Meningitis Caused by Echovirus Type 4 | CDC EID

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Volume 16, Number 1–January 2010
Research
Meningitis and a Febrile Vomiting illness Caused by Echovirus Type 4, Northern Territory, Australia
Peter G. Markey, Joshua S. Davis, Gerry B. Harnett, Simon H. Williams, and David J. Speers
Author affiliations: Centre for Disease Control, Darwin, Northern Territory, Australia (P.G. Markey); Royal Darwin Hospital, Darwin (J.S. Davis); and Queen Elizabeth II Medical Centre, Nedlands, Western Australia, Australia (G.B. Harnett, S.H. Williams, D.J. Speers)

Suggested citation for this article

Abstract
In July 2007, a cluster of meningitis cases caused by an echovirus 4 strain was detected in 1 indigenous community in the Top End of the Northern Territory of Australia. Illness was characterized by fever, vomiting, and headache. Over the next 4 months, additional cases of meningitis and the fever and vomiting syndrome emerged in other indigenous communities and subsequently in the major urban center of Darwin. We describe the epidemiology of 95 laboratory-confirmed meningitis cases and conclude that the epidemic fever and vomiting syndrome was caused by the same enterovirus. Nucleotide sequencing of the whole genome verified this enterovirus (AUS250G) as a strain of echovirus type 4. Viral protein 1 nucleotide sequencing demonstrated 96% homology with an echovirus 4 strain responsible for a large outbreak of meningitis in the Yanbian Prefecture of China in 1996.

Enteroviruses are among the most common human viral pathogens. Recent reports from the United States have documented >50 serotypes of enterovirus causing illness in humans (1). Illness syndromes with established causal links to enteroviruses include acute hemorrhagic conjunctivitis; viral meningitis; hand, foot, and mouth disease; and acute ascending paralysis (including poliomyelitis). In July 2007, a disease control unit in the Northern Territory of Australia reported a cluster of viral meningitis cases in a nearby community. Nucleic acid testing of the cerebrospinal fluid (CSF) of these patients detected an enterovirus.

The Northern Territory of Australia has a population of 210,000 living in an area of 1.35 million km2; the climate varies from desert and semiarid in the south to subtropical in the north. The northern part, known as the Top End, is characterized by several small urban centers and many small scattered indigenous communities with populations of 300–2,000. Darwin (population 100,000) is the major urban center. Approximately 30% of the population of the Northern Territory consists of indigenous Australians.

This cluster of meningitis cases coincided with an outbreak of another viral illness in the same and nearby communities; the illness affected mainly children and was characterized by fever, vomiting, and headache. During the next 4 months this epidemic febrile vomiting syndrome (EFVS) was reported in multiple communities in the northern part of the Northern Territory and eventually in Darwin. During the same period, clusters of viral meningitis were also being reported in some of the communities experiencing the EFVS.

We hypothesize that the cluster of viral meningitis cases and the EFVS were different manifestations of the same infection, caused by a strain of echovirus type 4 (E4) virus. This enterovirus was closely related to 2 E4 strains that caused a large outbreak of viral meningitis in the Yanbian prefecture of China in 1996 (2); ≈5,000 cases from a population of 2.16 million were identified.

Methods
Patients
A case of acute E4 enteroviral illness was defined as the detection, in 2007 in a resident of the Northern Territory, either of E4 in a CSF specimen or E4 in samples from another site during an illness characterized by fever and severe headache. Infants were children aged <1 year; children (including infants), <15 years of age; and adults, >15 years. Cases were included in our study only if specimens were collected in 2007 and samples from patients had a positive PCR or culture for E4 at the reference laboratory. A questionnaire was developed and details of cases were collected by a review of hospital case notes and, where possible, by telephone interviews with case-patients. The questionnaire documented clinical symptoms and signs together with laboratory results and risk factors, such as potential occupational exposure, child care, institutional exposure, or illness in the immediate family. Duration of illness was identified by discussion with the case-patient or, if the case-patient could not be contacted, was defined as the difference between date of symptom onset according to the medical record and date of hospital discharge.

The spread of the EFVS was investigated by asking senior clinic staff at all the remote community health centers in the regions affected about the local presence of a recent epidemic of fever, vomiting, and headache. If staff recalled such an epidemic, details were recorded about its timing, number of case-patients seen in the community health center, and the proportion of case-patients who were children. These interviews were all conducted within 2 months after the outbreak. Attack rates were calculated by using these estimates and the population of each community according to 2006 census data from the Australian Bureau of Statistics (3).

We collated data in Microsoft Excel 2000 (Redmond, WA, USA) and performed statistical analysis using STATA version 9.0 (StataCorp LP, College Park, TX, USA). Logistic regression was used to examine the relationship between the outcome variables (duration of illness, hospital admission, and length of stay) with the independent variables discussed below. Multivariate logistic regression models were built using a backwards stepwise approach. We compared categorical variables using the χ2 test and continuous variables using the Wilcoxon rank-sum test. A p value <0.05 was considered significant.

Virus Isolation, Detection, and Identification
Feces samples were cultured for enterovirus by using a human diploid fibroblast cell line. Enterovirus molecular testing was performed directly on CSF samples, dry throat swabs, feces samples, and fecal cell culture supernatants that demonstrated a typical cytopathic effect. In-house seminested reverse transcription–PCR (RT-PCR) was used, which was specific for 2 regions of the 5´ untranslated region (UTR) of the enterovirus genome (4,5). This 2-region RT-PCR method detects a wide range of enteroviruses. CSF samples were also routinely tested for bacterial pathogens by culture and for herpes simplex virus by PCR.

Enterovirus genotyping was performed by direct sequencing of the viral protein (VP) 1 capsid coding gene. Total RNA was extracted from cell culture supernatant followed by RT-PCR amplification by using primers previously described (6). The products were sequenced on both strands by using the ABI Prism BigDye Terminator v3.1 system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Sequencing reactions were interrogated on an ABI Prism 3130XL 16-channel Genetic Analyzer (Applied Biosystems). The deduced sequence was compared for identification by alignment with enterovirus sequences available in GenBank by using BLASTn (http://blast.ncbi.nlm.nih.gov). The whole viral genome was sequenced by use of primers designed from the obtained sequences and from aligned GenBank E4 sequences. We performed phylogenetic analysis for the VP1 gene using MEGA version 3.0 software (www.megasoftware.net) by the neighbor-joining method with the Kimura 2-parameter model and 1,000 bootstrap replicates.

Results
We identified 95 cases of acute E4 viral illness. Seventy-six of these cases had a positive PCR CSF result; 8 also had virus detected in throat or feces samples. In the remaining 19, E4 was detected by PCR from a throat swab, a fecal specimen, or both. Records were reviewed on all cases, and interviews were conducted with 48 case-patients or a parent if the case-patient was a child. Interviews were not possible for most indigenous case-patients who lived in remote communities because of a lack of home telephones.

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Suggested Citation for this Article
Markey PG, Davis JS, Harnett GB, Williams SH, Speers DJ. Meningitis and a febrile vomiting illness caused by echovirus type 4, Northern Territory, Australia. Emerg Infect Dis [serial on the Internet]. 2010 Jan [date cited]. Available from http://www.cdc.gov/EID/content/16/1/63.htm

DOI: 10.3201/eid1601.081519