Detection and genotyping of enteroviruses in cerebrospinal fluid in patients in Victoria, Australia, 2007-2013.

Genotyping by VP1 fragment polymerase chain reaction (PCR) and nucleic acid sequencing to detect enterovirus (EV) genotypes was performed directly on 729 EV PCR positive cerebrospinal fluid (CSF) samples collected between 2007 and 2012 from Victorian hospital inpatients. The overall genotype identification rate from CSF-positive material was 43%. The four most common genotypes identified were Echovirus 6 (24%), Echovirus 30 (17%), Echovirus 25 (10%), and Coxsackievirus A9 (10%), together comprising 61% of all EVs typed. The seasonal distribution of all EVs identified followed the recognized pattern of mainly summer epidemics. Three of the four predominant genotypes were present in each of the 6 years in which the study was conducted, with 20 other EV genotypes also detected, often in only a single year. Genotyping of EVs directly in CSF is faster, simpler and more sensitive than traditional virus neutralization assays performed on EV positive samples.

Update on molecular characterization of coxsackievirus B5 strains.

Among Coxsackie B viruses, Coxsckievirus B5 is one of the most predominant serotypes in human, it is frequently associated with cases of neurological diseases, epidemics of meningitis and is a common cause of cardiomyopathy and diabetes. In the present study 27 isolates of Coxsackievirus B5 from North Africa, obtained from cerebrospinal fluid and stool samples of healthy individuals, patients with acute flaccid paralysis or aseptic meningitis were investigated by partial sequencing in the 5' half of the VP1 region and compared to the up-to-date published Coxsackievirus B5 sequences in the same genomic region. Four distinct genomic groups and ten different clusters were individualized. Most of the isolates from Algeria and Tunisia belonged to two clusters. For both, the sequences from North Africa clustered mainly with sequences from European countries, the majority isolated recently during the 2000s. The analysis of the alignment of amino-acids sequences in the VP1 gene revealed four major substitutions in strains from different clusters, we also noticed changes in the BC-loop region; this region is associated with viral antigenicity. This study permit to better identify circulating Coxsackievirus B5 strains throughout the world and their genetic relationship. The protein analysis showed changes that could imply some antigenic significance. J. Med. Virol. 83:1247-1254, 2011. © 2011 Wiley-Liss, Inc.

[Relationship between variation of coxsackievirus B3 VP1 sequence from cerebrospinal fluid of children and severity of damage to central nervous system].

OBJECTIVE: To investigate the possible relationship between variation of coxsackievirus B3 (CoxB3) VP1 sequence from cerebrospinal fluid of children with severe and mild central nervous system (CNS) infection and damage to CNS in children from Shandong province. METHODS: The enteroviruses were detected using VP1 typing and sequencing primer for enteroviruses from 73 enterovirus-infected cases confirmed by detection of cerebrospinal fluid by enteroviruses common primer. VP1 sequences (450 nucleotides) were determined and analyzed for 21 CoxB3 enteroviruses strains isolated in Qingdao and Binzhou, and were compared with that of BLAST search procedures from GeneBank in NCBI. The variation of VP1 gene and amino acids sequence of CoxB3 enteroviruses was analyzed for severe and mild CNS infection. RESULTS: The nucleotide homogeneity of these CoxB3 appeared to be 97% - 99%, however, the homogeneity among different genotypes were 83% - 76%. Replacement of glutamine by histidine at amino acid locus 856 of VP1 CoxB3 was found in 4 cases with severe encephalitis. There were different variation in VP1 nucleotide sequence of CoxB3 in 3 cases with mild encephalitis and 14 cases with meningitis, but amino acids sequences had no regular variation. The modified Glasgow's coma score was below 7 in all the 4 cases with severe encephalitis. Of these 4 cases, 3 had consciousness disturbance for less than 3 days. Lethargy, restlessness and psychiatric symptoms were major manifestations, of whom 3 also had dysphagia, 1 had encephalatrophy obviously, Glasgow's coma score was 3, deep coma lasted for 9 days, and had concomitant fatal epileptic attacks. Of these 4 cases, 2 completely recovered, 1 had high muscle tone, 1 remained under anti-epileptic drug treatment at follow-up 6 months later. CONCLUSION: There were a small epidemic of CoxB3 CNS infection in children in 2005 in this area. The amino acid variation of CoxB3 VP1 possibly caused increased viral virulence and caused damage to CNS.

[Rapid enterovirus genotyping in cerebrospinal fluids: a two-year prospective study in a virology laboratory setting]. / Génotypage direct rapide des entérovirus dans le liquide céphalorachidien : étude prospective de deux ans au sein d'un laboratoire de virologie clinique.

UNLABELLED: Enterovirus (EV - 68 serotypes) infections comprise a wide spectrum of clinical presentations including infections of the central nervous system. In severe clinical presentation or epidemics, the precise identification of the involved serotype is necessary. OBJECTIVES: To perform enterovirus genotyping directly in cerebrospinal fluid (CSF) samples, and to assess its feasibility in a laboratory setting. METHODS: Enterovirus genotyping was carried out directly with CSF specimens tested for the diagnostic procedure by amplifying the complete 1D gene encoding the VP1 protein of the HEV-B serotypes (the most frequent) - providing results in two days. Secondly, sequences 1A/1B encoding the VP4/VP2 capsid proteins, respectively, were analysed (results in five days). RESULTS: Direct enterovirus genotyping allowed the identification of enterovirus involved in 77 out of 81 (95%) meningitis cases between January 2006 and December 2007. In combination with the indirect genotyping of enterovirus isolates, identification of the type was achieved in 94 out of 97 (96.9%) patients included in the study. The most frequent serotypes were echovirus 6 (E6) and 13 in 2006, coxsackievirus B2 and E30 in 2007. Four children presented an EV71 associated meningitis. CONCLUSION: When prospectively applied in a laboratory setting, direct enterovirus genotyping in CSF samples allows the identification of the involved enterovirus in two to five days. This time frame is relevant for an optimal patient management, the rapid identification of a new enterovirus variant or in the context of an epidemic alert.

sICAM-1 intrathecal synthesis and release during the acute phase in children suffering from Coxsackie A9 and S. pneumoniae meningoencephalitis.

The intercellular adhesion molecule is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. Serum and cerebrospinal fluid (CSF) soluble intercellular adhesion molecule 1 (sICAM-1) from normal control children as well as from children with Guillain-Barré syndrome (GBS), with Coxsackie A9 virus meningoencephalitis and with Streptococcus pneumoniae meningoencephalitis were studied. sICAM-1 was quantified using an immunoenzimatic assay and albumin using the immunodiffusion technique in both biological fluids. Increased sICAM-1 values in CSF in patients with GBS correspond to an increase of the albumin CSF/serum quotient. In contrast, in inflammatory diseases like S. pneumoniae and Coxsackie A9 virus meningoencephalitis an increased brain-derived fraction was observed. In particular cases these values are 60-65% and 70-75% respectively. The results indicate an additional synthesis of sICAM-1 in subarachnoidal space during central nervous system (CNS) inflammatory process. An important role of sICAM-1 in the transmigration of different cell types into CSF during CNS inflammation in children with S. pneumoniae and Coxsackie A9 meningoencephalitis may be suggested.

sICAM-1 intrathecal synthesis and release during the acute phase in children suffering from Coxsackie A9 and S. pneumoniae meningoencephalitis / Sintesis intratecal de sICAM-1 y liberación durante la fase aguda en niños con meningoencefalitis por Coxsackie A9 y S pneumoniae

The intercellular adhesion molecule is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. Serum and cerebrospinal fluid (CSF) soluble intercellular adhesion molecule 1 (sICAM-1) from normal control children as well as from children with Guillain-Barré syndrome (GBS), with Coxsackie A9 virus meningoencephalitis and with Streptococcus pneumoniae meningoencephalitis were studied. sICAM-1 was quantified using an immunoenzimatic assay and albumin using the immunodiffusion technique in both biological fluids. Increased sICAM-1 values in CSF in patients with GBS correspond to an increase of the albumin CSF/serum quotient. In contrast, in inflammatory diseases like S. pneumoniae and Coxsackie A9 virus meningoencephalitis an increased brain-derived fraction was observed. In particular cases these values are 60-65 percent and 70-75 percent respectively. The results indicate an additional synthesis of sICAM-1 in subarachnoidal space during central nervous system (CNS) inflammatory process. An important role of sICAM-1 in the transmigration of different cell types into CSF during CNS inflammation in children with S. pneumoniae and Coxsackie A9 meningoencephalitis may be suggested.

La molécula de adhesión intercelular es una glicoproteína que pertenece a la superfamilia de las inmunoglobulinas. Se estudiaron los niveles de molécula de adhesión intercelular tipo 1 soluble (sICAM-1) en suero y líquido cefalorraquídeo (LCR) de niños con meningoencefalitis por Streptococcus pneumoniae y por Coxsackie A9 al igual que en niños con sindrome de Guillain-Barré (SGB). sICAM-1 fue cuantificado por ensayo inmunoenzimático y la albúmina por inmunodifusión en ambos líquidos biológicos. Los valores incrementados de sICAM-1 en LCR en los pacientes con GBS corresponden a valores aumentados de razón LCR/suero de albúmina. En contraste, en las enfermedades inflamatorias como las meningoencefalitis por S. pneumoniae y por Coxsackie A9 se observa un incremento en la fracción derivada del cerebro. En casos particulares los valores se incrementan hasta un 60-65 por ciento y 70-75 por ciento respectivamente. Los resultados indican una síntesis adicional de sICAM-1 en el espacio subaracnoideo durante el proceso inflamatorio del sistema nervioso central (SNC). Esto puede sugerir un importante papel del sICAM-1 en la transmigración de diferentes tipos celulares en el LCR durante la inflamación del SNC en niños con meningoencefalitis por S pneumoniae y coxsackie A9.

Genetic variation of coxsackie virus B5 strains associated with aseptic meningitis in Greece.

In order to explore the genetic relationships among coxsackie virus B5 strains in Greece, the nucleotide sequences of the partial VP1 gene in strains isolated from aseptic cases of meningitis were determined and compared with those of strains isolated from other countries. Phylogenetic analysis showed a high degree of divergence (25%) among Greek strains isolated in different years, which clustered with high bootstrap values in a different subgroup of viruses, suggesting that enterovirus types vary with time rather than geographical distribution. A non-synonymous mutation present in the strains of this study was not observed in other coxsackie virus B5 strains.

[Etiology of aseptic meningitis prevalent in Xuzhou].

BACKGROUND: To find the pathogenic agents of aseptic meningitis prevalent in Xuzhou of Jiangsu province in 2001. METHODS: The enterovirus (EV) was cultured from CSF of the patients and identified with anti-serum by neutralization test. Neutralization titer of antibody in paired sera from meningitis children was determined. EV RNA was detected by RT-PCR. RESULTS: Four strains of Coxsackievirus B5, 2 strains of Coxsackievirus B3 and 1 strain of Echovirus 7 were isolated from 22 CSF specimens. The isolation rate of virus was 31.8% (7/22), 21 CSF were tested by RT-PCR, the positive rate of EV RNA was 52.4% (11/21); 57.9% (11/19) of patients paired-sera had over 4 folds antibody rise or became seroconverted. CONCLUSION: Enterovirus was the pathogenic agent of aseptic meningitis prevalent in Xuzhou of Jiangsu province, the main serotype of the virus was Coxsackievirus B5.

Acute transverse myelitis due to Coxsackie virus.

The case of a young adult male, who after a short upper respiratory illness presented with fever and alarming progressive neurological deficits, is reported. The diagnostic puzzle and the difficulty in establishing a diagnosis are reported. Acute transverse myelitis is a rare clinical manifestation of Coxsackie virus infection, and very few cases of transverse myelitis caused by serotype B have been reported in the English literature. This is a case report of an unusual acute transverse myelitis caused by Coxsackie B2 infection.

Acute transverse myelitis caused by Coxsackie virus B5 infection.

A 6-year-old boy developed symptoms of rapidly progressive paraplegia, associated with bowel and urinary dysfunction, but without sensory loss. Magnetic resonance imaging (MRI) examination showed diffuse swelling of the lower spinal cord on T1-weighted images. Based on the clinical presentation and MRI findings, a diagnosis of acute transverse myelitis was made. The serum titer of neutralizing antibody against Coxsackie virus B5 rose from 1/4 on admission to 1/256 1 month later and Coxsackie virus B5 was isolated from stool samples. This case serves as a reminder that acute transverse myelitis can be a rare clinical manifestation of Coxsackie virus B5 infection.

A fatal case of coxsackievirus B4 meningoencephalitis.

BACKGROUND: Coxsackieviruses and echoviruses are common causes of aseptic meningitis, but they rarely cause life-threatening illness. We report a fatal case of coxsackievirus B4 meningoencephalitis in a woman who developed extrapyramidal symptoms suggestive of encephalitis lethargica. The exact causative agent of encephalitis lethargica has rarely been found, but most cases of the syndrome are assumed to be of viral origin. CASE DESCRIPTION: A 33-year-old woman previously treated with methylprednisolone and cyclophosphamide for Henoch-Schönlein purpura was transferred from a referring hospital because of sore throat, fever, and chills. Her neurologic findings progressed from headache with mild photophobia to lethargy, cogwheeling, increased tone in all 4 limbs, and brisk reflexes. The patient was diagnosed as having coxsackievirus B4 meningoencephalitis and, despite treatment with the experimental antiviral agent pleconaril, died of an overwhelming central nervous system infection and myocarditis. Magnetic resonance imaging showed focal hyperintense lesions in the substantia nigra that corresponded to the location of pathological changes seen at autopsy. CONCLUSIONS: This patient had a fulminant coxsackievirus B4 viral meningoencephalitis with a clinical pattern reminiscent of encephalitis lethargica and striking focal abnormalities in the substantia nigra identified on magnetic resonance imaging. The magnetic resonance imaging findings correlated with pathological changes identified at autopsy that were similar to the pathological findings observed in patients with encephalitis lethargica and postencephalitic parkinsonism. It is likely that the patient's immunocompromised state led to an overwhelming infection from an otherwise relatively innocuous viral infection.

Development and evaluation of a nucleic acid sequence based amplification (NASBA) protocol for the detection of enterovirus RNA in cerebrospinal fluid samples.

A nucleic acid sequence based (NASBA) assay for the generic detection of enterovirus RNA in cerebrospinal fluid (CSF) samples was developed and compared with an established reverse transcription/nested polymerase chain reaction (PCR) protocol. The sensitivity of NASBA followed by detection of amplicons with a biotinylated oligonucleotide probe was < or = ten copies of enterovirus RNA, indicating that enterovirus NASBA achieves a similar sensitivity as nested PCR. Moreover, NASBA detected a panel of 22 different serotypes of the species poliovirus, human enterovirus A, human enterovirus B and human enterovirus C completely. For evaluating NASBA as a diagnostic tool, 61 CSF samples of patients suffering from aseptic meningitis were tested in parallel with NASBA and nested PCR. NASBA detected enterovirus RNA in four CSF samples, two of these were also positive by nested PCR and two other CSF samples were positive only by nested PCR (in total six positive samples). All other 55 of 61 CSF samples were concordantly enterovirus negative by both methods. In conclusion, the more simple to handle 'one step' NASBA is as sensitive as nested PCR and may be used as an alternative method for the detection of enterovirus RNA in CSF samples. Enterovirus NASBA is a 'one step' RNA amplification procedure that is less prone to cross-contamination compared to a three step nested PCR.

Viral isolation from cases of epidemic neuropathy in Cuba.

OBJECTIVE: To investigate the possibility of a viral agent in the central nervous system of patients with epidemic neuropathy. DESIGN: Virus isolation attempts, in cell cultures and suckling mice, from cerebrospinal fluid (CSF) of neuropathy patients and controls undergoing lumbar puncture for unrelated reasons. Serologic studies in patients, contacts, and controls. SETTING: An epidemic of optic and peripheral neuropathy affected more than 50,000 people in Cuba in 1991 through 1993. Illness was associated with dietary limitations and increased physical demands accompanying the shortages of food and fuel experienced in Cuba since 1989. Most patients responded to parenteral vitamin therapy, and the epidemic began to subside when oral vitamin supplementation was begun for the entire Cuban population. RESULTS: Coxsackievirus A9 (five isolates) and a similar, less cytopathic virus (100 isolates) were recovered from 105 (84%) of 125 CSF specimens from neuropathy patients. The strains with light cytopathic effect were antigenically related to Coxsackieviruses A9 and B4 by cross-neutralization and immunoblotting assays. Virus persisted in CSF of some patients for 1 to 12 months. Cerebrospinal fluid from patients and both types of virus from cell culture produced illness, including complete posterior flaccid paralysis, in newborn mice, and virus was reisolated from the mice. Mouse tissues and sural nerve biopsy specimens from patients were stained by immunoperoxidase and colloidal gold techniques using hyperimmune rabbit antisera against the virus with light cytopathic effect. CONCLUSIONS: Coxsackievirus A9 or an antigenically related agent with a light cytopathic effect was present in CSF of 84% of 125 patients with epidemic neuropathy. The role of these agents, probably in combination with nutritional factors, in the pathophysiology of the disease requires further investigation.

[Enterovirus meningoencephalitis in the last 5 years]. / Meningoencefalitis por Enterovirus en los últimos 5 años.

The results of the study of Enterovirus as viral meningoencephalitis producing agents, carried out from 1990 to 1994, are described, 546 feces samples, 95 cerebrospinal fluids and 1,058 matched sera were studied and obtained from 1,388 patients clinically diagnosed with this disease. Samples for viral isolation were inoculated into two different cellular systems. The highest number of isolation was found in diploid cells from human fibroblast. Antibody determinations were carried out by a neutralization test (micromethod) with 11 Enterovirus antigens (Echo 4, 6, 9, 11 and 30; and Coxsackie B1, 2, 3, 4, 5 and 6) and in epidemic periods with the isolated virus. During the years under study, 2 epidemic outbreaks took place: on caused by Coxsackie A9 (1990-1991) and the other one by Echo 30 (1994). A greater positivity to Echo 6 and 11 was found among the matched sera.

[Isolation of Coxsackieviruses B5 in a fatal case of meningoencephalitis]. / Aislamiento de virus Coxsackie B-5 en un caso fatal de meningoencefalitis.

A nine months old boy was admitted to the hospital with the diagnosis of meningoencephalitis 15 days after having a clinically diagnosed chickenpox. Lumbar puncture showed clear CSF with 0.23 g/l of proteins, 57 mg/dl of glucose, 30 red cells/mm3 and 5 leukocytes/mm3. Blood count showed a packed red cell volume of 22%, a hemoglobin of 7 g/dl, 14800 leukocytes with 1% eosinophils, 5% band and 39% segmented neutrophils, 50% lymphocytes and 5% monocytes and a decreased platelet count. On the fourth hospitalization day, the patient had vomiting, irritability and stiff neck. A new lumbar puncture showed a clear CSF that differed from the former only in the glucose level that increased to 102 mg/dl. The patient died and the necropsy showed a congestive and enlarged brain and congestive meninges infiltrated with lymphocytes. There was lymphoid follicle hyperplasia in the small bowel and enlarged mesenteric lymph nodes. Samples of brain, brain stem, spinal cord and stools were sent for virological study. A Coxsackie B-5 virus was isolated from the spinal cord sample.

[Inhibition of the multiplication of a Coxsackie A9 strain isolated from a patient with epidemic neuropathy by the interferons alfa and gamma]. / Inhibición de la multiplicación de la cepa de Coxsackie A9 aislada de un paciente con neuropatía epidémica frente a los interferones alfa y gamma.

We present the results of the in vitro action of alpha and gamma interferons and of Intacglobin and Igegam against the 47/93/IPK (Coxsackie A9) strain isolated from the cerebrospinal fluid of a patient with epidemic neuropathy. The in vitro studies showed that the two interferons inhibited the replication of this agent; they also showed the presence of antibodies to it in the Intacglobin and Igegam. The results attained demonstrated that the use of these compounds could be effective for the treatment of this entity.

[Viral infection in the neonatal period: diagnostic difficulties, the role of interferon alpha levels]. / Infection virale en période néonatale: difficultés diagnostiques, intérêt du dosage de l'interféron alpha.

Serum and/or cerebrospinal fluid (CSF) alpha interferon (aIFN) levels were determined in 31 neonates hospitalized for suspected sepsis. Final diagnosis was bacterial sepsis in 15, viral infection in 13 and no infection in 3. Among the 13 neonates with viral infection, enterovirus was isolated 5 times and coxsackievirus 4 times. No aIFN was found in cerebro-spinal fluid and/or serum among the neonates with bacterial sepsis or without infection. By contrast, in neonates with viral infection, CSF and/or serum aIFN levels were always elevated except in one case in which serum aIFN determination was not available. We conclude that in neonates, elevated levels of CSF and serum aIFN appear to be specific of viral infection, and that this might be helpful in the differential diagnosis of suspected sepsis.