Audrey Mirand

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. J. Med. Virol. 81:42–48, 2009.

Prospective identification of enteroviruses involved in meningitis in 2006 through direct genotyping in cerebrospinal fluid.

ABSTRACT Enterovirus infections were investigated with special emphasis on performing rapid molecular identification of enterovirus serotypes responsible for aseptic meningitis directly in cerebrospinal fluid (CSF). Enterovirus genotyping was carried out directly with specimens tested for the diagnostic procedure, using two seminested PCR assays designed to amplify the complete and partial gene sequences encoding the VP1 and VP4/VP2 capsid proteins, respectively. The method was used for identifying the enterovirus serotypes involved in meningitis in 45 patients admitted in 2005. Enterovirus genotyping was achieved in 98% of the patients studied, and we obtained evidence of 10 of the most frequent serotypes identified earlier by genotyping of virus isolates. The method was applied for the prospective investigation of 54 patients with meningitis admitted consecutively in 2006. The enterovirus serotypes involved were identified with the cerebrospinal fluid (CSF) of 52 patients (96%) and comprised 13 serotypes within the human enterovirus B species and 1 within the human enterovirus A species. The three most common serotypes were echovirus 13 (E13; 24%), E6 (23%), and coxsackievirus B5 (11.5%), a pattern different from that observed in 2005. Genotyping of virus isolates was also performed in 35 patients in 2006 (meningitis, n = 31; other diseases, n = 4). By comparison, direct genotyping in CSF yielded a more complete pattern of enterovirus serotypes, thereby allowing the detection of rare serotypes: three less common serotypes (CB2, E21, and E27) were not detected by indirect genotyping alone. The study shows the feasibility of prospective enterovirus genotyping within 1 week in a laboratory setting.

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.

Epidemiology of human enterovirus 71 infections in France, 2000-2009

BACKGROUND Human enterovirus 71 (EV-71) emerged as a significant pathogen able to cause large outbreaks involving severe neurological cases and children fatalities in Asia. OBJECTIVES To describe epidemiology of EV-71 infections in France. STUDY DESIGN Fifty-nine patients admitted in 12 different hospitals from 1994 to 2009 were included. The entire VP1 coding gene of 58 EV-71 strains was sequenced and phylogenetic analyses were performed to assign strains to genogroups/subgenogroups and to compare French isolates to European and worldwide isolates. RESULTS The median age of the patients was 1.04 years (9 days to 7 years). Among 46 documented EV-71 infections, 39 were self-limited. Seven children developed severe sepsis-like, respiratory or neurological complications. Among them, 2 children died from acute respiratory distress syndrome. All the EV-71 strains belonged to genogroup C: 31 isolates belonged to subgenogroup C1, 26 to subgenogroup C2 and 1 to subgenogroup C4. All the strains were genetically related to other European strains isolated at the same period of time. Although C1 isolates were predominant between 1994 and 2005, C2 strains have been predominant since 2007. No association was found between any genotype and the age or the clinical symptoms. CONCLUSIONS The C4 subgenogroup, which was associated with large outbreaks in China, did not spread in France. It is important to monitor more carefully the EV-71 strains circulating in France to detect the introduction of new genetic variants that could be associated with major outbreaks.

Fatal adenovirus infection in a neonate and transmission to health-care workers

BACKGROUND Human adenovirus (HAdV) infections, while common in infancy and childhood, occur rarely in the neonatal period but may be fatal. OBJECTIVES To describe a transmission of HAdV from a patient with fatal pneumonia to heath-care workers that could be considered as a model of respiratory virus transmission in a care unit. STUDY DESIGN Case report with virologic studies. RESULTS A 10-day-old neonate developed pneumonia with acute respiratory distress, external pulmonary bleeding and coagulopathy and died 36h after admission of multivisceral failure. An adenovirus was isolated from pulmonary biopsy and detected by PCR in blood and respiratory secretions. Ten days later, three members of medical staff in charge of this infant, who used neither masks nor glasses for close patient contact, developed keratoconjunctivitis. Molecular analysis of the infants and one of the pediatricians isolates identified a species D HAdv and showed 100% identity, thereby demonstrating viral transmission. CONCLUSION In view of the serious outcome, HAdV infections should be considered in the differential diagnosis of pneumonia in neonates. This case illustrates the epidemic potential of viruses with respiratory transmission and underlines the importance of complying with standard precautions to prevent viral spread in routine practice.

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.

Prospective genotyping of human rhinoviruses in children and adults during the winter of 2009–2010

BACKGROUND About 100 serotypes of human rhinovirus (HRV), classified into two species, have been identified by 1990. Uncultivable HRV variants have recently been identified and designated a new species. Recent improved diagnosis has led to a re-appraisal of the clinical impact of HRV infections in lower respiratory diseases. OBJECTIVES To characterise clinical features in hospitalised patients with positive HRV RNA detection and to determine the distribution of HRV species in respiratory infections diagnosed during the winter of 2009-2010. STUDY DESIGN Prospective virus typing was conducted by sequencing the VP4/VP2 genomic regions, and clinical data were collected. RESULTS Fifty-eight patients (for 63 respiratory specimens) were included. Phylogenetic analysis identified 52% of HRV species A, 6% of species B and 40% of species C, and revealed the co-circulation of 34 different HRV types during the study period. Three infants had successive infections with two or three different types. Five patients were admitted to an intensive care unit, four of them on arrival. Bronchiolitis, pneumonia and exacerbation of asthma were observed in 34/45 children. Pneumonia and severe exacerbation of chronic lung disease were observed in 8/13 adults, of whom 1, with immunocompromised status, died of multivisceral failure. CONCLUSIONS This study underlines the diversity of co-circulating strains and the potential severity of clinical presentations associated with HRV infections.

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

Outbreaks can be detected by syndromic surveillance, rapid enterovirus testing, and genotyping.

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.

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.