Enterovirus
Consistent with O. Reg. 671/92 of the French Language Services Act, laboratory testing information on this page is only available in English because it is scientific or technical in nature and is for use only by qualified health care providers and not by members of the public.
Testing Indications
Laboratory testing for Enterovirus should be considered for patients with severe respiratory illness, neurologic symptoms suspected to be associated with viral illness (e.g. meningitis/encephalitis or acute flaccid paralysis (AFP)/acute flaccid myelitis (AFM)) or myocarditis. Testing can be conducted on skin or mucosal lesions in patients with suspected Enterovirus illness (e.g. hand, foot, and mouth disease), though laboratory testing is not recommended in such patients, who usually experience a self-limiting illness.
This Test Information Sheet pertains to Enterovirus detection and molecular serotyping, including those associated with acute flaccid paralysis (AFP)/acute flaccid myelitis (AFM), e.g. serotypes D68 and A71.
For further information see the Enterovirus page.
Testing Guidelines for Patients with AFP/AFM or Other Acute Neurological Presentations of Possible Viral Cause
Persons of any age presenting with AFM, or any child less than 15 years of age with AFP should have laboratory investigations to detect a viral cause. Screening for viral pathogens may also be indicated for other neurological presentations where viral infection is on the differential diagnosis. Testing should include Enterovirus detection, as well as Enterovirus molecular serotyping if Enterovirus-positive. When submitting specimens from patients with neurological presentation, it is important to document the neurological symptoms on the laboratory requisition form, so the appropriate testing can be ordered. Specimen collection should include:
Stools
Collect two sets of stool samples - each stool sample is divided into a sterile container for viral testing and a bacterial (Cary-Blair) transport media container for Campylobacter testing. The clinician should write “Enterovirus / Enterovirus Molecular Serotyping” in the “Test(s) Requested” field of one requisition, and “Campylobacter Testing” in the “Test(s) Requested” of another requisition.
Respiratory specimens
Collect a nasopharyngeal (NP) swab and/or throat swab (NP swab preferred) in universal transport medium (UTM), or bronchoalveolar lavage in a sterile dry container. Always submit a throat swab from children under 15 years of age with acute flaccid paralysis. The clinician should write “Enterovirus / Enterovirus Molecular Serotyping" in the “Test(s) Requested” field of the requisition.
Cerebrospinal Fluid (CSF)
For all ages, collect cerebrospinal fluid (CSF) as appropriate for the investigation. If CSF is collected, the clinician should write “Enterovirus / Enterovirus Molecular Serotyping" in the “Test(s) Requested” field of the requisition.
Molecular serotyping
Enterovirus-positive samples and culture isolates is available at PHO laboratory and should be requested on Enterovirus-positive specimens from cases with complicated or atypical illness, such as severe respiratory infection (e.g. requiring ICU admission or fatal infection), neurological complications such as AFP/AFM, or myocarditis. If Enterovirus is suspected, and serotyping is required, please clearly indicate this on the requisition.
Specimen Requirements
| Test Requested | Required Requisition(s) | Specimen Type | Minimum Volume | Collection Kit |
Enterovirus PCR or Enterovirus culture or EV-D68 |
Stool |
1.0 gram |
||
Enterovirus PCR or EV-D68 |
Nasal, nasopharyngeal or throat swabs |
1 swab in transport media provided in the kit |
Virus Respiratory Kit order#: 390082 |
|
Enterovirus PCR or EV-D68 |
Bronchoalveolar lavage (BAL)/bronchial wash (BW), pleural fluid, sputum |
2.0 ml |
Sterile container |
|
Enterovirus PCR or EV-D68 |
Cerebrospinal fluid (CSF), blood, tissue, other sterile fluid |
1.0 ml |
Sterile container |
|
Enterovirus PCR or Enterovirus culture or EV-D68 |
Skin lesion swabs |
1 swab in transport media provided in the kit |
Submission and Collection Notes
During periods of special interest a selection of Enterovirus PCR-positive specimens will undergo molecular serotyping.
EV-D68 negative specimens may undergo further molecular serotyping.
Complete the following fields of the General Test Requisition Form if diagnostic testing is requested:
- patient setting
- relevant clinical findings including severe respiratory illness, neurological complication [e.g. acute flaccid paralysis (AFP)/myelitis (AFM)]
- onset date
- travel history
- sick contacts
- outbreak number if applicable
To collect stool samples, refer to the Virus – Enteric Kit Instruction Sheet for detailed specimen collection instructions.
To collect respiratory samples, refer to the Virus – Respiratory Instruction Sheet for detailed specimen collection instructions.
For suspected cases of Polio, contact the Public Health Ontario laboratory testing of suspected cases of polio or other causes of acute flaccid paralysis.
Storage and Transport
Label the specimen container with the patient’s full name, date of collection and one other unique identifier such as the patient’s date of birth or Health Card Number. Failure to provide this information may result in rejection or testing delay.
Special Instructions
Specimens should be stored at 2-8°C following collection and shipped to PHO laboratory on ice packs. Freeze the specimen at -70°C if more than 72 hours are expected between the time of specimen collection and testing.
Test Frequency and Turnaround Time (TAT)
Enterovirus testing is performed Monday to Friday.
Turnaround times (TAT) are listed for each of the following:
- TAT for Enterovirus PCR is up to 3 days after receipt at PHO – Toronto Laboratory
- TAT for EV-D68 is up to 3 days after receipt at PHO -Toronto Laboratory
- Virus Culture: specimens tested by virus culture are observed for up to 10 days prior to reporting a negative culture result; CSF specimens are observed for up to 17 days; specimens that are positive can be observed at any point during this period; TAT is up to 18 days
TAT will vary during the winter months.
Enterovirus real-time PCR methodology: this assay, developed by US CDC, amplifies a target in the 5’ untranslated region (5’UTR) of Enteroviruses. It may also cross react with rhinoviruses due to genetic similarity.
Enterovirus molecular serotyping methodology: Enterovirus-positive primary specimens will undergo molecular serotyping using gene sequencing of VP1, a viral capsid protein. The molecular serotyping methods were developed by CDC, USA. The Enterovirus molecular serotyping protocol involves amplification of a fragment (350 to 400 nucleotides) of the viral capsid protein 1 (VP1) by hemi-nested PCR, followed by Sanger sequencing of the resulting amplicon.
Molecular serotyping should be requested on Enterovirus-positive specimens from cases with complicated or atypical illness, such as severe respiratory infection (e.g. requiring ICU admission or fatal infection), neurological complications such as AFP/AFM, or myocarditis. In addition, PHOL may conduct molecular serotyping on a selection of Enterovirus-positive specimens to assist with Enterovirus molecular surveillance.
The EV-D68 real-time PCR protocol in use at PHO is available here.
Algorithm
Specimens requesting enterovirus (EV) D68 detection will be tested using a laboratory developed real-time EV-D68 PCR. EV-D68 negative specimens may undergo further molecular serotyping. If specific testing for EV-D68 is required this must be indicated on the PHO laboratory Requisition.
Request for Enterovirus
- Respriatory: Enterovirus PCR
- CSF, blood: Enterovirus PCR
- Stool: Enterovirus PCR, or Virus culture
- Tissue, skin scrapings and lesions: Enterovirus PCR and Virus culture
Request for EV-D68
- Respriatory: Enterovirus PCR and EV-D68 PCR
- CSF, blood: Enterovirus PCR and EV-D68 PCR
- Stool: Enterovirus PCR and EV-D68 PCR
- Tissue, skin scrapings and lesions: Enterovirus PCR and EV-D68 PCR
If “Enterovirus Detection/PCR" is requested on stool specimens, Enterovirus PCR will be performed. If “Virus Detection” or “Virus Culture” is requested, virus culture will be performed. Enterovirus-positive virus culture specimens are serotyped by immunofluorescence antibody (IFA) staining. IFA detects the following serotypes:
- Coxsackie A9, A16, A24, B1, B2, B3, B4, B5, B6
- Echo 4, 6, 9, 11, 30
- Entero 70, 71
- Polio 1, 2, 3
Specimens from patients of all ages with suspected poliomyelitis, or from children under 15 years of age with AFP/AFM, are tested at PHO laboratory and also forwarded to the National Microbiology Laboratory, Winnipeg, for further investigations.
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