Questions and Answers about Lab Testing
Questions in This Section
- What specimens should be collected from patients who meet the mumps clinical case definition?
- When is the optimal timing for collection of specimens for laboratory confirmation of mumps?
- What tests are available for laboratory confirmation of mumps?
- Where do I send laboratory specimens for testing?
- We have seen cases of “recurrent parotitis” in our state. Is this unusual and is it necessary to recommend isolation again from their last onset date?
A: CDC recommends collection of a buccal or oral swab specimen as soon after parotitis onset as possible from all patients with clinical features compatible with mumps. See Specimen Collection, Storage, and Shipment for detailed instructions regarding recommended samples for mumps testing.
A: A buccal or oral swab specimen for detection of virus should be collected as soon as possible upon suspicion of mumps disease. The early collection of buccal swab specimens provides the best means of laboratory confirmation, particularly among suspected mumps patients with a history of vaccination (Rota et al, 2013). If it has been <3 days since symptom onset, collect only a buccal swab specimen for detection of viral RNA by RT-PCR. If it has been >3 days since symptom onset, collect a buccal swab specimen for RT- PCR and a serum specimen for IgM detection. If the patient has orchitis/oophoritis, mastitis, pancreatitis, hearing loss, meningitis, or encephalitis, collect a buccal specimen for RT-PCR, a urine specimen for RT-PCR, and a serum specimen for IgM detection, regardless of days since symptom onset.
In an outbreak setting (see Figure below), the proportion of samples with positive results by RT-PCR (N target gene) decreased as the interval between onset and specimen collection increased (Rota et al., 2013). The trend was reversed for IgM detection with increasing time to sample collection the number of seropositive samples increased.
The percentage of positive results obtained from testing 296 confirmed mumps cases from New York City by day of sample collection after onset of symptoms. The serum samples were tested for presence of IgM using the CDC capture IgM EIA. The buccal swab samples were tested by RT-PCR using the mumps nucleoprotein (N) gene as the target.
*Done in collaboration with New York City Deparment of Health and Mental Hygiene Public Health Laboratory, New York, NY
Mumps virus was isolated from 209 (71%) of the 296 buccal swabs tested.
A: Viral detection methods include molecular assays, such as real time reverse transcription PCR (RT-PCR) to detect mumps viral RNA and standard methods of culturing virus in appropriate cell lines. RT-PCR is performed by almost all state and local public health laboratories, the Vaccine Preventable Disease Reference Centers (VPD-RC [2 pages])pdf iconexternal icon, and CDC. Genotyping, based on the sequence of the gene coding for the small hydrophobic (SH) protein can also be performed by the VPD-RCs and CDC and this is the only test to discriminate vaccine reactions from wild-type infections.
IgM can aid in diagnosis but is not confirmatory. The availability of assays to detect IgM to aid in the diagnosis of acute mumps infection and to measure IgG antibodies to document previous exposure to mumps vary among laboratories. The state health department can provide guidance regarding available laboratory services. At the direction of the state health department, healthcare providers and state and local health departments may send serum specimens from suspected mumps cases to CDC/VVPDB for IgM detection using a capture IgM enzyme immunoassay that incorporates a recombinant mumps nucleocapsid protein as the antigen
Q: We have seen cases of “recurrent parotitis” in our state. Is this unusual and is it necessary to recommend isolation again from their last onset date?
Questions in This Section
- Is collection of both a buccal/oral and a serum sample necessary?
- What does a mumps-negative RT-PCR result mean?
- What does a mumps-positive RT-PCR result mean?
- Why doesn’t a negative RT-PCR result and/or a negative IgM result rule out mumps as a diagnosis?
- Why attempt to isolate mumps virus in cell culture?
- What is a mumps genotype?
- What mumps virus genotype has been commonly detected in the United States?
- Are there small genetic changes that may be affecting our molecular diagnostics?
- Would genetic changes that are occurring cause the vaccine not to work against certain strains of the virus?
- If a clinical sample is determined to be positive by RT-PCR or by viral isolation at the state laboratory, is it necessary to send a sample to CDC for sequence analysis?
- Where can I find RT-PCR and real-time RT-PCR protocols for mumps?
- Where do I obtain material for RT-PCR and virus isolation controls?
A: CDC recommends collection of a buccal or oral swab specimen for RT-PCR if it has been <3 days since symptom onset. Buccal/oral swab samples enhance the ability to laboratory-confirm a mumps infection and can provide additional information (sequence analysis to obtain the mumps genotype) to aid epidemiologic investigations. In addition, because serologic tests cannot differentiate between an exposure to vaccine and an exposure to wild-type mumps virus, it is necessary to obtain buccal/oral samples to differentiate between wild-type and vaccine virus. If it has been >3 days since symptom onset, collect a buccal swab specimen for RT- PCR and a serum specimen for IgM detection. This may be particularly important in the beginning of an outbreak when it is unclear if mumps is the cause. If the patient has orchitis/oophoritis, mastitis, pancreatitis, hearing loss, meningitis, or encephalitis, collect a buccal specimen for RT-PCR, a urine specimen for RT-PCR, and a serum specimen for IgM detection, regardless of days since symptom onset.
A: Failure to detect mumps virus RNA by RT-PCR in samples from a person with clinically compatible mumps symptoms does not rule out mumps as a diagnosis. Successful detection of mumps virus depends primarily on the timing of collection and quality of the clinical sample. Vaccinated individuals may shed virus for a shorter period and might shed smaller amounts of virus, thus degradation of the sample has greater consequences for successful detection of virus. In outbreaks among two-dose vaccine recipients, mumps virus RNA was detected in samples from 30%–71% of case-patients if the samples were collected within 3 days following onset of parotitis. IgM was detected in 13 to 50% of these cases (Bitsko et al, 2008, Rota et al. 2009, Rota et al, 2013).
A: A positive RT-PCR signal indicates the presence of mumps virus RNA in the patient sample. A positive RT-PCR result provides laboratory confirmation of mumps infection in persons with symptoms consistent with mumps.
A: Successful detection of mumps virus or anti-mumps IgM antibody is dependent upon the timing of specimen collection and quality of the clinical sample. Vaccinated individuals may shed virus for a shorter period and may shed smaller amounts of virus. Viral RNA may not be detectable in samples that have been collected, stored or shipped improperly. IgM may be transient or absent and therefore not detected.
A: Virus isolation is considered among the best methods for confirming mumps infection. Virus can be detected when IgM antibodies or a rise in IgG titer are not detected. Often it is necessary to grow the virus in culture to have adequate material for viral sequencing. Sequence analysis allows the determination of the mumps genotype (there are currently 12 recognized genotypes). The sequence information may help to identify the source of the virus and can provide confirmation of suspected epidemiologic links.
A: Mumps strains are assigned to 1 of 12 genotypes based on the sequences of the gene coding for the short hydrophobic (SH) protein. In some circumstances, a genotype has been associated with endemic circulation of mumps virus in a country; however, routine genotype surveillance for mumps is limited to only a few countries. The genetic information from circulating mumps viruses is used to track the transmission pathways of the virus and can be used to suggest epidemiologic links, or lack thereof, between cases and outbreaks.
A: Since CDC initiated routine genotype surveillance for mumps in 2006, CDC and reference centers have detected mostly genotype G among people with mumps in the United States. A few of the other 11 genotypes were also detected, but they are usually associated with mumps importations into the United States and have not been associated with large outbreaks.
Mumps outbreaks are typically associated with only one genotype. There are no differences in the genotypes detected in vaccinated and unvaccinated people who have gotten mumps in the United States.
For more information, please see the “Genetic characterization of mumps viruses associated with the resurgence of mumps in the United States: 2015-2017external icon” study.
A: Viruses like mumps have very high rates of genetic change compared to other organisms. Our diagnostic assay, RT-PCR, is designed to detect a specific sequence that is conserved in many mumps genotypes. If genetic changes occur in the sequence that is being detected by RT-PCR, the assay may lose the ability to detect mumps virus with high sensitivity. CDC monitors the performance of the RT-PCR assay in collaboration with the four Vaccine Preventable Disease Reference Centers that are supported by the Association of Public Health Laboratories (APHL) and CDC. In addition, state laboratories performing the RT-PCR assay to detect mumps may participate in a proficiency testing program that was developed by CDC and the Reference Centers and is managed by the Wisconsin State Laboratory of Hygiene.
Q: Would genetic changes that are occurring cause the vaccine not to work against certain strains of the virus?
A: Not all genetic changes result in a change in viral proteins. “Silent mutations” change the genetic sequence without changing the predicted protein. Viral proteins are recognized by the host’s immune system. There are several viral proteins, each containing many recognition sites. Therefore, it would take many genetic changes to change the viral proteins to the extent that they are no longer recognized by the immune response in vaccinated individuals. CDC is developing tests to monitor the ability of serum from vaccinated individuals to neutralize currently circulating strains.
Q: If a clinical sample is determined to be positive by RT-PCR or by viral isolation at the state laboratory, is it necessary to send a sample to CDC or a VPD-RC for sequence analysis?
A: Laboratories are encouraged to send patient samples from positive sporadic cases of mumps as well as representative samples from an outbreak. The sequence of the mumps short hydrophobic (SH) gene is used to assign mumps viruses to one of 12 recognized genotypes. The sequence information may help to identify the source of the virus and can provide confirmation of suspected epidemiologic links. Samples for genotyping can be sent to the CDC or the VPD-RCs.
During ongoing outbreaks, it is not necessary to obtain a genotype on every RT-PCR positive specimen although it is recommended to obtain representative sequences on a weekly basis. However, if the outbreak spreads to another community, an effort should be made to obtain a genotype for specimen from the new outbreak. Additionally, specimens should be submitted for genotyping from confirmed cases that do not meet the case definition, that have an unusually long incubation period, or have travel history (domestic or international).
A: The following updated protocols are available online:
- Real-time RT-PCR protocol, targeting the nucleoprotein (N) gene pdf icon[3 pages] (Updated Aug 2021)
- Standard RT-PCR for mumps virus pdf icon[6 pages] (Updated Jun 2021)
For more information on changes made to the protocols, please see “Would genetic changes that are occurring cause the vaccine not to work against certain strains of the virus?”
A: CDC can provide a sample of synthetic RNA for real-time RT-PCR reactions (N gene) and for genotyping (endpoint) RT-PCR (SH gene). This material is ready to use in RT-PCR reactions. If laboratories would like to produce their own synthetic RNA samples or require a positive control for virus isolation, CDC can provide a sample of wild-type mumps virus, or viruses can be obtained from ATCCexternal icon or BEIexternal icon. Public health laboratories or laboratories affiliated with state public health laboratories may send request for mumps RNA or virus to email@example.com. A proficiency test for mumps RT-PCR can be obtained from the Wisconsin State Laboratory of Hygieneexternal icon.
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