A highly reliable method of diagnosis is virus isolation. Peripheral blood lymphocytes are prepared from fresh samples of heparinised blood and are co-cultivated with primary feline T cells for 2-3 weeks and the presence of virus in cultures is confirmed by measuring the levels of viral core proteins in the culture fluids. The procedure is laborious and is not used routinely.
Polymerase chain reaction (PCR)-based assays that detect proviral DNA, are available. However, it has been shown that such PCR tests are variable in performance and may in some cases be inferior to serological tests [Bienzle et al. 2004, Levy et al. 2004, MacDonald et al. 2004], with sensitivities and specificities ranging from 40 to 100%; PCR assays currently available detect clade A viruses well, but the other strains more variably. Strain variation may also explain discrepant results when identical samples are sent to different labs [Crawford et al. 2005 and 2007]. Discrepant results may also occur when serology and PCR are compared (seropositive, PCR negative), and may be explained by the presence of an FIV subtype not recognised by the PCR, rather than by the absence of FIV infection. This aspect is important when a cat may have been vaccinated against FIV. However, discrepant results (seronegative, PCR positive) may also be found: cats living in close contact with FIV-infected seropositive cats can become provirus positive without developing detectable levels of serum antibodies or disease [Dandekar et al. 1992]. These cats are infected and in most cases will seroconvert weeks to months later.
Routine tests for FIV infection detect antibodies recognizing viral structural proteins (such as the capsid protein p24 and a gp41 peptide) and may take the form of ELISA and immunochromatography tests. Western blot are considered the “gold standard” for FIV serology and are used to confirm questionable results.
In-house tests based on ELISA detect anti-FIV antibodies and are based on p24 and the transmembrane antigen [communication from Idexx, March 2008]. In contrast, immunochromatography tests only detect antibodies to short peptides corresponding to the transmembrane protein. In Western blots purified FIV is separated by gel electrophoresis into its constituent proteins. This allows the detection of antibodies to each individual FIV protein [Lutz et al. 1988a].
Both ELISA and immunochromatography tests are generally appropriate in most
situations, but do have their limitations because the diagnostic specificity of the commonly used test is below 100% which is especially important in low prevalence populations and when healthy cats test positive: for example, an FIV prevalence of 1% results in one positive test per 100 cats and a diagnostic specificity of 99% also results in one false positive in the same 100 cats. This gives two positive results in 100 cats only one of which is correct (positive predictive value equal to only 50%). Any positive result in a low prevalence population (e.g. young, indoor, pure bred cats) must therefore be confirmed e.g. by Western blot. A positive result in a cat from a high-risk group (e.g. a free roaming, aged, entire male) is likely to be a true positive because the frequency of true positives will exceed the frequency of false positives in this population. In contrast, negative results in low prevalence populations are generally very accurate, with the following exceptions. False negative results may be obtained early in infection, when cats become provirus positive but remain seronegative for several weeks to months. In addition, false negative results may be also obtained in the terminal stages of disease due to immunodeficiency and when high viral titres may lead to sequestration of anti-FIV antibodies in virus-antibody complexes.
Kittens born to FIV-infected queens may test seropositive as a result of passively acquired maternal antibodies (MDA). In such cases, kittens should be retested after approximately 16 weeks of age, by which time in most cases levels of MDA will have declined to undetectable levels so that a positive result is indicative of FIV infection in the kitten. However in rare cases antibodies may persist up to six months [Levy et al. 2003]. Therefore, a kitten testing seropositive at 16 weeks-of-age should be retested two months later. If it is still positive at six months it is infected. If an earlier result is required, PCR may be employed to detect virus negative kittens: in such cases, it is important that the queen is tested in parallel to ensure that the PCR can detect the infecting strain.
Vaccination of cats against feline immunodeficiency virus (FIV) with an inactivated, whole virus vaccine results in rapid and persistent production of antibodies that are indistinguishable from those used for diagnosis of FIV infection. Such vaccines are available in the USA but are not licensed in Europe and should therefore not be used. However, vaccinated cats may be imported. The diagnostic tests available at present do not distinguish vaccinated cats from infected cats, or from cats that are both vaccinated and infected [Richards 2005].
In research situations it is possible to stage the level of immune dysfunction by determining the number of CD4+ and CD8+ lymphocyte counts. However, due to the complexity of these assays and the fact that in a clinical situation pre-infection values are not available, means that these tests are not currently clinically useful.