Echovirus type 6 transmission clusters and the role of environmental surveillance in early warning, the Netherlands, 2007 to 2016.

BackgroundIn the Netherlands, echovirus type 6 (E6) is identified through clinical and environmental enterovirus surveillance (CEVS and EEVS). AimWe aimed to identify E6 transmission clusters and to assess the role of EEVS in surveillance and early warning of E6. MethodsWe included all E6 strains from CEVS and EEVS from 2007 through 2016. CEVS samples were from patients with enterovirus illness. EEVS samples came from sewage water at pre-specified sampling points. E6 strains were defined by partial VP1 sequence, month and 4-digit postcode. Phylogenetic E6 clusters were detected using pairwise genetic distances. We identified transmission clusters using a combined pairwise distance in time, place and phylogeny dimensions. ResultsE6 was identified in 157 of 3,506 CEVS clinical episodes and 92 of 1,067 EEVS samples. Increased E6 circulation was observed in 2009 and from 2014 onwards. Eight phylogenetic clusters were identified; five included both CEVS and EEVS strains. Among these, identification in EEVS did not consistently precede CEVS. One phylogenetic cluster was dominant until 2014, but genetic diversity increased thereafter. Of 14 identified transmission clusters, six included both EEVS and CEVS; in two of them, EEVS identification preceded CEVS identification. Transmission clusters were consistent with phylogenetic clusters, and with previous outbreak reports. ConclusionAlgorithms using combined time-place-phylogeny data allowed identification of clusters not detected by any of these variables alone. EEVS identified strains circulating in the population, but EEVS samples did not systematically precede clinical case surveillance, limiting EEVS usefulness for early warning in a context where E6 is endemic.

Laboratory diagnosis and genetic analysis of a family clustering outbreak of aseptic meningitis due to echovirus 30.

Echovirus 30 (E30) is a major pathogen associated with aseptic meningitis. In the summer of 2014, a family clustering aseptic meningitis outbreak occurred in urban-rural fringe of Ningbo city in Zhejiang Province in China. To identify the etiologic agent, specimens were tested by cell culture and reverse transcriptase-polymerase chain reaction. Pathogenic examination confirmed that the outbreak is caused by E30. The first case is a 6-year-old child, who studied in kindergarten in local, suffered from headache and fever. Same symptoms appeared in his parents, aunts, and other six relatives continuously. Meanwhile, vomiting occurred in majority of the patients and diarrhea in parts of them. White blood cells in cerebrospinal fluid (CSF) exceeded normal range in all patients. Protein levels in CSF were above normal range in half of the patients. Glucose levels in CSF were within normal range in all patients. We isolated six strains E30 in the stool specimens of patients, and carried out sequencing analysis to VP1 region. Sequencing results showed that 100% sequence identity was seen in both nucleotide and amino acid levels. Phylogenetic analysis discovered that isolate in this study was grouped into sublineage D2 together with sequences isolated from other areas of China in the 2000s and 2010s. Our study is the first family clustering outbreak of aseptic meningitis caused by E30 in Zhejiang Province in China. It is essential to establish an enterovirus molecular surveillance system in China to prevent mass outbreaks in Zhejiang.

Elucidation of echovirus 30's origin and transmission during the 2012 aseptic meningitis outbreak in Guangdong, China, through continuing environmental surveillance.

An aseptic meningitis outbreak occurred in Luoding City of Guangdong, China, in 2012, and echovirus type 30 (ECHO30) was identified as the major causative pathogen. Environmental surveillance indicated that ECHO30 was detected in the sewage of a neighboring city, Guangzhou, from 2010 to 2012 and also in Luoding City sewage samples (6/43, 14%) collected after the outbreak. In order to track the potential origin of the outbreak viral strains, we sequenced the VP1 genes of 29 viral strains from clinical patients and environmental samples. Sequence alignments and phylogenetic analyses based on VP1 gene sequences revealed that virus strains isolated from the sewage of Guangzhou and Luoding cities matched well the clinical strains from the outbreak, with high nucleotide sequence similarity (98.5% to 100%) and similar cluster distribution. Five ECHO30 clinical strains were clustered with the Guangdong environmental strains but diverged from strains from other regions, suggesting that this subcluster of viruses most likely originated from the circulating virus in Guangdong rather than having been more recently imported from other regions. These findings underscore the importance of long-term, continuous environmental surveillance and genetic analysis to monitor circulating enteroviruses.

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.

Detection of echovirus 18 in human breast milk.

We detected enteroviral RNA and cultured infectious virus from a series of banked breast milk samples from the mother of an infant with neonatal sepsis; sequencing of the enterovirus isolate identified it as echovirus type 18. In this case, it is possible that enterovirus transmission occurred through the breast milk.

An outbreak of viral meningitis associated with a public swimming pond.

From July to October 2001, 215 cases of aseptic meningitis occurred among the inhabitants of the German city of Kassel and neighbouring counties. A matched case-control study identified bathing in a public, nature-like pond during the beginning of the outbreak as a risk factor for disease [matched odds ratio (mOR) 44.8, 95% confidence interval (CI) 3.9-515.6]. Among bathers, patients with meningitis spent more time in the water (mOR 18.8, 95% CI 2.0-174.1) and swallowed water more frequently (mOR = 7.3, 95% CI 0.7-81.8). Of 30 cerebrospinal fluid samples tested, echovirus 30 was cultured from 16, and echovirus 13 from seven. An echovirus 30 sequence obtained from one pond water sample showed a 99% nucleotide and 100% amino-acid homology with patient isolates. This outbreak demonstrates the potential of nature-like swimming ponds to cause widespread community infection with substantial public health impact.

Neonatal Type I diabetes associated with maternal echovirus 6 infection: a case report.

AIMS/HYPOTHESIS: Neonatal diabetes mellitus is rare, and it has not been associated with beta-cell autoimmunity. Enteroviral infections during pregnancy have been implicated as a risk factor for the later development of Type I (insulin-dependent) diabetes mellitus. We now report of a baby girl who was born severely growth-retarded with neonatal insulin-deficient diabetes, and look for evidence of intrauterine enteroviral infections and beta-cell targeted autoimmunity. METHODS: Diabetes-associated autoimmunity was studied by measurement of several types of islet cell reactive autoantibodies. The infant's T-cell responses to insulin and enterovirus antigens were recorded and enterovirus antibodies were measured both from the mother and the child. RESULTS: Several types of diabetes-associated autoantibodies were detected postnatally, including insulin autoantibodies, conventional islet cell autoantibodies and glutamic acid decarboxylase antibodies, whereas no autoantibodies were observed in the mother. The infant's T-cells showed reactivity to insulin and purified enterovirus particles. Based on serological studies, the pathogenetic process could have been triggered by an echovirus 6 infection during pregnancy. The patient's diabetes has been permanent, although there were signs of endogenous insulin production for several months. Exocrine pancreatic insufficiency was diagnosed at the age of 1 year. CONCLUSION/INTERPRETATION: These observations suggests that enteroviral infections may induce beta-cell autoimmunity even in utero.

Nosocomial transmission of echovirus 30: molecular evidence by phylogenetic analysis of the VP1 encoding sequence.

We investigated six cases of enterovirus infection in a neonatal unit. The index patient, a 5-day-old boy, was admitted with aseptic meningitis due to echovirus 30 (E30). Secondary infections with E30 occurred in five babies. Comparison of the complete VP1 sequences showed that the isolates recovered from the index patient and his mother were closely related to those recovered from the five babies with secondary infections, demonstrating a nosocomial transmission of the virus. In the phylogenetic tree reconstructed from the VP1 sequences, the isolates formed a monophyletic cluster related to an E30 strain collected in June 1997 during an outbreak of aseptic meningitis.

Viral meningitis in child care center staff and parents: an outbreak of echovirus 30 infections.

OBJECTIVE: A report of five cases of viral meningitis among adults with children enrolled in a child care center prompted an investigation of risk factors for viral transmission from children to adult household members. METHODS: To determine recent echovirus 30 (E30) infections, the authors conducted a serologic survey. To determine risk factors for infection among adult household members, they conducted a retrospective cohort study using written questionnaires. RESULTS: Recent E30 infections were found in 84% of children tested, 57% of adult household members tested, and 47% of staff members tested. Infected adults were more likely than infected children to have clinical meningitis. Among adult household members, changing diapers was a risk factor for recent infection. Women who changed > or = 90 diapers per month had a higher infection rate than women who changed fewer diapers; in contrast, men who changed > or = 90 diapers per month had a lower infection rate than men who changed fewer diapers. Handwashing was protective: there was a negative correlation between handwashing after diaper changes and E30 infection among adults with infected children in diapers. CONCLUSIONS: Because child care centers can be a source of enteroviral infections among adult household members, adults with viral meningitis should be questioned about their children's day care or preschool attendance. The importance of handwashing should be stressed to adults with children in day care.

A community outbreak of echovirus infection associated with an outdoor swimming pool.

Forty-six people became ill with vomiting, diarrhoea and headache within days of an outdoor swimming pool opening for the summer season in a small seaside village. During the weekend of the outbreak, 185 tickets to the pool had been sold. It was found that 34 bathers were ill, and one subject had vomited into the pool. All other cases arose after this incident. The risk of infection was greatest among those who swallowed pool water (24/28 versus 10/17, p = 0.07). Echovirus 30 was isolated from the case who vomited into the pool and from six other cases. Normal chlorine levels had not been adequate to contain the infection risk from vomitus, and, in future, pool attendants witnessing such incidents should consider closing the pool to the public and seeking advice on superchlorination.

Echovirus 30 infection and aseptic meningitis in parents of children attending a child care center.

In July 1992, 13 parents with children attending a child care center (CCC) developed aseptic meningitis (AM) due to echovirus 30 (E30). To determine the extent of illness and risk factors for transmission, survey and blood specimens were collected from CCC families and teachers and from adult and pediatric controls. Infection was defined as the presence of anti-E30 IgM antibodies. CCC parents (60%, 67/111) and children (75%, 79/105) had significantly higher infection rates than did teachers (14%, 3/22), adult controls (24%, 10/41), and pediatric controls (24%, 17/70). Infected CCC parents had more severe illness (18% [12/65] had AM; 11% [7/65] were hospitalized) than did infected CCC children (3% [2/79] had AM and 1% [1/79] were hospitalized). More frequent handwashing among teachers compared with parents and among mothers of toddlers was associated with significantly lower rates of infection (P < or = .05). Education of parents about good handwashing practices may reduce transmission of E30 and other infectious agents from children to adults.

Enterovirus infections in neonates. A retrospective study of 21 cases.

OBJECTIVES: The aim of this study was to define the major features of enterovirus infections in the neonatal period based on our own experience. METHODS: Epidemiology, clinical manifestations and laboratory investigations concerning 21 neonates having experienced a Coxsackie B or an Echovirus infection between 1987 and 1991, were retrospectively reviewed. Aetiological diagnosis was made by classical viral isolation and/or by evidencing Coxsackie B-specific IgM antibodies with an immunocapture enzyme immunoassay. RESULTS: In 13 neonates the infection occurred between June and September. The onset of clinical signs ranged from day 1 to day 25 after birth with two separate periods: before 7 days of age, suggesting a perinatal transmission of the virus, or beyond this date, more likely connected with a postnatal transmission. Clinical manifestations included hyperthermia, gastroenteritis, meningitis, encephalitis, pneumonia and myocarditis, with a diphasic pattern in 6 cases. Most of the neonates improved gradually and developed normally. The Coxsackie B-specific IgM assay was the most rapid method whereas viral isolation, even though it took more time, was the most sensitive technique to establish the aetiological diagnosis in neonates. CONCLUSIONS: Enterovirus infections in neonates are difficult to diagnose and to differentiate from bacterial infections. A viral-like illness in the environment of the neonate allows the clinician to anticipate the clinical signs and a possibly fatal disease. Identification of the causal virus should be performed by both viral isolation and search for specific IgM antibodies. Treatment and prophylaxis are so far disappointing.

Congenital echo virus infection--morphological and virological study of fetal and placental tissue.

A prospective study of 78 pregnant women was undertaken to detect maternal enterovirus infection. Maternal faecal specimens and blood samples, placental and fetal tissue were taken for viral study, electron microscopy, histochemistry, and morphological examination. We present the post-mortem findings in three fetuses whose maternal infection was detected before delivery by isolation of ECHO virus type 33 and type 27 from faecal specimens and/or placental and fetal tissues. The morphological aspects were similar in all cases and included an acute infection of the placenta and hypoxic/hypotensive injury to fetal organs. In one case, viral particles were detected by electron microscopy of the fetal liver. This series of cases of intrauterine ECHO virus infection confirms the potential gravity of such infection during pregnancy and the need to prevent enteroviral disease.

Conservative management of an echovirus 11 outbreak in a neonatal unit.

Two babies in a neonatal unit presented on the same day with meningitis due to echovirus 11, which was thought to have been introduced by staff. At this time echovirus 11 was also isolated from the stools of eight other babies; five of them did not have signs of infection. No intervention was made except to emphasise the importance of handwashing. There was evidence of secondary spread to two babies who were both clinically well. The attack rate was twelve (29%) of forty-one babies exposed. Seven of the twelve infected babies were born before 30 weeks' gestation and would have had little or no maternal antibody, yet only two of the seven babies had signs of infection. Despite lack of special measures, all babies recovered. Most cases of horizontally acquired neonatal echovirus infection are mild: extreme measures in the management of outbreaks are unnecessary.

Vinyl versus latex gloves as barriers to transmission of viruses in the health care setting.

One type of vinyl and seven types of latex gloves without visual defects were tested with respect to their barrier function against high concentrations of three viruses of varying size: herpes simplex virus type 1 (HSV-1, 180 nm), human immunodeficiency virus type 1 (HIV-1, 100 nm), and echovirus type 9 (Echo 9, 25 nm). Viral suspensions of HSV-1 (10(8) TCD50/ml), HIV-1 (10(5) TCD50/ml), and echovirus type 9 (10(7.5)TCD 50/ml) were placed in an inverted glove finger immersed in media and maintained for 3 h at room temperature with sampling performed from outside the glove at 10 min, 30 min, 1 h, 2 h, and 3 h. No cytopathic effect (CPE) was identified after inoculation onto Vero cells or RhMK cells for HSV-1 and Echo 9, respectively, and reverse transcriptase activity was not detected in Hut 78 cells after inoculation of HIV-1 during any of the time intervals. Stretching of a glove finger for 18 h with repetition of the procedure with Echo 9 revealed no CPE. We conclude that under these experimental conditions, intact gloves act as effective barriers to the transmission of viral particles, including HIV in the health care setting.

Perinatal echovirus and group B coxsackievirus infections.

Enteroviral infections late in pregnancy are common, especially during periods of high prevalence of community infection. Most of these infections, however, are not associated with significant maternal or neonatal disease. Conversely, as many as 65 per cent of women who give birth to infants with proven enteroviral infection have symptomatic disease during the perinatal period. Maternal echovirus or coxsackievirus B infections are not associated with an increased risk of spontaneous abortions, but stillbirths late in pregnancy have been described. Although a slightly increased risk for congenital heart defects and urogenital anomalies has been reported for the offspring of women who seroconverted to the group B coxsackievirus during pregnancy, these data are highly tentative. Transmission of enteroviruses from mother to infant is relatively common (30-50 per cent) and may occur through contact with maternal secretions during vaginal delivery, blood, or upper respiratory tract secretions. Intrauterine transmission has been documented, but its frequency is unknown. Postnatal transmission from maternal or nonmaternal sources also occurs regularly. Neonatal disease may range from inapparent infection to overwhelming systemic illness and death. Common clinical syndromes associated with neonatal enteroviral infections are meningoencephalitis, pneumonia, myocarditis, and hepatitis. The severity and outcome of perinatally acquired enteroviral infection is influenced by several factors, including the virus strain involved, mode of transmission, and presence of passively acquired serotype-specific maternal antibody. Newborn nursery outbreaks of nonpolio enteroviral infections usually coincide with seasonal peaks of enteroviral disease in the community. These outbreaks have been due mostly to echovirus 11 or group B coxsackievirus serotypes 1 to 5 and are associated with attack rates of up to 50 per cent.