European Union Concerted Action on Virus Meningitis and Encephalitis conducted a multicenter retrospective study to evaluate the Amplicor Enterovirus PCR test [86]. 476 CSF samples were collected from 9 laboratories in 5 European countries and analysed via cul‐ tures and PCR [86]. Out of 476 samples, 50 were positive via cultures and 66 via PCR. Rela‐ tive increase in rate of positivity via PCR in relation to culture is thus 32%. Among the 50 samples positive by cultures, 38 were positive while 12 were negative by PCR. On repeat testing of the 12 samples that were culture positive but PCR negative with a different set of primers and probes, 4 of the samples became PCR positive [86]. On the other hand, in the 66 samples positive via PCR, 28 were negative via cultures [86]. Interestingly, in samples from patients with meningitis following the case definition of CSF pleocytosis (more than 10 leu‐ kocytes/mm3), 25 were positive via cultures and 45 via PCR, thus there were samples from patients who did not satisfy the criteria for pleocytosis and yet had enterovirus infection.

The California Encephalitis Project (CEP) conducted from 1998-2000 evaluated samples from 334 patients with case definition of encephalitis, which is encephalopathy requiring hospi‐ talization plus one of the following: fever, seizure, focal neurologic findings, cerebrospinal fluid pleocytosis and electroencephalographic or neuroimaging findings consistent with en‐ cephalitis [89]. Encephalopathy is defined as depressed or altered level of consciousness lasting 24 hours, lethargy and/or change in personality [89]. 9% of the cases had a confirmed

necrosis, stimulation of microglial cells and phagocytic destruction of neurons[83].

enables effective preventive measures and treatments to be implemented.

manifestation may be challenging as well.

**•** Investigations: Lumbar puncture

A common investigation in the presence of neurological symptoms would be a lumbar puncture. In aseptic meningitis, there is usually lymphocytic cerebrospinal fluid (CSF) pleo‐ cytosis [20] and normal glucose levels [79]. Yet in some of the patients, neutrophilia was ob‐ served with low glucose levels less than half of that of plasma glucose instead [5].

**•** Investigations: Magnetic resonance imaging

Magnetic resonance imaging of the brain is also a useful investigation. Reports of magnetic resonance imaging of polioencephalitis are rare as poliovirus is currently rarely seen in de‐ veloped countries. The few imaging reports of polioencephalitis reveal involvement of the midbrain and posterior medulla and pons [61].

During the 1998 Taiwan EV71 epidemic, magnetic resonance features were described by Shen et al [61]. Out of 15 patients classified in grade III with clinical encephalitis, 10 had ab‐ normal magnetic resonance imaging scans. Of the patients with abnormal scans, all 10 dem‐ onstrated hyperintense lesions of the posterior medulla and pons on T2 weighted images but not on T1 weighted images, which implies acute inflammation. The majority showed in‐ volvement of the mesencephalon and dentate nuclei of cerebellum, and in severe cases, the ventral horns of the spinal cord and deep supratentorial nuclei as well [61]. The inclusion of the brainstem is supported by pathological findings on autopsy [26, 80], with inflammation limited to the gray matter of the spinal cord and medulla as well as the tegmentum of the midbrain and pons [80]. It is of note that EV71 and poliovirus affect the same areas of the brain and the areas of involvement demonstrated on the nuclei correlated with the clinical symptoms and signs. A marked difference would be that although the inflammation of infe‐ rior olives is reported in EV71 infection, it is absent in bulbar poliomyelitis [80].

Newer techniques in magnetic resonance imaging such as fluid attenuated inversion recov‐ ery and diffusion weighted imaging allow detection of subtle meningeal and cortical abnor‐ malities that can occur in meningoencephalitis [81]. Consequently, in patients stable enough to undergo scans, magnetic resonance imaging can reveal areas of CNS involvement. This allows greater diagnostic accuracy and perhaps predicts the need for cardiothoracic support before patients deteriorate too rapidly.

### **9. Diagnostic methods**

Diagnosis normally depends on (a) rise in virus specific acute and convalescent antibody tit‐ ers, (b) isolation of the virus via viral cultures from throat swabs, stool specimens and CSF samples (c) visual identification of virus via (i) electron microscopy (ii) unique histological features and/or (iii) histochemical staining, (d) in situ hybridization assays or (e) polymerase chain reaction (PCR) to amplify viral nucleic acids [82, 83]. Since some enteroviruses such as coxsackievirus A [84] do not grow in standard cell cultures [85], the extensive use of PCR with its generally high specificity and sensitivity has greatly improved diagnosis for numer‐ ous pathogens. The overall sensitivity, specificity, positive and negative predictive values have been reported to be 85.7%, 93.9%, 61.7% and 98.3%,respectively, using viral culture as the gold standard [86]. However, the majority of clinically suspected viral encephalitis are still of unknown etiology. It is presumed as well that enterovirus will be detectable in the gastrointestinal (GI) tract, but in the case of chronic encephalitis, by the time the disease sur‐ faces, the virus may have cleared from the GI tract and be undetectable in the stool. Addi‐ tionally, due to low viral concentration, detection of viral RNA in the CSF early after manifestation may be challenging as well.

**8. Investigations and diagnosis**

270 Encephalitis

**•** Investigations: Lumbar puncture

**•** Investigations: Magnetic resonance imaging

midbrain and posterior medulla and pons [61].

before patients deteriorate too rapidly.

**9. Diagnostic methods**

In general, profound leucopenia is usually noted in patients with severe EV71 infections [56]. This is attributed to T-cell apoptosis as EV71 infection may increase FasL expression.

A common investigation in the presence of neurological symptoms would be a lumbar puncture. In aseptic meningitis, there is usually lymphocytic cerebrospinal fluid (CSF) pleo‐ cytosis [20] and normal glucose levels [79]. Yet in some of the patients, neutrophilia was ob‐

Magnetic resonance imaging of the brain is also a useful investigation. Reports of magnetic resonance imaging of polioencephalitis are rare as poliovirus is currently rarely seen in de‐ veloped countries. The few imaging reports of polioencephalitis reveal involvement of the

During the 1998 Taiwan EV71 epidemic, magnetic resonance features were described by Shen et al [61]. Out of 15 patients classified in grade III with clinical encephalitis, 10 had ab‐ normal magnetic resonance imaging scans. Of the patients with abnormal scans, all 10 dem‐ onstrated hyperintense lesions of the posterior medulla and pons on T2 weighted images but not on T1 weighted images, which implies acute inflammation. The majority showed in‐ volvement of the mesencephalon and dentate nuclei of cerebellum, and in severe cases, the ventral horns of the spinal cord and deep supratentorial nuclei as well [61]. The inclusion of the brainstem is supported by pathological findings on autopsy [26, 80], with inflammation limited to the gray matter of the spinal cord and medulla as well as the tegmentum of the midbrain and pons [80]. It is of note that EV71 and poliovirus affect the same areas of the brain and the areas of involvement demonstrated on the nuclei correlated with the clinical symptoms and signs. A marked difference would be that although the inflammation of infe‐

served with low glucose levels less than half of that of plasma glucose instead [5].

rior olives is reported in EV71 infection, it is absent in bulbar poliomyelitis [80].

Newer techniques in magnetic resonance imaging such as fluid attenuated inversion recov‐ ery and diffusion weighted imaging allow detection of subtle meningeal and cortical abnor‐ malities that can occur in meningoencephalitis [81]. Consequently, in patients stable enough to undergo scans, magnetic resonance imaging can reveal areas of CNS involvement. This allows greater diagnostic accuracy and perhaps predicts the need for cardiothoracic support

Diagnosis normally depends on (a) rise in virus specific acute and convalescent antibody tit‐ ers, (b) isolation of the virus via viral cultures from throat swabs, stool specimens and CSF samples (c) visual identification of virus via (i) electron microscopy (ii) unique histological In EV71 encephalomyelitis, inflammation is stereotypical. Common areas of involvement are the spinal cord, brainstem, hypothalamus, cerebellar dentate nucleus, cerebral cortex and meninges. The anterior pons, corpus striatum, temporal lobe, hippocampus and cerebel‐ lar cortex are spared. These areas of involvement facilitate the differentiation of encephalitis due to EV71 from Japanese encephalitis virus [87]. The nature of the lesions, however, is non-specific, with inflammatory infiltration of perivascular and parenchymal tissue, edema, necrosis, stimulation of microglial cells and phagocytic destruction of neurons[83].

The tests currently available have a low diagnostic yield, even in the case of PCR which has high specificity and high sensitivity. This is shown in a meta-analysis conducted in 2010 which reviewed 41 studies on the etiology of encephalitis [88]. In 26 of the studies, more than 50% of the cases were of unknown etiology [88]. Identifying the viral etiological agent enables effective preventive measures and treatments to be implemented.

The European Union Concerted Action on Virus Meningitis and Encephalitis conducted a multicenter retrospective study to evaluate the Amplicor Enterovirus PCR test [86]. 476 CSF samples were collected from 9 laboratories in 5 European countries and analysed via cul‐ tures and PCR [86]. Out of 476 samples, 50 were positive via cultures and 66 via PCR. Rela‐ tive increase in rate of positivity via PCR in relation to culture is thus 32%. Among the 50 samples positive by cultures, 38 were positive while 12 were negative by PCR. On repeat testing of the 12 samples that were culture positive but PCR negative with a different set of primers and probes, 4 of the samples became PCR positive [86]. On the other hand, in the 66 samples positive via PCR, 28 were negative via cultures [86]. Interestingly, in samples from patients with meningitis following the case definition of CSF pleocytosis (more than 10 leu‐ kocytes/mm3), 25 were positive via cultures and 45 via PCR, thus there were samples from patients who did not satisfy the criteria for pleocytosis and yet had enterovirus infection.

The California Encephalitis Project (CEP) conducted from 1998-2000 evaluated samples from 334 patients with case definition of encephalitis, which is encephalopathy requiring hospi‐ talization plus one of the following: fever, seizure, focal neurologic findings, cerebrospinal fluid pleocytosis and electroencephalographic or neuroimaging findings consistent with en‐ cephalitis [89]. Encephalopathy is defined as depressed or altered level of consciousness lasting 24 hours, lethargy and/or change in personality [89]. 9% of the cases had a confirmed viral agent, 3% a confirmed bacterial agent, 1% a confirmed parasitic agent, 10% a non-infec‐ tious etiology and 12% a possible etiology identified. 3% had a non-encephalitis infection identified. Nevertheless, CEP is not population based and the study group consisted of diag‐ nostically challenging cases. Therefore, the rate of unknown etiology cases may be an over‐ estimate when extrapolated to the general population.

enteroviruses as well, but the potential ADE phenomenon is an important consideration in the development of a safe and effective vaccine. The genetic diversity of enterovirus strains

Viral Encephalitis with Focus on Human Enteroviruses

http://dx.doi.org/10.5772/52574

273

Ribavirin, a broad-spectrum antiviral synthesized by ICN pharmaceuticals, Inc., USA inhib‐ its the replication of a variety of enteroviruses. Studies on EV71-infected mice has shown that ribavirin can reduce mortality by reducing the viral loads in tissues. The required dos‐ age of ribavirin is close to the initial dose of the drug administered intravenously to treat patients with encephalitis caused by Nipah virus [106]. Given these results, ribavirin may be, in combination with interferon, deployed to combat potentially fatal EV71 infection. In‐ terferon has a synergist effect and this combination is already adopted as a standard therapy

Pyridyl imidazolidinone is a novel class of EV71 inhibitor [108]. It was first identified using computer-assisted drug design. It targets EV71 capsid protein VP1 and time course experi‐ ments on one of the pyridyl imidazolidinones, BPR0Z-194, have shown that viral replication is effectively inhibited in early stages, suggesting that the compound inhibits adsorption of virions and/or viral RNA uncoating [108]. Resistant strains do exist, and sequence analysis has demonstrated that a single amino acid alteration at position 192 of VP1 confers resist‐

Pleconaril, an anti-viral produced by Sterling-Winthrop, Inc., USA incorporates itself into the capsid of enteroviruses and blocks the virus from docking to cellular receptors and uncoating to release RNA into the cell. It targets VP1 and has already passed the last stage of clinical trials [109]. Results are promising with pleconaril showing antiviral ef‐ fects for most enteroviruses [109, 110]. Presently, there is an ongoing study on the effica‐ cy of pleconaril in enteroviral sepsis syndrome in neonates [111]. The National Health Research Institutes (NHRI) in Taiwan has reported a number of virtual compounds with similar stable conformations and preliminary studies have identified a few promising

Another promising therapy is the use of RNA interference (RNA-i) in silencing viral gene expression [28, 112]. As aforementioned, viruses penetrate the BBB and the very nature of the BBB makes it difficult for large and charged molecules to cross it. RNA-i on the other hand, are small and have the potential to cross the BBB and exert a therapeutic effect. RNA-i can bind to specific viral mRNA, causing degradation and preventing the translation and synthesis of viral proteins that enable the virus to inhibit the host IFN-1 response [112]. To synthesize RNA-i for therapeutic use, the viral proteins and subsequent viral mRNA have to

**13. Potential treatments in the future – RNA interference**

therefore complicates the development of vaccines [105].

for HCV-infected patients [107].

ance to BPR0Z-194 [108].

imidazolidinone derivatives.

**12. Potential treatments in the future – anti-virals**

Diagnostic strategies that have emerged recently include MassTagPCR, panmicrobial DNA microarrays and high-throughput DNA pyrosequencing [82]. MassTag PCR is a multiplex PCR assay utilizing primer pairs targeting highly conserved gene sequences that represent a wide variety of potential pathogens. The primer pairs have been tagged with MassCodes that are used to identify the etiological agent. There are different MassTag PCR systems with different primers for different clinical specimens and presentations. Clinical use of this method has demonstrated effectiveness in identification of pathogens [90-93]. Panmicrobial DNA microarrays utilize a single chip with numerous highly conserved gene sequences, permitting the swift identification of pathogens similar to that of MassTag PCR [94, 95]. Clinical use of this diagnostic method has also demonstrated efficacy in pathogen identifica‐ tion [96-98].

High-throughput DNA pyrosequencing on the other hand, does not make use of highly con‐ served gene sequences. Instead, it uses random primers to amplify all RNA after removing human chromosomal DNA from the sample [99]. Amplification products are then se‐ quenced via pyrosequencing wherein DNA polymerases synthesize complementary strands to the amplified products and each enzymatic attachment of a complementary nucleotide re‐ sults in an emission of a light signal. The light signal is recorded and the sequences are iden‐ tified and subsequently analyzed to look for pathogens. This technique allows for identification of novel pathogens [100].
