Monitoring of enterovirus diversity in wastewater by ultra-deep sequencing: An effective complementary tool for clinical enterovirus surveillance.

In a one-year (October 2014-October 2015) pilot study, we assessed wastewater monitoring with sustained sampling for analysis of global enterovirus (EV) infections in an urban community. Wastewater was analysed by ultra-deep sequencing (UDS) after PCR amplification of the partial VP1 capsid protein gene. The nucleotide sequence analysis showed an unprecedented diversity of 48 EV types within the community, which were assigned to the taxonomic species A (n = 13), B (n = 23), and C (n = 12). During the same period, 26 EV types, of which 22 were detected in wastewater, were identified in patients referred to the teaching hospital serving the same urban population. Wastewater surveillance detected a silent circulation of 26 EV types including viruses reported in clinically rare respiratory diseases. Wastewater monitoring as a supplementary procedure can complement clinical surveillance of severe diseases related to non-polio EVs and contribute to the final stages of poliomyelitis eradication.

Multirecombinant Enterovirus A71 Subgenogroup C1 Isolates Associated with Neurologic Disease, France, 2016-2017.

In 2016, an upsurge of neurologic disease associated with infection with multirecombinant enterovirus A71 subgenogroup C1 lineage viruses was reported in France. These viruses emerged in the 2000s; 1 recombinant is widespread. This virus lineage has the potential to be associated with a long-term risk for severe disease among children.

Assessment of blood enterovirus PCR testing in paediatric populations with fever without source, sepsis-like disease, or suspected meningitis: a prospective, multicentre, observational cohort study.

BACKGROUND: Enteroviruses are the most frequent cause of acute meningitis and are seen increasingly in sepsis-like disease and fever without source in the paediatric population. Detection of enterovirus in cerebrospinal fluid (CSF) specimens by PCR is the gold standard diagnostic test. Our aim was to assess a method of detecting enterovirus in blood specimens by PCR. METHODS: We did a prospective, multicentre, observational study at 35 French paediatric and emergency departments in 16 hospitals. We recruited newborn babies (aged ≤28 days) and infants (aged >28 days to ≤2 years) with fever without source, sepsis-like disease, or suspected meningitis, and children (aged >2 years to ≤16 years) with suspected meningitis, who were admitted to a participating hospital. We used a standardised form to obtain demographic, clinical, and laboratory data, which were anonymised. Enterovirus PCR testing was done in blood and CSF specimens. FINDINGS: Between June 1, 2015, and Oct 31, 2015, and between June 1, 2016, and Oct 31, 2016, we enrolled 822 patients, of whom 672 had enterovirus PCR testing done in blood and CSF specimens. Enterovirus was detected in 317 (47%) patients in either blood or CSF, or both (71 newborn babies, 83 infants, and 163 children). Detection of enterovirus was more frequent in blood samples than in CSF specimens of newborn babies (70 [99%] of 71 vs 62 [87%] of 71; p=0·011) and infants (76 [92%] of 83 vs 62 [75%] of 83; p=0·008), and was less frequent in blood samples than in CSF specimens of children (90 [55%] of 163 vs 148 [91%] of 163; p<0·0001). Detection of enterovirus was more frequent in blood samples than in CSF specimens of infants aged 2 years or younger with fever without source (55 [100%] of 55 vs 41 [75%] of 55; p=0·0002) or with sepsis-like disease (16 [100%] of 16 vs nine [56%] of 16; p=0·008). Detection of enterovirus was less frequent in blood than in CSF of patients with suspected meningitis (165 [67%] of 246 vs 222 [90%] of 246; p<0·0001). INTERPRETATION: Testing for enterovirus in blood by PCR should be an integral part of clinical practice guidelines for infants aged 2 years or younger. This testing could decrease the length of hospital stay and reduce exposure to antibiotics for low-risk patients admitted to the emergency department with febrile illness. FUNDING: University Hospital Clermont-Ferrand.

Monitoring human enteric viruses in wastewater and relevance to infections encountered in the clinical setting: a one-year experiment in central France, 2014 to 2015.

BackgroundHuman enteric viruses are resistant in the environment and transmitted via the faecal-oral route. Viral shedding in wastewater gives the opportunity to track emerging pathogens and study the epidemiology of enteric infectious diseases in the community. Aim: The aim of this study was to monitor the circulation of enteric viruses in the population of the Clermont-Ferrand area (France) by analysis of urban wastewaters. Methods: Raw and treated wastewaters were collected between October 2014 and October 2015 and concentrated by a two-step protocol using tangential flow ultrafiltration and polyethylene glycol precipitation. Processed samples were analysed for molecular detection of adenovirus, norovirus, rotavirus, parechovirus, enterovirus (EV), hepatitis A (HAV) and E (HEV) viruses. Results: All wastewater samples (n = 54) contained viruses. On average, six and four virus species were detected in, respectively, raw and treated wastewater samples. EV-positive samples were tested for EV-D68 to assess its circulation in the community. EV-D68 was detected in seven of 27 raw samples. We collected data from clinical cases of EV-D68 (n = 17), HAV (n = 4) and HEV infection (n = 16) and compared wastewater-derived sequences with clinical sequences. We showed the silent circulation of EV-D68 in September 2015, the wide circulation of HAV despite few notifications of acute disease and the presence in wastewater of the major HEV subtypes involved in clinical local cases. Conclusion: The environmental surveillance overcomes the sampling bias intrinsic to the study of infections associated with hospitalisation and allows the detection in real time of viral sequences genetically close to those reported in clinical specimens.

[Enterovirus D68, a highly pathogenic and epidemic virus]. / Entérovirus D68 : un virus à haut potentiel pathogène et épidémique.

Since its discovery in 1962, enterovirus D68 (EV-D68) was one of the less frequently detected enteroviruses by the surveillance networks worldwide. In 2014, US pediatric hospitals reported increases in the number of children with severe respiratory illness. Following the alerts from the Center for Disease Control and Prevention, numerous cases of EV-D68 were reported in many countries. EV-D68 is associated with severe respiratory infections in children and adults, mostly in patients with underlying respiratory diseases. Like with poliovirus and EV-A71, rare but severe neurological complications may occur: acute flaccid myelitis is characterized by rapid onset of weakness and distinct abnormalities of the spinal cord gray matter on magnetic resonance imaging. Molecular epidemiology of strains isolated worldwide since the 90s shows a rapid evolution of the virus, reflecting its wide circulation in the general population. The recent emergence of EV-D68 underlines the unpredictable epidemic properties and the neurotropism of enteroviruses.

Phylogeography of Coxsackievirus A16 Reveals Global Transmission Pathways and Recent Emergence and Spread of a Recombinant Genogroup.

Coxsackievirus A16 (CV-A16; Picornaviridae) is an enterovirus (EV) type associated with hand, foot, and mouth disease (HFMD) in children. To investigate the spatial spread of CV-A16, we used viral sequence data sampled during a prospective sentinel surveillance of HFMD in France (2010 to 2014) and phylogenetic reconstruction. A data set of 168 VP1 sequences was assembled with 416 publicly available sequences of various geographic origins. The CV-A16 sequences reported were assigned to two clades, genogroup B and a previously uncharacterized clade D. The time origins of clades B and D were assessed in 1978 (1973 to 1981) and 2004 (2001 to 2007), respectively. The shape of the global CV-A16 phylogeny indicated worldwide cocirculation of genetically distinct virus lineages over time and across geographic regions. Phylogenetic tree topologies and Bayes factor analysis indicated virus migration. Virus transportation events in clade B within Europe and Asia and between countries of the two geographic regions were assessed. The sustained transmission of clade D viruses over 4 years was analyzed at the township level in France and traced back to Peru in South America. Comparative genomics provided evidence of recombination between CV-A16 clades B and D and suggested an intertype recombinant origin for clade D. Time-resolved phylogenies and HFMD surveillance data indicated that CV-A16 persistence is sustained by continuing virus migration at different geographic scales, from community transmission to virus transportation between distant countries. The results showed a significant impact of virus movements on the epidemiological dynamics of HFMD that could have implications for disease prevention.IMPORTANCE Coxsackievirus A16 is one of the most prevalent enterovirus types in hand, foot, and mouth disease outbreaks reported in Southeast Asia. This study is based on epidemiological and viral data on HFMD caused by CV-A16 in a European country. The phylogeographic data complemented the syndromic surveillance with virus migration patterns between geographic regions in France. The results show how viral evolutionary dynamics and global virus spread interact to shape the worldwide pattern of an EV disease. CV-A16 transmission is driven by movements of infected individuals at different geographic levels: within a country (local dynamics), between neighboring countries (regional dynamics), and between distant countries (transcontinental dynamics). The results are consistent with our earlier data on EV-A71 and confirm the epidemiological interconnection of Asia and Europe with regard to EV infections.

Ambulatory Pediatric Surveillance of Hand, Foot and Mouth Disease as Signal of an Outbreak of Coxsackievirus A6 Infections, France, 2014-2015.

The clinical impact of enteroviruses associated with hand, foot and mouth disease (HFMD) is unknown outside Asia, and the prevalence of enterovirus A71 (EV-A71) in particular might be underestimated. To investigate the prevalence of enterovirus serotypes and the clinical presentations associated with HFMD in France, we conducted prospective ambulatory clinic-based surveillance of children during April 2014-March 2015. Throat or buccal swabs were collected from children with HFMD and tested for the enterovirus genome. Physical examinations were recorded on a standardized form. An enterovirus infection was detected in 523 (79.3%) of 659 children tested. Two epidemic waves occurred, dominated by coxsackievirus (CV) A6, which was detected in 53.9% of enterovirus-infected children. CV-A6 was more frequently related to atypical HFMD manifestations (eruptions extended to limbs and face). Early awareness and documentation of HFMD outbreaks can be achieved by syndromic surveillance of HFMD by ambulatory pediatricians and rapid enterovirus testing and genotyping.

Epidemiological and clinical characteristics of patients infected with enterovirus D68, France, July to December 2014.

In 2014, the United States (US) experienced a nationwide outbreak of enterovirus D68 (EV-D68) infection with 1,152 cases reported mainly in hospitalised children with severe asthma or bronchiolitis. Following the US alert, 11 laboratories of the French enterovirus (EV) surveillance network participated in an EV-D68 survey. A total of 6,229 respiratory samples, collected from 1 July to 31 December 2014, were screened for EV-D68 resulting in 212 EV-D68-positive samples. These 212 samples corresponded to 200 EV-D68 cases. The overall EV-D68 positivity rates among respiratory samples were of 5% (184/3,645) and 1.1% (28/2,584) in hospitalised children and adults respectively. The maximum weekly EV-D68 positivity rates were of 16.1% for children (n = 24/149; week 43) and 2.6% for adults (n = 3/115; week 42). Of 173 children with EV-D68 infection alone, the main symptoms were asthma (n = 83; 48.0%) and bronchiolitis (n = 37; 21.4%). One child developed acute flaccid paralysis (AFP) following EV-D68-associated pneumonia. Although there was no significant increase in severe respiratory tract infections reported to the French public health authorities, 10.7% (19/177) of the EV-D68 infected children and 14.3% (3/21) of the EV-D68 infected adults were hospitalised in intensive care units. Phylogenetic analysis of the viral protein 1 (VP1) sequences of 179 EV-D68 cases, revealed that 117 sequences (65.4%), including that of the case of AFP, belonged to the B2 variant of clade B viruses. Continuous surveillance of EV-D68 infections is warranted and could benefit from existing influenza-like illness and EV surveillance networks.

Enterovirus meningitis in Tunisia (Monastir, Mahdia, 2011-2013): identification of virus variants cocirculating in France.

Acute enterovirus (EV) meningitis is a frequent cause of hospitalisation, and over 100 EV serotypes may be involved. A total of 215 patients of all ages with meningitis signs were investigated in 2 Tunisian hospitals. Their cerebrospinal fluid (CSF) was analysed retrospectively for EVs with a TaqMan real-time RT-qPCR. The virus strains were typed, and their evolutionary relationships were determined by Bayesian phylogenetic methods. An EV genome was detected in 21/215 patients (9.8%). The CSF viral loads ranged from 3.27 to 5.63 log10 genome copies/mL. The strains were identified in 13/21 patients and assigned to EV-B types. Viruses identified in Tunisian patients were genetically related to variants detected in France. The viral loads were similar in Tunisian and French patients for most EV types. The phylogenetic data and viral loads determined in Tunisian and French patients suggest that close EV variants were involved in aseptic meningitis in the 2 countries over a same period.

Enterovirus infections in hospitals of Ile de France region over 2013.

BACKGROUND: The monitoring and genotyping of Enterovirus (EV) infections can help to associate particular or severe clinical manifestations with specific EV types and to identify the aetiology of infectious outbreaks. OBJECTIVES: To describe the epidemiological features of EV infections diagnosed during the year 2013 in the Greater Paris area (Ile de France). STUDY DESIGN: During 2013, 2497 samples taken from 470 patients in 33 hospitals of Ile-de France were tested for EV genome by RT-PCR. EV genotyping was performed by the National Reference Centre (NRC) laboratories. EV infections were retrospectively reviewed by retrieving clinical and genotyping data from the NRC database. RESULTS: Of the 2497 samples, 490 (19.6%) was positive for EV genome detection. These EV infections represented 88.7% and 24.1%, respectively, of all reported regional and national infections. Twenty-seven different genotypes were identified. Echovirus 30 (E-30) accounted for 54.1% of all characterized strains and caused a large outbreak. Four severe neonatal infections were reported, of which two were caused by EV-A71. Respiratory infections involving EV-D68 were observed in two adults. One fatal case of Coxsackievirus A2-associated myocarditis was reported. CONCLUSION: Monitoring EV infections in combination with EV genotyping via the French EV network characterized the epidemiology of EV infections in the Ile de France region in 2013 and documented severe EV infections associated with EV-A71 or CV-A2.

Enterovirus Migration Patterns between France and Tunisia.

The enterovirus (EV) types echovirus (E-) 5, E-9, and E-18, and coxsackievirus (CV-) A9 are infrequently reported in human diseases and their epidemiologic features are poorly defined. Virus transmission patterns between countries have been estimated with phylogenetic data derived from the 1D/VP1 and 3CD gene sequences of a sample of 74 strains obtained in France (2000-2012) and Tunisia (2011-2013) and from the publicly available sequences. The EV types (E-5, E-9, and E-18) exhibited a lower worldwide genetic diversity (respective number of genogroups: 4, 5, and 3) in comparison to CV-A9 (n = 10). The phylogenetic trees estimated with both 1D/VP1 and 3CD sequence data showed variations in the number of co-circulating lineages over the last 20 years among the four EV types. Despite the low number of genogroups in E-18, the virus exhibited the highest number of recombinant 3CD lineages (n = 10) versus 4 (E-5) to 8 (E-9). The phylogenies provided evidence of multiple transportation events between France and Tunisia involving E-5, E-9, E-18, and CV-A9 strains. Virus spread events between France and 17 other countries in five continents had high probabilities of occurrence as those between Tunisia and two European countries other than France. All transportation events were supported by BF values > 10. Inferring the source of virus transmission from phylogenetic data may provide insights into the patterns of sporadic and epidemic diseases caused by EVs.

Transmission patterns of human enterovirus 71 to, from and among European countries, 2003 to 2013.

Enterovirus 71 (EV-71) is involved in epidemics of hand, foot, and mouth disease (HFMD) and has been reported to occur with severe neurological complications in eastern and south-east Asia. In other geographical areas, the transmission of this virus is poorly understood. We used large sequence datasets (of the gene encoding the viral protein 1, VP1) and a Bayesian phylogenetic approach to compare the molecular epidemiology and geographical spread patterns of EV-71 subgenogroups B4, B5, C1, C2, and C4 in Europe relative to other parts of the world. For the study, European countries considered were European Union (EU) Member States and Iceland, Norway and Switzerland. Viruses of the B4, B5, and C4 subgenogroups circulate mainly in eastern and south-east Asia. In Europe sporadic introductions of these subgenogroups are observed, however C1 and C2 viruses predominate. The phylogenies showed evidence of multiple events of spread involving C1 and C2 viruses within Europe since the mid-1990s. Two waves of sporadic C2 infections also occurred in 2010 and 2013. The 2007 Dutch outbreak caused by C2 and the occurrence of B5 and C4 infections in the EU between 2004 and 2013 arose while the circulation of C1 viruses was low. A transmission chain involving a C4 virus was traced from Japan to the EU and then further to Canada between 2001 and 2006. Recent events whereby spread of viruses have occurred from, to, and within Europe appear to be involved in the long term survival of EV-71, highlighting the need for enhanced surveillance of this virus.

Differential permissivity of human cerebrovascular endothelial cells to enterovirus infection and specificities of serotype EV-A71 in crossing an in vitro model of the human blood-brain barrier.

Human cerebral microvascular endothelial cells (hCMEC/D3 cell line) form a steady polarized barrier when cultured in vitro on a permeable membrane. Their susceptibility to enterovirus (EV) strains was analysed to investigate how these viruses may cross the blood-brain barrier. A sample of 88 virus strains was selected on phylogenetic features amongst 43 epidemiologically relevant types of the four EV species A-D. The EV-A71 genome was replicated at substantial rates, whilst the infectious virus was released at extremely low but sustained rates at both barrier sides for at least 4 days. EV-A71 antigens were detected in a limited number of cells. The properties of the endothelial barrier (structure and permeability) remained intact throughout infection. The chronic EV-A71 infection was in sharp contrast to the productive infection of cytolytic EVs (e.g. echoviruses E-6 and E-30). The hCMEC/D3 barriers infected with the latter EVs exhibited elevated proportions of apoptotic and necrotic cells, which resulted in major injuries to the endothelial barriers with a dramatic increase of paracellular permeability and virus crossing to the abluminal side. The following intracellular rearrangements were also seen: early destruction of the actin cytoskeleton, remodelling of intracellular membranes and reorganization of the mitochondrion network in a small cluster near the perinuclear space.

Evaluation of real-time RT-PCR Rhino&EV/Cc r-gene(®) (bioMérieux) kit versions 1 and 2 for rhinovirus detection.

BACKGROUND: Human rhinoviruses (HRVs) are frequent etiologic agents of tract infections, ranging from benign upper to potentially life-threatening lower respiratory tract infections. Diagnosis is based on molecular methods. 169 HRV types, belonging to species A, B and C, have been identified. This high genetic diversity makes it difficult to accurately detect circulating HRVs and to diagnose severe infection. OBJECTIVES: To comparatively assess the ability to detect HRV clinical isolates of the first version (V1) of the commercial real-time RT-PCR Rhino&EV/Cc r-gene(®) (bioMérieux) kit, of an in-house RT-PCR followed by genotyping, considered as the reference method, and of the second version of this commercial test (V2). STUDY DESIGN: From September 2011 to April 2013, HRVs were prospectively detected in 2525 respiratory specimens, using V1 in combination with the in-house reference RT-PCR. In November 2013, 85 specimens that had given initially false negative results with V1 were retested simultaneously with V1 and V2 and the in-house RT-PCR. In addition, 421 negative specimens with the in-house assay were prospectively tested with V2. RESULTS: Among the 2525 specimens, V1 detected 80.7% (502/622) of in-house RT-PCR positive isolates: 85.3% (220/258) of HRV-A, 84.4% (27/32) of HRV-B and 74.9% (176/235) of HRV-C. Among the 85 respiratory samples tested with V1, V2 and the in-house RT-PCR, V2 was more efficient than V1 in detecting 16 HRV isolates: 11/33 (33.3%) of HRV-A and 5/47 (10.6%) of HRV-C tested. The analytical sensitivity of V2 was greater for 8/18 HRV-A genotypes and 2/22 HRV-C genotypes. Relative to the in-house assay, the specificity of V2 was 100% (421/421). CONCLUSIONS: This study showed a slightly higher sensitivity of V2. However, diverse genotypes, especially HRV-C, were undetected.

Variations in cerebrospinal fluid viral loads among enterovirus genotypes in patients hospitalized with laboratory-confirmed meningitis due to enterovirus.

BACKGROUND: Acute enterovirus (EV) meningitis is a major cause of hospitalization among adults and children. It is caused by multiple EV genotypes assigned to 4 species (EV-A, EV-B, EV-C, and EV-D). METHODS: We determined viral loads in the cerebrospinal fluid (CSF) of 156 patients of all ages with EV meningitis during a 5-year observational prospective study. The virus strains were genotyped, and their time origin was determined with Bayesian phylogenetic methods. RESULTS: The CSF viral loads ranged between 3.4 and 7.5 log10 copies/mL (median, 4.9 log10 copies/mL). They were higher in neonates than in infants and children (P = .02) but were comparable in adults. Viral loads were associated with EV genotypes (P < .001). The EV strains were identified in 152 of 156 patients and assigned to 23 genotypes within the EV-A and EV-B species. The most frequent genotypes, echoviruses 6 and 30, were associated with different viral loads (P < .001). The highest viral loads were in meningitis cases caused by coxsackievirus A9, B4, and B5 genotypes. Most patients infected by a same genotype were infected by a major virus variant of recent emergence. CONCLUSIONS: The variations in CSF viral loads in patients at the onset of EV meningitis are related to genotypic differences in the virus strains involved.

Emerging and reemerging enterovirus diseases: From poliomyelitis to hand, foot and mouth disease.

Several picornaviruses (Picornaviridae) are currently attracting interest without the need of being "emergent". The Parechovirus genus, validated 40 years after the discovery of the first two members ("echoviruses 22 and 23") includes neurotropic viruses whose molecular diagnosis demonstrated the involvement in infant meningitis and newborn sepsis, in particular type 3. Improvements in multiplex molecular diagnosis of respiratory infections - thanks to the Influenza AH1N1pdm2009 pandemy - showed that rhinoviruses may be involved in severe forms. The risk of the re-emergence of poliomyelitis in Europe, after an 11-year period of elimination, is a serious threat, owing to the circulation of the wild-type poliovirus in the Middle East and Africa because of conflicts, population displacements and poverty. The current widespread epidemics of hand-foot-mouth disease and/or meningitis infections due to enterovirus 71, with fatal encephalitis and cardio-pulmonary failure, are clear evidence of its emergence in South-East Asia. Although uncommon in Europe and less frequently incriminated than coxsackieviruses A6 and A10 in hand-foot-mouth disease, EV71 represents a real risk for the future. Extensive genotyping of the enteroviruses by the Enterovirus Surveillance Network should ward off these two potential risks of emergence/reemergence.

Fatal subacute myocarditis associated with human bocavirus 2 in a 13-month-old child.

Human bocavirus has rarely been incriminated in fatal or life-threatening respiratory infections. We report a case of fatal disseminated infection with subacute lymphocytic myocarditis in a 13-month-old child. The human bocavirus 2 genome was detected by PCR analysis in nasal swab, plasma, urine, ascitic fluid, and mesenteric node, skeletal muscle, and lung tissue specimens.

Phylogenetic patterns of human coxsackievirus B5 arise from population dynamics between two genogroups and reveal evolutionary factors of molecular adaptation and transmission.

The aim of this study was to gain insights into the tempo and mode of the evolutionary processes that sustain genetic diversity in coxsackievirus B5 (CVB5) and into the interplay with virus transmission. We estimated phylodynamic patterns with a large sample of virus strains collected in Europe by Bayesian statistical methods, reconstructed the ancestral states of genealogical nodes, and tested for selection. The genealogies estimated with the structural one-dimensional gene encoding the VP1 protein and nonstructural 3CD locus allowed the precise description of lineages over time and cocirculating virus populations within the two CVB5 clades, genogroups A and B. Strong negative selection shaped the evolution of both loci, but compelling phylogenetic data suggested that immune selection pressure resulted in the emergence of the two genogroups with opposed evolutionary pathways. The genogroups also differed in the temporal occurrence of the amino acid changes. The virus strains of genogroup A were characterized by sequential acquisition of nonsynonymous changes in residues exposed at the virus 5-fold axis. The genogroup B viruses were marked by selection of three changes in a different domain (VP1 C terminus) during its early emergence. These external changes resulted in a selective sweep, which was followed by an evolutionary stasis that is still ongoing after 50 years. The inferred population history of CVB5 showed an alternation of the prevailing genogroup during meningitis epidemics across Europe and is interpreted to be a consequence of partial cross-immunity.

Improvement of the management of infants, children and adults with a molecular diagnosis of Enterovirus meningitis during two observational study periods.

Enteroviruses (EVs) are a major cause of aseptic meningitis, and RNA detection using molecular assay is the gold standard diagnostic test. The aim of this study was to assess the impact of an EV positive diagnosis on the clinical management of patients admitted for meningitis over the course of two observational study periods (2005 and 2008-09) in the same clinical departments. We further investigated in multivariate analysis various factors possibly associated with hospital length of stay (LOS) in all age groups (infants, children, and adults). The results showed an overall improvement in the management of patients (n = 142) between the study periods, resulting in a significantly shorter hospital LOS for adults and children, and a shorter duration of antibiotic use for adults and infants. In multivariate analysis, we observed that the time from molecular test results to discharge of patients and the median duration of antibiotic treatment were associated with an increase in LOS in all age groups. In addition, among adults, the turnaround time of the molecular assay was significantly correlated with LOS. The use of CT scan in children and hospital admission outside the peak of EV prevalence in infants tended to increase LOS. In conclusion, the shorter length of stay of patients with meningitis in this study was due to various factors including the rapidity of the EV molecular test (particularly in adults), greater physician responsiveness after a positive result (in adults and children), and greater experience on the part of physicians in handling EV meningitis, as evidenced by the shorter duration of antibiotic use in adults and infants.