Evaluation of Dirofilaria immitis antigen detection comparing heated and unheated serum in dogs with experimental heartworm infections

James Carmichael, Scott McCall, Utami DiCosty, Abdelmoneim Mansour, Linda Roycroft

To evaluate whether heated serum allows for earlier detection of Dirofilaria immitis antigen, dogs with experimental D. immitis infections underwent weekly blood sampling to compare antigen results using both heated and unheated serum. One of two isolates (JYD-34 or Big Head™) were used to infect naïve laboratory beagle dogs. Serum was collected from dogs weekly and divided into two aliquots, heated and unheated. The samples designated as heated were placed in a heat block at 104 °C for 10 min then centrifuged with collection of the resulting supernatant. Two commercial ELISAs, DiroCHEK® (Synbiotics Corporation, Zoetis) and PetChek® (IDEXX Laboratories, Inc.), were used to conduct D. immitis antigen testing on all serum samples. There was no statistical difference in the mean number of days from infection to positive D. immitis antigen status between the two commercial testing kits (DiroCHEK® versus PetChek®) with either heated or unheated serum. When unheated serum was utilized, very strong agreement between the two assays was demonstrated using Lin's concordance correlation coefficient (R c  = 0.98). However, when heated serum was compared, Lin's concordance correlation coefficient was only R c  = 0.64, showing a lesser agreement. There was a statistical difference in the mean number of days from infection to a positive test result for unheated serum when compared to mean days to positive status with heated serum. For DiroCHEK® the heated serum yielded a positive result 126.9 ± 18.9 days postinfection while the unheated serum yielded a positive result 162.6 ± 23.0 days postinfection; this was a significant 35.7 ± 32.2 days longer, on average, compared with heated serum. With PetChek® the heated serum yielded a positive result 131.5 ± 11.7 days postinfection while the unheated serum yielded a positive result 162.8 ± 23.8 days postinfection; this was a significant 31.3 ± 25.5 days longer, on average, compared with heated serum. The detection of D. immitis antigen earlier using heated serum was consistent for both heartworm isolates. Our results suggest heat treatment of serum may allow earlier detection of D. immitis antigen but with less consistency demonstrated across two testing platforms as compared with antigen detection using unheated serum.