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

Abstract

BackgroundTo 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.MethodsOne 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.ResultsThere 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.ConclusionOur 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.

Prime detection of Dirofilaria immitis: understanding the influence of blocked antigen on heartworm test performance

“…Evaluation of samples collected 150–152 days (5 months) after infection showed 6/14 (42.9%) were positive before heat pretreatment but all (14/14) were positive after heat pretreatment [ 29 ]. Another study showed that heat pretreatment of samples allowed detection of antigen in experimentally infected dogs an average of one month earlier than when using unheated samples [ 30 ].…”

“…We do not know the true heartworm infection status of the 17 amicrofilaremic dogs whose samples changed to positive with heat pretreatment but suspect some of these may represent early, prepatent infections. Heat pretreatment can allow earlier detection of heartworm infection in both dogs and cats [ 28 – 30 ].…”

“…Until recently it was thought that antigen was not detectable until six months after infection, and that in dogs given heartworm preventives, antigen detection may be delayed as long as nine months after infection [ 1 , 8 ]. More recent studies have documented that with heat pretreatment, antigen can be detected as early as 4.2 months in dogs or 5.6 months in cats [ 28 – 30 ]. In addition, recent work has shown that administration of preventives and doxycycline to an infected dog does interfere with antigen detection, but this false negative can be reversed with heat pretreatment of samples [ 33 ].…”

Heartworm prevalence in dogs versus cats: Multiple diagnostic modalities provide new insights

“…A total of nine dogs converted from antigen-negative to positive after heat treatment, but did not have detectable microfilariae. These dogs may have had early D. immitis infections; heat treatment has been shown to detect experimental infections as early as 4.2 months ( Carmichael et al, 2016 ). The results from this study support those from other studies demonstrating that heat treatment increases antigen test sensitivity, especially in dogs considered at risk of HW infection but with negative in-clinic antigen test results ( Little et al, 2018 ).…”

“…Infective 3rd stage larvae are transmitted by mosquito vectors and develop through tissue phases into adult nematodes in the pulmonary arteries ( Bowman and Atkins, 2009 , Lee and Atkins, 2010 ). Adult D. immitis reside in the main pulmonary artery approximately 4 months after initial infection, with antigen detected 4.2 months after infection if samples are heated prior to testing, or 6–9 months after infection if samples are not pre-treated ( Bowman and Atkins, 2009 , Carmichael et al, 2016 ). Mature worms reproduce sexually and release microfilariae into the circulation 6–9 months post infection, serving as a source of infection for mosquitoes to complete the life cycle ( Bowman and Atkins, 2009 , Ledesma and Harrington, 2011 ).…”