Major 5'terminally deleted enterovirus populations modulate type I IFN response in acute myocarditis patients and in human cultured cardiomyocytes.

Major 5'terminally deleted (5'TD) group-B enterovirus (EV-B) populations were identified in heart biopsies of patients with fulminant myocarditis or dilated cardiomyopathy suggesting that these 5'TD forms are key drivers of host-cell interaction in EV cardiac infections. To date, early emergence of EV-B 5'TD forms and its impact on type 1 IFN response during acute myocarditis remains unknown. Using quantitative RACE-PCR assay, we identified major EV-B 5'TD RNA populations in plasma or heart samples of acute myocarditis cases. Deletions identified within the 5' non-coding region of EV-B populations only affected secondary-structural elements of genomic RNA domain I and were distinguished in two major groups based on the extent of RNA structural deletions. Proportions of these two respective EV-B 5'TD population groups were positively or negatively correlated with IFN-ß levels in plasma samples of myocarditis patients. Transfection of synthetic CVB3/28 RNAs harboring various 5'terminal full-length or deleted sequences into human cultured cardiomyocytes demonstrated that viral genomic RNA domain I possessed essential immunomodulatory secondary-structural elements responsible for IFN-ß pathway induction. Overall, our results highlight the early emergence of major EVB-TD populations which deletions affecting secondary-structures of RNA domain I can modulate innate immune sensing mechanisms in cardiomyocytes of patients with acute myocarditis.

[Enteroviral infections in adults treated with rituximab: A new case of chronic meningitis and myofasciitis and literature review]. / Infections à entérovirus de l'adulte traité par rituximab : un nouveau cas de méningite et myofasciite chronique et revue de la littérature.

INTRODUCTION: Chronic enterovirus infections can occur in primary immunodeficiency with hypogammaglobulinemia. They usually associate meningitis and myofasciitis. Such infections have also been described in adults with rituximab-induced hypogammaglobulinemia. CASE REPORT: We report the case of a 33-year-old woman who was given rituximab for immune thrombocytopenia and developed rituximab-induced hypogammaglobulinemia (IgG 4.4g/L). One year after the last rituximab infusion, she developed lower limbs myofasciitis, followed two months later by a chronic lymphocytic meningitis. PCR in the serum and the cerebrospinal fluid at the time of the meningitis and the myofasciitis were positive to the same enterovirus (echovirus 11) while it was negative in the fascia biopsy. Under treatment with intravenous immunoglobulins, all symptoms and laboratory abnormalities improved and enterovirus PCR became negative. CONCLUSION: We report a case of chronic enterovirus infection associating meningitis and myofasciitis in an adult with rituximab-induced hypogammaglobulinemia. Outcome was favorable under treatment with intravenous immunoglobulins.

A European multicentre evaluation of detection and typing methods for human enteroviruses and parechoviruses using RNA transcripts.

Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in-house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV-A71, echovirus 30, coxsackie A virus 21, and EV-D68), HPeV3, and specificity controls. Reported results from 48 in-house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In-house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10-5 ). EV-specific PCRs showed low cross-reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in-house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point-of-care tests.

Emerging resistance mutations in PI-naive patients failing an atazanavir-based regimen (ANRS multicentre observational study).

Background: Atazanavir is a PI widely used as a third agent in combination ART. We aimed to determine the prevalence and the patterns of resistance in PI-naive patients failing on an atazanavir-based regimen. Methods: We analysed patients failing on an atazanavir-containing regimen used as a first line of PI therapy. We compared the sequences of reverse transcriptase and protease before the introduction of atazanavir and at failure [two consecutive viral loads (VLs) >50 copies/mL]. Resistance was defined according to the 2014 Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) algorithm. Results: Among the 113 patients, atazanavir was used in the first regimen in 71 (62.8%) patients and in the first line of a PI-based regimen in 42 (37.2%). Atazanavir was boosted with ritonavir in 95 (84.1%) patients and combined with tenofovir/emtricitabine or lamivudine (n = 81) and abacavir/lamivudine or emtricitabine (n = 22). At failure, median VL was 3.05 log10 copies/mL and the median CD4+ T cell count was 436 cells/mm3. The median time on atazanavir was 21.2 months. At failure, viruses were considered resistant to atazanavir in four patients (3.5%) with the selection of the following major atazanavir-associated mutations: I50L (n = 1), I84V (n = 2) and N88S (n = 1). Other emergent PI mutations were L10V, G16E, K20I/R, L33F, M36I/L, M46I/L, G48V, F53L, I54L, D60E, I62V, A71T/V, V82I/T, L90M and I93L/M. Emergent NRTI substitutions were detected in 21 patients: M41L (n = 2), D67N (n = 3), K70R (n = 1), L74I/V (n = 3), M184V/I (n = 16), L210W (n = 1), T215Y/F (n = 3) and K219Q/E (n = 2). Conclusions: Resistance to atazanavir is rare in patients failing the first line of an atazanavir-based regimen according to the ANRS. Emergent NRTI resistance-associated mutations were reported in 18% of patients.

Performance of genotypic algorithms for predicting tropism for HIV-1 CRF01_AE recombinant.

OBJECTIVES: There is no consensus about the performances of genotypic rules for predicting HIV-1 non-B subtype tropism. Three genotypic methods were compared for CRF01_AE HIV-1 tropism determination. METHODS: The V3 env region of 207 HIV-1 CRF01_AE and 178 B subtypes from 17 centers in France and 1 center in Switzerland was sequenced. Tropism was determined by Geno2Pheno algorithm with false positive rate (FPR) 5% or 10%, the 11/25 rule or the combined criteria of the 11/25, net charge rule and NXT/S mutations. RESULTS: Overall, 72.5%, 59.4%, 86.0%, 90.8% of the 207 HIV-1 CRF01_AE were R5-tropic viruses determined by Geno2pheno FPR5%, Geno2pheno FPR10%, the combined criteria and the 11/25 rule, respectively. A concordance of 82.6% was observed between Geno2pheno FPR5% and the combined criteria for CRF01_AE. The results were nearly similar for the comparison between Geno2pheno FPR5% and the 11/25 rule. More mismatches were observed when Geno2pheno was used with the FPR10%. Neither HIV viral load, nor current or nadir CD4 was associated with the discordance rate between the different algorithms. CONCLUSION: Geno2pheno predicted more X4-tropic viruses for this set of CRF01_AE sequences than the combined criteria or the 11/25 rule alone. For a conservative approach, Geno2pheno FPR5% seems to be a good compromise to predict CRF01_AE tropism.

[Acute hemorrhagic edema of infancy associated with Coxsackie virus infection]. / Œdème aigu hémorragique du nourrisson associé à une infection à Coxsackie virus.

Acute hemorrhagic edema of infancy is a rare but benign vasculitis occurring in infants aged from 4 to 24 months. Skin lesions can take various forms, including extensive hemorrhagic purpura, and can therefore be mistaken for purpura fulminans if associated with fever, which leads to initiating broad-spectrum antibiotic treatment. In the present case, we describe a 7-month-old boy with acute hemorrhagic edema of infancy and rapidly extensive purpura lesions that led to intravenous cefotaxime and amikacin treatment. Diagnosis was made on the next day by a dermatologist, based on the typical aspect of skin lesions, hemodynamic stability, and negative bacteriological samples. Coxsackie virus B5, a pathogenic enterovirus, was found by specific PCR in cerebrospinal fluid. The outcome was spontaneously favorable after discontinuation of antibiotics on day 2. We discuss the imputability of the enterovirus in triggering this case of acute hemorrhagic edema of infancy.

Breast cancer is associated to impaired glucose/insulin homeostasis in premenopausal obese/overweight patients.

The association between breast cancer (BCa) presence and altered glucose/insulin metabolism is controversial likely due to an inaccurate insulin resistance (IR) assessment and inappropriate stratification of patients by body-mass index (BMI) and menopausal state. 148 women with suspect of sporadic BCa were stratified by BMI and menopause. Fasting levels of glucose, insulin, glycohemoglobin and selected IR-related and tumor-derived markers were measured. Glucose/insulin levels during OGTT were used to calculate insulin resistance/sensitivity indexes. Analysis of 77 BCa-bearing patients and 71 controls showed an association between BCa and IR as demonstrated by impaired glucose/insulin homeostasis (increased fasting- and OGTT-induced glucose levels) and deteriorated IR indexes, which was especially patent in premenopausal women. The association between BCa presence and IR was markedly influenced by BMI, being obese BCa patients significantly more insulin resistant than controls. BCa presence was associated to elevated levels of IR (glucose, triglycerides) and tumor-derived (VEGF) markers, especially in overweight/obese patients. BCa presence is associated to IR in overweight/obese premenopausal but not in premenopausal normal weight or postmenopausal women. Our data support a bidirectional relationship between dysregulated/imbalanced glucose/insulin metabolism and BCa, as tumor- and IR-markers are correlated with the impairment of glucose/insulin metabolism in overweight/obese premenopausal BCa patients.

[Fatal case of enterovirus A71 hand, foot, and mouth disease infection]. / Décès d'un nourrisson dans un contexte de syndrome pieds-mains-bouche associé à l'entérovirus A71.

Hand, foot, and mouth disease associated with enterovirus (EV) infections is a common pediatric pathology that is usually considered as benign. However, neurological complications of varying severity, sometimes fatal, are possible, particularly when EV-A71 is involved. Several Asian countries are regularly affected by large-scale epidemics of EV infections with substantial morbidity and mortality, where early screening and appropriate therapeutic management are a public health challenge. In 2016, Europe experienced an epidemic of unusual magnitude, associated with increasing cases of severe neurological complications in Spain and France, mainly affecting children. Virological diagnosis is based on EV genome detection in peripheral clinical specimens (vesicles or oral ulcerations, throat, nasopharyngeal aspirate, stool) in addition to cerebrospinal fluid and blood. EV-A71 is rarely detected in cerebrospinal fluid, which renders the diagnosis of EV-A71-associated encephalitis challenging. We report the case of a 27-month-old child with hand, foot, and mouth disease turning into rapidly progressive and fatal cardiopulmonary failure associated with EV-A71 infection, in France in 2016. EV infections associated with hand, foot, and mouth disease warrant specific epidemiological surveillance outside the Asian region.

[Clinical characteristics and course of hand, foot, and mouth disease]. / Symptomatologie et évolution de la maladie « pieds-mains-bouche ¼.

Hand, foot and mouth disease (HFMD) and herpangina (HA) are common childhood diseases mostly associated with human enteroviruses (EV). Although usually benign illnesses, neurological complications may be observed during large epidemics when enterovirus A71 (EV-A71) is involved, as observed in the Asia Pacific Region and in China since the late 1990s. The occurrence of these complications warrants reinforcing the surveillance of the emergence of EV-A71 infections in France and Europe. Monitoring EV infections associated with HFMD can be considered as an effective tool to detect an upsurge of EV-A71 infections in a timely manner. In 2014, a national sentinel surveillance system for HFMD/HA was set up in France through a network of volunteer pediatricians and coordinated by the National Reference Center for Enteroviruses and Parechoviruses. Although classical manifestations of HFMD/HA can be easily recognized, there are several atypical presentations of the disease that can be confused with other skin conditions. Delayed cutaneous manifestations, such as onychomadesis and acral desquamation, may also occur and should prompt consideration of HFMD in the preceding weeks. Severe complications following HFMD include neurological manifestations (mainly rhombencephalitis) or less frequently cardiopulmonary failure and can sometimes be fatal. In China, the case severity rate has been estimated at 1%, with a case fatality rate at 0.03%. EV-A71 was involved in more than 90% of the fatal cases. Diagnosis of EV infections associated with severe neurological manifestations is based on the molecular detection of the EV genome in vesicles, cerebrospinal fluid (CSF), throat and stool given that EV-A71 is rarely recovered from the CSF. Positive EV genome detection should be followed by EV genotyping to identify the type of the EV. In temperate-climate countries, outbreaks of HFMD occur mostly but not exclusively during summer and autumn months. Adults may also present with HFMD. In 2016, an upsurge of severe neurological manifestations was reported in France; EV-A71 accounted for 50% of the cases. No specific treatment is available, but two inactivated EV-A71 vaccines are currently available in China.

Prevalence of HIV-1 drug resistance in treated patients with viral load >50 copies/mL: a 2014 French nationwide study.

Background: Surveillance of HIV-1 resistance in treated patients with a detectable viral load (VL) is important to monitor, in order to assess the risk of spread of resistant viruses and to determine the proportion of patients who need new antiretroviral drugs with minimal cross-resistance. Methods: The HIV-1 protease and reverse transcriptase (RT) and integrase genes were sequenced in plasma samples from 782 consecutive patients on failing antiretroviral regimens, seen in 37 specialized centres in 2014. The genotyping results were interpreted using the ANRS v24 algorithm. Prevalence rates were compared with those obtained during a similar survey conducted in 2009. Results: The protease and RT sequences were obtained in 566 patients, and the integrase sequence in 382 patients. Sequencing was successful in 60%, 78%, 78% and 87% of patients with VLs of 51-200, 201-500, 501-1000 and >1000 copies/mL, respectively. Resistance to at least one antiretroviral drug was detected in 56.3% of samples. Respectively, 3.9%, 8.7%, 1.5% and 3.4% of patients harboured viruses that were resistant to any NRTI, NNRTI, PI and integrase inhibitor (INI). Resistance rates were lower in 2014 than in 2009. Resistance was detected in 48.5% of samples from patients with a VL between 51 and 200 copies/mL. Conclusion: In France in 2014, 90.0% of patients in AIDS care centres were receiving antiretroviral drugs and 12.0% of them had VLs >50 copies/mL. Therefore, this study suggests that 6.7% of treated patients in France might transmit resistant strains. Resistance testing may be warranted in all treated patients with VL > 50 copies/mL.

Acute flaccid paralysis following enterovirus D68 associated pneumonia, France, 2014.

Human enterovirus D68 (EV-D68) is known to be associated with mild to severe respiratory infections. Recent reports in the United States and Canada of acute flaccid paralysis (AFP) in children with detection of EV-D68 in respiratory samples have raised concerns about the aetiological role of this EV type in severe neurological disease. This case study is the first report of AFP following EV-D68 infection in Europe.

[Severe human parechovirus-3 sepsis in a 6-week-old infant]. / Infection sévère à paréchovirus de type 3 chez un nourrisson de 6semaines.

Febrile infants under 3 months of age are often treated with broad-spectrum intravenous antibiotics while awaiting culture results, to prevent mother-to-child bacterial infections. Human parechoviruses (HPeV) have recently been described as etiologic agents of meningitis and severe sepsis in neonates and young infants. They are rarely investigated and are therefore probably underestimated. They cause acute clinical symptoms that can incorrectly suggest a bacterial infection. In the present case, a 6-week-old infant infected with HPeV developed severe sepsis, complicated by hepatic cytolysis, meningitis, acute renal failure, and mild hemophagocytic lymphohistiocytosis. HPeV type 3 was found by routine specific RT-PCR in cerebrospinal fluid, stools, and plasma. The outcome was spontaneously favorable after 4 days. Early diagnosis of the HPeV infection by routine specific RT-PCR reduces unnecessary antibiotic use and extended hospitalization in febrile young infants.

A case of congenital Echovirus 11 infection acquired early in pregnancy.

Enterovirus (EV) maternal infection during pregnancy and its relation to fetal developmental pathology are seldomly described. When reported, the main manifestations of EV congenital infections are myocarditis or intra-uterine fetal demise (IUFD). No information on intrauterine Echovirus 11 infection or the effect of transplacental Echovirus 11 infection on development of the fetus has been described in literature up to date (excluding late-pregnancy infections). We report here a case of an extreme form of pulmonary hypoplasia in a neonate, characterized by total failure of development of terminal respiratory units. This pregnancy was marked by spontaneous demise of a co-twin at 14 weeks of gestation (WG), as well as by positive PCR for EV (Echovirus 11 serotype) in the amniotic fluid, performed for moderate pericardial effusion at 22WG. No signs of cardiac disease were further observed, but at 32WG a bilateral abnormal lung development was noticed After spontaneous delivery at 38WG, the child could not be resuscitated, and died at one hour after birth. Pulmonary hypoplasia is usually described following decrease intrapulmonary pressure due to oligohydramnios or compression due to intrathoracic mass of variable cause. However, rare cases of primary pulmonary hypoplasia are also described and usually of unknown etiology. The coexistence in our case of a congenital EV infection and a severe primary pulmonary hypoplasia with congenital acinar aplasia, challenges our understanding of the pathogenesis of this severe pulmonary growth arrest.

[Epidemiology of parechovirus infections of the central nervous system in a French pediatric unit]. / Épidémiologie des infections neuroméningées à parechovirus dans un service de pédiatrie générale.

UNLABELLED: Human parechoviruses (HPeV), like their counterpart enteroviruses (EV), are associated with clinical manifestations ranging from asymptomatic disease to infections of the central nervous system. Newborn and young infants are particularly at risk for severe infection. In the last 5 years, the molecular diagnosis of HPeV infection in cerebrospinal fluid (CSF) and the identification of the associated HPeV type revealed the specific association between HPeV3 and meningitis or sepsis-like illness in neonates and infants. HPeV infection is not yet routinely diagnosed in clinical virology laboratories. OBJECTIVES: To determine the clinical, biological, and epidemiological characteristics of HPeV infections of patients hospitalized at the Centre Hospitalier de Versailles, France. METHODS: A total of 380 CSF samples originally referred to our laboratory for enterovirus testing between January 1st, 2007 and August 31st, 2011, were selected and retrospectively screened for the genome detection of HPeV. All HPeV detected were identified by amplification and sequencing of the complete 1D sequence encoding the VP1 protein. RESULTS: The HPeV genome was detected in CSF samples from nine (2.8%) patients. All were young infants under 18 months of age (median, 30 days; age range, 8 days to 18 months). Fever was observed for all children and eight out of nine (89%) presented with irritability. No pleiocytosis and normal glucose and protein levels were observed. The mean duration of the hospital stay was 4 days (range, 2-7 days) and antibiotics were administered to five patients (55.6%). Yearly frequency of genome detection varied remarkably: 1.1% in 2007, 0% in 2008 and 2011, 2.9% in 2009 and 7.1% in 2010. All genotyped viruses were HPeV3. CONCLUSION: This study confirmed the importance of the HPeV genome detection in CSF samples from patients presenting with sepsis-like illness or suspected infection of the central nervous system, particularly in children under 2 years of age. The introduction of the molecular diagnosis of HPeV infection broadens the panel of diagnosis of neonatal sepsis and central nervous system symptoms in young children. Rapid identification of HPeV by PCR could also contribute to shorter duration of both antibiotic use and hospital stay.

[Enterovirus nosocomial infections in a neonatal care unit: from diagnosis to evidence, from a clinical observation of a central nervous system infection]. / Infections nosocomiales à entérovirus en néonatologie : du diagnostic à la preuve, à propos d'une observation d'infection neuroméningée.

Although enteroviruses generally cause asymptomatic or mild disease, neonates are at higher risk for severe illnesses, among which systemic disease characterized by multiorgan involvement is a potentially fatal condition. Enterovirus neonatal infections may be the source of nosocomial infections in neonatology or in pediatric intensive care units. We report central nervous system infections due to Echovirus 11 in two neonates and the molecular evidence of nosocomial transmission of this strain in a neonatal unit by enterovirus genotyping and phylogenetic analysis. This report illustrates the importance of including enterovirus genome detection in the sepsis screening concomitantly with bacteriological investigations performed at admission of a neonate. Rapid diagnosis and subsequent genotyping could have a beneficial impact on clinical practices at the individual level (reducing the length of antibiotic therapy) and public health policy at the collective level by reinforcing hygiene measures to prevent nosocomial infections, with nurseries and neonatal units being at greater risks.

Outbreak of hand, foot and mouth disease/herpangina associated with coxsackievirus A6 and A10 infections in 2010, France: a large citywide, prospective observational study.

Hand, foot and mouth disease (HFMD) and herpangina (HA) are frequently caused by several distinct serotypes belonging to the human enterovirus A species (HEVA). Enterovirus 71 is considered as a significant public health threat because of rare but fatal neurological complications. A sentinel surveillance system involving paediatricians from Clermont-Ferrand (France) was set up to determine the clinical and epidemiological characteristics of HFMD/HA associated with enterovirus infections. A standardized report form was used to collect demographic and clinical data. Throat or buccal specimens were obtained prospectively and tested for the presence of enteroviruses. The frequency of HEVA serotypes was determined by genotyping. Phylogenetic relationships were analysed to identify potential new virus variants. From 1 April to 31 December 2010, a total of 222 children were enrolled. The predominant clinical presentation was HA (63.8%) and this was frequently associated with clinical signs of HFMD (48%). An enterovirus infection was diagnosed in 143 (64.4%) patients and serotype identification was achieved in 141/143 (98.6%). The predominant serotypes were coxsackievirus A10 (39.9%) and A6 (28%), followed by coxsackievirus A16 (17.5%) and enterovirus 71 (6.3%). Fever was observed in 115 (80.4%) children. No patient had neurological complications. Coxsackievirus A10 and A6 strains involved in the outbreak were consistently genetically related with those detected earlier in Finland and constituted distinct European lineages. Although several enterovirus serotypes have been involved in HFMD/HA cases, the outbreak described in this population survey was caused by coxsackievirus A6 and coxsackievirus A10, the third dual outbreak in Europe in the last 3 years.

Repeated genomic transfers from echovirus 30 to echovirus 6 lineages indicate co-divergence between co-circulating populations of the two human enterovirus serotypes.

Human echovirus types 6 (E-6) and 30 (E-30) cause seasonal epidemics of aseptic meningitis. These two enteroviruses are frequently observed in co-circulation, an epidemiological pattern that is prerequisite for the occurrence of dual infections, which can lead to recombination between co-infecting virus strains. Viral sequences were determined at loci 1D (VP1 capsid protein) and 3CD (non structural proteins) in 49 E-6 strains recovered in a single geographical region in France from 1999 to 2007, during the epidemiological survey of enterovirus infections. They were compared with previously recorded sequences of E-30 strains to investigate their evolutionary histories and possible recombination patterns. Phylogenetic analyses identified two distinct E-6 populations and different subpopulations. Assuming a relaxed molecular clock model and a Bayesian skyline demographic model in coalescent analyses with the BEAST program, the substitution rate in E-6 was estimated at 8.597×10(-3) and 6.252×10(-3) substitution/site/year for loci 1D and 3CD respectively. Consistent estimates of divergence times (t(MRCA)) were obtained for loci 1D and 3CD indicating that two distinct E-6 populations originated in 1997 and 1999. Incongruent phylogenetic patterns inferred for the two loci were indicative of recombination events between the two populations. Phylogenies including the E-30 3CD sequences showed close genetic relationships between E-6 and discrete E-30 subpopulations. Recombination breakpoints were located with statistical significance in E-6 and E-30 genomes. Estimates of t(MRCA) of phylogenetic recombinant clades indicated directional genetic transfers from E-30 to E-6 populations and their co-divergence over the time period studied.

Phylogenetic evidence for a recent spread of two populations of human enterovirus 71 in European countries.

Human enterovirus 71 (EV-71) is a cause of seasonal epidemics of hand, foot and mouth disease, and of less common but severe neurological manifestations. Uncertainty persists regarding the circulation of virus populations in several geographical areas and the timescale of their dissemination. We determined EV-71 sequences at loci 1D (VP1 capsid protein) and 3CD (non-structural proteins) in 86 strains recovered in Austria, France and Germany and performed an evolutionary genetic study of extant virus populations. Phylogenetic analyses positioned 78 of the 86 sequences within two clades among subgenogroups C1 and C2. A minor sequence cluster was assigned to subgenogroup C4. Analyses incorporating the available sequences estimated the substitution rate in genogroup C at 3.66 x 10(-3) and 4.46 x 10(-3) substitutions per site year(-1) for loci 1D and 3CD, respectively, assuming a relaxed molecular-clock model for sequence evolution. Most of the 'European' strains belonged to clades C1b and C2b, which originated in 1994 [95 % confidence interval (CI), 1992.7-1995.8] and 2002 (95 % CI, 2001.6-2003.8), respectively. Estimates of divergence times for locus 3CD were consistent with those measured for locus 1D. Intertwining between clades representing EV-71 subgenogroups and clades corresponding to other enterovirus types (notably early coxsackievirus A prototype strains) in the 3CD phylogeny is highly indicative of ancestral recombination events. Incongruent phylogenetic patterns estimated for loci 1D and 3CD show that a single tree cannot model the epidemic history of circulating EV-71 populations. The evolutionary timescale of genogroup C estimated for both loci was measured only in decades, indicating recent dissemination.

Impact of rapid enterovirus molecular diagnosis on the management of infants, children, and adults with aseptic meningitis.

Enteroviruses (EV) are the main etiological agents of aseptic meningitis. Diagnosis is made by detecting the genome using RT-PCR. The aim of the study was to evaluate the impact of a positive diagnosis on the management of infants, children, and adults. During 2005, 442 patients were admitted to hospital with suspected meningitis. Clinical and laboratory data and initial treatment were recorded for all patients with enteroviral meningitis. The turnaround time of tests and the length of hospital stay were analyzed. The results showed that EV-PCR detected EV in 69 patients (16%), 23% (16/69) were adults. About 18% of CSF samples had no pleocytosis. After positive PCR results, 63% of children were discharged immediately (mean 2 hr 30 min) and 95% within 24 hr. Infants and adults were discharged later (after 1.8 and 2 days, respectively). The use of antibiotics was significantly lower in children than in infants and adults. The PCR results allowed discontinuation of antibiotics in 50-60% of all patients treated. Patients received acyclovir in 16% of cases (7% children vs. 50% adults) and 23% (11% vs. 69%) underwent a CT scan. Clinical data were compared between patients whose positive EV-PCR results were available within 24 hr (n = 32) and those whose results were available > 24 hr after collection of CSF (n = 14). Duration of antibiotic treatment (difference: 2.3 days; P = 0.05) was reduced between the two groups. No statistical difference in the length of stay was observed. The EV-PCR assay should be performed daily in hospital laboratory practice and considered as part of the initial management of meningitis.

Phylogeography of circulating populations of human echovirus 30 over 50 years: nucleotide polymorphism and signature of purifying selection in the VP1 capsid protein gene.

A comprehensive set of 443 1D gene sequences (encoding the VP1 capsid protein) was analyzed to investigate the phylogenetic relationships and evolutionary patterns among strains of human echovirus 30 (E30; genus Enterovirus, family Picornaviridae) characterized over 50 years. Maximum-likelihood (ML) phylogenetic trees of complete and nonredundant 1D gene sequences (total length=876 nucleotides) showed evidence of distinct lineages related to the isolation period of virus strains. Virus transportation was confirmed as a major epidemiological factor in the appearance of epidemics since recurrence of aseptic meningitis outbreaks in a given geographic area was associated with distinct E30 variants detected earlier in distant regions. Detection of the codon changes associated with E30 evolution was investigated with methods implemented in the Datamonkey web server. Evolution of the 1D gene was dominated by continual negative (purifying) selection against nonsynonymous substitutions at most codon sites, as determined by dN/dS ratio. Amino acid polymorphism was maintained at a limited number of sites (10/292) in the VP1 protein (within loops connecting beta strands and C-terminus). Amino acid changes are allowed at these sites because they are likely exposed on the virion particle and nonsynonymous substitutions are observed in the corresponding codons because negative selection is relaxed.