In experimental infections, antigen detection in urine was consistent with blood products for infections 7 months in length and greater. As a screening test, urine was specific with a high negative predictive value, and was positive in some cases where antigenemia was not detected. In both experimental and clinical populations, percent change in OD after heat treatment was positive in blood products and negative in urine.
Antigenuria following antigenemia is a common result of many infectious processes in humans and animals [21, 22]. Therefore, urine has been used as a diagnostic template for antigen detection in other infectious diseases in dogs and humans, including histoplasmosis and blastomycosis [23]. Parasitic nematode infections of the urinary tract can be diagnosed via detection of eggs with sedimentation and urinalysis [24, 25, 26], but urine has not been extensively explored for detection of nematode infections present outside the urinary tract.
The potential use of urine as a diagnostic sample for heartworm antigen detection is based on the presence of adult D. immitis and microfilariae in the cardiovascular system and the role of the kidneys in filtering the blood. Previous studies have demonstrated glomerular changes secondary to heartworm disease, including thickening and vacuolation of the glomerular basement membrane, deposition of immune complexes in the glomerular basement membrane leading to immune complex-mediated glomerulonephritis, proliferation of mesangial cells and matrix and occasionally the presence of microfilariae lodged in the glomeruli [10, 11, 12]. Rarely, microfilariae have be detected on urine sedimentation examinations, and can be associated with hematuria, proteinuria, or other signs of renal disease [27].
To date, only a few studies have investigated heartworm-associated antigenuria in dogs, but our knowledge on the use of urine as an alternative diagnostic template for routine heartworm screening is limited. In experimental heartworm studies, D. immitis antigenuria was seen in infected dogs by day 398 post-infection [28], and in infected cats by day 240 post-infection [9]. However, our results show that antigenuria may be detected as early as 4 months post-infection as seen in samples of experimentally infected dogs assessed in the present study.
During the 5-6-month post-infection period, antigen detection in blood or blood products has been described as “inconsistent” [5]. Previous studies have also demonstrated the variability in detection of serum/plasma heartworm antigen during this period [29]. One explanation is that during this time there are not many adult worms present, so the amount of antigen available for detection is low [30]. Given that the soonest antigen is commonly detected is between 5–7 months post-infection [4], or as early as 3 months post-infection with ICD methods [5], discrepancies in results are possible earlier in infection. These discrepancies are potentially responsible for the “moderate” agreement demonstrated in this study. In the present study, samples of all biofluids from infections fewer than 4 months in duration were antigen negative, pre- and post-ICD. All experimental serum/plasma samples tested for antigen at 5 months post-infection (n = 11) and 6 months post-infection (n = 3) were positive for antigen, regardless of antigenuria status. During the same time, 7 urine samples tested positive at 5-months post-infection (n = 11), and 2 urine samples tested positive at 6-months post-infection (n = 3). Urine samples from one dog (Dog #3) in this population were negative for antigen at both 5- and 6-months post-infection, but positive at 7-months both pre- and post-heat-treatment. The change in this sample’s positivity from 6 to 7-months post-infection highlights the variability during this period as the developing larvae finish maturing into adults and establishing infection in the pulmonary arteries (Table 5). All serum/plasma and urine samples were positive from 7 months onward, supporting the use of urine for antigen detection when following the AHS recommendations. Data from this population also demonstrates that samples ≥ 5 months post infection typically contained adequate antigen levels for detection in the urine.
Seven urine samples in the experimental population were negative. Infection duration of these samples ranged from 3–6 months post-infection. Two of the negative samples were 3- and 4-months post-infection and the associated serum was also negative for antigen with or without the addition of heat treatment. Both samples were collected before the expected time for antigen to be in high enough concentrations for detection. The other 5 negative urine samples were either 5-months post-infection (4/5) or 6-months post-infection (1/5), where antigen was detected in serum in all instances, 4 without heat treatment, and 1 only after the addition of heat treatment.
In the clinical population, urine had high specificity and low sensitivity for detecting heartworm antigen, which may limit its use as a clinical screening test but might still be useful in a research setting, as few false positive results would be expected. The low sensitivity was a result of 18 urine samples testing negative for antigen pre- and post-ICD, with matching serum samples testing positive pre- or post-ICD. The low PPV in this population was a result of 12 urine samples testing positive for antigen while matching serum samples were negative for antigen, pre- and post-ICD. While these results are difficult to interpret in a diagnostic and clinical context and further investigations are warranted, there might be plausible biological explanations for such results. If these results are true negatives, the urine positivity could have been due to cross-reactivity of antigens associated with other parasites (Spirocerca lupi, Dirofilaria repens, etc.) that may have been altered during the heat treatment protocol as seen in serum and plasma of dogs infected with other nematodes [31, 32], even if matching sera tested negative. Alternatively, in the case that all or some of these were true positives, heartworm antigen present in urine could have been detectable because of an increased urine concentration due to time of collection or dehydration, while antigen levels in serum were still present at low concentrations. Adequate urine volume was not available to measure USG on all samples; however, in the experimental population, samples had an adequate sample volume, and no significant difference was detected in the urine of animals with positive or negative serum/plasma samples. According to the manufacturer, false positives in serum and plasma may occur in animals infected with 3 or fewer adult females. This, coupled with the detection of heartworm antigenuria in experimental samples as early as 4 months post-infection could suggest that these are true positive results.
Our combined results of antigen detection in serum pre- and post-ICD was comparable to that found in previous studies in a similar population from the same geographic area [33], and higher than the expected prevalence for client-owned dogs in Texas [3]. While heat treatment of urine did not prove to be beneficial for antigen detection, our study further supports the value of heat-treatment of serum or plasma samples from dogs in endemic areas, or with an unknown history of heartworm prevention [6]. However, it is possible that in a different biofluid, such as urine, heartworm antigens are less stable and more prone to degradation, rendering them undetectable post-heat treatment. This was demonstrated in both populations in the present study when urine positivity changed from positive to negative post-heat treatment. This change in urine antigen positivity was more evident in the clinical samples compared with the experimental samples.
In the experimental sample population, urine was positive when serum/plasma was positive in all but 4 samples. The percentage of urine antigen-positive samples (i.e., pre- or post-ICD) out of all serum/plasma positive samples was 81.4% (22/27) in this population.
In the clinic sample population, urine was most often negative when serum was negative, except for the 12 samples discussed previously. The percentage of urine positive samples out of all serum positive samples was 51.4% (19/37).
Regarding the agreement between antigen detection in the serum/plasma and the presence of antigen in the urine, the results were variable depending on the population: experimental or clinical, and whether urine had been subjected to ICD via heat treatment. In both populations, the average optical density of serum samples increased with the addition of heat, indicating the presence of more antigen for detection, and decreased in urine after the addition of heat, indicating less antigen available for detection. In experimental samples, agreement was higher between post-ICD serum and pre-ICD urine (kappa = 0.77) when each fluid should have maximum antigen available for detection, vs pre-ICD serum and pre-ICD urine (kappa = 0.45). Agreement was lowest between pre-ICD serum samples and post-ICD urine (k = 0.39), when antigen for detection in each fluid should theoretically be lowest. In the clinic population, agreement was similar between post-ICD serum and pre-ICD urine (kappa = 0.54) and pre-ICD serum and urine (kappa = 0.58). In both of these groups, antigen in urine should be highest, but antigen in serum would be expected to be higher after heat treatment. Therefore, the finding of only moderate agreement between post-ICD serum and pre-ICD urine was unexpected and is due to a higher number of positive urine samples compared with serum.