Detection of virus in CSF from the cases with meningoencephalitis by next-generation sequencing

We screened for viral DNA in cerebrospinal fluid samples using next-generation sequencing (NGS) technology to diagnose CNS viral infections. We collected CSF samples from four cases with clinically suspected viral meningoencephalitis. DNA extracted from the samples was analyzed with NGS, and the results were further validated using PCR. Herpes simplex virus 1 (HSV-1) was detected in the CSF of two patients, HSV-2 and human herpes virus type 3 (HHV-3, VZV) in the CSF of two other patients separately. The number of unique reads of the identified viral genes ranged from 144 to 44205 (93.51 to 99.57 %). The coverage of identified viral genes ranged from 12 to 98 % with a depth value of 1.1 to 35, respectively. The results were further confirmed using PCR in three cases. The clinical presentation and outcomes of these four cases were consistent with the diagnostic results of NGS. NGS of CSF samples can be used as a diagnostic assay for CNS viral infection. Its further application for “pan-viral” or even “pan-microbial” screening of CSF might influence the diagnosis of CNS infectious diseases.     

Intrathecal synthesis of virus-specific oligoclonal antibodies in patients with enterovirus infection of the central nervous system

Summary Cerebrospinal fluid (CSF) and serum samples from six patients with enterovirus infections were investigated by isoelectric focusing (IEF) and affinity-mediated immunoblot (AMI) for the clonal distribution of entervirus-specific antibodies. In two patients with either acute meningitis or encephalitis and in one patient with a relapse of multiple sclerosis, oligoclonal IgG bands specific for enteroviruses were found predominantly in the CSF, revealing intrathecal synthesis of these antibodies. In three other patients with neurological symptoms probably unrelated to a current enterovirus infection, IEF and AMI disclosed nearly identical patterns of coxsackievirus-B-specific oligoclonal bands in the CSF and serum, indicating diffusion of these antibodies from the serum into the CSF. Although the number of patients in this study is small, the results suggest that intrathecally synthesized enterovirus-specific antibodies may be used as a means of identifying an enterovirus infection of the CNS.     

Factors influencing PCR detection of viruses in cerebrospinal fluid of patients with suspected CNS infections

BACKGROUND: Polymerase chain reaction (PCR) is used to detect viruses in the cerebrospinal fluid (CSF) of patients with neurological disease. However, data to assist its use or interpretation are limited. OBJECTIVE: We investigated factors possibly influencing viral detection in CSF by PCR, which will also help clinicians interpret positive and negative results. METHODS: CSF from patients with was tested for human herpesviruses types 1-6, JC virus, enteroviruses, and Toxoplasma gondii. The likelihood of central nervous system (CNS) infection was classified as likely, possible, or unlikely. PCR findings in these categories were compared using single variable and logistic regression analysis. RESULTS: Of 787 samples tested, 97 (12%) were PCR positive for one or more viruses. Of episodes likely to be CNS viral infections, 30% were PCR positive compared to 5% categorised as unlikely. The most frequent positive findings were Epstein Barr virus (EBV), enteroviruses, and herpes simplex virus (HSV). Enteroviruses and HSV were found predominantly in the likely CNS viral infection group, whereas EBV was found mainly in the unlikely group. Positive PCR results were more likely when there were 3-14 days between symptom onset and lumbar puncture, and when CSF white cell count was abnormal, although a normal CSF did not exclude a viral infection. CONCLUSIONS: The diagnostic yield of PCR can be maximised by using sensitive assays to detect a range of pathogens in appropriately timed CSF samples. PCR results, in particular EBV, should be interpreted cautiously when symptoms cannot readily be attributed to the virus detected.     

Intrathecal viral replication and cerebral deficits in different stages of human immunodeficiency virus disease

The objectives of this study is to clarify whether there are phases critical for the infection of the central nervous system (CNS) as defined by active viral replication in the cerebrospinal fluid (CSF) in human immunodeficiency virus (HIV) infection. One hundred and nine HIV-1-positive homo- and bisexual patients in early and late disease stages with or without highly active antiretroviral therapy (HAART) were included in the cross-sectional, diagnostic (phase I) multicenter study. No patients had any overt neurological deficits; all underwent venous and lumbar puncture as well as neuropsychological testing. In untreated early-stage patients, cerebrospinal fluid (CSF) viral load correlated with inflammatory parameters, but not significantly with neuropsychological abnormalities. CSF viral load and inflammatory reactions were suppressed in HAART-treated early-stage patients. In HAART-treated late-stage patients, there was a weak correlation between CSF viral load and CSF cell count as well as a moderate correlation with immune activation markers and with distinct cerebral deficits independent of CSF viral load. Seventeen of the 109 patients had higher CSF than plasma viral loads and marked inflammatory reactions and immune activation. In patients with greater plasma than CSF viral loads, the factors contributing to cerebral deficits still need to be identified. The results suggest not only that there is an early "set point" for CSF/central nervous system (CNS) infection, but also that there is a subgroup of patients in whom intrathecal viral replication correlates with cerebral deficits. Lumbar puncture should be performed in all positive patients to identify members of this subgroup and to ascertain what characteristic factors they have in common in order to improve therapy.     

Involvement of the central nervous system in patients with dengue virus infection

The findings of a neurological evaluation in 85 patients with confirmed, acute, dengue virus infection are described. Signs of central nervous system involvement were present in 18 patients (21.2%). The most frequent neurological symptom was mental confusion. The frequency of neurological involvement did not differ between patients with primary and secondary dengue infection, and the prevalence of central nervous system involvement in dengue fever and dengue hemorrhagic fever also did not differ significantly. The presence of CNS involvement did not influence the prognosis of dengue infection. Dengue viral CSF RNA was found in 7 of 13 patients submitted to a spinal tap, the CSF viral load being less than 1000 copies/ml. PCR was negative in serum samples obtained from three patients on the same day as the CSF samples, suggesting that the dengue virus actively enters the CNS and that the presence of the virus in the CNS does not result from passive crossing of the blood-brain barrier.     

Neuro-Intensive Care of Patients with Acute CNS Infections

Infections in the central nervous system (CNS) are caused by a wide range of microorganisms resulting in distinct clinical syndromes including meningitis, encephalitis, and pyogenic infections, such as empyema and brain abscess. Bacterial and viral infections in the CNS can be rapidly fatal and can result in severe disability in survivors. Appropriate identification and acute management of these infections often occurs in a critical care setting and is vital to improving outcomes in this group of patients. This review of diagnosis and management of acute bacterial and viral infections in the CNS provides a general approach to patients with a suspected CNS infection and also provides a more detailed review of the diagnosis and management of patients with suspected bacterial meningitis, viral encephalitis, brain abscess, and subdural empyema.     

Early penetration of the blood-brain-barrier by HIV

CNS dysfunction occurs frequently in patients with HIV infection. To better define the role of HIV in the pathogenesis of neurologic dysfunction, HIV isolation and antibody studies were investigated from the CSF in 52 seropositive patients, 29 with and 23 without neurologic signs and symptoms, in various stages of disease development ranging from asymptomatic to ARC to AIDS. HIV was recovered from the CSF of 5 of 29 (17%) patients with neurologic signs and symptoms and 5 of 23 (22%) neurologically asymptomatic patients. All patients with positive CSF HIV cultures had antibodies directed against HIV p24 and gp41 in serum and CSF by Western blot analysis and elevated intra-blood-brain-barrier total IgG and HIV-specific IgG synthesis rates. The frequency of CSF HIV isolation from the group of seropositive patients without AIDS, 9 of 32 (28%), exceeded that of patients with AIDS, 1 of 20 (5%) (p less than 0.05). These findings indicate that HIV infects the CNS early in the course of viral infection and prior to the development of HIV-associated neurologic abnormalities.     

Anti-galactocerebroside testing in Mycoplasma pneumoniae-associated encephalitis

Mycoplasma pneumoniae (Mp) is the most frequently identified pathogen in the California Encephalitis Project, but the role and mechanism of Mp is unclear. Since auto-antibodies to anti-galactocerebroside (anti-GalC) have been reported in patients with evidence of acute Mp infection of the central nervous system (CNS), serum and cerebrospinal fluid (CSF) samples from 26 patients with evidence of Mp were tested for anti-GalC antibodies. Anti-GalC antibody was found in the CSF of only eight (31%) of 21 Mp patients indicating that CSF anti-GalC antibody is not a specific marker for Mp CNS disease.     

Soluble urokinase receptor (uPAR,CD 87) is present in serum and cerebrospinal fluid in patients with neurologic diseases

BACKGROUND: The receptor for urokinase plasminogen activator (uPAR) promotes invasion by neoplastic or inflammatory cells by focusing proteolysis of urokinase to the cell surface. In pathologic conditions, soluble forms of the receptor (suPAR) are released, and activate cell receptors to promote chemotaxis. In the CNS, suPAR and other components of the plasminogen activation system (PAS) could be associated with an increase of the blood-brain barrier (BBB) permeability and subsequent neural damage. OBJECTIVE: To detect suPAR in the serum and cerebrospinal fluid (CSF) of patients with diverse neurologic conditions. PATIENTS AND METHODS: Serum and CSF from 121 patients with cancer, bacterial and viral infection, stroke, demyelinating disease and peripheral neuropathy were examined for the presence of suPAR. RESULTS: suPAR was elevated in the serum of patients with paraneoplastic syndromes, and carcinomatous meningitis and infections, but less in stroke and demyelinating disease patients. CSF suPAR was present in the cancer and CNS infection groups, but not in the other groups. The levels of serum and CSF suPAR were correlated, and CSF suPAR correlated with the albumin index. CONCLUSIONS: suPAR is present in serum and CSF of patients with carcinomatous meningitis, paraneoplastic disorders and bacterial and viral infection of the CNS. suPAR could be associated with BBB disruption and with promotion of CNS invasion by chemotactically active cells, macromolecules, and microbes.     

Severe post-EBV encephalopathy associated with myelin oligodendrocyte glycoprotein-specific immune response

The mechanisms leading to CNS disorders after EBV infections are unclear. We report the case of a patient who developed a severe, but reversible, encephalopathy following an infectious mononucleosis. We detected no EBV DNA in the blood or in the cerebrospinal fluid (CSF) and no EBV-specific antibodies in the CSF. However, we found a potent MOG-specific cellular and humoral immune response. Interestingly, MOG-specific cellular immune response rapidly decreased, paralleling the improvement of clinical condition. In conclusion, this detailed study shows that acute EBV infection can trigger a potent auto-inflammatory response in the CNS, without evidence of an overt infection.     

Reactivity of locally produced CSF antibodies in patients with neurosyphilis against antigens ofTreponema pallidum

The reactivity and specificity of locally produced cerebrospinal fluid (CSF) antibodies against antigens ofTreponema pallidum were assessed by Western blotting in patients with clinical signs of parenchymal or meningovascular neurosyphilis. All nine patients showed local production of treponeme-specific antibodies in the central nervous system (CNS). In most of the patients serum and CSF antibodies were bound to the same antigens: the common treponemal 48/45 kDa protein and the putative specificT. pallidum protein in the range of 12-14 kDa. In some patients the intensity of staining obtained by CSF antibodies was higher than that derived from serum, indicating locally produced antibodies. In contrast to other more acute inflammatory CNS diseases, no expanded or different antigen binding of the CSF antibodies compared with serum antibodies was found in neurosyphilic patients. The results presented are discussed with regard to the role of the blood-brain barrier in antibody concentrations of CSF and serum.     

Confirmed primary HHV-6 infection in children with suspected encephalitis

HHV-6 infection has been associated with neurological symptoms in children. Two variants of human herpes virus 6, HHV-6A and HHV-6B, have been identified. Their role in neurological infections is poorly understood. We studied 53 children with suspected encephalitis for HHV-6A (strain GS) and HHV-6B (strain Z29) antibodies using an indirect immunofluorescence test. Primary infection was separated from past infection by an IgG-avidity test. The identified primary infections were studied for HHV-6 specific DNA by PCR. Forty-one children of 53 had IgG antibodies to HHV-6. Six children had low avidity of HHV-6 IgG antibodies indicating acute primary infection; four to type A, one to B, and one to both types. By serology, HHV-6 viral etiology was suggested in 6/53 (11.3%) of cases. One of the six patients with primary infection had HHV-6 DNA in serum and two in CSF. The children with primary HHV-6 infection were significantly younger than the whole series, 2.3 years vs. 6.4 years. We conclude that primary HHV-6 infection appears to be an important associated or causative agent in neurological infections of young children, and it can be confirmed from a single serum specimen using the IgG-avidity test.     

Early diagnosis of central nervous system (CNS) viral infections in the neonatal period and early infancy]

The neurological sequelae of CNS viral infections in the neonatal period and early infancy are one of the serious causes of brain dysfunction in children. It is often difficult to diagnose the CNS viral infection because of lack of neurological symptoms. But clinical manifestations are very important for early diagnosis of herpes simplex virus encephalitis and cytomegaloviral infections. We established early diagnosis of CNS viral infections by screening of serum IgM and determining 2' 5' oligoadenyl (2-5A) synthetase activity in the cerebrospinal fluid (CSF). The results showed high serum IgM levels in 75% and abnormal high levels of 2-5 A synthetase activity in CSF of all cases of the young babies with viral meningitis.     

Immunoreactive IFN-gamma in CSF in neurological disorders.

Interferon gamma (IFN-gamma) was measured in the CSF of neurological patients using a highly specific and sensitive immunoradiometric assay. It was detected in 52% of patients presenting as suspected meningitis or encephalitis and in 83% with proven viral meningitis. In contrast IFN-gamma was detected in only 26% of patients who did not have an acute infection at the time of presentation. Only 15% of patients with multiple sclerosis had detectable CSF IFN-gamma. The presence of IFN-gamma in CSF in response to acute viral infections of the central nervous system may be of importance in relation to the pathophysiology of immunologically mediated neurological disorders.     

Central nervous system infections in cancer patients.

In the immunocompromised patient, even mild forms of any combination of headache, meningismus, altered mental status, or focal neurologic signs should initiate an evaluation for possible CNS infection. The limited signs and symptoms of acute CNS infection are not due to specific organisms but to pathologic changes at the neuroanatomic site of infection. The initial clinical history, examination, laboratory, and neuroradiographic data will narrow the problem to one of several groups of agents, although it may not be possible to specify a single causative agent. It should be remembered that several concurrent infections (i.e., CMV and toxoplasmosis, aspergillosis, and bacterial sepsis) may be present. Thus, the clinician should rely on broad antibiotic coverage appropriate to the suspected causative agent or agents at the site of infection. It may be necessary to offer broad-spectrum antibiotic coverage for a CSF presentation that is subsequently found to result from a viral illness or from a noninfectious cause. However, one should avoid undertreating those infections for which specific therapy can be offered, and broad-spectrum treatment usually will not be regretted. Uncertainty in diagnosis following noninvasive procedures should lead to a brain biopsy. Although many of the infections discussed in this article have a poor prognosis, some of the most common pathogens, such as Cryptococcus, Listeria, and Toxoplasma, have effective specific therapies to which the patient should have access as rapidly as possible. The clinician who has successfully treated a patient with CNS infection should remain vigilant for late sequelae or recurrence of infection. Chronic treatment of some infections, such as toxoplasmosis or aspergillosis, may be necessary. The reintroduction of steroids for the treatment of an underlying cancer may reactivate previously treated disease, such as cryptococcosis, and periodic CSF surveillance is appropriate under these circumstances. Recurrence of the symptoms should raise the suspicion of recurrent or new infection, and the patient also should be evaluated with CT or MRI for the development of hydrocephalus or for new metastatic disease. In patients who have had varicella-zoster infection, postherpetic neuralgia and delayed arteritis may develop. Seizures, hearing loss, and neuropsychologic sequelae may follow any meningoencephalitis. The patient should always be reevaluated for the possibility of infection with a different opportunistic organism. CNS infections remain a major cause of morbidity and mortality in immunosuppressed patients with malignancies. In one series, 60% of such patients died as a result of their CNS infection, many at a time when the underlying disease had an otherwise good prognosis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Long-term persistence of intrathecal virus-specific antibody responses after herpes simplex virus encephalitis

Summary Paired sera and cerebrospinal fluids (CSF) from nine surviving patients were collected 4.5 to 8 years after acute herpes simplex (HS) virus encephalitis. Oligoclonal bands of IgG were detected in the CSF of all, and seven patients had an elevated CSF IgG index. Antibodies to HS, varicella-zoster (VZ), measles, and cytomegalo viruses were analysed by enzyme-linked immunosorbent assay (ELISA) and by imprint immunofixation (IIF) of specimens separated by electrophoresis and by thin-layer electrofocusing. Intrathecal synthesis of HS and VZ IgG antibodies was demonstrated in all and of measles IgG antibodies in one patient by both methods. Intrathecal synthesis of HS IgA antibodies was demonstrated by ELISA in three and by IIF in seven patients; the latter method also disclosed intrathecal synthesis of VZ IgA antibodies in two. No patient had intrathecal synthesis of viral IgM antibodies. The intrathecally synthesized antibodies demonstrated by IIF displayed oligoclonal characteristics. The IIF analyses as well as virus absorption tests indicated that the intrathecally synthesized VZ IgG and IgA antibodies could be explained as HS antibodies cross-reacting with VZV. The results indicate that a long-term persistence of intrathecal antibody reesponses to HS virus is a common feature after acute HS encephalitis. The intrathecal production of measles IgG antibodies in one case may reflect a similar persistence of non-specific immune responses induced during the acute infection.     

CSF in 85 patients with AIDS and CNS cryptococcosis.

In an eight years time period (July 1984-June 1992) CSF samples of 40718 patients were studied, and 610 were from patients with AIDS clinically diagnosed and immunologically confirmed through HIV antibodies detection. Among opportunistic infections detected in them 85 were CNS cryptococcosis. For the purpose of this study the CSF of these 85 patients are the AIDS group of CNS cryptococcosis. For comparison, CSF data from 50 patients with CNS cryptococcosis but without AIDS were taken (non-AIDS group); in this group, 22 patients were immunosuppressed after renal transplant. In AIDS group, the more frequent CSF findings were: yeast presence at direct exam (Fuchs-Rosenthal cell counting chamber), growing of the yeast in cultures, and gamma globulins increase. In non-AIDS group were more frequent: hypercytosis, neutrophil cells presence, and total protein increase. Differences between the two groups are discussed taking into account CNS/CSF immune changes induced by HIV infection. It is concluded that in CNS cryptococcosis of patients with AIDS the CSF evidenced more extensive signs of the fungal opportunistic infection than signs of inflammatory response to the infection. The latter were more prominent among patients of the non-AIDS group of CNS cryptococcosis.     

Acute multiple sclerosis exacerbations are characterized by low cerebrospinal fluid suppressor/cytotoxic T-cells

The present study describes peripheral blood (PB) and CSF T-lymphocyte subpopulations in 55 MS patients with and without acute exacerbation and compares the results with those obtained from 8 with CNS infections and 45 with other neurological disorders or symptoms (OND). The MS patients were most strongly characterized by a decline of their CSF suppressor/cytotoxic T-cells, which was most profound during acute exacerbations. The proportional amount of CSF helper/inducer T-cells (Th) was higher in both MS group than in the OND group, but not different from that of the CNS infection group. No statistically significant change in the CSF Th-cells during exacerbations was seen. MS patients without an exacerbation had somewhat higher levels of their PB Th-cells than the patients with OND, but otherwise no differences in the PB T-cell subsets were seen.     

Cerebrospinal fluid biomarkers in patients with varicella-zoster virus CNS infections

Varicella-zoster virus (VZV) is one of our most common viruses causing central nervous system (CNS) infection with sometimes severe neurological complications. Glial fibrillary acidic protein (GFAp), light subunit of neurofilament protein (NFL) and S-100β protein are cerebrospinal fluid (CSF) biomarkers that have been used to estimate the severity of brain damage and outcome in various CNS diseases. So far, these biomarkers have not been utilised to investigate glial pathology and neuronal damage in patients with VZV CNS infections. In this prospective study, we measured CSF GFAp, NFL and S-100β as markers of brain damage in 24 patients with acute neurological manifestations and VZV DNA detected in CSF by PCR and compared with a control group (n = 14). Concentrations of CSF NFL and GFAp were increased in patients with VZV CNS infection compared with controls (p = 0.002 and p = 0.03) while levels of S-100β were reduced. In patients with VZV encephalitis the elevations of CSF NFL and GFAp were more pronounced compared with patients with other VZV CNS syndromes. No correlations between the levels of biomarkers and viral load, neurological sequels or clinical outcome were found in this limited number of patients. These results indicate that VZV induces neuronal damage and astrogliosis with more severe brain damage in patients with VZV encephalitis than in patients with other neurological complications caused by this virus.     

OKT8-binding lymphocytes in diseases of the nervous system

OKT8-binding lymphocytes, which consist mainly of suppressor-cytotoxic T cells, were demonstrated in blood and CSF by an immunochemical method. In patients with non-inflammatory diseases, the mean value was 18% in blood and 19% in CSF. In acute viral infections of the CNS, on the other hand, the percentage was significantly lower in the initial days of the disease but then increased up to the 5th week. Eleven patients suffering from multiple sclerosis had similar percentages of OKT8-binding cells, whether or not they had acute symptoms.