National Reference Center for Measles, Rubella and Mumps Viruses

National Reference Centers ()are NRCs expert laboratories in the microbiology of communicable diseases . They are appointed by decree of the Ministry of Health for a period of four years. Their renewal is subject to an evaluation by a NRC Committee under the responsibility of the Director General of the French Institute for Public Health Surveillance (InVS).

It is responsible, in close collaboration with Public Health France (SpF), for contributing to the diagnosis and epidemiological surveillance of human infections caused by the measles virus (Measles virus, MeV), the human respiratory syncytial virus (hRSV, RSV), the human metapneumovirus (hMPV), the 4 human parainfluenza viruses (hPIV) and the mumps virus (MuV, mumps virus).

Virological diagnosis of measles generally accompanies the "DO" (Mandatory Measles Reporting) process. It is based either on direct diagnosis: detection of the virus or its components (RNA, antigens), or on the detection of antibodies: indirect diagnosis or serodiagnosis.

The samples:

• The saliva sample is collected by the physician using a saliva kit provided by the ARS), which includes the collection system (salivatte), transport container (box and envelope), and information (clinical identification). The sample allows for the detection of the viral RNA genome and antiviral antibodies in cases of negative RNA. It is then sent by mail to the National Reference Center (CNR).
• In a hospital setting, a nasal or nasopharyngeal swab on virological transport medium is recommended. This allows for the detection of viral RNA.
• Blood sampling is most often used to test for measles virus antibodies (serology). This is currently the recommended approach in community medicine (see Recommendations Sheet). Peripheral blood is collected in a dry tube. This is not necessary if a saliva sample has already been taken. The presence of the virus in the blood (viremia) can be detected in an EDTA blood tube.
• In certain complications, other samples are useful: tracheobronchial secretions, urine, cerebrospinal fluid…

The techniques:

• The basic technique used for virus detection is the search for viral RNA using a molecular technique: real-time RT-PCR (reverse transcription polymerase chain reaction). In the event of a positive detection, the viral genotype is identified by sequencing a portion of the viral genome.
• Isolation of measles virus strains is carried out at the CNR on Vero Slam cell cultures. It is indicated for the study of certain viral strains.
• The detection of measles virus antibodies, of the IgG and IgM classes, is routinely performed using commercial enzyme-linked immunosorbent assays (ELISAs). The detection of neutralizing antibodies in cell culture (protective antibodies) is possible, but more complex. Above a certain level, they correlate with the antibody levels detected by ELISA.

Response, interpretation and transmission times for results:

• Viral RNA is detectable in approximately 4-5 hours using RT-PCR. This indicates an ongoing viral infection. It is present in saliva or nasopharynx during the invasion and eruptive phases. Detection in the blood (viremia) is early and transient. The presence of the virus in urine can persist for a few days.
• Isolating the virus in culture requires a minimum of one week of culture.
• Measles virus antibodies can be measured by ELISA in approximately 2 hours. The presence of IgM and IgG antibodies indicates infection (except in cases of recent measles vaccination). IgM and IgG antibodies appear with the rash; IgM antibodies persist for about 2 months, and IgG antibodies for a much longer time
• Any transport of samples to the CNR may cause false negative results in tests for the detection of RNA, virus or IgM antibodies.

The results of the tests carried out at the CNR are immediately transmitted to the prescribing physician or biologist, who, within the framework of the DO, must transmit them to the local ARS

For all viruses (RSV, hMPV, hPIV, MuV, NDV), the recommended sample is a collection of nasal secretions:

• They are collected by firm swabbing of the anterior part of the nasal cavities or by aspiration using a pump. They must be placed in a specific medium (Virological Transport Medium) and sent to the CNR.
•   Other samples can be used: tracheobronchial secretions in intubated patients, CSF: mumps.
• Blood sampling is unnecessary in RSV , hMPV, hPIV, and NDV infections (there is no viremia, and specific antibodies appear with a delay). It is useful in mumps: detection of IgM and IgG antibodies by ELISA in cases of primary infection.

    The techniques used for direct searching:

• The detection of RSV, hMPV and hPIV1,2 and 3 antigens is performed by immunofluorescence on a smear of nasal secretions.
• Viral isolation is possible on MRC5 cells for RSV, and on certain human or animal continuous cell lines for hPIV, hMPV, and MuV. This technique is not recommended for diagnosis.
• Viral RNA detection is performed by RT- PCR for all viruses: RSV , hPIV, hMPV, MuV, NDV. Depending on the case, these are real-time or conventional RT- PCR tests , uni- or multiplex

    Response, interpretation and transmission times for results:

• The direct immunofluorescence test provides a result in approximately 60 minutes. It may appear negative if the sample was taken late, if it contains few cells, or was transmitted under poor conditions.
• Viral RNA is detectable in approximately 4-5 hours using RT-PCR, and in 24 hours using conventional RT-PCR . It indicates an ongoing viral infection. For all viruses, it is always present during the acute phase of the illness and can persist for several days after recovery.

The results of the tests carried out at the CNR are transmitted directly to the prescribing physician or biologist.

• The CNR contributes to the knowledge of seroprevalence and the level of protection of the population against measles, mumps, RSV , hHMPV, hPIV and NDV viruses through serological surveys.
• The CNR ensures the epidemiological tracing of infections linked to these different viruses occurring in communities (identification of the index case, nosocomial epidemics in hospital services, epidemics in communities of elderly people, etc.) through the use of specific molecular tools.
• The CNR establishes and maintains a strain library including reference strains, strains representative of successive epidemics and strains isolated from serious or particular pathologies.

• The CNR ensures the maintenance and dissemination of reference biological material: viral strains, cell lines, positive RNA controls.
• He is involved in the development, evaluation and dissemination of diagnostic techniques, both commercial and academic.
• He participates in university and hospital teaching on measles, mumps, RSV, hMPV and hPIV viruses.
• It contributes to the training of foreign biologists by hosting them for internships or by associating them with the transfer of technologies.

The CNR is part of the European laboratories accredited for the diagnosis of measles "Euro Measles/Rubella laboratory Network", by theWHO.

The CNR has established a regular collaboration with three organizations:

• InVS : bi-monthly sending of epidemiological information: number of strains, genotypes.
• “European Regional Reference laboratory for Measles and rubella”, at the Institute of Immunology (Luxembourg): sending of viral sequences for comparison with other European genotypes.
• “WHO Regional Office for Europe, Communicable Disease Unit” in Copenhagen: sending epidemiological information.

The National Reference Centre (CNR), located in the Human and Molecular Virology Laboratory of the Caen Normandy University Hospital, is open Monday to Friday from 8 a.m. to 5 p.m.Samples can be received on Saturday mornings from 8 a.m. to 12 p.m.

Viral RNA testing is performed every working day.

The information sheet accompanying the sample must include, in addition to administrative information (surname, first name, age, sex, etc.), the following information:

• the name, address, telephone number and email address of the prescribing physician,
• the date the rash began and the date the saliva sample was taken,
• an overall assessment of the severity of the disease: typical measles, mild measles, complicated measles,
• The vulnerable population: infants, pregnant women, immunocompromised individuals,
• the notion of childhood vaccination (1 or 2 doses) or recent vaccination (1 to 4 weeks), or recent travel,
• the existence or not of clustered cases and/or an identified contact,
• The result can be expected within 24-48 hours of receiving the sample. It is immediately sent by fax or email to the prescribing physician or laboratory. A postal shipment, including the viral genotype in the case of a positive RNA test, follows a few days later
• Viral genotype identification by sequencing is performed twice a week.