EFNS-ENS guidelines for the use of PCR technology for the diagnosis of infections of the nervous system.

BACKGROUND: Polymerase chain reaction (PCR) as a means to amplify nucleic acids has become an essential element in diagnosis of infections. It has evolved into a simple and rapid, easy- to- use approach. At present there are no published guidelines for the usage of PCR technology for the diagnosis of infections of the nervous system. METHODS: We reviewed the advantages and pitfalls of PCR in order to guide neurologists and infectious diseases experts in its application for the diagnosis of infections of the nervous system. Medical reference systems were searched, and original papers, meta-analyses, review papers, book chapters and guidelines recommendations were reviewed. The final literature search was performed in May 2012. Recommendations were reached by consensus. RECOMMENDATIONS: The reliability of PCR technology for the diagnosis of neurological infections is currently based on the pathogens. The main contribution of PCR is to the diagnosis of viral infections followed by bacterial CNS infections with the notable exception of tuberculous meningitis. Efficacy for the diagnosis of protozoal infections and helminthic infestations has also been established in many instances. Unfortunately, current molecular PCR technology is far from becoming routine in resource-poor countries where such infections are prevalent. Despite the importance of fungal infections in the context of the immune-compromised host, there is not enough data to recommend the routine use of PCR. CONCLUSIONS: PCR technology is currently a reliable method for the diagnosis of viral and bacterial (except tuberculosis) infections, and only for some protozoal infections and helminthic infestations.

Viral meningoencephalitis: a review of diagnostic methods and guidelines for management.

BACKGROUND: Viral encephalitis is a medical emergency. The prognosis depends mainly on the pathogen and host immunologic state. Correct immediate diagnosis and introduction of symptomatic and specific therapy has a dramatic influence upon survival and reduces the extent of permanent brain injury. METHODS: We searched the literature from 1966 to 2009. Recommendations were reached by consensus. Where there was lack of evidence but consensus was clear, we have stated our opinion as good practice points. RECOMMENDATIONS: Diagnosis should be based on medical history and examination followed by CSF analysis for protein and glucose levels, cellular analysis, and identification of the pathogen by polymerase chain reaction amplification (recommendation level A) and serology (level B). Neuroimaging, preferably by MRI, is essential (level B). Lumbar puncture can follow neuroimaging when immediately available, but if this cannot be performed immediately, LP should be delayed only under unusual circumstances. Brain biopsy should be reserved only for unusual and diagnostically difficult cases. Patients must be hospitalized with easy access to intensive care units. Specific, evidence-based, antiviral therapy, acyclovir, is available for herpes encephalitis (level A) and may also be effective for varicella-zoster virus encephalitis. Ganciclovir and foscarnet can be given to treat cytomegalovirus encephalitis, and pleconaril for enterovirus encephalitis (IV class evidence). Corticosteroids as an adjunct treatment for acute viral encephalitis are not generally considered to be effective, and their use is controversial, but this important issue is currently being evaluated in a large clinical trial. Surgical decompression is indicated for impending uncal herniation or increased intracranial pressure refractory to medical management.

EFNS guideline on the management of community-acquired bacterial meningitis: report of an EFNS Task Force on acute bacterial meningitis in older children and adults.

Acute bacterial meningitis (ABM) is a potentially life-threatening neurological emergency. An agreed protocol for early, evidence-based and effective management of community-acquired ABM is essential for best possible outcome. A literature search of peer-reviewed articles on ABM was used to collect data on the management of ABM in older children and adults. Based on the strength of published evidence, a consensus guideline was developed for initial management, investigations, antibiotics and supportive therapy of community-acquired ABM. Patients with ABM should be rapidly hospitalized and assessed for consideration of lumbar puncture (LP) if clinically safe. Ideally, patients should have fast-track brain imaging before LP, but initiation of antibiotic therapy should not be delayed beyond 3 h after first contact of patient with health service. In every case, blood sample must be sent for culture before initiating antibiotic therapy. Laboratory examination of cerebrospinal fluid is the most definitive investigation for ABM and whenever possible, the choice of antibiotics, and the duration of therapy, should be guided by the microbiological diagnosis. Parenteral therapy with a third-generation cephalosporin is the initial antibiotics of choice in the absence of penicillin allergy and bacterial resistance; amoxicillin should be used in addition if meningitis because of Listeria monocytogenes is suspected. Vancomycin is the preferred antibiotic for penicillin-resistant pneumococcal meningitis. Dexamethasone should be administered both in adults and in children with or shortly before the first dose of antibiotic in suspected cases of Streptococcus pneumoniae and H. Influenzae meningitis. In patients presenting with rapidly evolving petechial skin rash, antibiotic therapy must be initiated immediately on suspicion of Neisseria meningitidis infection with parenteral benzyl penicillin in the absence of known history of penicillin allergy.

The effect of different pine hosts on the sampling of Rhizopogon spore banks in five Eastern Sierra Nevada forests.

Our primary goal was to determine whether detection of Rhizopogon internal transcribed spacer (ITS) groups is affected by the pine species used in seedling bioassays. Our secondary goal was to investigate composition of Rhizopogon spore banks in the Eastern Sierra Nevada of California, a previously unsampled region. We used seedlings of Pinus contorta, Pinus jeffreyi, Pinus lambertiana, and Pinus muricata as bioassay plants and identified the Rhizopogon retrieved by internal transcribed spacer (ITS) sequence analysis. We found that each of the pine species retrieved all of the abundant Rhizopogon ITS groups, but there were significant differences among pines in the richness of Rhizopogon ITS groups recovered. Pinus muricata recovered all ITS groups found in this study and was significantly better than P. lambertiana. Rhizopogon communities from the five sampled sites contained six to eight ITS groups per site, with two unique sequence groups and a higher abundance of the Rhizopogon ellenae and Rhizopogon arctostaphyli groups than at previously sampled sites. These results show high cross-receptivity between Rhizopogon and pine species, and regional patterns in spore bank composition.

Viral encephalitis: a review of diagnostic methods and guidelines for management.

Viral encephalitis is a medical emergency. The spectrum of brain involvement and the prognosis are dependent mainly on the specific pathogen and the immunological state of the host. Although specific therapy is limited to only several viral agents, correct immediate diagnosis and introduction of symptomatic and specific therapy has a dramatic influence upon survival and reduces the extent of permanent brain injury in survivors. We searched MEDLINE (National Library of Medicine) for relevant literature from 1966 to May 2004. Review articles and book chapters were also included. Recommendations are based on this literature based on our judgment of the relevance of the references to the subject. Recommendations were reached by consensus. Where there was lack of evidence but consensus was clear we have stated our opinion as good practice points. Diagnosis should be based on medical history, examination followed by analysis of cerebrospinal fluid for protein and glucose contents, cellular analysis and identification of the pathogen by polymerase chain reaction (PCR) amplification (recommendation level A) and serology (recommendation level B). Neuroimaging, preferably by magnetic resonance imaging, is an essential aspect of evaluation (recommendation level B). Lumbar puncture can follow neuroimaging when immediately available, but if this cannot be obtained at the shortest span of time it should be delayed only in the presence of strict contraindications. Brain biopsy should be reserved only for unusual and diagnostically difficult cases. All encephalitis cases must be hospitalized with an access to intensive care units. Supportive therapy is an important basis of management. Specific, evidence-based, anti-viral therapy, acyclovir, is available for herpes encephalitis (recommendation level A). Acyclovir might also be effective for varicella-zoster virus encephalitis, gancyclovir and foscarnet for cytomegalovirus encephalitis and pleconaril for enterovirus encephalitis (IV class of evidence). Corticosteroids as an adjunct treatment for acute viral encephalitis are not generally considered to be effective and their use is controversial. Surgical decompression is indicated for impending uncal herniation or increased intracranial pressure refractory to medical management.

The importance of seed reserves for seedling performance: an integrated approach using morphological, physiological, and stable isotope techniques.

To investigate how seed reserves affect early seedling performance, we conducted a factorial greenhouse experiment using Lithocarpus densiflora (Tanoak). Seedlings were grown from large (5.8+/-0.7 g) and small (3.2+/-0.4 g) seeds and, following shoot emergence, seeds were either removed or left attached. Seedlings were harvested for quantification of biomass and delta13C at seven time periods following seed removal (2, 4, 8, 16, 32, 64, 128 days) and seedling photosynthesis was measured three separate time periods (2-4, 49-82, 95-128 days after seed removal). Biomass increased for all seedlings, but the increase was significantly larger for seedlings with attached seeds than with removed seeds. Seed removal just after shoot emergence significantly decreased seedling biomass, but seed removal 64 days after shoot emergence had no effect on seedling biomass. Seedling photosynthesis per unit leaf area varied by time and seed presence, but not by seed size. At the first period, seedlings with attached seeds had significantly higher photosynthetic rates than seedlings with removed seeds, at the second period there was no effect of seed removal, and at the third time period seedlings with attached seeds had significantly lower photosynthetic rates than seedlings with removed seeds. Despite temporal variation in photosynthesis per unit leaf area, seedlings with attached seeds always had significantly greater leaf area than seedlings with removed seeds, resulting in significantly higher total plant photosynthesis at all three time periods. The delta13C values of both the leaves and roots were more similar to that of the seed for seedlings with attached seeds than for seedlings with removed seeds, however, seed removal and seed size strongly affected root delta13C. This study demonstrates that seed reserves have important effects on the early growth, physiology, and delta13C of L. densiflora seedlings.

Guidelines for the diagnosis and management of neurological complications of HIV infection.

The spectrum of neurological complications of HIV-infection has remained unchanged through the years, but its epidemiology changed remarkably as a result of the introduction of highly active antiretroviral therapy (HAART). Guidelines for the diagnosis and treatment of cerebral toxoplasmosis, cryptococcal meningitis, progressive multifocal leukoencephalopathy, CMV encephalitis, CMV polyradiculomyelitis, tuberculous meningitis, primary CNS lymphoma, HIV dementia, HIV myelopathy and HIV polyneuropathy are given with a grading of evidence and recommendations.

Neurovirological methods and their applications.

Over the last 30 years neurovirology has emerged as a major discipline which has much relevance to both human disease and many aspects of neuroscience. This overview of the field aims to define briefly most of the major neurovirological techniques, both "classical" and more recent, and to indicate how these have been used to gain knowledge about the pathogenesis, clinical investigation, and treatment of viral infections of the central nervous system.

Diagnosis and treatment of viral encephalitis.

Acute encephalitis constitutes a medical emergency. In most cases, the presence of focal neurological signs and focal seizures will distinguish encephalitis from encephalopathy. Acute disseminated encephalomyelitis is a non-infective inflammatory encephalitis that may require to be treated with steroids. Acute infective encephalitis is usually viral. Herpes simplex encephalitis (HSE) is the commonest sporadic acute viral encephalitis in the Western world. Magnetic resonance imaging of brain is the investigation of choice in HSE and the diagnosis may be confirmed by the polymerase chain reaction test for the virus in the cerebrospinal fluid. In this article, we review the diagnosis, investigations, and management of acute encephalitis. With few exceptions (for example, aciclovir for HSE), no specific therapy is available for most forms of viral encephalitis. Mortality and morbidity may be high and long term sequelae are known among survivors. The emergence of unusual forms of zoonotic encephalitis has posed an important public health problem. Vaccination and vector control measures are useful preventive strategies in certain arboviral and zoonotic encephalitis. However, we need better antiviral therapy to meet the challenge of acute viral encephalitis more effectively.

Varicella-Zoster virus gene expression in latently infected rat dorsal root ganglia.

Latent infection of human ganglia with Varicella-Zoster virus (VZV) is characterized by a highly restricted pattern of viral gene expression. To enhance understanding of this process we used in situ hybridization (ISH) in a rat model of VZV latency to examine expression of RNA corresponding to eight different VZV genes in rat dorsal root ganglia (DRG) at various times after footpad inoculation with wild-type VZV. PCR in situ amplification was also used to determine the cell specificity of latent VZV DNA. It was found that the pattern of viral gene expression at 1 week after infection was different from that observed at the later times of 1 and 18 months after infection. Whereas multiple genes were expressed at 1 week after infection, gene expression was restricted at the later time points when latency had been established. At the later time points after infection the RNA transcripts expressed most frequently were those for VZV genes 21, 62, and 63. Gene 63 was expressed more than any other gene studied. While VZV DNA was detected almost exclusively in 5-10% of neurons, VZV RNA was detected in both neurons and nonneuronal cells at an approximate ratio of 3:1. A newly described monoclonal antibody to VZV gene 63-encoded protein was used to detect this protein in neuronal nuclei and cytoplasm in almost half of the DRG studied. These results demonstrate that (1) this rat model of latency has close similarities in terms of viral gene expression to human VZV latency which makes it a useful tool for studying this process and its experimental modulation and (2) expression of VZV gene 63 appears to be the single most consistent feature of VZV latency.

Adeno-associated virus-mediated delivery of glial cell line-derived neurotrophic factor protects motor neuron-like cells from apoptosis.

Motor neuron disorders including amyotrophic lateral sclerosis may benefit from the induction of neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) that are known to be trophic and protective for motor neurons. However, the application of such factors is limited by an inability to successfully target their expression in the nervous system. In this study we investigate the potential of using adeno-associated virus (AAV) as a vector for gene delivery into motor neuron-like cells. In initial experiments on the motor neuron cell line NSC-19 using a recombinant AAV vector expressing the reporter gene beta-galactosidase (AAV-LacZ), we successfully demonstrate the utility of AAV for gene transfer. In addition, a recombinant AAV vector expressing GDNF was shown to express and secrete high levels of the neurotrophic factor into the surrounding media of NSC-19 infected cells. Finally, the AAV-GDNF vector is demonstrated to act in a neuroprotective fashion. Withdrawal of trophic support from NSC-19 cells through serum deprivation results in a subsequent increase in the number of cells entering apoptosis. However, the percentage of apoptotic cells are significantly reduced in cells infected with the AAV-GDNF vector, as compared to AAV-LacZ or uninfected controls. This work demonstrates the potential of using AAV as a vector in motor neuron-like cells and should prove important in devising future gene therapy strategies for the treatment of in vivo motor neuron disorders.

Cytokines and the acute phase response in post-treatment reactive encephalopathy of Trypanosoma brucei brucei infected mice.

Stimulation of the acute phase response during infection of mice with Trypanosoma brucei brucei (T. b. brucei) was investigated in an experimental model of the post-treatment reactive encephalopathy (PTRE), a common side-effect of anti-trypanosome therapy. Plasma levels of the acute phase proteins (APP), haptoglobin (Hp) and serum amyloid P (SAP) increased by day 7 post-infection, but by day 20 had fallen to an intermediate level. This was accompanied by induction of the cytokines, interleukin (IL)-6 and tumour necrosis factor-alpha (TNFalpha) in both liver and brain. Treatment of mice on day 21 with a subcurative dose of diminazene aceturate (Berenil), a procedure known to induce a mild PTRE, cleared the parasite from the circulation with plasma APP and liver expression of mRNA for IL-6 and TNFalpha returning to the levels in the controls. Cytokine mRNA for both IL-6 and TNFalpha was detected in the brains of animals with developing PTRE although TNFalpha was not significantly greater than in the control group. A further subcurative dose of Berenil, leading to a more severe PTRE, was associated with elevated serum concentrations of Hp and SAP, increased TNFalpha mRNA in the liver and detectable IL-6 and TNFalpha mRNA in the brain. mRNA for IL-1alpha was expressed in brain and liver samples from all animals. A severe PTRE caused a systemic acute phase response which was not apparent with a mild PTRE. The pattern of cytokine mRNA induction was similar following both drug treatments. However, the difference in APP production could be caused by a breakdown in the blood-brain barrier during severe PTRE allowing cytokine synthesised in the brain to enter the circulation and maintain a systemic response.

Involvement of apolipoprotein E in herpes simplex encephalitis.

APOE polymorphism may influence risk for cold sores and, by interacting with latent HSV-1, risk for Alzheimer's disease (AD). APOE genotype also influences outcome after brain injury. We sought to determine whether APOE genotype influences risk for herpes simplex encephalitis (HSE), whether apoE is involved in the response to HSE and if APOE genotype influences outcome from HSE. There was increased immunoreactivity of neurons, neuropil and glia for apoE areas of brain damaged by HSE. APOE genotypes for cases of HSE (n = 57) were similar to those of controls (n = 41). APOE genotypes for survivors of HSE were similar to those of patients who died. We conclude that apoE is involved in the response to damage associated with HSE, as in other forms of brain injury. However, APOE genotype does not appear to influence either the risk of developing HSE or subsequent mortality.

Varicella-zoster virus gene expression in latently infected and explanted human ganglia.

A consistent feature of varicella-zoster virus (VZV) latency is the restricted pattern of viral gene expression in human ganglionic tissues. To understand further the significance of this gene restriction, we used in situ hybridization (ISH) to detect the frequency of RNA expression for nine VZV genes in trigeminal ganglia (TG) from 35 human subjects, including 18 who were human immunodeficiency virus (HIV) positive. RNA for VZV gene 21 was detected in 7 of 11 normal and 6 of 10 HIV-positive subjects, RNA for gene 29 was detected in 5 of 14 normal and 11 of 11 HIV-positive subjects, RNA for gene 62 was detected in 4 of 10 normal and 6 of 9 HIV-positive subjects, and RNA for gene 63 was detected in 8 of 17 normal and 12 of 15 HIV-positive subjects. RNA for VZV gene 4 was detected in 2 of 13 normal and 4 of 9 HIV-positive subjects, and RNA for gene 18 was detected in 4 of 15 normal and 5 of 15 HIV-positive subjects. By contrast, RNAs for VZV genes 28, 40, and 61 were rarely or never detected. In addition, immunocytochemical analysis detected the presence of VZV gene 63-encoded protein in five normal and four HIV-positive subjects. VZV RNA was also analyzed in explanted fresh human TG and dorsal root ganglia from five normal human subjects over a period of up to 11 days in culture. We found a very different pattern of gene expression in these explants, with transcripts for VZV genes 18, 28, 29, 40, and 63 all frequently detected, presumably as a result of viral reactivation. Taken together, these data provide further support for the notion of significant and restricted viral gene expression in VZV latency.

Replication of the herpes simplex virus type 1 RL1 mutant 1716 in primary neuronal cell cultures--possible relevance to use as a viral vector.

The herpes simplex virus type 1 (HSV-1) RL1 deletion mutant 1716 has properties that make it a promising candidate as a viral vector for gene therapy in the human nervous system. These properties include its ability to spread along neural pathways and establish a latent infection in post-mitotic neurons, while retaining a non-virulent phenotype in vivo and an inability to cause a lytic infection in stationary or fully differentiated cells. In this study, we used viral replication assays and indirect immunofluorescence to investigate the ability of 1716 to bind to, enter, express genes and produce progeny virus in dissociated neuronal cell cultures prepared from rat hippocampal, medial septal and dorsal root ganglion (DRG) tissues and in primary rat astrocyte cultures. Both heterogeneous cultures and those that had been enriched for neurons were employed. Following both low and high multiplicities of virus infection, the behaviour of 1716 was compared with its wild-type parent HSV-1 strain 17 in these cultures. It was found that the growth of 1716 was significantly impaired compared to wild type HSV-1, with these differences being magnified at lower multiplicities of viral infection as well as in neuron-enriched cultures: this impairment is likely to be due to decreased replication, as immunofluorescence assays showed that 1716 bound to, entered and expressed genes in all neuronal cell types and astrocytes with similar efficiency to the wild type virus. This ability of 1716 to enter and express genes in different neuronal populations demonstrates its potential suitability as a viral vector.

Identification of a human olfactory ensheathing cell that can effect transplant-mediated remyelination of demyelinated CNS axons.

The olfactory ensheathing cell (OEC) has attracted much interest recently because of its potential for transplantation-based therapy of CNS disease. Rat OECs are able to remyelinate demyelinated axons and support regeneration of damaged axons. Although OECs can be grown readily from the rat, a macrosmatic species, it has been uncertain whether it would be similarly straightforward to obtain these cells from the human, a microsmatic species with a relatively poorly developed olfactory system. In this study, we have identified a human OEC which shares many properties with its rat counterpart, including expression of the low-affinity nerve growth factor receptor (L-NGFr) and similar growth factor requirements. Purified populations of human OECs obtained by selection with L-NGFr antibodies have extremely high viability in tissue culture, and are capable of remyelinating persistently demyelinated CNS axons following transplantation into experimentally induced demyelinating lesions in the rat spinal cord. Thus, the human OEC represents an important new cell for the development of transplant therapy of CNS diseases.

Latent Varicella-zoster virus in human dorsal root ganglia.

To understand further the molecular events underlying the process of Varicella-zoster virus (VZV) latency in human ganglionic tissues, in situ hybridisation (ISH) for VZV RNA and DNA, and PCR in situ amplification for VZV DNA were used in human dorsal root ganglia from 12 individuals (3 normal and 9 who had died with AIDS). The results showed that (a) two separate regions of the VZV genome, represented by genes 4 and 40, were detected in neurons in two normal and three AIDS ganglia, (b) evidence of transcription of VZV genes 4, 21, 29, and 63 was found in normal and AIDS cases, and (c) VZV DNA and RNA for the same gene (gene 29) was detected in neurons in serial tissue sections in three cases. Thus more than one region of the VZV genome is present in neurons during VZV ganglionic latency, and the presence of both a VZV gene and its corresponding RNA transcript can be shown to occur in the same localised region of DRG tissue.