Abstract
This retrospective study was performed on 71 dogs which had been admitted for heartworm screening or with clinical suspicion of heartworm disease. The examination methods included polymerase chain reaction (PCR) to identify Dirofilaria immitis and/or Dirofilaria repens infections and a heartworm antigen (Ag) test (VetScan). By using PCR, 26 dogs were found positive only for Dirofilaria immitis (Group 1), while 21 dogs for both D. immitis and D. repens (Group 2). Group 3 included 24 dogs with D. repens infection only according to the PCR results. The sensitivity of the VetScan Ag test for the Group 1 and 2 animals proved to be 97.7% (95% Blaker confidence interval; CI 89.0%–99.9%). The specificity of the VetScan Ag test, calculated from the results of Group 3, was found to be 66.7% (95% CI 45.6%–83.1%), which was lower than that reported from the USA, where D. repens does not occur. In cases when PCR results were positive for D. repens but negative for D. immitis, the occult dirofilariosis was the likely explanation for the positive D. immitis Ag tests. These observations highlight the importance of performing more Ag tests simultaneously in those areas where both Dirofilaria species are present.
Introduction
Heartworm disease (HWD) is a worldwide distributed parasitosis caused by Dirofilaria immitis being present in Europe, America, Australia, and Asia. Several regions of the Central and Southern European countries are endemic (McCall et al., 2008; Morchon et al., 2012; Simon et al., 2012). Until 2000, HWD had been diagnosed in Hungary only in dogs imported from the USA (Boros et al., 1982; Vörös et al., 2000). The first autochthonous D. immitis infection was detected in a Hungarian Vizsla from the eastern part of Hungary (Jacsó et al., 2009). Since then, some papers have been published about dogs and wild canids infected with D. immitis in the country (Farkas et al., 2014, 2020; Tolnai et al., 2014; Bacsadi et al., 2016; Trájer et al., 2016). The other Dirofilaria species, Dirofilaria repens is present in Africa, Europe, and Asia, but not in the Americas (Simon et al., 2012; Genchi and Kramer, 2017). The occurrence of this parasite has been known in Hungary for many decades (Kotlán, 1951; Fok et al., 1998; Széll et al., 1999; Jacsó and Fok, 2006; Jacsó, 2014; Farkas et al., 2020) as well as in other countries of Europe (Capelli et al., 2018).
Dirofilaria immitis can cause fatal cardiorespiratory consequences and even death without proper treatment, whilst D. repens infection is less pathogenic. Their differentiation is especially important during the diagnostic work-up of clinical cases (ESCCAP Guideline 05, 2019). For detecting microfilariae (mf) of both Dirofilaria species, the modified Knott test is used mostly (Jacsó and Fok, 2006; Majoros and Juhász, 2015; Genchi et al., 2018). However, the Knott technique is rather applicable for diagnosing microfilaraemia without definitive distinction of D. immitis and D. repens (Magnis et al., 2013).
The polymerase chain reaction (PCR) technique as a molecular biological method can be considered the most sensitive and reliable diagnostic technique for in vivo diagnosis of blood filaroids (Favia et al., 1996; Rishniw et al., 2006; Latrofa et al., 2012; Rojas et al., 2015). This method allows definitive determination and distinction of D. immitis and D. repens if there are at least 4 mf in 1 mL peripheral blood (Gioia et al., 2010).
The circulating antigens are produced only by the adult female D. immitis worms (Roth et al., 1993; Goodwin, 1998; Atkins, 2003). The sensitivity and specificity of the serological methods are affected by some factors, e.g., by the number of adult female worms and by the amount of the circulating antigens (Roth et al., 1993; Lee et al., 2011; Aron et al., 2012; Burton et al., 2020). The application of the Ag tests has its distinguished importance in cases of occult dirofilariosis (more precisely occult heartworm infection) when no mf can be detected in the peripheral blood despite a persisting HW infection with the presence of adult heartworms. Occult dirofilariosis can have various causes, e.g., previous macrocyclic lactone application for prevention or unisex infections. Another important phenomenon is the so-called true occult dirofilariosis, which occurs when the mf are eliminated from the peripheral blood by the immune system of the infected dog (Wong et al., 1973; Rawlings et al., 1982; Bagi et al., 2017; Vörös et al., 2017). For occult dirofilariosis, the PCR assay is not indicated as D. immitis DNA is extracted from mf in case of in vivo determination. Therefore, the diagnostic accuracy of the Ag tests is of utmost importance in occult dirofilariosis, as the sole laboratory diagnostic method in these cases. This is especially true in those regions, including several European countries, where both D. immitis and D. repens may occur either separately or simultaneously in infected dogs (Ionica et al., 2015; Ciuca et al., 2016a, b; Bagi et al., 2017; Farkas et al., 2020).
We have found only a few publications reporting on the application of these Ag tests in those regions where both D. immitis and D. repens are present (Ionica et al., 2015; Ciuca et al., 2016a, b). Genchi et al. (2018) have reported their experiences regarding the sensitivity and specificity of the following Ag tests: Speed Diro™(BVT-Virbac); PetChek® HTWMPF (IDEXX); Witness Dirofilaria Ag Test (Zoetis); Idexx SNAP Heartworm RT test (IDEXX). However, Genchi et al. (2018) have not compared their Ag test results with molecular biological (i.e., PCR) methods to identify circulating mf but rather performed morphological analysis by using the Knott method. Trájer et al. (2016) and Farkas et al. (2020) have described the detection of occurrence of D. immitis and D. repens alone or concomitantly in Hungary. They used PCR without comparison with the serological diagnostic methods. It is also worth to mention that no serological tests are available to detect adult D. repens worms (Tarello, 2011; Jacsó, 2014; Genchi and Kramer, 2017).
The VetScan Ag test (Abaxis VetScan® Canine Heartworm Rapid Test Kit) is an immunochromatographic sandwich assay test for the detection of adult D. immitis worms (Lee et al., 2011). Its performance in terms of sensitivity and specificity has been compared with those of numerous similar tests (Atkins, 2003; Lee et al., 2011; Aron et al., 2012; Rojas et al., 2015; Henry et al., 2018). However, these reports included canine populations from the Americas where D. repens does not occur.
In the literature, we did not find reports on the validation of D. immitis Ag tests in dogs from regions where both Dirofilaria species are present. Therefore, the main goal of our study was to determine the sensitivity and specificity of the VetScan Ag test – as an example for Ag tests - in dogs originating from D. repens affected areas i.e., dogs with known single D. immitis or D. repens or with mixed infections. For these purposes, the PCR assay served as the most accurate method of our study. As a second goal, we aimed to demonstrate if occult dirofilariosis could be the explanation for positive Ag tests in spite of a negative PCR result of D. immitis. By answering these questions, we intended to demonstrate the diagnostic difficulties and possibilities of HWD in regions where concomitant infections by D. immitis and D. repens as well as occult dirofilariosis occur.
Materials and methods
Altogether 71 dogs were included retrospectively into the study. These dogs were randomly selected from dogs arriving for HW screening examinations or with clinical suspicion of HWD. The examinations were done at the Clinic of Internal Medicine of the University of Veterinary Medicine Budapest (UVMB), Hungary, at the Budatétény Animal Hospital, Budapest, Hungary, and at the Rákosliget Small Animal Ambulatory Clinic, Budapest, Hungary. Selection criteria included performed PCR assays, with positive results of D. immitis and/or D. repens as well as VetScan Ag test studies to detect adult female HWs.
EDTA-preserved peripheral blood taken by the veterinarians of the clinics was examined freshly or preserved at 4 °C for maximum two days. Blood samples were taken at different times of the day. These timings were not recorded in the study. The modified Knott test was applied at the Department of Parasitology and Zoology, UVMB to detect circulating mf (Majoros and Juhász, 2015).
DNA was isolated from 0.2 mL of blood from each sample using NucleoSpin® Tissue kit (Macherey-Nagel GmbH, Germany). Multiplex and conventional PCRs targeting fragments of 12S rDNA of both Dirofilaria spp. and 16S rRNA of D. immitis were used, respectively (Liu et al., 2005; Gioia et al., 2010). The majority of the PCRs were done at the Department of Parasitology and Zoology, UVMB, whilst some of them have been performed at the Praxislab Kft. and at the Vet-Med-Labor Zrt. in Budapest, Hungary, where similar PCR assays were applied. PCRs were done in 68 dogs with microfilaraemia detected by the modified Knott test. Three dogs were examined by PCR despite the negative Knott test, as they had a positive VetScan Ag test result, and their history and clinical findings suggested the possibility of HW infection. This was performed keeping in mind that the PCR is more sensitive than the Knott test, i.e., it can be positive in Knott negative cases (Gioa et al., 2010; Rojas et al., 2015).
All samples were tested for the presence of D. immitis antigen with the VetScan Ag test (Abaxis VetScan® Canine Heartworm Rapid Test Kit, ABAXIS Europe GmbH, Germany). In addition to a positive VetScan Ag test, serologic test kits from other manufacturers were used in six dogs, which had negative PCR results for D. immitis, but positive for D. repens, in order to define the possibility of an occult dirofilariosis. To this end, the following Ag tests were applied: DiroCHEK® Canine Heartworm Antigen Test Kit (Synbiotics Corporation, San Diego, USA); Witness Dirofilaria Ag Test (Zoetis Hungary); Idexx SNAP Heartworm RT test (IDEXX Germany). The Ag tests were done from serum samples in the laboratory of the Department of Parasitology and Zoology, UVMB or from whole blood at the Department and Clinic of Internal Medicine, UVMB as well as at the two external clinics according to the prescriptions of the manufacturers.
Statistical methods
The sensitivity and the specificity of the VetScan Ag test were estimated in comparison to PCR results. Thus, only those cases were included in the analysis where VetScan Ag test as well as PCR results were available. The examined patients were selected as described above, therefore sensitivity and specificity of the VetScan Ag test is related to this population. The sensitivity was calculated by including cases with positive D. immitis results. The specificity was determined by using patients with negative D. immitis and positive D. repens PCR results as detailed in Results. Point estimates of sensitivity and specificity (Stevenson, 2008) were accompanied by 95% Blaker confidence intervals (CI) (Blaker, 2000).
Results
The dogs were divided into three groups based on the PCR results: Group 1: 26 (36.6%) dogs were positive only for D. immitis. Group 2: 21 (29.6%) dogs proved to be positive for both D. immitis and D. repens. Group 3: 24 dogs (33.8%) were positive only for D. repens.
The sensitivity of the VetScan Ag test was 46/47 = 97.7% (95% CI 89,0–99,9%) based on the compiled results of Group 1 and 2 where the dogs had PCR-proven D. immitis infections. For the specificity of the VetScan Ag test, the results from Group 3 were used, in which dogs were D. immitis negative and D. repens positive by PCR. The specificity of the test was 19/24 = 66.7% (95% CI 45.6–83.1%).
Examining only Group 1 (n = 26), where all animals were positive only for D. immitis with PCR, the VetScan Ag test gave positive reactions in all cases except one dog. As such, the sensitivity in this group was 25/26 = 96.2% (95% CI 81.7–99.8%). When analysing Group 2 (n = 21), where all animals were PCR-positive both for D. immitis and D. repens, the sensitivity within this group was 100% (95% CI 84.8–100%).
The sensitivity and the specificity of VetScan Ag test results were compared with previous studies performed in the USA where D. repens does not occur. The sensitivity of the used Ag test, calculated based on the D. immitis PCR-positive samples of Group 1 and Group 2, was excellent (97.7% in average). These results were similar to those of other authors (Table 1). The specificity of the Ag test, calculated based on Group 3 with only PCR-positive D. repens samples, was lower compared to the reports from the USA. Atkins (2003) examined blood samples of dogs infected experimentally with a known number of adult female HWs. Aron et al. (2012) as well as Henry et al. (2018) verified the Ag positivity of their blood samples by necropsy. However, Dirofilaria PCR detections were not performed in these previous studies (Table 1).
Comparison of the sensitivity and specificity values of the VetScan Ag test obtained in our study with those published by others
| Present study | Atkins (2003) | Aron et al. (2012)** | Henry et al. (2018) | |
| Sensitivity % (95% CI) | 97.7 (89.0–99.9) n = 47 | 78.0 (72.0–84.0) n = 208 | 92.0 n = 25 | 98.5 (95.7–99.7) n = 200 |
| Specificity % (95% CI) | 66.7 (45.6–83.1)* n = 24 | 97.0 (84.0–100) n = 32 | 100 n = 24 | 94.0 (83.4–98.7) n = 50 |
CI: Blaker 95% confidence interval; n: number of tested animals; *: Applied to patients found infected with D. repens and negative for D. immitis by PCR. **: CI was not provided.
The samples of 6 dogs in Group 3 were positive with VetScan Ag test, despite that their PCR results were negative for D. immitis and positive only for D. repens. Of these 6 cases, 4 dogs were also seropositive when the Witness Dirofilaria Ag test was done additionally. The other 2 dogs were tested also with the Witness Dirofilaria Ag test, furthermore with the Idexx Snap Heartworm RTtest, as well as with the DiroCHEK® Canine Heartworm Ag test, and all gave positive results.
Discussion
Several publications can be found in the literature on the evaluation of various Ag tests detecting HW infections. The sensitivity of these tests varies between 77% and 100%, while the specificity yields between 90% and 100% (Atkins, 2003; Aron et al., 2012; Starkey et al., 2017; Genchi et al., 2018; Burton et el., 2020). As such, they are important parts of HW screening examinations and the diagnosis of HWD. However, it is known that Ag tests of D. immitis infections may give false positive reaction for Spirocerca lupi (Aroch et al., 2015), Angiostrongylus vasorum (Schnyder and Deplazes, 2012), and Acanthocheilonema dracunculoides (Szatmári et al., 2020). False positive cross-reaction may also occur for D. repens. Genchi et al. (2018) did not find cross-reaction when they used several Ag tests in 21 cases, which were microfilaremic with Knott test. However, there was no PCR examination in that study. Ciuca et al. (2016b) performed PCRs to detect D. immitis and D. repens on 108 Knott-positive canine blood samples as well as DiroCHECK®, the Zoetis Ag test to demonstrate the presence of adult HWs. The antigen testing for D. immitis showed conflicting results in the examined 12 dogs with only D. repens mf. Two dog samples (16%) were antigen negative both before and after the heat treatment of blood sera. Six dog samples (50%) demonstrated antigen positivity after heating and four samples (30%) were antigen positive both before and after heat treatment. These results might suggest that dogs infected with D. repens can produce false D. immitis positivity, especially after preheat treatment, or may have also an occult infection with D. immitis.
Dirofilaria repens cross-reaction with HW Ag tests has been indirectly proven by Venco et al. (2017). They reported on two experiments in the same publication: One study included live adults of D. immitis, D. repens, Toxocara canis, Toxocara cati, Dipylidium caninum, Taenia taeniaeformis, and Mesocestoides spp. larvae which were washed and incubated with saline solution. These solutions containing excretory/secretory antigens were tested with different HW Ag tests. In their second study, sera from dogs with natural infections by An. vasorum or D. repens, living in areas free of heartworm disease, were tested with the same Ag tests before and after preheat treatment. They found that cross-reactions with An. vasorum and D. repens might happen, and it can even potentially occur also with other helminths. Dirofilaria repens appeared to release more antigens – responsible for cross-reaction – than the other worms examined. Preheat treatment reduced the specificity of the test by increasing cross-reactivity.
False negative Ag test results might also occur, mainly due to the low number of adult female heartworms, which can influence sensitivity (Atkins, 2003). It is known that the negative Ag test may became positive after heating the blood sample (Velasquez et al., 2014; Drake et al., 2015; Ames et al., 2017; Bendas et al., 2017; Savadelis et al., 2017). Other authors have also found that the occurrence of false positive reactions increased with sera preheating, thereby heating seems to decrease specificity (Venco et al., 2017; Savadelis et al., 2018; Gruntmeir et al., 2020). Sobotyk et al. (2021) performed D. immitis Ag test with DiroCHEK®Canine Heartworm Ag test on dogs experimentally infected with D. repens. No cross-reaction (i.e., false positivity) was found in any dogs prior to heat treatment, whilst the antigen test became positive after heat treatment of the samples. They concluded that the preheat treatment of the serum possesses limited clinical value and should be applied with caution. The heating of blood samples is not recommended by the American Heartworm Society (AHS) for routine heartworm screening (Nelson et al., 2014, 2020). In addition, preheating is not included in the procedure manual of any manufacturers. For these reasons, we did not perform the preheat Ag test in our cases.
In the present study, we used the VetScan heartworm Ag test as an example for the in-clinic antigen tests. The results of this Ag test were compared with PCR results of known D. immitis and/or D. repens infected dogs. The sensitivity of the VetScan Ag test performed on D. immitis positive dog samples (Group 1 and 2) was excellent, in correlation to the relevant literature (Atkins, 2003; Aron et al., 2012; Henry et al., 2018). However, its specificity was lower in Group 3, where dogs were infected with D. repens but negative for D. immitis, compared to the relevant data of these reports, all from the USA where D. repens does not occur.
In our study, D. immitis and D. repens infection occurred alone or as mixed infection in a similar proportion regarding the number of cases within the three groups. This observation was confirmed by PCR albeit it differs from the previous findings. Previous studies reported lower rate of mixed infections than the rate of D. immitis or D. repens cases alone (Ionica et al., 2015; Ciuca et al., 2016a, b; Trájer et al., 2016; Farkas et al., 2020). Ionica et al. (2015) screened 390 dogs with a multiplex PCR assay. Of them, 46 (11.8%) were positive for dirofilariosis and 8 (2%) for Acanthocheilonema reconditum infection. Coinfections with both Dirofilaria species and D. repens and Ac. reconditum were found in 11 (23.9%) and 2 (4.3%) dogs in their study, respectively. Ciuca et al. (2016a) examined 45 stray dogs with Knott test and 23 (51.1%) patients were positive for circulating mf. Nineteen dogs were positive for D. immitis and 4 for both D. immitis and D. repens using another type of multiplex PCR system. Ciuca et al. (2016b) have performed PCR examination using the same multiplex assay on 24 dogs, which were positive for circulating mf. Six out of 24 dogs have been found infected with D. immitis, 12 with D. repens and 6 animals with both D. immitis and D. repens. In one of these cases with ‘mixed’ dirofilariosis, Ac. reconditum infection has also been detected. Based on the positive DiroCHEK® Canine Heartworm Ag test before and after heat treatment of the samples, the authors have hypothesised that dogs being infected with D. repens might have had also an occult infection with D. immitis. A similar hypothesis has been suggested by Rojas et al. (2015) who found D. immitis positivity with the Vetscan Ag test in five dogs negative by both the Knott test and PCR.
One of the explanations of the lower specificity obtained in our study could be the possible occurrence of occult dirofilariosis (i.e., occult heartworm disease). We suspected occult infections for 6 dogs in Group 3 which were D. immitis negative with PCR, but positive for D. immitis antigen with several Ag tests of different manufacturers. The in vivo proof of occult dirofilariosis could be the demonstration of adult heartworms in the main pulmonary arteries during echocardiography. However, no adult heartworms were detected by this imaging method in these dogs. The explanation could be that these cases belonged to the 1st and 2nd clinical categories of HWD, where adult worms rarely occur in those vessels (Bowman and Atkins, 2009; Nelson et al., 2020). Nevertheless, our results seem to provide a stronger argument for the coinciding presence of D. repens infection and occult dirofilariosis compared to those of Rojas et al. (2015) and Ciuca et al. (2016b), as these authors applied only one Ag test in their studies. Ionica et al. (2015) also performed only one Ag test and found 7 Ag-positive cases with sole D. repens infection i.e., without D. immitis PCR positivity. In our opinion, the chance for a cross-reaction caused by D. repens is less when several Ag tests are applied, and all are positive. This phenomenon rather speaks for occult dirofilariosis as it is supposed in our cases. The same scenario might be valid to exclude potentially false positive cross-reactions with other filarioid worms like S. lupi (Aroch et al., 2015) and An. vasorum (Schnyder and Deplazes, 2012).
In those continents/countries where both helminths occur, the parasitological diagnosis of D. immitis including its differentiation from a sole D. repens infection is inevitable before starting any therapy. In dogs with a positive Knott test demonstrating microfilaraemia, PCR should be performed to identify these parasites with great certainty (Favia et al., 1996; Rishniw et al., 2006; Latrofa et al., 2012; Rojas et al., 2015). However, Ag tests are also important, especially in occult dirofilariosis cases (Vörös et al., 2017; Genchi et al., 2018; Nelson et al., 2020). This issue raises significant diagnostic dilemmas when a decision should be made for the treatment of HWD (Maxwell et al., 2014; Nelson et al., 2020). At the Clinic of Internal Medicine of UVMB, we use three or sometimes even four Ag tests from different manufacturers simultaneously in cases of occult dirofilariosis (Vörös et al., 2017). The complex treatment is applied only if all tests are positive and/or adult HWs are seen in the main pulmonary arteries. However, in the D. immitis PCR-negative cases, even multiple positive Ag tests cannot fully exclude the possibility of a false positive cross-reaction with other blood filaroids. Nevertheless, it can be assumed that the proper treatment of HWD is better than not to perform it because of the lack of an all-round diagnostics (Nelson et al., 2020). In our patients with equivocal results, we offer the moxidectin-doxycycline therapy i.e., without melarsomine as it has been recently described (Ames et al., 2017; Bendas et al., 2017; Savadelis et al., 2017; Genchi et al., 2019).
Our study has several limitations. Unfortunately, we have not found Ag-test-positive dogs with D. repens positive and D. immitis negative PCR results, and echocardiographically detectable adult heartworms that could have been an in vivo proof of occult dirofilariosis. The PCR of blood samples, collected from our patients, was performed in three different laboratories for both financial and organisational reasons. Another limitation of our study is that we did not apply any methods to exclude S. lupi or An. vasorum infections. A cross-reaction with D. repens might be directly proven with D. immitis Ag tests on D. repens PCR positive dogs, as well as performing necropsy to exclude a simultaneous D. immitis infection. No such reports exist in the literature as far as we know. Thus, the occult dirofilariosis can be confirmed definitely by post-mortem pathological examination, which was not an option as our patients healed after the successful treatments.
In conclusion, the sensitivity of the Ag test performed on D. immitis positive dog samples was excellent, compared to the previous reports on this subject. However, its specificity was lower in the examined dogs, which were infected with D. repens but negative for D. immitis, when compared to the related reports, all from the USA, where D. repens does not occur. In cases of positive D. repens and negative D. immitis PCR results, the occult dirofilariosis could be the explanation for the positive D. immitis Ag tests. These observations highlight the importance of performing multiple Ag tests in those areas where both Dirofilaria species are present. This has special importance when a PCR cannot be performed in the lack of mf in the peripheral blood or when this technique reveals only the presence of D. repens. Further research would be required on a larger population to explore the occurrence rate of occult dirofilariosis, as well as the antigen cross-reaction caused by D. repens in cases of canine heartworm infections in those regions where both Dirofilaria species are present.
Acknowledgements
Our research was supported by the KK 69P02RM06 research project (2017) and by the NKB project (2018), both provided by the University of Veterinary Medicine Budapest, Hungary. This study was supported by the Doctoral School of the UVMB as part of the doctoral thesis of the first author. The authors thank the Praxislab Kft. and the Vet-Med Labor Zrt. for performing some of the PCRs.
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