Molecular Detection and Characterization

Virus isolation and molecular characterization by real-time RT-PCR and VP1 sequencing.

• Stool –Required specimen type for poliovirus diagnostics as the likelihood of poliovirus isolation is the highest from stool. To increase the probability of poliovirus isolation, obtain at least two stool specimens 24 hours apart from patients with suspected poliomyelitis. These samples should be collected ideally within 14 days after onset. Minimum volume required: 1gram; 2-3 grams preferred.
• Serum – Not recommended as this sample type is not likely to yield poliovirus in culture.
• CSF – Not recommended as detection of poliovirus in this specimen type is uncommon.
• Nasopharyngeal/Oropharyngeal Swab – Not recommended as respiratory specimens are not as likely as stool to yield poliovirus in culture. However they may be useful in ruling out non-polio causes of acute flaccid paralysis (AFP).
  

Note: If CSF, serum or respiratory swabs are sent for poliovirus testing, they will not undergo poliovirus isolation, they will be referred for Enterovirus and Human Parechovirus molecular detection and typing

Specimens may be subject to rejection if they are not shipped frozen, if they have insufficient volume, or are not accompanied by relevant patient information.

• Stool – Collect within 14 days of illness onset and place in a sterile leak proof container, no special medium required.  Isolation rates are increased by obtaining 2 samples collected 24 hours apart.

Store samples, as soon as possible following collection, frozen at =-20°C until shipped for testing.  Ship frozen on dry ice.  Package Poliovirus samples in a separate shipping container from other samples being sent to the NML.

Shipping of specimens shall be done by a TDG certified individual in accordance with TDG regulations. For additional information regarding classification of specimens for the purposes of shipping, consult either Part 2 Appendix 3 of the TDG Regulations or section 3.6.2 of the IATA Dangerous Goods Regulations as applicable.

Cases of acute flaccid paralysis (AFP) in children less than 15 years of age and cases of suspected poliomyelitis in patients of any age.

Completed Enteroviruses and Enteric viruses' requisition. If possible include relevant clinical findings, travel history, vaccination history, and any relevant lab results that have been completed at local or provincial laboratories.

N/A

Detection and molecular characterization of polioviruses involves three WHO accredited protocols:

• Virus Isolation in Cell Culture: Supernatant from processed stool samples are inoculated onto two WHO recommended cell lines: L20B cells (a transgenic mouse cell line expressing the human poliovirus receptor, CD155) and RD-A cells (human rhabdomysosarcoma). Inoculated cell cultures are observed microscopically for 14 days for the presence of cytopathic effect, that likely indicates an infection with a poliovirus (L20B positive cultures) or a non-polio enterovirus (RD-A only positive cultures). L20B positive cultures are referred for further molecular characterization as described below.
• Poliovirus(PV) Intratypic Differentiation (ITD) by Real Time PCR: Polio group, serotype, and vaccine strain-specific molecular reagents that target defining PV properties within the capsid region (VP1) are used in a series of real time RT-PCR assays to determine the serotype and to distinguish vaccine-like polioviruses from wild and vaccine derived polioviruses (intratypic differentiation). non-vaccine like, ITD discordant and all poliovirus type 2 (PV2) are referred for VP1 sequencing to confirm if they are Sabin/Sabin-like, nOPV2/nOPV2-like, vaccine derived (VPDV) or wild polioviruses (WPV).
• Poliovirus VP1 Sequencing: Classification of poliovirus isolates are confirmed based on the extent of sequence divergence of the full VP1 region of the isolate from the corresponding Sabin or nOPV2 reference strains - PV1 (AY184219); PV2 (AY184220); PV3 (AY184221); nOPV2 (MZ245455):
  • Sabin-Like - < 1% divergent (< 10 nt differences) for PV types 1 and 3; < 0.6% divergent (< 6 nt differences) for PV type 2.
  • Novel Oral Poliovirus Vaccine type 2 (nOPV2-like) - < 0.6% divergent (< 6 nt differences).
  • VDPV - = 1% divergent (= 10 nt differences) for PV types 1 and 3; = 0.6% divergent (= 6 nt differences) for PV type 2. When vaccine origin is determined to be from novel oral poliovirus vaccine (nOPV), the corresponding type includes -n in its acronym (e.g. VDPV2-n).
  • Wild Poliovirus (WPV) - = 15% divergent
    

Further testing if required may be referred to the CDC in Atlanta, Georgia, which is the WHO GPLN Reference Laboratory.

29 calendar days for the complete test algorithm.

1. World Health Organization. Polio Laboratory Manual. WHO/IVD/04.10,2004.
2. S1. Supplement to the WHO Polio Laboratory manual-an Alternative Test Algorithm for Poliovirus Isolation and Detection.
3. Kilpatick DR, Iber JC, Chen Q, Ching K, Yang S, De L, Mandelbaum MD, Emery B, Campagnoli R, Burns CC, Kew O. Poliovirus serotype-specific VP1 sequencing primers. J of Virol. Methods 174(2011) 128-130