Detection of Feline Leukemia virus p27 antigen by microELISA vs
immunochromatographic tests in cats from Guayaquil
Detección de antígeno
p27 del virus de la Leucemia Felina por método de microELISA vs test inmunocromatográficos
en gatos de Guayaquil
Melissa Joseth Carvajal Capa1
Milena Paulet Vélez Sarabia2
José Alberto Echeverría Alcívar3
Irina Trejo Cedeño4
Fabiola Lissette Jiménez Valenzuela5
Published Instituto
Superior Tecnológico Edwards Deming. Quito - Ecuador Periodicity January - March Vol. 1, Num. 24, 2025 pp. 42-52 http://centrosuragraria.com/index.php/revista Dates of receipt Received: July 02, 2024 Approved: November 04, 2024 Correspondence author melissa.carvajal01@cu.ucsg.edu.ec Creative Commons License Creative Commons License,
Attribution-NonCommercial-ShareAlike 4.0
International.https://creativecommons.org/licenses/by-nc-sa/4.0/deed.es
Abstract: The domestic cat is one of the pets of choice,
which leads to the evaluation of diagnostic methods for diseases such as feline
viral leukemia or Vilef, where the efficacy and speed of diagnosis is important
because it is a highly fatal disease. The aim of this study was to determine
the percentage of sensitivity of two immunochromatography tests commonly used
in veterinary offices to diagnose Vilef and to check their usefulness by making
a comparison with the results of the microELISA method, as a reference
technique. Using a quantitative, experimental, applied approach, the research
was developed from the collection of blood sera from cats in a clinical
laboratory in the city of Guayaquil, Ecuador. Forty blood sera were analyzed
for detection of feline leukemia virus p27 antigen by the microELISA method and
later with immunochromatography tests. The results determined that when
reaching a kappa value range of 0.8, the tests present a very good concordance
degree, resulting in a 93 % of sensitivity. Concluding that the tests evaluated
are reliable and can be considered as tools for rapid diagnosis.
1 Veterinary
Medicine Engineer, Universidad Católica Santiago de Guayaquil,
melissa.carvajal01@cu.ucsg.edu.ec, https://orcid.org/0009-0001-5343-9506 2 Engineer in
Veterinary Medicine,Milena.velez@hotmail.com ,
https://orcid.org/0009-0008-7439-6403 3 Veterinary
Medicine Engineer, Universidad Católica Santiago de Guayaquil,
jose.echeverria05@cu.ucsg.edu.ec, https://orcid.org/0009-0007-8319-5091 4 Veterinary
Medicine Engineer, Universidad Católica Santiago de Guayaquil,
Irina.trejo@cu.ucsg.edu.ec, https://orcid.org/0000-0003-0295-4273 5 Engineer in
Veterinary Medicine, Universidad Católica Santiago de Guayaquil,
Fabiola.jimenez01@cu.ucsg.edu.ec, https://orcid.org/0009-0004-1521-7437
Resumen: El gato doméstico es uno de los animales de
compañía de preferencia, lo que conduce a la evaluación de los métodos de
diagnóstico de enfermedades que padece como La leucemia viral felina o Vilef,
donde la eficacia y rapidez del diagnóstico es importante por ser una
enfermedad altamente mortal. El presente estudio tuvo como objetivo determinar
el porcentaje de sensibilidad de dos test de inmunocromatografía de uso común
en consultorios veterinarios para diagnosticar Vilef y de comprobar su utilidad
haciendo una comparativa con los resultados del método de microELISA, como
técnica de referencia. Mediante un enfoque cuantitativo, experimental, de tipo
aplicado, se desarrolló la investigación, a partir de la recolección de sueros
sanguíneos de gatos en un laboratorio clínico de la ciudad de Guayaquil,
Ecuador. Se analizaron 40 sueros sanguíneos para detección de antígeno p27 del
virus de leucemia felina por el método de microELISA y posteriormente con los
test de inmunocromatografía. Los resultados determinaron que al alcanzar un
rango de valor kappa de 0.8, los test presentan un grado concordancia muy
bueno, resultando así mismo con un 93 % de sensibilidad. Concluyendo que los
test evaluados son confiables y pueden ser considerados como herramientas para
un diagnóstico rápido.
Palabras clave: Gatos, antígeno p27, virus de Leucemia Felina,
microELISA, inmunocromatográficos
Introduction
Feline
leukemia, or FIV, is one of the main viral threats of major recurrence in
domestic cats. VileF is produced by a rotavirus, whose transmission is
influenced by population density and exposure to the virus by the approach of
infected cats with persistent viremic picture, being the main way of contagion
is through saliva (Hwang et al., 2018; Perharic et al., 2018).
The
severity of infection depends on factors such as the age of the cat and the
strength of the immune system, determining the possible elimination or latency
of the virus and the resulting VileF genotype (Aiyaranoi et al., 2018). In
addition, of the four stages of the disease, which are: abortive, regressive,
progressive and focal, the progressive phase is the one with the least
favorable prognosis, where the age of the cat at the time of infection is a
determining factor for clinical outcomes and the process of evolution of the
disease (Calle-Restrepo et al., 2013).
The
use of diagnostic tests for VileF has become essential for the determination of
a positive patient. The diagnosis will depend on the stage of the disease, with
p27 antigen detection being the first test to be performed, using methods such
as microELISA and immunochromatography.
The importance of a diagnosis lies in the speed of the data provided and
the effectiveness of the results obtained. This allows an adequate management
of infected and uninfected animals, in order to extend or prolong the days of
these felines with a good quality of life (Campbell et al., 2020).
In
daily practice in veterinary care centers, immunochromatography tests are
frequently used because of their rapid response time, with a result obtained in
a few minutes, as opposed to laboratory tests such as the microELISA, which can
take up to 72 hours. It is important to mention that in spite of the time
advantages obtained with a test, these can yield false negatives, while
laboratory tests such as the microELISA test are more sensitive and specific.
Immunological
tests use antigen to detect antibody or antibody to detect a pathogen antigen
in a patient sample (Vazquez, 2020). Sensitivity to antigen or antibody
detection is a factor that validates the performance of the laboratory test.
The immunological microELISA test that detects the p27 antigen in plasma or
serum has a high sensitivity and specificity, without considering vaccination,
lactation or colostrum intake as interference (Muñoz, 2005; Palmero and
Carballés, 2010; Calle et al., 2013; Esquivel, 2019). The present study aims to
reflect the validity of the results, measuring the percentage of sensitivity of
imunochromatography tests used in veterinary centers, detecting the p27 antigen
for feline leukemia diagnosis, using the microELISA method as a reference test.
Methodology
The methodology applied in the
present study presents a quantitative approach, experimental design, applied
type, with correlational objectives, with experimental variables, diachronic
transversal type, developed in a controlled environment during a limited period
of time. The study was carried out from October 2022 to January 2023, 40 blood
samples were chosen from cats received at the Diagnovet Clinical Veterinary
Laboratory in the city of Guayaquil.
The larger samples were selected for
further processing with the microELISA technique. Once this procedure was
finished, they were analyzed with the two immucromatography tests of different
laboratories of manufacture and of greater use in veterinary centers, which are
represented as Test IC 1 and Test IC 2, for their identification. As described
by Krecic, these two tests are the most requested in veterinary practice for
the diagnosis of feline leukemia by detection of p27 antigen (Krecic et al.,
2017).
In previous years the diagnosis of
feline leukemia was made based on clinical symptoms, since there were very few
guardians who authorized laboratory tests to determine whether their cat was
positive or not to the disease, currently the ease of testing makes them become
frequently used for diagnosis in veterinary centers. Campbell in his
comparative study of immunochromatography tests establishes the importance of
assessing the sensitivity of these tests, to ensure an effective diagnosis that
helps in the management of infected animals and their treatment (Campbell, et
al., 2020). Under this sense, all the points were examined and the necessary
resources were gathered for the methodological structure to comply with the
objectives set out in the research.
For the comparative evaluation of
results, the 2x2 contingency table (Table 1) was used, taking the microELISA
test as a reference and relating the results with the two results obtained with
the serological tests, in this case the two immucromatography tests referred
to. The degree of concordance between the two techniques was evaluated and then
the kappa index or kappa value was calculated to obtain the final data,
categorized according to the ranges of values in Table 2.
Table 1. Cohen's 2x2 contingency table
|
Reference diagnostic tests |
||||
|
Positive |
Negative |
|||
|
Diagnostic tests evaluated |
Positive |
a |
c |
f1 |
|
Negative |
b |
d |
f2 |
|
|
c1 |
c2 |
N |
||
Source: Cohen 1960
N
N
Actual
or non-random concordance CR = CO - CA
Maximum
match that can occur beyond chance MCMA = 1 - CA
MCMA
Source: Cohen 1960
Table
2. Ranges of
Kappa Values
|
Kappa value ranges |
Degree
of agreement between tests |
|
<0.00 |
No agreement |
|
0.00-0.20 |
Insignificant |
|
0.21-0.40 |
Under |
|
0.41-0.60 |
Moderate |
|
0.61-0.80 |
Good |
|
0.81-1.00 |
Very good |
Source:
Cohen, 1960
a + b
Source:
Cohen, 1960
Results
Forty blood serum samples from cats
received at the reference laboratory were analyzed and were subjected to the
microElisa test at , resulting in 30 positive and 10
negative results. Subsequently, they were analyzed with the selected
immunochromatography tests, resulting in 28 positive and 12 negative for Test
IC 1 and 28 positive and 12 negative for Test IC 2, as shown in Table 3.
Table 3. Results obtained in IC Test 1 and IC
Test 2
|
Variable |
microElisa |
% |
Test IC 1 |
IC Test 2 |
% |
Sig. |
|
Positive |
30 |
75 |
28 |
28 |
70 |
0.1573 |
|
Negative |
10 |
25 |
12 |
12 |
30 |
|
|
Total |
40 |
40 |
40 |
IC Test 1: Immunochromatography
Test 1; IC Test 2: Immunochromatography Test 2
According to the results obtained,
the contingency table (Table 4) was made and the formulas of observed
concordance, concordance due to chance, real concordance or not due to chance,
maximum concordance that can occur beyond chance were performed, reaching the
Kappa value range of 0.8, determining that the degree of concordance between
the diagnostic tests evaluated is very good, as can be seen in the following
approach.
Table 4. Contingency table microELISA vs IC
Test 1 and IC Test 2
|
Reference diagnostic tests |
||||
|
+ |
- |
|||
|
Diagnostic tests evaluated |
+ |
28 |
0 |
28 |
|
- |
2 |
10 |
12 |
|
|
30 |
10 |
40 |
||
N
N
True concordance or not due to
chance CR = 0.95 - 0.6 = 0.35
Maximum match that can occur beyond
chance MCMA = 1 - 0.6 = 0.40
0.40
With respect to the sensitivity
percentage obtained based on Table 4 and applying the formula referred to
above, the resulting value is 93 % out of 100.
28 + 2
There are no previous research
reports detailing the degree of sensitivity of immunochromatography tests
comparing the results with the microELISA reference test, because it is highly
reliable and specific. (2020) on the diagnosis of ViLeF, a comparison is made
between the PCR test as a reference test and the immunochromatography tests as
tests to be evaluated, resulting from the total number of samples 39 positive
by PCR and one positive by immunochromatography, concluding that the
pathogenesis of the infectious agent must be taken into consideration to make a
correct test and make associations with the clinical symptomatology in order
not to make a wrong diagnosis.
Similarly a study conducted by Westman et al., (2017), focused on a comparison of
the PCR technique with immunochromatography tests, determining false positives
with gerontic cats being the ones that mostly obtained these results. He
concluded that feline leukemia virus is a viral disease that is difficult to
diagnose due to the complex relationship between the pathogen - feline host,
evidencing in his research the unreliability of immunochromatography tests as
diagnostic tests.
Evidently, the results obtained in
this research are in contrast to the studies of Campbell and Westman in
previous years, in the results that they present, there is little sensitivity
in the serological tests for detection of p27 antigen with immunochromatography
tests, unlike the current data where the degree of sensitivity in the same
tests was very good. This differentiation is attributed to the improvements of
the manufacturing laboratories in offering tests with rapid and better
sensitivity, due to the demand for reliable diagnostic tests in veterinary
centers that collaborate in the timely care of patients with serious or
life-threatening diseases.
Conclusions
After the analysis of the 40 samples
of blood serum of cats prescribed in the clinical veterinary laboratory
Diagnovet of the city of Guayaquil, 30 positive and 10 negative results were
obtained for the microElisa test. Unlike the results obtained with the selected
immunochromatography tests, giving as a result for the IC 1 test 28 positive
and 12 negative, and with the IC 2 test, 28 positive and 12 negative. In
addition, the serological tests evaluated for the immunochromatography test, to
detect p27 antigen for the feline leukemia virus, presented concordance with a
range of Kappa value of 0.8 that evaluates them as very good and presenting a
degree of sensitivity of 93%. Therefore, it is concluded that the
immucromatographic tests used in this research are of very good reliability and
sensitivity, which added to the rapidity with which the results are obtained,
are a useful diagnostic tool for feline leukemia in veterinary centers. It was
also evidenced the low probability of false negatives in the tests used in the
study.
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