TESTING OF RAPID ANIGEN RABIES Ag DETECTION KITS

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1 TESTING OF RAPID ANIGEN RABIES Ag DETECTION KITS REPORT PREPARED BY THE AUSTRALIAN ANIMAL HEALTH LABORATORIES DIAGNOSTIC SERVICES RESPONSE GROUP INTRODUCTION The Australian Animal Health Laboratory was approached by BioNote, Inc. (ANIMAL GENETICS, INC.) Korea, to test their Anigen Rabies Ag detection rapid test kit for sensitivity and specificity against Australian bat lyssavirus. All the positive bat brains were obtained form Pteropus spp., while the negative brains were obtained from a variety of bat species. METHOD Using the method outlined by Kang et al (2007) 23 known Lyssavirus positive brains and 19 known negative brains determined by Fluorescence Antibody staining technique (FAT) were tested on the Rapid Rabies Anigen kits provided. Briefly, a 10-20% brain homogenate of each brain was prepared in Phosphate Buffered Saline ph 7.2 (Kang et al, 2007). The homogenate was diluted with an equal volume of the diluent provided in the kit. The suspension was incubated at room temperature for 10 minutes. Four drops or approximately 125µl were loaded into the device, using the transfer pipette supplied with the kit and incubated for 5 minutes at room temperature for the sample to be drawn into the device as specified by the manufacturer. RESULTS There was 100% correlation between the FAT and Rapid kit test results for all the positive and negative brains tested (Table 1). The negative brains demonstrated no non-specific cross reactivity and therefore the kit demonstrated a specificity of 100% on the limited number of samples tested. Although the positive results also correlated with the FAT, the level of reactivity observed with the devices varied between samples (figure 1 and 2). It should be noted that sample was weakly positive and the reaction is not visible in figure 2 but was visually observed. All the positive brains tested were from Pteropus spp. of bats. These are the most common samples submitted to our laboratory.

2 Table 1. List of all the positive and negative samples tested in this study. SPECIES SAN FAT RESULT KIT RESULT POSITIVE BRAINS Pteropus poliocephalus (+ve control)** O Positive Positive Pteropus poliocephalus (GHFF) Positive Positive Pteropus poliocephalus Positive Positive Pteropid alecto (Black flying fox) Positive Positive Pteropid poliocephalus Positive Positive Pteropus alecto Positive Positive Pteropus alecto Positive Positive Pteropus poliocephalus O Positive Positive Pteropus poliocephalus O Positive Positive Pteropus poliocephalus O Positive Positive Pteropus poliocephalus O Positive Positive Pteropus poliocephalus O Positive Positive Pteropus alecto O Positive Positive Pteropus alecto O Positive Positive Pteropus sp. O Positive Positive Pteropus scapulatus O Positive Very Weak Pos* Pteropus poliocephalus O Positive Positive Pteropus poliocephalus OO-0573 Positive Positive Pteropus sp. O Positive Positive Pteropus poliocephalus O Positive Positive Pteropus poliocephalus Positive Positive Pteropus poliocephalus Positive Positive Pteropus alecto Positive Positive Pteropus scapulatus Positive Positive NEGATIVE BRAINS Pteropus scapulatus (-ve control)** Negative Negative 2 [Insert footer if required]

3 Miniopterus schreibersii (Large bent winged bat) Negative Negative Chalinolobus morio (Chocolate wattled bat) Negative Negative Pteropus poliocephalus Negative Negative Microbat (unidentified) Negative Negative Pteropus scapulatus Negative Negative Microbat (unidentified) Negative Negative Microbat (unidentified) Negative Negative Pteropus poliocephalus Negative Negative Microbat (unidentified) Negative Negative Nyctophilus geoffroyi Negative Negative Pteropus sp Negative Negative Pteropus poliocephalus Negative Negative Long Eared bat Negative Negative Nyctophilus geoffroyi (Lesser long eared bat) Negative Negative Pteropus poliocephalus Negative Negative Pteropus sp O Negative Negative Chalinolobus gouldii O Negative Negative Pteropus poliocephalus O Negative Negative GHFF Grey headed flying fox; * not visible on photo; ** lab controls; SAN sample admission number; FAT fluorescent antibody test CONCLUSIONS All Lyssavirus positive brains originally detected by FAT were positive using the device. The level of reactivity observed with the devices varied between samples. This could be partly due to variability during the preparation of the 10-20% brain homogenate as the amount of diagnostic material available was limited. It was not possible to relate these observed differences in reactivity to differences in virus titre. There were no false positives detected as no reactivity was detected by the FAT negative samples that were available from different species of bats. It should be noted that all the positive brains tested were from Pteropus spp. of bats. These are the most common samples submitted to our laboratory. The use of this kit according to manufacturer s guidelines would significantly reduce the sensitivity of the kit, due to the additional dilution of the homogenate. When the kits were performed according to Kang et al (2007) they accurately detected Australian bat lyssavirus. Therefore, the correct preparation of the brain homogenate is essential for the accurate performance of the kit. 3 [Insert footer if required]

4 We have yet to test the kits against the insectivorous ABLV strain. We are trying to source some insectivorous ABLV samples for our reference collection. In the future it may be possible for us to test these kits against the insectivorous ABLV. BIBLIOGRAPHY BoKyu Kang, JinSik Oh, ChulSeung Lee, Bong-Kyun Park, YoungNam Park, KyungSoo Hong, KyungGi Lee, ByungKi Cho, DaeSub Song Evaluation of a rapid immunodiagnostic test kit for rabies virus. Journal of Virological Methods 145: REGARDS ANDREA CERTOMA Contact Dr Wilna Vosloo Phone ( ) Fax ( ) Wilna.vosloo@csiro.au 4 [Insert footer if required]

5 Figure 1: NEGATIVE BRAINS RESULTS Negative Brains 4 drops (125µl) of 10% brain homogenate applied to the test window, 5min incubation at room temperature.

6 Figure 2: POSITIVE BRAINS RESULTS Positive Brains 4 drops (125µl) of 10% brain homogenate applied to the test window, 5min incubation at room temperature.