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). 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. Table 2: CALAS vs IMMY assay results *Two samples tested negative on CALAS assay had confirmed cryptococcosis and tested positive on the IMMY assay. 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). Table 3: Cryptococcal testing history on discordant samples 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 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. 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. CALAS assay IMMY assay Serum Negative 47 45* Positive 8 10* CSF 16 5 Sample type CALAS result Number Serum Negative 47 Positive 8 CSF 16 5 Collection dates Sample type CALAS result IMMY Result Patient 1: 16.03.11 19.09.11 Serum 1:4 Negative 1:256 Patient 2: 25.02.11 27.02.11 02.03.11 CSF 1:2 1:64 Results 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. Table 1: Comparison of CALAS and IMMY Methods CALAS Assay IMMY assay Test type Latex Agglutination Dipstick sandwich immunochromatographic Kit Storage Refrigerated (2 – 8 ºC) Room temperature (22 – 25 ºC) Controls required Positive Control Antibody Control Negative Control Latex Control Positive Control Negative Control Pre-treatment of Negative control 30mins at 56ºC Nil 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 exclude prozone effect Recommended Test procedure 5mins on rotator 10mins on bench Result determination Subjective reading of agglutination pattern (1+ reaction is negative) Qualitative and semi-quantitative results Presence or absence of control and test line Discussion 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. 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