Evaluation of the IMMY CrAg Lateral Flow Assay for Detection of
Cryptococcal Antigen
Susan Clarke and Robert Gibb
Pathology Queensland – Central, Royal Brisbane and Women’s Hospital, Brisbane, Australia
Introduction
Immunocompromised individuals, particularly AIDS patients, are most at risk of
infection with Cryptococcus species complex (Cryptococcus neoformans and
Cryptococcus gattii). Accurate, reliable and timely cryptococcal antigen results are
required to ensure diagnosis and effective treatment of this opportunistic and
occasionally fatal fungal infection. Testing for cryptococcal antigen is undertaken in
many pathology laboratories with the Meridian Cryptococcal Antigen Latex System
assay (CALAS) which has been the test kit of choice for many years. An evaluation
was performed on the new IMMY CrAg Lateral Flow Assay (IMMY).
Table 2: CALAS vs IMMY assay results
Serum
CSF
Method
The CALAS and IMMY assays were compared for their ease of use in a clinical
laboratory setting and for their sensitivity and specificity in determining cryptococcal
antigen status in patient samples.
Serum and CSF samples were chosen for evaluation based on the original results
obtained on the CALAS assay at the Pathology Queensland Central Laboratory.
CALAS assay
IMMY assay
Negative
47
45*
Positive
8
10*
Negative
16
16
Positive
5
5
*Two samples tested negative on CALAS assay had confirmed cryptococcosis
and tested positive on the IMMY assay.
Sample type
CALAS result
Number
Serum
Negative
47
Positive
8
Of the 76 samples tested, 61(80%) gave a negative result and 13 (17%) gave a
positive result on both the CALAS and IMMY assays (Table 2). Of particular
interest were the 2 (3%) serum samples which gave discordant results between the
two assays. In both cases the serum tested negative for cryptococcal antigen on the
CALAS assay and positive on the IMMY assay. Further investigation revealed that
these two discordant samples came from patients with a previous history of
cryptococcosis (Table 3).
Negative
16
Table 3: Cryptococcal testing history on discordant samples
Positive
5
Collection dates
CSF
Patient 1:
Results
16.03.11
19.09.11
25.02.11
27.02.11
02.03.11
Sample type
Serum
Serum
CSF
Serum
CSF
CALAS result
1:4
Negative
1:2
Negative
1:2
IMMY Result
1:256
1:4
1:64
1:4
1:64
Comparison of the CALAS and IMMY methods (Table 1) showed that both assays
can be performed in five to ten minutes. However, the IMMY assay proved to be
less time consuming overall as it required no pre-treatment of either samples or
controls.
Patient 2:
Table 1: Comparison of CALAS and IMMY Methods
The IMMY assay was found to be a more sensitive assay (100%) when compared
to the CALAS assay (87%). However, the CALAS assay sensitivity may be
underestimated due to sample bias as a result of limited sample numbers
Test type
CALAS Assay
Latex Agglutination
IMMY assay
Dipstick sandwich
immunochromatographic
Kit Storage
Refrigerated (2 – 8 ºC)
Room temperature
(22 – 25 ºC)
Controls required
Positive Control
Negative Control
Antibody Control Positive Control
Latex Control
Negative Control
Pre-treatment of Negative 30mins at 56ºC
control
Pre-treatment of Sample Serum: incubated with pronase
for 15mins at 56ºC
plus 5mins at 100 ºC
CSF:
5mins at 100 ºC
Dilution of sample to Recommended
exclude prozone effect
Test procedure
5mins on rotator
Result determination
Nil
Nil
and
One CSF sample gave particularly interesting results. The original CALAS assay
gave a titre of 1:2 which was considered unusually low for a patient with
confirmed cryptococcosis. The IMMY assay was positive with a titre of 1:64.
The CSF sample had a very high protein level of 3800mg/L. Following treatment
of the CSF with pronase (not required for CSF), the CALAS assay produced a titre
of 1:32. The protein level in the CSF appears to have affected the sensitivity of the
CALAS assay. As the IMMY assay requires no pretreatment, the titre obtained
was not compromised.
Recommended
Discussion
10mins on bench
An in-house evaluation of the new IMMY CrAg Lateral Flow Assay was
undertaken and results compared with the currently used CALAS assay.
The evaluation showed that the IMMY assay is quick and easy to use. The test can
be performed on the bench using a single micronic tube per sample without the
requirement of sample or control pretreatments. Titrations can be performed in
further tubes if necessary and the lateral flow strips inserted to obtain an endpoint.
Reading of results is less subjective, requiring only the identification of a control
and test line versus the interpretation of an agglutination pattern.
The sensitivity of the IMMY assay was found to be greater than that of the
CALAS assay. Positive results were identified in two samples from confirmed
cryptococcosis patients with the IMMY assay that were negative with the CALAS
assay. Interfering agents such as protein were also found to have less effect on test
results with the IMMY assay.
The IMMY assay proved to be a rapid, sensitive and user-friendly assay that will
make a valuable contribution to the testing protocol for cryptococcal antigen in
the laboratory.
Subjective reading of agglutination Presence or absence of
pattern (1+ reaction is negative)
control and test line
Qualitative
results
Titres obtained using the IMMY assay were found to be 2 to 64 times higher than
the corresponding titres from the CALAS assay. This difference may be explained
by the increased sensitivity of the IMMY assay compared with the CALAS assay
as well as the less subjective interpretation of the IMMY assay results.
semi-quantitative Qualitative and semiquantitative results
The IMMY method was much simpler to perform. One drop of diluent is added to 40ul
of sample in a micronic tube and the lateral flow dipstick is inserted in the tube.
Titration of the sample can be performed in additional tubes as required and dipsticks
inserted for result determination.
The CALAS assay requires pre-treatment of samples to remove rheumatoid factor and
titration of samples in tubes before transfer to a reaction card. Testing with both
detection and control latex must be performed for each sample and all dilutions.
Reading of results was less subjective with the IMMY assay. A
control line must be present for the test to be valid. Presence of a
test line indicates a positive result. Absence of a test line
indicates a negative result (Fig. 1).
In contrast, reading the CALAS assay result requires
interpretation of an agglutination pattern where only 2+ or
greater reactions are positive. A positive reaction with the control
latex indicates non-specific interference which complicates the
reading of results.
Figure 1: IMMY assay result Interpretation