HHV-6-positivity in diseases with demyelination.

BACKGROUND: The triggering agent of multiple sclerosis is still unknown and many viruses, including human herpesvirus-6 (HHV-6), are under suspicion. In earlier study we found patients who had HHV-6 reactive OCBs in their CSF. We wanted to investigate whether HHV-6 has an active role in diseases with demyelination. OBJECTIVE: To analyze the HHV-6-reactive cases in detail and investigate the possible independent role of HHV-6 in the development of central nervous system involvements with demyelination. STUDY DESIGN: We studied serum and CSF samples that were collected over a period of one year, from all patients who had oligoclonal bands (OCB) in cerebrospinal fluid (CSF) and were examined in the Department of Neurology, University Central Hospital of Helsinki, Finland. Clinical evaluation was accomplished blinded of HHV-6 analysis and follow-up time was two years. All patients underwent MRI of the head and clinically indicated CSF analysis. RESULTS: The 17 patients with HHV-6-reactive OCBs were significantly younger and had significantly more IgG-OCBs in comparison to patients without HHV-6-reactive OCBs. Initial diagnoses in patients with HHV-6-reactive OCBs remained the same during the follow-up time. CONCLUSION: Patients with HHV-6-positive OCBs appear to form a separable group. In progressive neurological diseases HHV-6 may have a role in long-term infection with demyelination.

First two cases of neonatal human parechovirus 4 infection with manifestation of suspected sepsis, Finland.

Human parechoviruses are a family of viruses closely related to enteroviruses, and associated with neonatal sepsis-like syndrome, respiratory symptoms and gastrointestinal infection. Here we present clinical details of two neonatal sepsis cases suspected to be caused by HPeV4 infection. The patients were hospitalized in October, 2012. No other causative agents were detected.

Human parechovirus seroprevalence in Finland and the Netherlands.

BACKGROUND: Human parechoviruses (HPeVs) are RNA viruses associated with mild gastrointestinal and respiratory infections in children, but may also cause neonatal sepsis and CNS infections in infants. While the prevalence of HPeVs is known mostly among hospitalized populations, the knowledge of HPeV seroprevalence in the general population is poor. OBJECTIVES: The aim of this study was to identify and compare the HPeV1-6 seroprevalence in Finnish and Dutch populations. STUDY DESIGN: A type specific microneutralization assay was set up for detecting neutralizing antibodies (nABs) against HPeV types 1-6. Altogether 616 serum samples from Finnish and Dutch population were analyzed for antibodies against HPeVs. The samples were collected from Finnish children aged 1, 5 or 10 years, Finnish adults, 0- to 5-year-old Dutch children, Dutch women of childbearing age and Dutch HIV-positive men. RESULTS: In both adult populations, seropositivity was high against HPeV1 (99% in Finnish and 92% in Dutch samples) and HPeV2 (86% and 95%). Against HPeV4, the seropositivity was similar (62% and 60%). In Dutch adults, nABs against HPeV5 and 6 (75% and 74%) were detected more often than in Finnish adults (35% and 57%, respectively). In contrast, seropositivity against HPeV3 was as low as 13% in the Finnish and 10% in the Dutch adults. The seroprevalence of all HPeV types increased with age. CONCLUSIONS: The seroprevalence of HPeVs is high in Finnish and Dutch populations and HPeV type 2 and types 4-6 are significantly more prevalent compared to earlier reports. The seroprevalence of antibodies observed against HPeV3 was low.

Human parechoviruses are frequently detected in stool of healthy Finnish children.

BACKGROUND: Human parechoviruses (HPeVs) are common viruses mainly infecting young children. Most infections are mild, but HPeV3 causes severe CNS infections in new-born infants. OBJECTIVES: The aim was to study the epidemiology of HPeVs in Finnish general population in the period 1996-2007, with special emphasis on the different types circulating in Finland. STUDY DESIGN: A total of 2236 stool specimens were collected from 200 healthy Finnish children in a prospective birth cohort study, most before the age of 2 years. Samples were tested for the presence of HPeV RNA using a specific RT-PCR. The genotype of HPeV was determined by sequencing the VP1 genomic region. RESULTS: HPeV RNA was detected in 144 (6.4%) specimens from 78 (39%) children. The dominant type was HPeV1 (93% of the type-identified 105 samples), although types 3 and 6 were also identified. HPeV was found sequentially in more than one sample in 43 infections lasting up to 93 days. The positive findings were distributed equally in young ages and declined towards the age of 2 years. Infections clustered around the autumn months with no obvious change between years. No significant differences were seen between boys and girls. CONCLUSIONS: HPeV is a common virus infecting Finnish children under 2 years of age. HPeVs circulate throughout the year with clear accumulation on autumn, also seen in individual years over the 11-year study period. The virus deserves increased attention and should be included in the test panel of clinical virus laboratories.

Intrathecal human herpesvirus 6 antibodies in multiple sclerosis and other demyelinating diseases presenting as oligoclonal bands in cerebrospinal fluid.

Demyelinating diseases of the central nervous system (CNS) often include elevated IgG production in intrathecal space presenting as oligoclonal bands (OCBs) in cerebrospinal fluid (CSF). In most demyelinating diseases, e.g. in multiple sclerosis (MS), the underlying cause is not known. We used isoelectric focusing and affinity immunoblot to study the specificity of CSF OCBs to human herpesvirus-6 (HHV-6) in patients with demyelinating diseases of the CNS including MS. Eighty patients with positive OCB finding were included in the study. The OCBs reacted with the HHV-6 antigen in 18 cases (23%). Twelve of 46 MS patients (26%), 5 of 24 other demyelinating diseases (21%) and 1 of 10 other neurological disorders (10%) had HHV-6 specific OCBs in CSF. A specific intrathecal HHV-6 A and B antibody production was shown in a proportion of patients with demyelinating diseases and might suggest a role in the pathogenesis of these diseases.

Distribution of varicella-zoster virus (VZV) wild-type genotypes in northern and southern Europe: evidence for high conservation of circulating genotypes.

Phylogenetic analysis of 19 complete VZV genomic sequences resolves wild-type strains into 5 genotypes (E1, E2, J, M1, and M2). Complete sequences for M3 and M4 strains are unavailable, but targeted analyses of representative strains suggest they are stable, circulating VZV genotypes. Sequence analysis of VZV isolates identified both shared and specific markers for every genotype and validated a unified VZV genotyping strategy. Despite high genotype diversity no evidence for intra-genotypic recombination was observed. Five of seven VZV genotypes were reliably discriminated using only four single nucleotide polymorphisms (SNP) present in ORF22, and the E1 and E2 genotypes were resolved using SNP located in ORF21, ORF22 or ORF50. Sequence analysis of 342 clinical varicella and zoster specimens from 18 European countries identified the following distribution of VZV genotypes: E1, 221 (65%); E2, 87 (25%); M1, 20 (6%); M2, 3 (1%); M4, 11 (3%). No M3 or J strains were observed.

IgM and avidity of IgG antibodies in primary HHV-6 infections.

Primary HHV-6 infection could be diagnosed by enzyme immunoassay from a single serum using IgG avidity test based on results obtained from 43 patients, 26 with seroconversion and 17 with variable antibody levels. The avidity was less than 30% in all patients with seroconversion. HHV-6 IgM appeared non-specific.

Primary human herpesvirus-6 infection in the central nervous system can cause severe disease.

Human herpesvirus-6 (HHV-6) infection is common in infancy, and symptoms are usually mild. However, encephalitis and other neurologic complications have been reported. Primary HHV-6 infection has been rarely confirmed in the central nervous system. We studied 21 children with suspected HHV-6 infection, drawn from a prospective, large-scale study of neurologic infections in Finland. Human herpesvirus-6 polymerase chain reaction was performed on cerebrospinal fluid samples, and antibody tests were performed on serum and cerebrospinal fluid. We identified nine children, aged 3 to 24 months, who had HHV-6-specific nucleic acid in cerebrospinal fluid. Primary infection was confirmed by seroconversion of specific antibodies in six, whereas one had a fourfold increase, and one had a fourfold decrease, in the antibody titer supporting recent infection. Generalized and prolonged seizures appeared in six children, four had a rash, four had ataxia, and four had gastroenteritis. All but two had a high fever. At follow-up, four children had evident neurologic sequelae, ataxia, and developmental disability, and needed special education. Primary HHV-6 infection may invade the central nervous system, and can cause neurologic symptoms and potentially permanent disability in children aged

Genotypic analysis of varicella-zoster virus and its seroprevalence in Finland.

We evaluated the seroprevalence of varicella-zoster virus (VZV) in the Finnish population among various age groups and genetically characterized VZV strains from documented cases of varicella and zoster. VZV-specific immunoglobulin G was measured in 2,842 serum samples that had been submitted for virological studies to the Department of Virology, University of Helsinki, from 1995 to 1996. Specimens for VZV genotyping were obtained from vesicular lesions from two pediatric patients and 26 adult patients. Seroprevalence to VZV varied markedly by age: 45% in children aged < or = 2 months, 12.5% in children aged 6 to 8 months, and > 90% in children near 10 years of age, plateauing thereafter into advanced age. The seroprevalence rates indicate that in Finland, as in other countries with temperate climates, primary VZV infection usually occurs during the first decade of life. Twenty-eight VZV DNA-positive specimens were analyzed to identify VZV vaccine and wild-type genotypes. All analyzed specimens were wild type and the European (E) genotype.

Multiplex-PCR and oligonucleotide microarray for detection of eight different herpesviruses from clinical specimens.

BACKGROUND: Human herpesviruses cause clinically important diseases, e.g. infections of the central nervous system. New diagnostic tools are required for rapid and reliable detection of these viruses. OBJECTIVES: A microarray-based method was designed for detection of eight human herpesviruses in cerebrospinal fluid (CSF), whole blood, plasma, serum and proficiency-testing specimens. STUDY DESIGN: Herpes simplex type 1 and 2, varicella-zoster, cytomegalo-, Epstein-Barr and human herpes viruses 6A, 6B and 7 were amplified from clinical specimens by two multiplex-PCRs and transcribed to single-stranded RNAs which were hybridized to oligonucleotides on microarray. The results were compared to those from conventional PCR. In total, 227 specimens were tested including 23 CSF, 10 whole blood, 73 plasma, 10 proficiency-testing samples and 111 negative control samples. RESULTS: Concordant results were obtained in 214/227 (94%). Microarray detected 10 possible double and one triple infection. Negative control samples (70 serum, 30 CSF and 11 proficiency-testing samples) were all negative. CONCLUSIONS: Microarray is suitable for detection of multiple herpesviruses in clinical samples.

Plasminogen activators and their inhibitors in human saliva and salivary gland tissue.

The purpose of this study was to identify plasminogen activators (PA) and their specific inhibitors in human cell-free saliva and to investigate their expression in salivary gland tissue. Saliva samples were obtained from 34 patients visiting a neurological out-patient department. The activities of tissue and urokinase plasminogen activators (tPA and uPA, respectively), the relative inhibition of tPA, and the amounts of plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2, respectively) in cell-free saliva were studied. The activities of tPA and uPA, and tPA inhibition, were measured using in-house microtiter plate assays, and PAI-1 and PAI-2 levels were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits. Immunohistochemistry was used to evaluate the expression of PAs and PAIs in the salivary gland. Tissue plasminogen activator activity was found in most samples, with a mean activity of 0.63 IU ml(-1). uPA was observed in only a few samples. PAI-1 was not detected, but PAI-2 was present in all samples (with a mean value of 11.1 ng ml(-1)). The mean PAI-2 level in women was 12.4 and in men was 7.6 ng ml(-1). The activity of tPA and the relative inhibition of tPA seemed to be inversely associated. Tissue plasminogen activator, PAI-1, and PAI-2 were evident in salivary gland tissue, whereas the expression of uPA was low. The tPA activity in saliva suggests an active proteolysis. Plasminogen activator inhibitor 2 was found to be the main inhibitor of PAs in saliva.

Co-localization of human herpes virus 6 and tissue plasminogen activator in multiple sclerosis brain tissue.

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system of unknown etiology. Several viruses have been suggested as playing a role in the pathogenesis of MS. The aim of this study was to investigate the interrelationship of human herpesvirus 6 (HHV-6) and plasminogen activation at the cellular level in MS plaques. MATERIAL/METHODS: Brain tissue specimens obtained from autopsies of 15 patients with MS and 10 controls were studied immunohistochemically for HHV-6 and cytomegalovirus (CMV) antigen and tissue plasminogen activator (tPA) protein. The presence of Ebstein-Barr virus (EBV) EBER RNA was studied using RNA in situ hybridization. RESULTS: HHV-6 antigen was identified in the cells of 67% (10/15) of MS brain sections and 30% (3/10) of the control sections. All samples were negative for CMV antigen and all samples with intact RNA were negative for EBV EBER RNA as demonstrated by in situ hybridization. tPA expression was found to be increased in MS plaques compared with the control samples. Interestingly, in 5 MS samples both HHV-6 antigen and tPA stained clearly, compared with none in the controls, but HHV-6 and tPA only occasionally co-localized in the same cells. CONCLUSIONS: At the cellular level, HHV-6 and plasminogen activation seem to co-localize in MS.

Varicella zoster and Borrelia burgdorferi are the main agents associated with facial paresis, especially in children.

BACKGROUND: The etiology of facial paresis (FP) often remains unresolved. Yet, a microbial association is frequently suspected. OBJECTIVE: To evaluate the infectious etiology of FP by using sensitive tests. STUDY DESIGN: We studied the serum and cerebrospinal fluid of 42 patients diagnosed with idiopathic peripheral facial paresis using sensitive serological methods and nucleic acid detection and for reference, 42 patients with other neurological disorders (OND) matched for age, sex, season and geographical area. RESULTS: Varicella zoster virus and Borrelia burgdorferi accounted for 56% of all associated agents in children with FP compared with 11% of OND (P=0.01). In adults, the respective numbers were 29 and 13%. Other treatable etiological agents, Chlamydia pneumoniae and Mycoplasma pneumoniae, accounted for 11% in children and 8% in adults and with the same prevalence between patients with FP and OND. CONCLUSIONS: Microbes, with specific therapy available accounted for 52% of all associated agents in the patients with FP when compared with 26% in controls with OND (P=0.04). Based on this, we conclude that the patients with FP may benefit from antimicrobial therapy.

CSF findings in neonates with seizures; infectious and noninfectious.

In order to evaluate parameters of cerebrospinal fluid (CSF) we studied 105 CSF samples of 50 neonates with seizures of unknown origin for cell count and chemistry (protein, albumin, glucose, IgG-index and albumin-ratio). Viral studies for 13 different microbes were performed from serum and CSF. CSF parameters of the babies with a suggested viral infection (n = 13) were compared with those without any viral findings (n = 37), and followed up to the age 45 weeks since conception. CSF mononuclear white blood cell count was < or = 20 x 10(6)/l at the age of < or = 40 weeks since conception, and thereafter i.e. at term it was < or = 10 x 10(6)/l in all neonates without viral infection, whereas mononuclear cell count was above these limits in 8 of 13 neonates with viral infection. The rate of IgG-index remained high only in the neonates with a viral infection when studied at the age of over 43 weeks since conception. We conclude that studies of CSF are a valuable diagnostic aid in CNS viral infections of neonates when evaluated in reference to the age since conception, and the limits of mononuclear white blood cells in normal CSF of neonates are in lower limits than reported before.

Viral infections in neonates with seizures.

The aim of this study was to determine the incidence of viral infections in the central nervous system (CNS) of neonates with seizures of unknown origin. Serum and cerebrospinal fluid (CSF) samples of 50 neonates were studied for antibodies to 13 different microbes by enzyme immunoassay, and CSF samples were investigated for four specific nucleic acids by polymerase chain reaction. For viral cultures throat, stool and CSF samples were obtained. CNS viral infection was suggested in 13 of the 50 neonates (26%). Antibodies to varicella, herpes simplex 1, influenza A or B, parainfluenza 1, adeno or enteroviruses were detected in the CSF of nine children; and herpes simplex 2 specific DNA was found in one child. Cytomegalovirus was cultured from the urine of two children. In one child unspecified, but evident pleocytosis in the CSF was observed. The incidence of CNS viral infections in association with neonatal seizures may be much higher than previously reported.

Acute central nervous system complications in varicella zoster virus infections.

BACKGROUND: In a previous multicenter study on central nervous system (CNS) viral infections varicella zoster virus (VZV) appeared the most frequent etiologic agent and appeared often without rash. OBJECTIVE: To evaluate the appearance and diagnostics of VZV in CNS more thoroughly, we studied the cases systematically by using sensitive and specific methods to learn the best diagnostic approach in order to start specific therapy. STUDY DESIGN: We analyzed all serum and cerebrospinal fluid samples of 174 patients, 88 females and 86 males, with acute CNS symptoms associated with VZV infection diagnosed in the multicenter study on viral CNS infections. RESULTS: About 38 patients (22%) had chickenpox, 59 (34%) had shingles, and 77 (44%) had no cutaneous symptoms at all. The mean age of chickenpox patients was 8.6 years, of the others 46.6 and 41.4 years. VZV-specific nucleic acid was detected in the CSF in one fourth of the patients in all groups, primarily during the first week of illness. In serum specimens, specific IgM was present in two thirds of the patients with chickenpox, whereas in the others in one third of the cases. In CSF, specific IgM was present in 15-17% of patients with skin manifestations, compared with 6% of those without rash. CONCLUSIONS: The role of VZV infections in CNS complications seems remarkable, often presenting without rash. Even these cases should be promptly recognized in order to conduct proper antiviral therapy. In children, a combination of PCR and IgM tests is the best approach. In adults, PCR, together with the measurement of intrathecal antibody production yields best results.