Wilkes RP, Anis E, et al. Rapid and sensitive insulated isothermal PCR for point-of-need feline leukemia virus detection. J Feline Med Surg. 2018 Apr;20(4):362-369.
Feline leukemia virus is a gamma retrovirus that is known to cause various malignant, proliferative and degenerative disorders of the feline hematopoietic system. The usual result of infection by the virus is what is referred to as a regressive infection where cats clear infectious virus from the blood but retain proviral DNA in infected cells and their progeny. In other alternative situations as a progressive infection, FeLV replication is unchecked, and infectious virus or viral RNA can be recovered persistently from the bloodstream. Cats that are progressively infection are a major source of infection to other cats and most die within three years from a related hematopoietic disorder.
The article states that the prevalence of FeLV is in decline worldwide through widespread use of effective vaccines as well as test and removal policies. Accurate and quick identification of FeLV-infected cats at point-of-need (PON) can support management strategies leading to virus eradication.
The most common rapid screening tests used at PON are immunoassays that detect circulating p27 antigen. Due to the declining prevalence of FeLV and consequent increase of false-positive results and the significance of such a positive result, recommendations are that antigen-positive samples are tested again using a second methodology. What are other options for testing? Virus isolation (VI), considered the gold standard in FeLV infection diagnosis, is not readily available and also is expensive. Molecular assays such as RT-qPCR and qPCR are considered reasonable alternatives.
RT-qPCR assays detect viral RNA which is considered to correlate well with viremia and progressive infection. qPCR assays detect proviral DNA that integrates into the genome of infected cells. In both instances of regressively and progressively infected cats, proviral DNA is harbored and quantitative assays can help in distinguishing between the populations. These molecular assays are recommended to detect FeLV-exposed cats, to investigate obscure clinical cases and to screen potential blood donors. Currently this method of testing is restricted to commercial labs and the authors concur that a rapid, affordable and user-friendly platform is needed for PON detection of FeLV viral RNA or proviral DNA.
The investigators in this study evaluated the performance of two systems developed commercially for FeLV detection at PON: an insulated isothermal PCR (iiPCR) that detects proviral DNA and a reverse transcription (RT)-iiPCR that detects both viral RNA and proviral DNA. These reagent sets were assessed in this study by testing significant feline pathogens (M haemofelis, FCoV, FHV, FCV, and FIV).
In reporting their results, the authors state both FeLV iiPCR and RT-iiPCR performed similarly to the reference qPCR and VI when testing clinical samples with few discrepancies. With the RT-iiPCR methodology, one can consider testing different blood fractions to assist in distinguishing progressively and regressively infected cats – i.e. testing serum for viral RNA and testing buffy coat for both viral RNA and proviral DNA. A positive result on serum would suggest a progressive infection.
Testing for FeLV status is recommended before introduction of cats into group housing, before administration of the FeLV vaccine and before blood donation. As the authors state that while regressively infected cats appear to have a low risk of developing FeLV-related disease, and have been considered to not pose a significant infection risk to healthy cats, more recent studies have determined that regressive infections can be reactivated to resume virus replication, shedding and disease.
While p27 antigen immunoassays serve as an initial screening test at patient side, these tests cannot identify cats with regressive infection, and a portion of p27-negative cats are regressively infected. Also, FeLV RNA could be detected in the circulation or tissues of some regressively infected cats, even in absence of detectable proviral DNA.
Therefore the authors state that the FeLV RT-iiPCR, with an excellent sensitivity for both RNA and DNA in its template, is the recommended method to identify all FeLV-exposed cats at patient side, but with the preferred buffy coat sample.
Integrated FeLV proviral DNA cannot be eliminated by the host’s immune system. Both progressively and regressively infected cats will test positive for proviral DNA using the iiPCR test methodology. However, this test method could work with the p27 immunoassay as a patient-side tool to aid in FeLV infection confirmation and diagnosis. This combination of a proviral PCR method and p27 antigen immunoassay have shown to achieve 100% diagnostic sensitivity.
p27-positive + proviral DNA (iiPCR positive results) = progressive infection
p-27-negative + proviral DNA (iiPCR positive results) = regressive infection (with potential for reactivation).
Final conclusions are that both the RT-iiPCR and proviral iiPCR/POCKIT systems could help rule out prior exposure to FeLV at point-of-need such as veterinary clinics, shelters and customs inspection at ports of entry. (VLT)
Wilkes RP, Kania SA, et al. Rapid and sensitive detection of feline immunodeficiency virus using an insulated isothermal PCR-based assay with a point-of-need PCR detection platform. J Vet Diagn Invest. 2015 Jul;27(4):510-5.