Enterovirus

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 Collection and Handling

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

Virus – Enteric Kit order#: 390087

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

Virus Culture Kit order#: 390081

Submission and Collection Notes

1

During periods of special interest a selection of Enterovirus PCR-positive specimens will undergo molecular serotyping.

2

EM is performed from mid-December to mid-April in addition to culture. Further investigation of EM positive specimens may be available upon request.

3

EV-D68 negative specimens may undergo further molecular serotyping.

4

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
5

To collect stool samples, refer to the Virus – Enteric Kit Instruction Sheet for detailed specimen collection instructions.

6

To collect respiratory samples, refer to the Virus – Respiratory Instruction Sheet for detailed specimen collection instructions.

7

For suspected cases of Polio, contact the Public Health Ontario laboratory testing of suspected cases of polio or other causes of acute flaccid paralysis.

Preparation Prior to 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.

Requisitions and Kit Ordering

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
  • TAT for rapid respiratory virus culture (R-Mix TOO) is up to 4 days.
  • 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.

Reporting

Results are reported to the ordering physician or health care provider as indicated on the requisition.

Specimens that are positive for Poliovirus are reported to the Medical Officer of Health as per Health Protection and Promotion Act.

Test Methods

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 +/- electron microscopy (EM)4
  • Tissue, skin scrapings and lesions: Enterovirus PCR and Virus culture3

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 +/- EM 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

EM is performed from mid-December to mid-April in addition to culture. Further investigation of EM positive specimens may be available upon request.

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.

Additional Information

For any suspected cases of Polio, contact the Medical Microbiologist or your local Public Health Laboratory. Please refer toPoliovirus for laboratory testing of suspected cases of polio or other causes of acute flaccid paralysis.

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Updated 12 Feb 2019