RFFIT Tests

About the RFFIT Test

Current Turnaround Time: RFFIT test results are normally available in 3 to 4 weeks from the date that the sample is received here at the Kansas Rabies Laboratory.

RFFIT stands for Rapid Fluorescent Foci Inhibition Test. It is a serum neutralization (inhibition) test, which means it measures the ability of rabies specific antibodies to neutralize rabies virus and prevent the virus from infecting cells. These antibodies are called rabies virus neutralizing antibodies (RVNA).

In RFFIT, the serum (the non-cellular portion of blood) is first diluted fivefold (1 part serum in 4 parts diluent). Further (serial fivefold) dilutions are performed, each of which contain less and less serum. These serum dilutions are mixed with a standard amount of live rabies virus and incubated. Whatever RVNA is present will neutralize the virus. Next, cultured cells are added and the serum/virus/cells are incubated together. Whatever rabies virus is left (i.e., that which has not been neutralized by the RVNA), will infect the cells and this can be seen under the microscope through the use of specific staining. Calculation of the endpoint titer is made from the percent of virus infected cells observed on the slide.

We provide both the World Health Organization (WHO) guideline of 0.5 International Units per milliliter (IU/mL) as an adequate response to rabies vaccination, as well as the ACIP level of “complete neutralization at a serum dilution of 1:5 in the RFFIT test” as evidence of adequate response in a person on our RFFIT antibody test reports. We provide both guidelines to better assist individuals and healthcare providers in their decision-making about how to interpret our tests results in light of vaccination status and the possible necessity of booster vaccination(s).

Frequently Asked Questions

Animal Result Interpretations

What is measured?
Rabies Virus Neutralizing Antibodies (RVNA) are antibodies with the ability to neutralize rabies virus and prevent the virus from infecting cells. In the test, serum (the non-cellular portion of blood) is first diluted in a diluent (e.g. 1 part serum in 4 parts diluent). Further (serial fivefold) dilutions are performed, each of which contain less and less serum. These serum dilutions are mixed with a standard amount of live rabies virus and incubated. Whatever RVNA is present will neutralize the virus. Next, some tissue culture cells are added and the serum/virus/cells are incubated together. Whatever rabies virus is left (i.e., that which has not been neutralized by the antibody in serum), will infect the cells and this can be seen under the microscope through the use of specific staining. Calculation of the endpoint titer is made from the percent of virus infected cells observed on the slide.

What does the result mean?
The result of this test can be expressed in two ways: as a Rabies virus neutralizing antibodies (RVNA) endpoint titer (e.g., 1:50) or as a value for RVNA potency (e.g., 0.5 IU). The IU stands for international unit and is calculated from the titer by comparing it against the titer of a standard reference serum. We use the following formula: sample titer divided by the reference serum titer, multiplied by the IU/mL value of the reference serum.

Example: The sample titer is 1:100, the reference serum titer is 1:200, and the value of the reference serum is 2.0 IU/mL. Using the formula: (100/200) X 2.0 IU/mL = 1.0 IU/mL the potency of RVNA in the sample serum is therefore 1.0 IU/mL.

Because the RFFIT test is a biological system using live cells, infectious virus, and antibodies, the reference serum can vary in titer level for each batch of testing (within an established acceptable range). Therefore the calculation of IU/mL depends on the titer of the reference serum measured in the batch tested. In general, you can take the titer value divided by 100 to get a rough estimate of the IU/mL value. To obtain the exact value you must use the calculation with the measured reference serum titer value.

What does the level of Rabies virus neutralizing antibodies (RVNA) mean?
When we measure antibodies to rabies virus in serum (the liquid part of blood), this measurement provides an indication of an immune response to rabies. The test is not able to differentiate whether the immune response is in response to rabies virus exposure or to rabies vaccination. Titer levels or IU/mL values equal to or above 0.5 IU/mL (~1:50), provide evidence of a robust immune response after rabies vaccination. Although the presence of antibodies after vaccination is important, a specific level above 0.5 IU/mL does not give complete assurance of protection against the disease in every individual because there are other immunological factors which are involved in the protection from rabies infection. An overview of rabies challenge studies indicates RVNA levels predict survival on more a qualitative rather than quantitative basis [1]. Protective levels can differ per species and per assay used for measurement [2]. Currently, whether an animal requires a rabies booster vaccination is not determined by the level of RVNA in the serum, but by local regulations [3]. Regulations are routinely under review with consideration of new information. Kansas State University is part of this process and is working to influence change that will provide the best guidelines for rabies booster requirements.

References

Aubert MF. Practical significance of rabies antibodies in cats and dogs. Rev Sci Tech 1992;11:735-60.
Moore SMG, A.; Vos, A.; Freuling, C.M.; Ellis, C.; Kliemt, J.; Muller, T. Rabies Virus Antibodies from Oral Vaccination as a Correlate of Protection against Lethal Infection in Wildlife. Tropical Medicine and Infectious Disease. 2017;2(31). Epub 21 July 2017 http://www.mdpi.com/2414-6366/2/3/31
Centers for Disease Control and Prevention. Compendium of animal rabies prevention and control., National Association of State Public Health Veterinarians, Inc. (NASPHV), 2016 - Link

Human Result Interpretations

What is “the RFFIT”?
The acronym RFFIT stands for Rapid Fluorescent Focus Inhibition Test. The test measures the ability of antibodies that may be present in a sample to neutralize and block rabies virus from infecting the cells used in the test. These antibodies are called rabies virus neutralizing antibodies (RVNA). In the test, serum (the non-cellular portion of a blood sample) is first diluted fivefold (1 part serum in 4 parts diluent). Further (serial fivefold) dilutions are performed, each of which contain less and less of the sample. The serum dilutions are mixed with a standard amount of live rabies virus and incubated. If RVNA are present in the sample, they will bind to the virus. Tissue culture cells are then added and incubated with the test sample and virus. Whatever rabies virus that may not have been neutralized by the antibody in the sample will then infect the cells. These foci of infection in the cells can then be seen under the fluorescent microscope. If there are a lot of infected cells, there is very little antibody; conversely, if antibodies are high, the cells will have very little evidence of infection. The endpoint titer is calculated from the percent of virus-infected areas observed within the wells containing the various dilutions of the sample on the slide.

What does your result tell you?
The RFFIT result can be reported in either a titer which is a ratio (e.g., 1:50) or as a standardized concentration represented as international units (IU) per mL of serum (e.g., 0.5 IU/mL). The IU value is calculated from the titer by comparing it against a standard reference serum. We use the following formula: sample titer divided by the reference serum titer, multiplied by the IU/mL value of the reference serum.

According to World Health Organization guidelines, a rabies antibody level of greater than or equal to 0.5 IU/mL demonstrates an adequate response to vaccination (1). If the level falls below this value, a booster dose of rabies vaccine may be recommended for people who are at frequent risk of rabies virus exposure. In contrast, the ACIP guidelines state that evidence of complete neutralization at a serum dilution of 1:5 in RFFIT testing (corresponding to 0.1-0.2 IU/mL in our laboratory) is considered an adequate response to rabies vaccination (2). The lowest antibody level that can be accurately and precisely measured by the RFFIT in our laboratory is 0.1 IU/mL; below this level, there is uncertainty as to the specificity of the result. Because the ACIP level is close to the assay threshold, the level of 0.5 IU/mL is more conservative for guiding human vaccination decisions and applicable in most situations.

Points that should be considered as to whether a person should receive a booster dose of rabies vaccine when their antibody level falls below 0.5 IU/mL are:

Anticipated risk of exposure (i.e., routinely handling sick animals or rabies reservoir species in enzootic areas)
Length of time until the next antibody measurement
Previous rabies antibody levels and the probability of decay to low or undetectable levels in the intervening period
Individual health status (consider immunocompromising conditions or a history of poor vaccine response)
Timely access to vaccine and administration should a potential exposure occur

For further information, see “Level of rabies protection”.

References
ACIP Recommendations

Prospective Serological Monitoring

For Prospective Serological Monitoring (PSM) per the Compendium for Animal Rabies Prevention and Control recommendations to evaluate a rabies exposed dog or cat for previous rabies vaccination, contact the laboratory directly (785)532-4483 or at rabies @vet.k-state.edu for sample submission information and form.