It is recommended that clinical specimens for the molecular surveillance of measles and rubella are collected in parallel with samples for antibody testing for case classification. Routine testing by RT-PCR in addition to antibody detection enhances the ability to rapidly confirm suspected cases of measles and rubella but will require additional resources.

An oropharyngeal (throat swab) is the recommended sample for both RNA detection and virus isolation for suspected cases of measles or rubella. Nasopharyngeal (NP) swabs will serve as good samples for both virus isolation and RNA detection but are more difficult to collect. NP aspirates and nasal swabs are variations that have been used successfully to detect measles virus.

Note: Some surveillance programmes include an option to use swabs to collect oral secretions (mouth swabs) for measles virus RNA detection as an alternative to a throat swab. Mouth swabs are often easier than throat swabs to obtain from young children. Saliva and crevicular secretions are collected by swabbing the inside cheek near the gum line.

If measles- and rubella-specific IgM is tested using OF rather than serum, the OF can also provide a sample for virus-specific RNA detection. However, OF is considered not suitable for virus isolation. For the purpose of RNA detection by molecular testing, OF can be collected up to 21 days after rash onset. As with other types of samples for molecular testing, the likelihood of obtaining a positive signal by RT-PCR is much improved when samples are collected closer to rash onset. However, it is also true that at any interval post rash onset, a negative RT-PCR result may be obtained from true cases of measles or rubella. Therefore, negative RT-PCR results alone are not sufficient to rule out a suspected case and serologic testing is generally required to corroborate negative RT-PCR results.

Countries that use OF as the primary specimen for antibody detection and virus-specific RNA detection should collect throat swabs to provide an opportunity to make virus isolates from representative cases during an outbreak (see chapter 7). Serum specimens have been used in rare instances when better samples for genotyping are not available, and archival samples of serum have been used to aid in identification of circulating rubella strains prior to routine surveillance.

Urine specimens have been used successfully for both measles and rubella detection and virus isolation, but throat swabs are much more sensitive compared to urine samples, particularly for rubella. Throat/oropharyngeal swabs, NP swabs, and urine samples must be collected within 5 days of rash onset to be considered an adequate sample for virus isolation. While virus may be present through at least day 5 after rash onset, the success rate for virus isolation is much higher with specimens collected within 3 days from rash onset.

Note: Specimens for virus isolation and RNA detection from congenital rubella syndrome (CRS) and congenital rubella infection (CRI) cases are described in section 3.6.

Throat swabs and urine can be collected up to 14 days after rash onset for RNA detection by RT-PCR although RNA detection rates are much lower after 7 days post rash onset. Both measles and rubella virus-specific RNA can be detected in throat swab samples from a few days before onset of rash to several days afterwards. Prompt collection of samples at first contact with a suspected case of measles is the best approach to ensure a quality sample. Collection of both types of samples (throat swab and urine) may enhance the successful detection of RNA or the ability to isolate virus. The collection of both samples may be particularly useful and appropriate in elimination settings when a sporadic case is detected.

Both measles and rubella viruses are sensitive to heat, and infectivity decreases with time, even when samples are refrigerated for extended periods of time. While RNA detection by RT-PCR can often be successful despite disruptions in the maintenance of a cold chain, every effort should be made to transport and maintain all clinical specimens for RNA detection or virus isolation to freezers (-40 to -70°C) to best preserve RNA and the viability of the virus. Clinical samples transported at -20°C may preserve the integrity of the viral RNA for RT-PCR testing however, the viability of virus may be diminished or lost.

3.5.1 Collection, processing, and transport of upper respiratory specimens
The preferred sample for both measles and rubella virus detection is the throat (oropharyngeal) swab. Swabs should be collected using only synthetic fibre swabs with plastic shafts. Do not use calcium alginate swabs or swabs with wooden shafts, as they may contain substances that inactivate viruses and/or inhibit PCR testing. The throat swab is collected by swabbing the posterior pharynx, avoiding the tongue. A tongue depressor may be used. Light pressure should be applied to collect the epithelial cells around the tonsil area. Similar swabs should be used for mouth swabs for measles RNA. For this sample, the swab is rubbed on the inside of the cheek area and the tongue.

The nasopharyngeal swab (NP) is often collected in a hospital or clinic setting and provides a good specimen but may be less acceptable to the patient. A NP swab has a flexible shaft and the patient’s head should be tilted back. The swab is inserted into the nostril parallel to the palate and should contact the mucosal surface. The swab in held in place for a few seconds to absorb secretions. Both nostrils can be swabbed and the two swabs can be combined.

The throat and/or NP swabs are placed immediately into sterile tubes containing 2-3 ml of viral transport media (VTM) or PBS. It is important to prevent the swabs from drying out. (See Annex 3.4, Composition of viral transport media, antibiotics and reagents). A throat swab can be combined with an NP swab in one tube when both specimens are collected from the same patient. The throat and/or NP swabs may be refrigerated at 4-8°C for up to 48 hours and shipped on ice/frozen cold packs. If an interval of greater than 3-5 days will elapse prior to arrival at the receiving laboratory, it is best to preserve the sample at -70°C and transport on dry ice.

Note: Prior to freezing and the removal of the swab, the tubes should be vigorously mixed using a tube vortex for about 15 seconds to dissociate the cells from the swab material. The fluid should be drained from the swab by pressing the swab tip along the side of the tube before removal of the swab.

Efforts should be taken to avoid defrosting as a single defrosting step may render the specimen useless for virus isolation and may also affect the ability to extract intact RNA for RT-PCR. Upon arrival in the testing laboratory, the throat, nasal, or NP swab sample can be frozen at -70°C if the volume of the fluid is sufficient (2-3 ml) and the swab has been removed. If the swab has not been removed prior to freezing, follow the steps provided (see Note, above) to maximize recovery of the sample. Cell culture medium (MEM or DMEM) is added to the sample to bring the total volume to 2-3 ml. The swab is discarded as potentially infectious material. If there is a large amount of debris in the fluid, the contents can be centrifuged at low speed and the supernatant removed to a labelled sample tube.

3.5.2 Collection, processing, and transport of urine samples
The first-voided urine of the day is preferred as it contains the highest concentration of cells sloughed off in the urinary tract, although in practice the sample is collected at first contact with the suspected case. The volume of urine collected should be at least 10 ml. A larger volume (up to 50 ml) will generally provide more infected cells. The urine is collected in a suitable sterile container that is leak-proof and should be stored at 4-8°C until the urine can be centrifuged. The original urine sample should not be frozen prior to centrifugation.

Whole urine samples may be shipped in sealed containers at 4°C, but centrifugation within 24 hours of collection is recommended. The urine is centrifuged at 500 × g (approximately 1500 rpm) for 5 to 10 minutes, preferably at 4°C and the supernatant is discarded. Sterile VTM, tissue culture medium or phosphate-buffered saline (PBS) is added to the urine sediment, to bring the final volume to 2 ml. If a pellet is not visible, remove all but the last 1-2ml at the bottom of the centrifuge tube and mix with an equal volume of sterile, isotonic fluid (VTM, PBS etc). The processed urine sample may be stored at +4°C and shipped within 48 hours to a laboratory that conducts measles virus isolation or RNA detection. Alternatively, the urine sample may be frozen at -70°C in viral transport medium and shipped on dry ice.