Detection of Feline Leukemia Virus RNA in Saliva from Naturally Infected Cats and Correlation of PCR Results with Those of Current Diagnostic Methods

Abstract

A novel diagnostic test for feline leukemia virus (FeLV) RNA in saliva from naturally infected cats is described in this study. We evaluated different diagnostic tests and compared them with the widely used enzyme-linked immunosorbent assay (ELISA) for the detection of p27 in the diagnosis of FeLV. Blood samples from 445 cats were tested for the presence of provirus by real-time PCR and plasma and saliva specimens from those cats were tested for the presence of viral RNA by real-time reverse transcription (RT)-PCR and for the presence of p27 by ELISA. In comparison to conventional ELISA, the diagnostic sensitivity and specificity of the detection of salivary FeLV RNA by real-time RT-PCR were found to be 98.1 and 99.2%, respectively. Detection of viral RNA in saliva had a positive predictive value of 94.6% and a negative predictive value of 99.7%. The kappa value was 0.96, demonstrating an almost perfect agreement between both tests. Furthermore, we confirmed previous results showing that a number of cats which tested negative for the presence of p27 in plasma were in fact positive for the presence of DNA provirus in blood specimens (5.4%). However, 96.4% of these latently infected cats did not shed viral RNA in saliva; therefore, we assume that these cats are of relatively low clinical importance at the time of testing. This study shows considerable diagnostic value of the detection of saliva FeLV RNA in naturally infected cats. This new diagnostic method has advantages over the conventional ELISA, such as less invasive sample collection and no requirement for trained personnel.

Development and application of a quantitative real-time PCR assay to detect feline leukemia virus RNA

“…A similar correlation was found by Tandon et al (2005). Consistent with other investigators, we identified 22 samples which had detectable, albeit low, viral DNA levels and undetectable viral RNA (Tandon et al, 2005;Gomes-Keller et al, 2006a;Gomes-Keller et al, 2006b;Hofmann-Lehmann et al, 2006). Several possible explanations for discordant RNA and DNA qPCR results exist.…”

“…The finding of previously covert viral DNA in some cats which ostensibly totally resisted FeLV infection was not restricted to FeLV-61E-A, as other investigators found a similar phenomenon in cats challenged with the FeLV-A/Glasgow-1 Flynn et al, 2002;Tandon et al, 2005;Cattori et al, 2006;Gomes-Keller et al, 2006a;Gomes-Keller et al, 2006b;. It remained unknown, however, whether the viral DNA we detected by our qPCR assay represented intact, replication-competent provirus or replication-defective viral DNA sequences.…”

“…Application of qPCR to detect FeLV DNA in conjunction with p27 capsid capture ELISA has resulted in diagnostic sensitivity of 100% Flynn et al, 2002;Tandon et al, 2005;Torres et al, 2005;Cattori et al, 2006;Gomes-Keller et al, 2006a;Gomes-Keller et al, 2006b;Hofmann-Lehmann et al, 2006). In addition we, and others, found animals in which viral DNA was detected in the absence of detectable antigenemia, demonstrating that qPCR sensitivity was greater than p27 ELISA and that viral DNA burden was an accurate predictor of FeLV infection outcome.…”

Feline Immunodeficiency

“…PCR is being used currently for detection of viral nucleic acid (RNA or proviral DNA) and is highly strain specific. PCR positive for FeLV proviral DNA indicates the presence of exogenous but not necessarily can be used as a diagnosis for viremia (Gomes-Keller et al, 2006). In these cases the RT-PCR detects the presence of viral RNA and informs the development of viremia in infected animals, but current reagents and testing protocols should be well standardized.…”

“…Recommend annual retesting after any discordant test result until agreement. A positive test doubtful a healthy animal, it should be done further confirmatory tests such as direct immunofluorescence and PCR for provirus (Gomes-Keller et al, 2006;Hartmann, 2006;Lutz et al, 2009). …”