Molecular Detection and Genotyping

Molecular detection of the rubella virus by real-time RT-PCR and genotyping of positive specimens.

Nasopharyngeal (NP), throat swabs or urine collected as soon as possible after rash onset (within 5 days). Note: specimens collected later will still be accepted however the assay sensitivity will not be optimal. For CRI/CRS cases, collect urine, NP or throat swabs within the first few months after birth. Viral isolates may also be sent. Other specimen types may be accepted for research purposes; please contact the Viral Exanthemata laboratory prior to submission.

Collect 50 mL (minimum 10 mL) of urine in sterile container. For swabs, use sterile swabs approved for virus isolation to swab the throat or nasal passages to collect epithelial cells. Place swabs in 2-3 mL viral transport medium (VTM) for a minimum of 1 hour. For viral isolate, collect infected cells and media and submit 1 mL.

Process urine by centrifuging at 2500 x g for 15 minutes at 4?C. Resuspend the sediment in 1-2 mL VTM. For small volumes of urine (CRI/CRS cases), resuspend sediment in a minimum of 0.5 mL of VTM. For swab specimens, remove the swab. For all specimens, store at 4°C and ship on wet ice for arrival at the NML within 48 hours. Otherwise, freeze at -70°C and ship frozen on dry ice. Viral Isolates should be shipped frozen (-70°C) on dry ice.

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.

1. Suspected cases of rubella (typically with fever, rash and at least one of the following: arthralgia/ arthritis or lymphadenopathy or conjunctivitis) or serological laboratory confirmation in the absence of recent (1-14 days) rubella vaccination. 2. Suspected congenital rubella infection (CRI) / congential rubella syndrome (CRS).

Completed Measles, Mumps, and Rubella requisition form. Include date of rash onset, date of fever onset, number of doses of rubella vaccine received, date of last rubella vaccination, travel history, date of possible rubella exposure, as applicable.

Labs that perform their own rubella RT-PCR testing should submit positive samples for genotype surveillance (which includes reporting to the WHO). It is important to provide a complete patient history (including travel) for accurate genotype surveillance. The NML is a WHO/PAHO accredited Measles and Rubella Regional Reference Laboratory.

Extracted RNA from samples is amplified by real-time RT-PCR for the E1 gene and analyzed by melting curve analysis. A Tm within a pre-defined range is a positive rubella RT-PCR result and is laboratory confirmation of rubella virus. A portion of the E1 gene of positive samples is amplified by conventional RT-PCR and a WHO standardised region is sequenced to determine the genotype of the rubella virus.

Detection: 7 calendar days. Genotyping: 21 calendar days. Stat testing available upon request.

1. WHO. Standardization of the nomenclature for genetic characteristics of wild-type rubella viruses. WER 2005;14:126-132. 2. Tipples G, J Hiebert. Detection of measles, mumps and rubella viruses. Methods Mol Biol. 2011; 665: 183-193. 3. World Health Organization. Manual for the laboratory diagnosis of measles and rubella virus infection. 2007. WHO/IVB/07.01