Questions and Answers About Lab Testing
General Laboratory Testing
Q: What specimens should be collected from patients who meet the mumps clinical case definition?
A: CDC recommends that a buccal or oral swab specimen and a blood specimen be collected from all patients with clinical features compatible with mumps. See Materials and Methods for Specimen Collection, Storage, and Shipment for detailed instructions regarding recommended samples for mumps testing.
Q: When is the optimal timing for collection of specimens for laboratory confirmation of mumps?
A: The acute-phase serum and clinical samples 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.
Q: What tests are available for laboratory confirmation of mumps?
A: Standard serologic assays that detect mumps IgM are commercially available in both EIA and IFA formats. Viral detection methods include standard methods of culturing virus in appropriate cell lines and techniques, such as real time RT-PCR to detect mumps viral RNA.
Q: Where do I send laboratory specimens for testing?
A: First, contact your health department to determine where to submit specimens and how to ship them.
To send specimens to CDC, use the CDC Specimen Submission Form.
Please be sure to provide:
- Contact name with telephone number and/or email address
- Type of virus specimen and date collected
- Date serum sample collected
- MMR vaccination history [date(s) if known]
- Clinical signs and symptoms
- Patient’s date of birth or age
- Onset date of parotitis or jaw swelling if applicable
Centers for Disease Control and Prevention
STAT Unit #81
Attn: Dr. William J. Bellini
1600 Clifton Road NE
Atlanta, GA 30333
Contact Jennifer Rota by telephone at 404-639-1156 or by e-mail at email@example.com for additional information and to provide shipment details.
Q: Are new tests to detect mumps infection in previously vaccinated persons being developed?
A: Yes, such tests are being developed. Standard diagnostic methods, such as IgM EIA and RT-PCR, work well for diagnosing mumps infection in immunologically naïve individuals; however, the duration and intensity of the IgM response and viral shedding are reduced in persons with a prior immune response to mumps virus, making laboratory confirmation a challenge. Other tests (detection of a 4-fold rise in acute- and convalescent-phase IgG titer or detection of IgM in a convalescent-phase serum sample) have limited usefulness since they identify mumps infection too late to be helpful clinically.
The ELISpot assay appears to be more sensitive than the EIA for detection of activated mumps-specific, antibody-secreting B cells (ASC) in whole blood, and the ELISpot assay is not influenced by pre-existing antibodies in the serum. Preliminary studies demonstrated that mumps-specific ASC could be detected by ELISpot assay in 7 of 7 patients who were part of a confirmed outbreak in the northeastern United States. Before this assay is routinely used, additional research needs to be done to determine the optimal time for blood collection following symptom onset as well as the optimal conditions for specimen transport and testing. If you are interested in additional information about the ELISpot assay technique and possible training opportunities, please contact Don Latner, PhD (firstname.lastname@example.org) or Carole Hickman, PhD (email@example.com).
Mumps Serologic Testing
Q: What type of serologic assay is recommended for mumps IgM testing?
A: Both EIA and IFA assays can perform well for diagnosis of primary mumps infection. Acute-phase mumps specimens may contain significant levels of mumps IgG, especially among persons with a history of 1 or 2 doses of MMR. The IFA format is particularly susceptible to interference by high levels of mumps IgG. Treatment of serum with an agent to remove human IgG antibody, such as Gullsorb or a similar IgG inactivation reagent, is necessary to avoid false-positive IgM test results.
Q: What serologic tests are used at CDC to diagnose mumps?
A: A capture IgM EIA (non-quantitative) that incorporates a recombinant mumps nucleocapsid protein as the antigen is used to detect mumps IgM. A commercial, indirect EIA (non-quantitative) is used for detection of IgG.
Q: What does a positive mumps IgG test result mean?
A: A single serum sample tested for mumps-specific IgG is not useful for diagnosing acute mumps infections. The presence of mumps-specific IgG, as detected using a serologic assay (EIA or IFA), does not necessarily predict the presence of neutralizing antibodies or protection from mumps disease. See related questions regarding measurement of rise in IgG titer.
Q: What is the protective neutralizing antibody titer for mumps?
A: There is no known protective level of neutralizing antibody (antibody titer) for mumps, and there are no other immune parameters that correlate with protection from mumps disease.
Q: What etiologic agents are likely to interfere with serologic assays for mumps (i.e., produce false-positive results)?
A: Parainfluenza viruses 1, 2, and 3, Epstein-Barr virus, adenovirus, and human herpesvirus 6 have all been noted to interfere with mumps serologic assays (Davidkin et al. 2005).
Q: My patient specimen was positive by the monspot test (Epstein-Barr) but also gave a positive result for mumps IgM antibody. What is the explanation for this?
A: The initial immune response to Epstein-Barr produces a polyclonal B cell stimulation; the antibodies are broadly reactive and can result in a positive mumps IgM result. However, the monospot test should be considered less susceptible to a false-positive result with serum collected from a true case of mumps.
Q: If the IgM result is negative and IgG is positive, can mumps be ruled out?
A: Absence of a mumps IgM response in a vaccinated or previously infected individual presenting with clinically compatible mumps does not rule out mumps as a diagnosis. A positive IgG result is expected among previously vaccinated persons. Older persons or foreign nationals with no history of mumps illness or vaccination may have detectable mumps IgG due to a previous subclinical infection.
Q: Can serologic tests differentiate between a recent or prior exposure to mumps virus and a response to mumps vaccine?
A: The presence of mumps-specific IgG indicates a recent or a prior exposure to mumps virus or mumps vaccine. Serologic tests cannot differentiate between an exposure to vaccine and an exposure to wild-type mumps virus. See question regarding utility of viral samples for genetic analysis.
Q: The acute-phase serum was negative for mumps IgM. Is it appropriate to collect additional serology specimens?
A: Unvaccinated persons or unknown vaccination history
If an acute-phase serum sample collected ≤3 days after parotitis onset in an unvaccinated person is negative for IgM, testing a second sample collected 5–7 days after symptom onset is recommended since the IgM response may require more time to develop. A second negative IgM result does not rule out mumps unless the IgG result is also negative.
Previously vaccinated persons
There is some evidence that serum collected ≥10 days after parotitis onset may improve the ability to detect IgM among persons who have received only 1 or 2 doses of MMR (Krause et al. 2007; CDC, unpublished data). However, persons with a history of mumps vaccination may not have detectable mumps IgM antibody regardless of the timing of specimen collection. See Overview of Laboratory Confirmation by IgM Serology.
Q: What does a positive IgM result mean?
A: Mumps is confirmed by detecting mumps IgM antibody in serum samples collected as soon as possible after symptom onset. A positive IgM test result indicates current or very recent infection or reinfection. A positive IgM test result may also be observed following mumps vaccination.
Q: What are the recommendations for collection of a convalescent-phase serum sample?
A: A second (convalescent-phase) serum sample is collected about 2–3 weeks after the first (acute-phase) sample. A 4-fold increase in IgG titer between acute- and convalescent-phase samples, as measured in plaque-reduction neutralization assays or similar quantitative assays, or a seroconversion from negative to positive from acute to convalescent phase, as determined by EIA, is considered a positive diagnostic result for mumps. Paired serum samples from vaccinated persons, even if appropriately timed, may not show a rise in IgG titer.
Q: Why is it difficult to demonstrate a rise in titer (seroconversion) from persons with a history of vaccination?
A: In vaccinated persons, the existing IgG will begin to rise soon after exposure and infection. At the time of symptom onset and collection of the acute-phase serum sample, IgG may already be quite elevated, which would obviate the 4-fold rise in titer expected when comparing acute- and convalescent-phase titers. For this reason, it is recommended to obtain the acute-phase specimen as soon as mumps infection is suspected.
Q: What is the experience at CDC with paired serum samples from previously vaccinated persons with mumps?
A: A 4-fold rise in IgG titer is rarely demonstrated between paired serum samples from persons who have received one or two doses of MMR vaccine. In our experience using a plaque neutralization assay, we have only seen a 4-fold rise in neutralizing antibody titer between paired samples in persons who were also IgM positive.
Q: What method can be used to demonstrate a 4-fold rise in IgG titer between properly spaced serum samples?
A: In order to measure a 4-fold rise in titer, it is recommended that laboratories use an established assay or test protocol that provides a quantitative or semi-quantitative measure of antibody. Moreover, it is recommended that laboratories pay close attention to the parameters of the testing protocol to make certain that the acute- and convalescent-phase serum specimens fit the criteria of the protocol in terms of both timing of collection and parameters of data analysis. When a qualitative EIA is used for IgG titer determination, it is essential to do end-point titrations to determine if there is a 4-fold or greater difference in titers between acute- and convalescent-phase serum samples.
Mumps Viral Testing
Q: Why should I ask for buccal or oral swab specimens in addition to the serum sample?
A: Buccal and 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. Because serologic tests cannot differentiate between an exposure to vaccine and an exposure to wild-type mumps virus, it is necessary to obtain other clinical samples to determine the genotype (wild-type vs. vaccine).
Q: What does a mumps-negative RT-PCR result mean?
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%–35% of case-patients if the samples were collected within the first 3 days following onset of parotitis; IgM was detected in 13%–15% of these cases (Bitsko et al. 2008; Rota et al. 2009).
Q: What does a mumps-positive RT-PCR result mean?
A: A positive RT-PCR signal indicates the presence of mumps virus RNA in the patient sample. The positive result should be used only to support a clinical diagnosis of mumps. A positive RT-PCR result provides laboratory confirmation of mumps infection in persons with symptoms consistent with mumps who have not been vaccinated within the preceding 45 days.
Q: Why attempt to isolate mumps virus in cell culture?
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 can help to identify the source of the virus and can provide confirmation of suspected epidemiologic links. In addition, virus isolation is less likely than PCR assays to give false-positive results due to contamination.
Q: What is a mumps genotype?
A: Mumps strains are assigned to 1 of 12 genotypes on the basis of the sequence of the gene coding for the SH (short hydrophobic) protein. In some cases, a genotype has been associated with endemic circulation of mumps virus in a country or region; however, routine virologic 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. The Jeryl-Lynn vaccine strain is a member of genotype A, so identification of genotype A suggests an association with recent vaccination.
Q: What is the gold standard for laboratory confirmation of mumps?
A: Virus culture is the gold standard for mumps confirmation. However, sample quality must be maintained to ensure viability of the virus. Laboratories are strongly encouraged to perform cell culture isolation of mumps from buccal or oral swab specimens. Primary monkey kidney cells and Vero cells are frequently used to isolate virus. Detection of mumps in culture can be done using immunofluorescent antibody staining or standard RT-PCR.
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 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 will help to identify the source of the virus and can provide confirmation of suspected epidemiologic links.
Q: Where can I find RT-PCR and real-time RT-PCR protocols for mumps?
A: The following protocols are available online:
- Real-time RT-PCR protocol, targeting the nucleoprotein (N) gene (120 KB, 8 pages)
- Standard RT-PCR for mumps virus (61 KB, 6 pages)
Q: Where do I obtain material for RT-PCR and virus isolation controls?
A: CDC can provide a sample of RNA that has been purified from cells infected with mumps virus. This material is ready to use in RT-PCRs with any set of primers. If laboratories would like to produce their own RNA samples or require a positive control for virus isolation, CDC can provide a sample of wild-type mumps virus. Public health laboratories or laboratories affiliated with state public health laboratories may send request for mumps RNA or virus to firstname.lastname@example.org.
- Bitsko RH, Cortese MM, Dayan GH, Rota PA, Lowe L, Iversen SC, Bellini WJ. Detection of RNA of mumps virus during an outbreak in a population with a high level of measles, mumps, and rubella vaccine coverage. J Clin Microbiol 2008;46:1101–3.
- Briss PA, Fehrs LJ, Parker RA, Wright PF, Sannella EC, Hutcheson RH, Schaffner W. Sustained transmission of mumps in a highly vaccinated population: assessment of primary vaccine failure and waning vaccine-induced immunity. J Infect Dis 1994;169:77–82.
- Davidkin I, Jokinen S, Paananen A, Leinikki P, Peltola H. Etiology of mumps-like illnesses in children and adolescents vaccinated for measles, mumps, and rubella. J Infect Dis 3005;191:719–23.
- Gut JP, Lablache C, Behr S, Kirn A. Symptomatic mumps virus reinfections. J Med Virol 1995;45:17–23.
- Krause CH., Molyneaux PJ, Ho-Yen DO, McIntyre P, Carman WF, Templeton KE. Comparison of mumps-IgM ELISAs in acute infection. J Clin Virol 2007;38:153–6.
- Narita M, Matsuzono Y, Takekoshi Y, Yamada S, Itakura O, Kubota M, Kikuta H, Togashi T. Analysis of mumps vaccine failure by means of avidity testing for mumps virus-specific immunoglobulin G. Clin Diagn Lab Immunol 1998;5:799–803.
- Sakata H, Tsurudome M, Hishiyama M, Ito Y, Sugiura A. Enzyme-linked immunosorbent assay for mumps IgM antibody: comparison of IgM capture and indirect IgM assay. J Virol Methods 1985;12:303–11.
- Sartorius B, Penttinen P, Nilsson J, Johansen K, Jönsson K, Arneborn M, Löfdahl M, Giesecke J. An outbreak of mumps in Sweden, February–April 2004. Euro Surveill 2005;10 (9):pii=559. Available at http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=559
- Rota JS, Turner JC, Yost-Daljev MK, Freeman M, Toney DM, Meisel E, Williams N, Sowers SB, Lowe L, Rota PA, Nicolai LA, Peake L, Bellini WJ. Investigation of a mumps outbreak among university students with two measles-mumps-rubella (MMR) vaccinations, Virginia, September–December 2006. J Med Virol 2009;81:1819–25.
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