of antibodies that are indistinguishable from those
accination of cats against FIV with a whole-virus
vaccine results in rapid and persistent production
used for diagnosis of FIV infection.1,2As such, veteri-
nary practitioners can no longer reliably determine the
FIV infection status of cats that have positive antibody
results in ELISA, western blot, or immunofluorescent
antibody tests.1These cats may have antibodies attrib-
utable to vaccination against FIV, infection with FIV, or
both. The inability of present serologic tests to distin-
guish between antibodies induced by infection or vac-
cination has created a diagnostic dilemma for FIV.
The polymerase chain reaction (PCR) assay has
been promoted as a potential solution for confirming
the true FIV status of cats.3-5Polymerase chain reaction
assays exponentially amplify specific viral DNA
sequences until they are present at detectable concen-
trations. Because diagnostic PCR assays can detect as
few as 1 to 10 copies of viral DNA in a given sample,
they are much more sensitive than many other testing
methodologies.6However, the high sensitivity of PCR
assays may lead to false-positive results if minute
amounts of DNA contamination occur during collec-
tion, storage, or processing of samples.6
Like other lentiviruses, FIV has a high intrinsic
mutation rate in the envelope (env) and capsid (gag)
genes, which has led to the evolution of several distinct
genetic subtypes. Sequence divergence in the env and
gag genes ranges as much as 26% both within a sub-
type and between subtypes.5,7There are 5 well-charac-
terized subtypes of FIV (A to E) based on genetic diver-
gence in the env and gag genes.5The most common
subtypes found in FIV-infected cats in the United
States and Canada are subtypes A, B, and C.5Recently,
a sixth subtype (TX) has been described in cats from
Texas.8Additional subtypes may emerge as more
strains of FIV are fully characterized.
The PCR assay depends on precise matching
between the genetic sequences of the virus and the
primer sequences selected for detection of the genome.
Primers are often selected on the basis of env and gag
genetic sequences of a few well-characterized FIV
strains. How well these env and gag primers detect the
wide variety of genetically divergent FIV strains found
in nature is unknown, but false-negative results rang-
ing from 10% to 100% have been reported.5-7,9,10
Several reference laboratories offer unlicensed
PCR assays for diagnostic purposes, but virtually noth-
ing is known about the sensitivity, specificity, and over-
all performance of these assays. Accurate diagnosis of
FIV is important for both uninfected and infected cats.
Failure to identify infected cats may lead to inadvertent
exposure and transmission of FIV to uninfected cats.
Misdiagnosis of FIV in uninfected cats may lead to
inappropriate euthanasia. This is especially a problem
in cats for which complete medical histories are not
JAVMA, Vol 226, No. 9, May 1, 2005Scientific Reports: Original Study1503
Accuracy of polymerase chain reaction assays
for diagnosis of feline immunodeficiency
virus infection in cats
P . Cynda Crawford, DVM, PhD; Margaret R. Slater, DVM, PhD; Julie K. Levy, DVM, PhD, DACVIM
Objective—To determine the sensitivity, specificity,
and overall diagnostic accuracy of polymerase chain
reaction (PCR) assays offered by commercial diag-
nostic laboratories for diagnosis of FIV infection in
Design—Prospective clinical trial.
Procedure—Blood was collected from cats that were
neither infected with nor vaccinated against FIV, unin-
fected cats that were vaccinated with a licensed FIV
vaccine, and cats experimentally and naturally infect-
ed with FIV representing subtypes A, B, and C. Coded
blood samples were submitted to 3 laboratories in the
United States and Canada offering PCR assays for
diagnosis of FIV infection to veterinary practitioners.
All laboratories tested fresh blood samples, and 1 lab-
oratory also tested samples submitted as dried blood
smears. The FIV infection status in all cats was con-
firmed by virus isolation. Sensitivity, specificity, and
correct results were calculated for each PCR assay.
Results—Sensitivity ranged from 41% to 93%.
Specificity ranged from 81% to 100% in unvaccinated
cats and 44% to 95% in cats vaccinated against FIV.
Correct results were obtained in 58% to 90% of 124
cats tested. All tests misidentified both uninfected
and infected cats. False-positive results by all labora-
tories were higher in cats vaccinated against FIV than
in unvaccinated cats, suggesting that vaccination
interferes with the performance or interpretation of
PCR assays used for diagnosis of FIV infection.
Conclusions and Clinical Relevance—PCR assays
used for diagnosis of FIV infection presently marketed
to veterinary practitioners in North America vary signifi-
cantly in diagnostic accuracy and did not resolve the
diagnostic dilemma resulting from vaccination of cats
against FIV. (J Am Vet Med Assoc 2005;226:1503–1507)
From the Department of Small Animal Clinical Sciences, College of
Veterinary Medicine, University of Florida, Gainesville, FL 32610
(Crawford, Levy); and the Department of Veterinary Anatomy and
Public Health, College of Veterinary Medicine, Texas A&M
University, College Station, TX 77843-4458 (Slater).
Supported by a grant from the George Sydney and Phyllis Redman
Miller Trust in cooperation with the Winn Feline Foundation.
IDEXX Laboratories Inc contributed testing reagents and services.
The authors thank Drs. Christian Leutenegger, Susan Little, Leslie
Sinclair, Ellen Collisson, and Pamela Berlinski for providing blood
samples from cats infected with FIV and Marc Salute, Karen Scott,
and Alex Trapp for technical assistance.
Address correspondence to Dr. Levy.
available, such as those in animal shelters or cats
adopted as strays. The purpose of the study reported
here was to determine the sensitivity, specificity, and
overall diagnostic accuracy of PCR assays offered by
commercial diagnostic laboratories to veterinary prac-
titioners in the United States and Canada for diagnosis
of FIV infection in cats.
Materials and Methods
Cats—The study design was a prospective clinical trial
involving 124 cats. Sensitivity and specificity of PCR assays
used for diagnosis of FIV infection were determined with 42
specific pathogen-free (SPF) cats neither infected with nor
vaccinated against FIV, 41 SPF cats not infected with but vac-
cinated against FIV, 19 SPF cats experimentally infected with
FIV, and 22 client-owned cats with natural FIV infection.
Mean ± SD age of vaccinated cats was 0.5 ± 0.4 years (range,
8 weeks to 1.1 years) at the time the first dose of vaccine
against FIV was administered. The experimentally infected
cats included 9 with FIV subtype A (4 NCSU1, 4 PPR, and 1
Stroker), 4 with FIV subtype B (Mt Airy), and 6 with FIV
subtype C (PGammer). The client-owned cats with natural
FIV infection were recruited from Florida, Maryland, and
Ontario. The FIV subtypes were known for only 6 of those
cats and included 3 with subtype A, 2 with subtype B, and 1
with subtype C.
The SPF cats were housed in facilities accredited by the
Association for Assessment and Accreditation of Laboratory
Animal Care. The study was approved by the University of
Florida Institutional Animal Care and Use Committee and
the Clinical Research Review Committee.
Vaccination against FIV—The licensed FIV vaccinea
used in this study is a dual subtype vaccine containing inac-
tivated subtype A and subtype D viruses, cells in which the
virus was propagated, and adjuvant. The vaccine was admin-
istered SC in the right hind limb every 2 weeks for a total of
3 doses as recommended by the manufacturer. Blood samples
from vaccinated cats were collected 5.0 ± 1.5 months (range,
3.5 to 7.2 months) after the first vaccine dose.
Blood collection—Cats were briefly anesthetized for
blood collection with isoflurane administered by face mask.
Blood (21 mL) was collected from the jugular vein into
potassium EDTA tubes from each cat. Separate tubes were
collected for each laboratory participating in the study to
avoid the risk of contamination associated with preparation
of multiple aliquots from 1 sample. Because 1 laboratory
solicits submission of blood smears as a convenient alterna-
tive to collection of blood samples, 2 blood smears were
made on glass slides for each cat by withdrawing 0.5 mL of
blood from 1 of the EDTA tubes and allowing 0.25 mL of
blood to air dry on each slide. The dried slides were packaged
in cardboard carriers. Each blood tube and slide carrier was
enclosed in an individual plastic bag to further reduce risk of
leakage and contamination during shipping. All samples
were coded so that laboratory personnel were unaware of the
FIV status of the cats. Samples were shipped overnight with
ice packs so that they arrived at the diagnostic laboratories
within 24 hours of collection.
To determine whether the vaccine viruses could be
detected by the PCR assays, each laboratory was asked to
assay the vaccine itself. In addition, the vaccine was diluted
1:5 into a blood sample from an uninfected, unvaccinated cat
and was coded and submitted to the laboratories along with
the other blood samples.
To determine the potential for detection of the vaccine
viruses in blood samples from recently vaccinated cats, a sin-
gle dose of the vaccine was administered to 4 SPF cats. Blood
samples were collected as previously described immediately
prior to vaccination, then daily for 6 days, then every 3 days
for a total of 24 days after vaccination. The samples were
coded and submitted to the laboratories as for the other
PCR laboratories—Three laboratories in the United
States and Canada that routinely offer PCR assays for diag-
nosis of FIV infection agreed to participate in the study. Each
laboratory provided basic information about their propri-
etary PCR reagents and assay.
Laboratory 1b(PCR1 assay) extracted genomic DNA
from WBCs harvested from 4.5 mL of blood by use of a com-
mercial kit. A real-time PCR assay was used to assess the
quality and quantity of the extracted genomic DNA by ampli-
fication of a housekeeping gene.11For detection of FIV
sequences in the genomic DNA, a real-time PCR assay was
used to detect the gag gene for subtypes A, B, C, D, and
TX.6,12,13,cResults were reported as positive or negative.
Laboratory 2d(PCR2 assay) isolated genomic DNA from
WBCs in 0.25 mL of blood by use of a commercial reagent.
The genomic DNA was subjected to conventional PCR assay
by use of proprietary primer sequences for both the env and
gag gene for the first round of amplification, followed by a
second round of amplification using internal (nested)
primers as described.14The PCR assay products were resolved
by agarose gel electrophoresis and the outcome reported as
positive or negative based on the presence or absence of a
band of the expected size.
Laboratory 3e(PCR3 assay) isolated genomic DNA from
75 µL of blood by use of a commercial kit. In addition, this
laboratory rehydrated the dried blood smears with 200 µL of
saline (0.9% NaCl) solution and used 75 µL of the rehydrat-
ed sample for DNA isolation (PCR4 assay). The genomic
DNA samples were subjected to conventional PCR amplifica-
tion by use of a mixture of 2 sets of primers for the gag gene
as previously described.9The PCR assay products were
resolved by agarose gel electrophoresis and the outcome
reported as positive or negative based on the presence or
absence of a band of the expected size for either primer set.
Samples that were negative after the first round of amplifica-
tion were subjected to a second round of amplification with
the same primers.
FIV culture—The FIV status of each cat was verified by
culture of peripheral blood mononuclear cells for virus as
previously described.15Briefly, mononuclear cells were iso-
lated by discontinuous density gradient centrifugation of
blood. The CD8+T cells, which have been found to inhibit
FIV replication in culture,15-17were removed from the
mononuclear cells by immunomagnetic separation by use of
monoclonal anti-feline CD8 antibody conjugated to phyco-
erythrin,fanti-phycoerythrin microbeads,gand magnetic
separation columnshas described.15The remaining mononu-
clear cells were cocultured with a feline CD4+T-cell line per-
missive for FIV replication.15All cultures were performed in
triplicate. Culture supernatants were collected at 7, 14, and
21 days and tested for FIV p24 antigen by ELISA.iCats were
considered as being infected with FIV if the culture super-
natant was positive for FIV antigen at any of the 3 time
Statistical analyses—Data analysis was performed by
use of a commercial software program.jData with normal dis-
tribution were described as mean ± SD and range. Data that
were not normally distributed were summarized as median
and range. Sensitivity of the PCR assays for FIV infectionwas
calculated as the percentage of cats infected with FIV cor-
rectly identified as positive by the assay. The effects of viral
subtype and of experimental versus natural infection on sen-
sitivity were tested by use of χ2analysis. Specificity was cal-
1504 Scientific Reports: Original Study JAVMA, Vol 226, No. 9, May 1, 2005
culated as the percentage of cats not infected with FIV cor-
rectly identified as negative by the assay. Ninety-five percent
confidence intervals were used to determine the precision of
the sensitivity or specificity estimate. Assays with sensitivity
or specificity outside the 95% confidence interval of another
assay were considered significantly different at values of P
< 0.05. The overall accuracy of each PCR assay was defined
as the number of correct results for all 124 cats.
Virus culture confirmed the FIV status of all 124
cats. Laboratory 1 (PCR1 assay) correctly identified 31
of the 41 FIV-infected cats (76% sensitivity; Table 1).
All 3 FIV subtypes (A, B, and C) were found among the
detected and undetected infections. There was no sig-
nificant difference in the sensitivity when results were
analyzed by viral subtype or by experimental versus
natural infection. The PCR1 assay correctly identified
42 of 42 uninfected, unvaccinated cats (100% speci-
ficity) and 39 of 41 uninfected, vaccinated cats (95%
specificity). Specificity was not significantly different
between the 2 groups (unvaccinated vs vaccinated) of
uninfected cats. Overall, the PCR1 assay correctly
identified 112 of 124 cats tested (90% correct results;
Laboratory 2 (PCR2 assay) correctly identified 38
of 41 FIV-infected cats (93% sensitivity; Table 1). The
PCR2 assay detected all 3 subtypes of FIV but also
missed some cats with subtype A or B infection. There
was no significant difference in the sensitivity when
results were analyzed by viral subtype or by experi-
mental versus natural infection. The PCR2 assay cor-
rectly identified 34 of 42 uninfected, unvaccinated cats
(81% specificity). In contrast, only 27 of the 41 vacci-
nated cats were correctly identified as not having FIV
(66% specificity). Thus, false-positive results were sig-
nificantly more likely in vaccinated cats (34%), com-
pared with unvaccinated cats (19%). Overall, the PCR2
assay correctly identified 99 of 124 cats tested (80%
correct results; Figure 1).
Laboratory 3 correctly identified only 21 of 41 FIV-
infected cats (51% sensitivity) when blood samples
were used for analysis (PCR3 assay) and 17 of 41 FIV-
infected cats (41% sensitivity) when blood smears were
used (PCR4 assay; Table 1). All 3 subtypes of FIV (A, B,
and C) were found among both the detected and missed
infections, and there were no significant differences in
the sensitivity when results were analyzed by viral sub-
type or by experimental versus natural infection. Both
PCR3 and PCR4 assays correctly identified 34 of 42
uninfected, unvaccinated cats (81% specificity).
However, only 18 of 41 vaccinated cats were correctly
reported as not having FIV by the PCR3 assay (44%
specificity) and 21 of 41 by the PCR4 assay (51% speci-
ficity). Thus, false-positive results were significantly
more likely in vaccinated cats (56% for PCR3 and 49%
for PCR4 assays), compared with unvaccinated cats
(19% for both PCR3 and PCR4 assays). Overall, the
PCR3 assay correctly identified 73 of 124 cats tested
(59% correct results), and the PCR4 assay correctly
identified 72 of 124 cats (58% correct results; Figure 1).
In summary, the PCR2 assay was the most sensi-
tive assay, followed by PCR1. The PCR1 assay was the
most specific assay for both unvaccinated cats and cats
vaccinated for FIV. Overall, the PCR1 assay was the
most accurate, followed by PCR2.
Laboratory 3 used the same PCR assay for blood
samples (PCR3) and blood smears (PCR4). As such,
the results for each cat would be expected to match
regardless of the sample format. However, discordant
results between the assays used for blood samples and
blood smears were reported for 24% of uninfected and
unvaccinated cats, 61% of uninfected and vaccinated
cats, and 34% of FIV-infected cats. For all 124 cats, 49
samples were discordant (40%), 27 samples matched
but were inaccurate (22%), and 48 samples matched
and were correct (39%).
To determine the effect of recent FIV vaccination
on PCR testing, each laboratory performed its PCR
JAVMA, Vol 226, No. 9, May 1, 2005 Scientific Reports: Original Study 1505
Status of cats No. of catsPCR1PCR2PCR3PCR4
FIV-infected cats (sensitivity)41 76a
Unvaccinated cats (specificity) 42
Vaccinated cats (specificity) 41
a,b,c Within a row, values with different superscript letters are significantly different (P ? 0.05).
Table 1—Sensitivity and specificity (95% confidence intervals) for 4 commercial polymerase chain
reaction (PCR) assays used for detection of FIV infection in cats.
Figure 1—Overall diagnostic accuracy of polymerase chain reac-
tion (PCR) assays from Laboratory 1 (PCR1), Laboratory 2
(PCR2), and Laboratory 3 (PCR3 and PCR4). The PCR assays
were performed on blood samples (PCR1, PCR2, and PCR3) and
blood smears (PCR4) from cats neither infected with nor vacci-
nated against FIV (n = 42), cats not infected with but vaccinated
against FIV (41), and cats infected with FIV (41). Overall diag-
nostic accuracy is defined as the number of correct results for
the total population of cats (n = 124) and is expressed as a per-
centage. *Significantly (P < 0.05) different from PCR2, PCR3,
and PCR4. †Significantly (P < 0.05) different from PCR1, PCR3,
assay on the vaccine, blood samples spiked with the
vaccine, and blood samples from recently vaccinated
cats. Results of all PCR assays (PCR1, PCR2, and
PCR3) were positive when the vaccine alone was
assayed. Two of the assays (PCR1 and PCR3) also
detected the vaccine virus in the blood sample spiked
with the vaccine. The PCR1 assay reported negative
results for all blood samples collected from 4 cats 1 to
24 days after a single vaccination. The PCR2 assay
reported a single positive result in 1 cat 2 days after
vaccination. The PCR3 assay reported positive results
for several blood samples collected prior to vaccination
and on days 1, 2, 3, 9, 12, and 15 after vaccination.
Performance of PCR diagnostic assays for detec-
tion of FIV infection presently marketed to veterinary
practitioners in North America varied significantly
among different laboratories. The PCR assays used by
the 3 reference laboratories that participated in this
study misidentified both infected and uninfected cats.
Sensitivity (identification of truly infected cats) for FIV
infection ranged from 41% to 93% (ie, 7 to 59 of every
100 FIV-infected cats tested would be incorrectly iden-
tified as not being infected with FIV, depending on the
particular laboratory that was used). All PCR assays
detected cats that were infected either experimentally
or naturally with FIV subtypes A, B, and C, which are
the predominant subtypes in North American cats.
However, the PCR assays also missed cats infected with
these subtypes. Sensitivity was not associated with the
type of PCR assay (conventional vs real-time) that was
used for detection of FIV infection or the primer speci-
ficity for env or gag gene sequences. For example, the
real-time PCR assay used by Laboratory 1 (PCR1
assay) can reportedly detect as few as 1 to 10 copies of
proviral DNA and uses reagents that identify gag gene
sequences in 5 subtypes of FIV (A, B, C, D, and TX).6,12,c
Despite the capacity for detection of only a few copies
of viral DNA from a wide range of FIV subtypes, this
assay failed to detect nearly 25% of cats infected with
Specificity (identification of truly uninfected cats)
ranged from 81% to 100% when unvaccinated cats were
tested. Laboratory 1 (PCR1 assay) correctly identified
all uninfected, unvaccinated cats as not being infected
with FIV; however, the other 2 laboratories (PCR2,
PCR3, and PCR4 assays) identified 19% of the cats as
being infected with FIV. In addition, these 2 laborato-
ries reported significantly higher false-positive results
for cats vaccinated against FIV than unvaccinated cats,
such that 33 to 56 of every 100 vaccinated cats tested
would be misidentified as being infected with FIV. In
contrast, the specificity of the PCR assay used by labo-
ratory 1 (PCR1 assay) was not significantly affected by
the vaccination status of the cat.
A potential reason for the high rate of false-positive
results in vaccinated cats is detection of virus adminis-
tered in the vaccine. The PCR assays for all 3 laborato-
ries detected FIV in the vaccine itself, but 1 laboratory
(PCR2 assay) did not detect the vaccine virus added
directly to whole blood. The most accurate PCR assay
(PCR1) did not detect the vaccine virus in blood col-
lected from cats during the first 3 weeks after a single
vaccination against FIV was administered. The least
accurate PCR assay (PCR3) intermittently reported pos-
itive results for blood collected prior to vaccination and
on days 1 to 15 after vaccination. These results, coupled
with the fact that cats were tested 2 to 6 months after
completion of vaccination, indicated that PCR detection
of the vaccine virus was not a plausible explanation for
the high rate of false-positive results in vaccinated cats.
Another possible explanation for the high rate of
false-positive results in cats vaccinated for FIV may
have been related to the type of PCR assay used by the
laboratories. Vaccination did not significantly interfere
with identification of uninfected cats by laboratory 1
(PCR1 assay). This laboratory uses a real-time PCR
assay in which results are generated by a computer,
thereby decreasing subjectivity inherent with human
interpretation. Vaccination significantly interfered with
identification of uninfected cats by laboratories 2 and
3. The conventional PCR assays used by these labora-
tories rely on visual assessment of agarose gels to diag-
nose FIV; thus, interpretation of results is influenced
by human subjectivity. Although the blood samples
were coded so that laboratory personnel would be
unaware of the cats’ FIV status, some laboratories pre-
screen samples for FIV antibodies prior to performing
PCR assays. If personnel are aware of serologic test
results prior to viewing the gels, their subjective assess-
ment could be biased. However, all laboratories denied
performing any serologic screening tests before PCR
assays were performed. Another possible explanation
for decreased specificity observed by Laboratories 2
and 3 may have been the use of 2 rounds of PCR ampli-
fication, compared with use of only 1 round of real-
time PCR by laboratory 1. Although multiple rounds of
PCR amplification may decrease specificity overall, it
should not have a greater effect on samples from vacci-
nated than from unvaccinated cats.
When results from all 124 cats were combined, the
percentage of cats in which the FIV status was correctly
identified by the PCR assays ranged from 58% to 90%.
The PCR assay has been promoted as a solution to the
diagnostic dilemma created by FIV vaccination because
serologic tests can no longer accurately identify FIV-
infected cats. However, PCR assays used for detection of
FIV infection presently marketed to veterinary practi-
tioners in North America vary significantly in diagnostic
accuracy and do not resolve the diagnostic issues result-
ing from FIV vaccination against FIV in cats.
Fel-O-Vax FIV, Fort Dodge Animal Health, Fort Dodge, Iowa.
Lucy Whittier Molecular Core Facility, School of Veterinary
Medicine, University of California, Davis, Calif.
Leutenegger CM, Lucy Whittier Molecular Core Facility,
School of Veterinary Medicine, University of California, Davis,
Calif: Personal communication, 2004.
Vita-Tech Laboratories, Buffalo, NY.
DyNAgenics Veterinary Diagnostics Parasitology Research Lab
LLC, Denver, Colo.
Mouse anti-feline CD8 α/β clone vpg9, Serotec Inc, Raleigh, NC.
Miltenyi Biotec Inc, Auburn, Calif.
MiniMACS, Miltenyi Biotec Inc, Auburn, Calif.
PetChek FIV Antigen Test, IDEXX Laboratories, Westbrook, Me.
Intercooled Stata 8, Stata Corp, College Station, Tex.
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