Results and Discussion
Of the 97 blood samples of ideal or close to ideal quality (samples from
animal trials and samples H81-H91, see supporting table 1), 79 were
positive by qPCR. The INGEZIM ASFV CROM Ag LFA detected 61 positives,
resulting in a sensitivity of 77.2%. No false positives occurred, hence
100% specificity was observed on this dataset. The performance was
therefore in line with the study published by Sastre et al. (2016) where
field samples of unimpaired quality were detected with roughly 67%
sensitivity when compared to an OIE listed qPCR. Specificity was also
close to 100%. Sastre et al. (2016) evaluated the LFA only with fresh
samples, as represented by our group of samples derived from animal
trials or hunted wild boar. With the ongoing circulation of ASF in
European wild boar, however, virus detection in carcasses has become an
important issue. Sample quality is then usually reduced due to
decomposition effects, an aspect that has not yet been elucidated for
the ASFV antigen LFA. All our 80 carcass-derived blood samples were
obtained from ASF-positive wild boar and confirmed by qPCR with cq
values ranging from 14 to 38 (see supporting table 2). Here, significant
differences were observed between the samples that were previously
frozen, and those that were not: in native samples tested without any
modifications (C1-C16, n=16, see supporting table 3), the LFA delivered
only two positive results (sensitivity of 12.5%). After freeze-thawing,
testing of the same 16 samples in the LFA yielded seven positives
(sensitivity of 43.75%). Surprisingly, one of the samples that had
yielded a positive result in the native context was now tested negative.
The increase of overall positive results is in accordance with the
sensitivity of 48.75% we observed in all of the previously
freeze-thawed carcass-derived samples (C1-C80), where 39 positives were
detected by the LFA (see supporting table 2). Interestingly, however, we
did not observe a better sensitivity after freeze-thawing in EDTA-blood
samples of high quality in a previous study by our group (Pikalo et al.,
2021). The positive effect of freeze-thawing is probably due to the fact
that most of the virus in blood is associated with erythrocytes (Wardley
& Wilkinson, 1977), and therefore, the destruction of blood cells
during freeze-thawing results in a higher antigen availability for
detection in the test, a process especially effective when erythrocytes
are bound to clots in samples of reduced quality. No false positive
reactions occurred with any sample types.
In our study, the INGEZIM ASFV CROM Ag assay could not deliver reliable
results with native blood from carcasses. Particular samples with cq
values as low as 15 (C4, see supporting table 3), indicating a
considerable virus load in the carcass, still delivered negative results
in the LFA.
While we observed increased sensitivity after erythrocytolysis by
freeze-thawing (12.5% vs 44% sensitivity, samples C1 to C16, see
supporting table 3), for the practical implementation of the assay in
the field, of course, freezing cannot be an option due to the technical
requirements not fitting a point-of-care application. Possible
alternatives to freeze-thawing for erythrocytolysis could be the
dilution of blood in aqua dest . or lysis buffer. On a very
limited dataset (n=4), hypotonic lysis seemed to improve the results (3
FN native, 1 FN after water lysis, no FN after freeze-thawing; data not
further shown). While both methods could be feasible under field
conditions, the effects of this deviation from the manufacturer’s
instructions on the assay should be elucidated and could be the basis of
future optimization of the assay. After all, it must be noted that even
with erythrocytolysis through freeze-thawing, we could only achieve a
sensitivity of roughly 50% in carcass-derived samples, a value not fit
fur purpose. Considering the negative impact of immune-complexes,
samples were screened for the presence of antibodies. Only seven samples
were positive for ASFV-specific antibodies and three delivered doubtful
results in the antibody ELISA (see supporting tables 1 and 2). Eight of
these samples were positive and two were false negative in the
antigen-specific LFA (see supporting tables 1 and 2). While the small
number does not allow for evaluation of possible interference, the
principal functionality of the test in the presence of antibodies is
indicated.
In general, the LFA was more reliable using samples with cq values below
30, indicating a rather high viral load. Of those samples derived from
animal trials (n=64), 56 were true positive according to the rapid test,
resulting in a sensitivity of 87.5% in that group. This goes along with
observations in a previous study performed in our group, when the LFA
was most sensitive during the clinical phase of ASF, at the peak of
viral replication (Pikalo et al., 2021). In the present study, however,
it was observed that the influences of clotting and decay in the
carcass-derived samples seemed be able to outweigh the effects of higher
viral loads, since here no clear correlation even with very low cq
values and positive results in the LFA were observed (see supporting
table 2).
Taking into consideration the differences between the highly amplifying
qPCR and native antigen detection by LFA, the marked lower sensitivity
in the later is to be expected. Still, the possibility for point-of-care
testing holds a considerable advantage and on-site assays can provide a
valuable additional diagnostic tool under certain circumstances. An
acceptable sensitivity of the LFA was confirmed during the clinical
phase of the disease, when fresh samples can be obtained from live
animals or immediately after death. Here, the application of a rapid
test could be of value in domestic pig holdings, when ASF is clinically
suspected and live animals can be picked for sampling (given a careful
interpretation of negative results in the LFA and still immediate
initiation of laboratory diagnosis). Furthermore, epidemiological
investigations can benefit from antigen assays for the on-site analysis
of infected populations, when weaknesses in sensitivity are considered.
However, with a sensitivity of roughly 50%, or even well below when no
erythrocytolytic procedure is applied as proposed by the manufacturer’s
instructions, our findings imply that the LFA has only very limited use
for antigen detection in blood from carcasses after extended post
mortem intervals. When resources are scarce and prioritization of
diagnostic workflows is needed, the high specificity may allow for
positive on-site results in the LFA to surrogate a laboratory
confirmation. Negative results, however, must always be rated with high
caution due to the low sensitivity we observed in samples of reduced
quality. OIE listed methods such as qPCR remain the only safe and proven
methods for the unreserved detection of an ASFV infection. Therefore,
the on-site assay should be regarded as a complimentary option rather
than a substitute to laboratory diagnosis for carcass testing.