CDC Report on Missouri H5N1 Serology Testing | Bird Influenza

Topline summary of survey results

In August 2024, health care workers in Missouri saw a patient with gastrointestinal symptoms who had a history of chronic respiratory disease. The person was hospitalized and tested for multiple respiratory pathogens, which tested positive for influenza A. He recovered from his illness and was discharged from the hospital. Follow-up testing was conducted at the Missouri Department of Health and Senior Services Public Health Laboratory using polymerase chain reaction (PCR), which is part of routine national influenza surveillance. Testing revealed that the patient had a presumptive positive test for avian influenza A(H5) (“H5 avian influenza”). There was no history of contact with infected animals or humans. The specimen was transferred to the CDC as part of routine protocol. CDC confirmed this specimen to be H5N1 avian influenza and reported it as a case on September 6, 2024.

Missouri state and local officials conducted a long-term retrospective investigation of all close contacts of the patient, including seven individuals (including six health care workers) who exhibited symptoms that warranted further investigation due to possible exposure to the patient. 1 person and 1 family member) were identified. CDC is supporting this investigation and testing blood samples from six of these people (and the original patient) to identify signs of previous infection or exposure to H5N1 after symptoms have fully resolved. A series of tests were conducted. This was not possible for the others, whose suggestive symptoms had completely resolved at the time of the interview. None of the results of this extensive testing and investigation support that human-to-human transmission has occurred. None of the healthcare workers tested showed any signs of previous H5N1 infection. They were all “seronegative.” This finding excludes person-to-person transmission between MO case patients and tested healthcare workers. Both the Missouri case and the household contacts have some, although conflicting, evidence suggesting exposure to H5N1 or previous infection with H5N1 using these serological tests. There was.

To date, no human-to-human transmission of H5 avian influenza has been confirmed in the United States. CDC believes that although the immediate risk to the general public from H5N1 avian influenza remains low, the risk of infection for people who have come into contact with infected animals is high.

Following these tests, CDC continues to assess that the risk of H5N1 infection for people who have not had contact with infected animals remains low.

Background of serological testing methods:

Serological tests for H5N1 are retrospectively identified after sufficient time has elapsed for the immune system to produce antibodies, and are tested on serum samples from a single point in time from previously symptomatic exposed individuals. It was carried out against. These tests assess whether antibodies produced by a patient or a patient's close contacts can neutralize or bind to the H5N1 virus. If they can neutralize or bind the virus, it suggests a previous infection or exposure to the virus.

Samples were collected by state and local authorities from:

• 1 H5 case confirmed (reported) September 6, 2024) People who test positive for H5 by PCR through regular influenza surveillance.
• One household contact of this case was unable to be tested at the time because his symptoms had resolved.
• Five healthcare workers who may have been exposed to the infected person in the course of their work did not get tested until their symptoms subsided.

Note that there was also one previously identified healthcare worker exposed to a confirmed H5 case. This person was tested for influenza at the time and was found to be negative, so no serology was performed on this person. The amount of virus detected in clinical specimens from confirmed cases in Missouri was so low that it was not possible to recover the complete genome sequence of the virus. The CDC was able to obtain some sequence data from the few viruses present in clinical samples. The sequence data, published as “A/Missouri/121/2024”, is a hemagglutinin (HA) compound that can influence how a patient's immune system responds to infection (antigenic). ). These antigenic changes may mean that antibodies generated by a person's immune response after exposure to this virus may not bind to H5N1 viruses isolated from other H5N1 cases used in the CDC's H5 serology test. It meant something.

To ensure accurate serological results, CDC experts developed two reverse genetics (RG) viruses with the same antigenic changes recorded in the A/Missouri/121/2024 HA sequence . These viruses took three weeks to create. CDC then used these RG viruses to test blood samples from seven people in Missouri for immune responses to specific viruses that could indicate exposure/infection.

Humans already have a complex immunity to influenza viruses. Public health surveillance of potential influenza infection recommends blood specimens collected at two different time points for serological testing to assess the presence of infection. One sample is taken within seven days of onset of symptoms, and the second sample is taken at least 21 days after the patient has recovered. After the onset of symptoms (ideally 21-28 days). The increase in antibodies measured between the two specimens provides conclusive evidence of an immune response to the infection. In the Missouri study, only one specimen was collected several weeks after the first case was identified. Given the retrospective nature of this study, serological analysis in this setting is further complicated.

Inspection method:

Currently, there are no commercially available H5N1 serology tests as they have no clinical role in patient care. CDC primarily conducts influenza serology testing to inform public health research and policy. Unlike clinically available serology for other infectious diseases, non-clinical serology testing for seasonal influenza viruses takes approximately 1 week. H5 serology tests require additional biosafety level 3 enhanced (BSL-3E) processing and therefore take longer. Testing in this particular situation required additional steps to ensure valid results. Tests were performed against four different target viruses. The two RG viruses developed for this study (above), the H5N1 virus isolated from the first human case during this outbreak (posted as “A/Texas/37/2024”), and the seasonal A(H1N1 )pdm09 virus. Both RG viruses developed by the CDC were used in the test to provide additional assurance regarding the validity of the results.

The following tests were conducted in multiple iterations.

• Microneutralization (MN) assay: The main serology test is called the microneutralization (MN) test. This is a sensitive and specific test for identifying influenza virus-specific antibodies in serum. MN assay was performed to measure neutralizing antibodies against RG H5 virus.
• Hemagglutinin inhibition (HI) assay: The HI assay uses red blood cells (RBCs) to test for antibodies against the hemagglutinin of a specific influenza virus. Multiple types of RBC were evaluated in the HI assay to detect antibodies in serum.
• Multiplex Antibody Detection Assay (MIADA): A test that looks for antibodies that bind to 28 different influenza antigen targets (28-plex) and also measures total immunoglobulin (Ig), IgG, IgM, and IgA antibody responses.
• Seasonal Influenza Serum Absorption: Prior to testing for the A(H5N1) virus, an additional step was also performed to remove potentially cross-reactive antibodies to seasonal influenza viruses. This step helps eliminate the possibility of cross-reactivity due to previous exposure to seasonal influenza viruses (through infection or vaccination), which could result in false-positive H5N1 serology test results.

Serological findings and interpretation:

CDC and historic WHO criteria for H5N1 seropositivity indicative of infection require two positive tests: the MN assay and either HI or another immunological assay such as MIADA (see end note) .

• All five healthcare workers in contact with the MO case patient were seronegative using MN, HI, and MIADA assays. this means they did do not have There is serological evidence of past infection with H5N1 avian influenza.
• The Missouri case and her household contacts had some evidence of a humoral immune response to H5N1 avian influenza virus, suggesting possible infection, but testing did not detect it overall.
  • The MN assay detected neutralizing antibodies against RG virus in the index patient and his family contacts. (Neutralizing antibodies are antibodies that protect cells from pathogens by interfering with the cell's ability to infect.)
  • HI testing was negative for both this patient and his family.
  • Neither MO's case nor her household contacts tested positive for MIADA.

Conclusions of the Missouri study:

Five HCWs had unequivocally negative serology for H5 on all tests. This means they are not infected with H5. This finding excludes human-to-human transmission between the patient with the index MO case and any of the five health care workers. Symptoms reported by these contacts were not caused by contact with this patient.

Test results for sera from MO case patients and their household contacts were similar, with both showing evidence of an antibody immune response to H5 in only one assay (detecting H5-neutralizing antibodies), but No other serological assays used to detect . The weak immune signal suggests that both of these people may have been exposed to H5 avian influenza despite not meeting the accepted threshold for seropositivity. These similar immunological results and the epidemiological data of the same date of onset of symptoms in these two individuals suggest a single common avian influenza exposure rather than person-to-person transmission within the household. I support it. Intensive epidemiological studies have not identified exposures to animals or animal products that account for the potential for these infections, and these serological data do not further elucidate the exposures that may cause these infections. You can't.

End note:

World Health Organization historical serological case definition using a single serum

A single convalescent serum collected at least 21 days after onset of symptoms (or last exposure to an infected animal/person). Microneutralizing antibody titer ≥ 1:40 For A(H5) virus,

and Either

• 1) Positive results using different serological assays (e.g. Hemagglutination inhibition (HI) antibody titer ≥40) or
• 2) H5-specific positive result from another immunoassay such as ELISA Multiple binding antibody assay.