ARCHIVED - Guidelines for the Prevention and Control of Mumps Outbreaks in Canada
Appendix 4: Laboratory Guidelines for the Diagnosis of Mumps
The purpose of these laboratory guidelines is to provide information on the collection, transportation, laboratory testing, and interpretation of laboratory test results of specimens for suspected mumps cases. A comprehensive description of mumps diagnostics can be found in the Manual of Clinical Microbiology(1). The information presented here is based on recent experiences in mumps diagnostics both in Canada and in the United States.
- The reverse transcriptase polymerase chain reaction (RT-PCR) assay is reliable for the definitive diagnosis of an acute mumps infection, but its sensitivity can be influenced by the following:
- timing of the specimen collection in relation to onset of illness;
- specimen integrity (rapid specimen processing).
- Buccal swab or saliva from the buccal cavity collected within the first 3 to 5 days of parotitis or symptom onset is the preferred specimen.
- Testing for mumps-specific IgM class antibody has been shown to be poorly predictive for the diagnosis of acute mumps in a partially immunized population (may be detectable in only 30% of acute cases).
- Collection of an acute serum specimen and a convalescent serum specimen 10 to 14 days later may show a seroconversion for IgM and/or IgG antibody in those cases in which the mumps RT-PCR assay and IgM antibody were negative or indeterminate at onset of illness, thus identifying additional cases.
- Testing by RT-PCR and IgM class antibody detection is not sufficiently reliable to rule out mumps infection
- In the absence of another diagnosis for parotitis, persons with symptoms clinically compatible with mumps (clinical illness) AND an established epidemiologic link to a laboratory-confirmed case should be reported as confirmed cases (epidemiologically confirmed case). As well, those with clinical illness but no established epidemiologic links should be managed (for public health purposes) as a probable mumps case, particularly during periods of known outbreak activity.
3.0 National Surveillance Case Definition for Mumps2
- Confirmed Case
Laboratory confirmation of infection* in the absence of recent history of administration of mumps vaccine by
- isolation of mumps virus from an appropriate clinical specimen
- detection of mumps virus RNA
- significant rise or seroconversion in mumps IgG antibody titre by a standard serologic assay
- detection of mumps IgM class antibody in a person with compatible illness
- clinical illness in a person who can be epidemiologically linked to a laboratory-confirmed case
- isolation of mumps virus from an appropriate clinical specimen
- Probable Case
Clinical illness in the absence of appropriate laboratory tests and no epidemiological link to a laboratory-confirmed case
4.0 Specimen Collection
4.1 For virus detection (RT-PCR) or isolation (culture)
Buccal swabs or saliva swabs particularly in the area around Stensen's duct adjacent to the swollen salivary glands are optimal (see <http://www.cdc.gov/nip/diseases/mumps/detection_IL.pdf>).
Buccal specimens should be collected using a swab approved for virus isolation and placed in virus transport media. Swabs may be dacron, nylon, or rayon tipped, and either flocked or non-flocked. Calcium alginate swabs are not acceptable as they inhibit PCR reactions. Charcoal swabs or swabs in Ames media used for swabbing for bacterial pathogens (such as group A Streptococcus) are not acceptable. Swabs with wooden or aluminum shafts are also not acceptable. Oral samples should be collected within 5 days of onset of symptoms. Swabs should be placed in standard viral transport media (VTM). Swabs must be left in VTM for a minimum of 1 hour to allow the virus to elute.
Typically, mumps has been found in the urine by culture up to 14 days after the onset of symptoms. However, experiences in the Nova Scotia and U.S. outbreaks suggest that mumps cannot be detected in the urine with the same sensitivity as in oral specimens. Limited data suggest that virus can be detected in the urine slightly later (> 4 days post-onset) than in oral specimens.
- A minimum volume of 50 mL urine should be collected in a sterile container.
- Centrifuge urine at 2500 x g for 15 minutes at 4°C.
- Resuspend the sediment in 2 mL of residual urine before processing.
The first (acute) serum specimen should be collected as soon as possible upon presentation of mumps. A second (convalescent) serum specimen should be collected ideally at least 10 days after the first sample.
4.3 Specimen Storage and Transport
Unprocessed samples can be shipped at 4ºC within 48 hours of collection. If subsequent testing is delayed, processed samples can be frozen at -70°C and shipped on dry ice. Transport urine, buccal or saliva samples, and sera as diagnostic specimens, category B.
Although the presence of mumps-specific IgM antibodies is typically indicative of a primary acute mumps infection, recent data show that in a partially vaccinated population (recipients have received only one dose of mumps-containing vaccine) the IgM antibody response is delayed or absent. In addition, when the prevalence of mumps is low, the positive predictive value of the IgM test is correspondingly lower, and the potential for generating false-positive results is considerably greater. In sporadic suspected mumps cases without an established epidemiologic link to a confirmed case or without travel history to an area with known/likely mumps activity, one should be suspicious of positive IgM results, which are potentially false positives. In such sporadic suspected mumps cases, additional laboratory tests, such as paired acute/convalescent IgG serology or detection of mumps virus by RT-PCR, should be highly encouraged to confirm the mumps infection and subsequent genotyping of the strain if positive by RT-PCR.
The presence of mumps-specific IgG is indicative of a recent or prior exposure to mumps virus. Seroconversion (i.e., negative to positive result) or a fourfold or greater rise in titre between the acute and convalescent sera is indicative of an acute mumps infection. However, this does require a delay in the collection of the second (convalescent) serum sample of 10 days or more after the collection of the first (acute) serum sample. When using enzyme immunoassays (EIA) for IgG titre determination, it is important to do end-point titrations as opposed to single dilution runs of a sample in order to conclusively determine the fourfold or greater difference in titres between acute and convalescent sera.
Limitations to IgG serology:
- Serology cannot differentiate between vaccine and wild-type mumps strain exposure.
- The presence of mumps-specific IgG, as determined using an EIA, does not necessarily predict the presence of neutralizing antibodies and thus immunity. Conversely, the absence of detectable mumps IgG using EIA may reflect the lower sensitivity of the EIA in comparison to a more sensitive assay, such as a neutralization assay, in which IgG may be detectable.
- A single serum sample tested for mumps-specific IgG has no value in diagnosing an acute mumps infection.
- Experience from the recent outbreaks in the United States suggests that when investigating suspected mumps in previously vaccinated individuals, in particular those who have received two doses of mumps-containing vaccine, the absorbance or quotient EIA values of IgG class antibody to mumps in the first (acute) serum may be quite high, and thus documentation of a fourfold or greater rise in titre/absorbance quotient may not be possible. Moreover, detection of appreciable levels of IgG class antibody in the acute serum does not rule out mumps infection.
The National Microbiology Laboratory can assist with the determination of mumps IgG titres if requested.
6.0 Mumps Virus Detection
6.1 Reverse transcriptase polymerase chain reaction (RT-PCR)
Because of the limited sensitivity of IgM testing discussed above, RT-PCR has been employed as the primary diagnostic approach for the laboratory confirmation of mumps cases.
Mumps virus is an RNA virus, and thus RT-PCR is the common approach for mumps virus detection. Commercial mumps RT-PCR assays are not available, and so conventional hemi-nested3 and real-time in-house assays have been developed and validated. The National Microbiology Laboratory uses the LightCycler real-time PCR platform for both the CDC Taqman probe-based method for detection of the SH gene, as well as the real-time method developed by Uchida et al.4 based on detection of the F gene. Appropriate positive and negative controls are essential for inclusion in RT-PCR runs to control for inhibition, extraction and contamination issues. Protocols and controls for mumps RT-PCR are available from the National Microbiology Laboratory on request.
Although the analytic sensitivity of RT-PCR is between 10 and 100 genome copies, the overall clinical sensitivity is affected by pre-analytical factors (see Section 3.0 above), namely the following:
- specimen type and quality;
- timing of specimen collection, in relation to onset of illness;
- rapid transportation of the specimen to the laboratory;
- rapid processing of specimens:
- It is recommended that specimens for virus detection be processed within 48 hours after sample collection. Further delays result in significant reduction in sensitivity.
- appropriate storage of specimens; and
- avoid freeze thawing unprocessed specimens.
Mumps cases with a history of immunization may also present challenges for RT-PCR. Such cases may have a shorter period of viral shedding and shed potentially lower amounts of virus, resulting in a higher incidence of false-negative results by the RT-PCR assay.
6.2 Mumps virus isolation
Mumps virus can be isolated in several cell lines, including primary monkey kidney, human neonatal kidney, HeLa, and Vero cells. Cytopathogenic effect is usually detected after 6 to 8 days post-inoculation. Accordingly, the RT-PCR assay is a more rapid approach for diagnostic purposes. Refer to the Manual of Clinical Microbiology1 for further details on mumps virus isolation protocols.
6.3 Mumps virus genotyping
Mumps genotyping has been standardized3 and is available at the National Microbiology Laboratory. Mumps virus genotyping is useful for molecular epidemiologic purposes such as differentiating vaccine and wild-type strains, especially if the individual developed a mumps-compatible presentation within 28 days of receiving the vaccine. In addition, genotyping can be helpful in linking cases, linking outbreaks, tracking importations, and documenting the elimination of a particular strain from a geographic area.
7.0 Interpretation of Laboratory Results
In an outbreak, a negative test, either RT-PCR or IgM antibody, cannot be interpreted to rule out mumps infection. In the absence of another diagnosis to rule out mumps infection, persons with clinically compatible mumps (clinical illness) AND an established epidemiologic link to a laboratory-confirmed case should be reported as confirmed cases (clinical confirmed case). As well, those with clinical illness but no established epidemiologic links should be managed (for public health purposes) as a probable mumps case, particularly during periods of known outbreak activity.
In order to properly interpret laboratory results and to assess the performance of mumps diagnostic assays, both clinical and epidemiologic information needs to be considered along with the laboratory information. As outlined above, prior vaccination history, travel history, and timing of sample collection relative to disease onset are all factors that must be considered in the interpretation of laboratory results for the purpose of confirming mumps cases. As such, communication and information sharing between public health and the laboratory are essential.
8.0 Mumps Testing at the National Microbiology Laboratory
Information on mumps testing available at the National Microbiology Laboratory can be found through the online guide to services at http://www.nml-lnm.gc.ca/english/guide/default.asp.
- Leland DS. 2007. Parainfluenza and mumps viruses. In: PR Murray, EJ Baron, JH Jorgensen et al, eds., ML Landry and MA Pfaller (ed.), Manual of clinical microbiology, 9th ed, Washington, D.C.: ASM Press, 2007.
- Laboratory Centre for Disease Control. Case definitions for diseases under national surveillance. CCDR 2000;26(S3). <http://www.phac-aspc.gc.ca/publicat/ccdr-rmtc/00vol26/26s3/index.html>.
- Jin L, B Rima, D Brown et al. Proposal for genetic characterisation of wild-type mumps strains: preliminary standardisation of the nomenclature. Arch Virol 2005;150:1903-9.4.
- Uchida K, M Shinohara, S Shimada et al. Rapid and sensitive detection of mumps virus RNA directly from clinical samples by real-time PCR. J Med Virology 2005;75:470–4.
Graham Tipples, Jennifer Beirnes, and Joanne Hiebert (Viral Exanthemata Section, National Microbiology Laboratory, Public Health Agency of Canada)
Todd Hatchette and Janice Pettipas (QEII Health Sciences Centre, Halifax, Nova Scotia)
Kevin Fonseca (ProvLab Alberta, Calgary, Alberta)
Martin Petric (BC-CDC, Vancouver, British Columbia)
Jeannette Macey, Shelley Deeks, and Tammy Lipskie (Centre for Immunization and Respiratory Infectious Diseases, Public Health Agency of Canada)
William Bellini, Paul Rota, and Jennifer Rota (Measles, Mumps, Rubella and Herpes Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, USA)
11.0 Contact Information, Public Health Agency of Canada
11.1 National Microbiology Laboratory
Graham Tipples, PhD, FCCM
Director, Surveillance and Reference Services
Chief, Viral Exanthemata Section
1015 Arlington Street
Winnipeg, MB, Canada R3E 3R2
Phone: (204) 789-6080
Fax: (204) 789-7039
Pager: (204) 935-4712
Joanne Hiebert or Jennifer Beirnes
Viral Exanthemata Section
Phone: (204) 789-6082 or (204) 789-7055
Fax: (204) 789-5009
Email: Joanne.Hiebert@phac-aspc.gc.ca or Jennifer.Beirnes@phac-aspc.gc.ca
11.2 Centre for Immunization and Respiratory Infectious Diseases
The Public Health Agency of Canada continues to post general information about mumps and a national summary of the outbreak(s) on its Web site: www.phac-aspc.gc.ca/mumps-oreillons/index.html.
Until further notice, please notify the Centre for Immunization and Respiratory Infectious Diseases (which compiles the national summary) of any confirmed mumps cases in your jurisdiction that are linked to the outbreaks.
*A laboratory-confirmed case does not have to meet the clinical illness description.
The Canadian Public Health Laboratory Network has endorsed the addition of mumps RT-PCR testing as a standard approach for mumps virus RNA detection.
Clinical illness is characterized by acute onset of unilateral or bilateral tender, self-limited swelling of the parotid or other salivary gland, lasting > 2 days, and without other apparent cause.
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