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Detection of circulating antigen of Dirofilaria immitis has been a mainstay of identifying heartworm infection in clinical practice for the past three decades. Several validated commercial antigen tests have very good sensitivity, specificity, and positive predictive values, especially when used in patients for which heartworm infection is likely. In some dogs and cats infected with heartworm, antigen may not be available for detection although present in the patient sample; heat pretreatment of these samples reveals the antigen, changing the false negative to positive. This phenomenon was documented in the literature in the 1980s but subsequently overlooked by the heartworm research community for many years. In this review, we provide a summary of the current understanding of the role of heat reversal in diagnosing heartworm infection. This additional diagnostic step is most important for patients in which heartworm infection is likely, such as dogs or cats in an endemic area with an inconsistent history of heartworm preventive use, or dogs with a prior diagnosis of heartworm infection that were recently treated. To illustrate the concept, we share a summary of results from canine samples tested at the state veterinary diagnostic laboratory in Oklahoma, USA in 2017 by modified Knott test and by commercial antigen test before and after heat treatment of samples; in this sample set, heat treatment changed all D. immitis microfilaria-positive but antigen-negative samples to antigen-positive. Pet dogs with a history of consistent preventive use are unlikely to become positive with heat pretreatment; for that reason, routine pretreatment of all samples tested in a veterinary practice is not recommended. We also review known causes of false negative and false positive results on heartworm antigen tests that, although uncommon, can complicate accurate diagnosis in individual patients. Together, this review provides a primer to aid understanding of strategies that can enhance accurate diagnosis of heartworm infection in veterinary practice and clinical research.

The increase in reports of resistance to macrocyclic lactones in the canine heartworm, Dirofilaria immitis is alarming. While DNA based tests have been well-validated, they can be expensive. In a previous study, we showed that two biochemical tests adapted to a 96- well plate format and read in a spectrophotometer could detect differences among lab validated D. immitis isolates. The two tests- Resazurin reduction and Hoechst 33342 efflux-detect metabolism and P-glycoprotein activity respectively in microfilariae isolated from infected dog blood. Our objective was to optimize the two assays further by testing various assay parameters in D. immitis isolates not tested previously. We tested microfilarial seeding density, incubation time and the effect of in vitro treatment with ivermectin and doxycycline in five other D. immitis isolates-JYD-34, Big Head, Berkeley, Georgia III and LOL. All assays were performed in 3 technical replicates and 2-4 biological replicates. To understand the molecular basis of the assays, we also performed qPCR for selected drug metabolism and elimination associated genes of the ABC transporter and cytochrome P450 gene families. Metabolism and ABC transporter activity as detected by these assays varied between strains. Anthelmintic status (resistant or susceptible) did not correlate with metabolism or P-gp efflux. Basal transcriptional variations were found between strains in ABC transporter and cytochrome P450 genes. These assays provide a greater understanding of the biochemical variation among isolates of D. immitis, which can be exploited in the future to develop in vitro diagnostic tests capable of differentiating susceptible and resistant isolates.

Background The current American Heartworm Society guidelines recommend the concomitant use of an antigen detection test and a microfilariae detection test (MFDT) for diagnosing heartworm infection in dogs. The modified Knott’s (MK) test is the preferred MFDT for determining the morphological characteristics of Dirofilaria immitis microfilariae, but it requires extensive microscopy training and can be time-consuming in a clinical setting. The Pluslife Mini Dock is a point-of-care diagnostic platform that uses RNase HII-assisted amplification (RHAM) to eliminate the need for DNA extraction, with results available within 30 min. This study aimed to assess the performance of the Pluslife Mini Dock duplex Dirofilaria immitis/Dirofilaria repens assay in dog blood compared with the MK. Methods Archival, frozen whole-blood samples collected from 250 dogs at shelters in central Texas, USA, were used. Samples were subjected to the MK on the day of collection and stored at 2°C until further processing. The samples were then thawed and subjected to the Pluslife Mini Dock D. immitis/D. repens duplex assay. The results were analyzed using Cohen’s kappa coefficient and McNemar’s Chi-squared test. Results Overall, 93.6% of results matched between the two tests; however, the Pluslife assay detected a higher proportion of D . immitis -positive samples (32.4%; 81/250) than the MK (30.0%; 75/250). There was no statistical significance between tests ( p  = 0.2113). Cohen’s kappa statistic indicated almost perfect agreement between the two tests (0.85). Additionally, Acanthocheilonema reconditum was detected in 11 samples in the MK test, without generating false-positive results with the Pluslife assay, indicating its specificity. Conclusions Our data suggest that the Pluslife Mini Dock D. immitis/D. repens duplex assay provides a novel diagnostic platform and is a suitable option for point-of-care MFDT. Graphical Abstract

Background Canine heartworm disease is associated with various clinicopathologic abnormalities; however, most previous studies have focused on comparisons between infected and non-infected dogs rather than differences according to disease severity. The American Heartworm Society (AHS) clinical classification reflects disease severity in clinical practice, but clinicopathologic changes based on this classification have not been fully characterized. This study aimed to evaluate clinicopathologic variables according to disease severity and to assess their associations with heartworm class in dogs with heartworm disease. Methods This retrospective study included 35 dogs diagnosed with heartworm disease. Dogs were classified according to Amearican Heartworm Society guidelines into mild (class 1–2) and advanced (class 3–4) groups. Clinicopathologic variables, including complete blood count, electrolyte, serum chemistry, and thromboelastography (TEG) parameters, were compared between groups, and correlations with heartworm class were evaluated. Results A total of 35 dogs were retrospectively analyzed and categorized into mild (class 1–2, n  = 17) and advanced (class 3–4, n  = 18) groups. Compared with the mild group, dogs in the advanced group had significantly higher RDW, BUN, AST, CREA, and thromboelastography reaction time (R), and significantly lower Na⁺, Ca²⁺, and Cl⁻ concentrations. Spearman correlation analysis showed that heartworm class was positively correlated with RDW (ρ = 0.550), BUN (ρ = 0.601), AST (ρ = 0.788), and R (ρ = 0.730), and negatively correlated with Na⁺ (ρ = -0.430), Ca²⁺ (ρ = -0.422), and Cl − (ρ = -0.392) (all P  < 0.05). CREA was significantly different between groups but was not significantly correlated with heartworm class. Conclusions Clinicopathologic variables differed according to disease severity in dogs with heartworm disease. These findings suggest that selected variables may reflect increasing clinical severity and provide adjunctive information for clinical assessment. Further studies are needed to validate these associations.

Background The nematodes Dirofilaria immitis and D. repens are enzootic in Greece. In the light of evidence of dirofilariosis spreading to new areas around the world, the aim of the present study was to update and enrich the current knowledge on the prevalence of Dirofilaria infections in dogs in Greece, to assess the risk factors of heartworm infection, and to initiate the drawing of the epizootiological map of canine dirofilariosis, investigating Dirofilaria infections in five locations along the north-south axis of the country, i.e. municipalities of Thessaloniki, Larissa, Achaia, Attica and Heraklion, associated with the five largest urban centres of Greece. Methods Blood samples collected from 750 dogs in total, were examined by the modified Knott’s method and by serology. A questionnaire including information about each examined dog was filled in and statistical analysis of the results was performed using the Chi-square test and a Binary Multiple Univariate Generalized Linear Model. Results In total, 31 (4.1 %) out of 750 examined animals were found positive for D. immitis by any of the tests applied (Knott’s method and serological examination). Moreover, microfilariae of D. repens and Acanthocheilonema reconditum were detected by the Knott’s method in 17 (2.3 %) and 10 (1.3 %) of the animals, respectively. At the municipality level, the prevalence of infection was 14, 7, 5.3, 0.7 and 0 % for D. immitis , 1, 2, 8.7, 0.3 and 0 % for D. repens , and 0, 3, 2.7, 0.7 and 1 % for A. reconditum in Thessaloniki, Larissa, Achaia, Attica and Heraklion, respectively. In addition, in three dogs (one each in Thessaloniki, Achaia and Attica) mixed D. immitis - D. repens infections were detected by the Knott’s method. The area of the country, dog’s usage and age were determined as risk factors for heartworm infection. Conclusions Northern areas of Greece have higher Dirofilaria prevalence and the prevalence in a western province (Achaia) is recorded for the first time. The mosquito population dynamics recorded in the past is likely to play an important role in the distribution of Dirofilaria infections in Greece, and needs further investigation. Similarly, the role of wild reservoirs of filarial parasites in different areas needs to be clarified. Promotion by veterinarians of preventive treatment and compliance by pet owners is essential in all parts of Greece, regardless of the recorded prevalence of infection.

Abstract Background Platelet surface–associated immunoglobulin (PSAIG) occurs in thrombocytopenic dogs with vector-borne diseases and immune thrombocytopenia (ITP) and may be associated with thrombocytopenia severity and inflammatory markers, including neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios (NLR, PLR). Hypothesis/Objectives Assess associations between PSAIG positivity, hematologic parameters, thrombocytopenia severity, and vector-borne status in thrombocytopenic dogs. Animals Sixty-nine client-owned thrombocytopenic dogs (<200 × 103/μL) were enrolled between June 2022 and June 2023. Methods Dogs were prospectively enrolled. Platelet surface–associated immunoglobulin was measured using flow cytometry. Vector-borne pathogens were assessed by serology (Ehrlichia spp., Anaplasma spp., Borrelia burgdorferi, Dirofilaria immitis) and PCR for Ehrlichia canis. Hematologic parameters were compared between PSAIG groups (Mann–Whitney U), and associations tested by univariable logistic regression. Results Dogs positive for PSAIG (n = 16) had lower median automated platelet counts (16.5 × 103/μL; interquartile range [IQR]: 8.25-40.75) than PSAIG-negative dogs (n = 53; 64 × 103/μL; IQR: 25.0-92.5; P = .001), with similarly lower manual platelet counts (48 × 103/μL; IQR: 20-86 vs 96 × 103/μL; IQR: 55–138; P = .01) and automated PLR (7.14; IQR: 3.30-15.28 vs 21.82; IQR: 9.42–38.99; P = .01). In logistic regression, PSAIG positivity was associated with lower platelet counts and automated PLR, E. canis PCR positivity, and Anaplasma seropositivity, with the strongest association for concurrent E. canis PCR and Anaplasma seropositivity (odds ratio [OR]; 15.3; 95% confidence interval [CI]: 2.69-86.99; P = .002). Conclusions and clinical importance Lower platelet counts and automated PLR were associated with PSAIG positivity in thrombocytopenic dogs. Associations between PSAIG, E. canis infection, and co-exposure to Anaplasma spp. support immune-mediated platelet destruction in infected dogs.

Background Canine vector-borne diseases (CVBD) have become a major concern for canine and human public health. The aim of the study described here is to add epidemiological data regarding four pathogens responsible for CVBD, namely anaplasmosis, borreliosis, dirofilariosis and ehrlichiosis in a national survey conducted in Mexico. Methods Seventy-four veterinary centres located in 21 federal Mexican states were asked to test dogs with clinical signs suspect for CVBD and healthy dogs, for detection of Dirofilaria immitis antigen and antibodies against Anaplasma spp., Borrelia burgdorferi ( sensu lato ) and Ehrlichia canis using the SNAP® 4DX® from IDEXX® Laboratories. Results A total of 1706 dogs were tested, including 943 apparently healthy and 722 CVBD-suspect dogs. Infected dogs were 36.7 %. The highest percentages of infection with E. canis (51.0 %) and Anaplasma spp. (16.4 %) were obtained in the northwestern region, while D. immitis was most frequently found in the northeastern region of the country (8.9 %). Four dogs from the northwestern, northeastern, eastern and southeastern regions, respectively, were positive for B. burgdorferi ( sensu lato ). Northcentral regions showed lowest overall prevalence of infection (2.4 %). Co-infections were detected in 8.8 % of the dogs tested. Statistically significant lower positivity was found among dogs aged less than one year (23.2 %) and small-sized dogs (27.6 %), while higher prevalence of infection was found in dogs living outdoors (42.0 %), dogs with detectable tick infestation (43.3 %) and dogs that received treatment for tick-transmitted infections (58.8 %). Seropositivity was a risk factor for the presence of clinical signs as follows: Anaplasma spp. (OR = 2.63; 95 % CI: 1.88–3.67; P  < 0.0001), D. immitis (OR = 2.52; 95 % CI: 1.61–3.95; P  < 0.0001), E. canis (OR = 3.58; 95 % CI: 2.88–4.45; P  < 0.0001), mixed infections (OR = 4.08; 95 % CI: 2.79–5.96; P  < 0.0001), one or more agents (OR = 3.58; 95 % CI: 2.91–4.42; P  < 0.0001). Conclusions Canine serological evidence supports that dogs from Mexico are at risk of acquiring Anaplasma spp., D. immitis and/or E. canis , while B. burgdorferi ( sensu lato ) transmission is minimal in the country. Practitioners play a fundamental role in the detection and control of these diseases to protect dogs and humans.

Background Dirofilaria immitis is responsible for heartworm disease in dogs in endemic areas worldwide. Screening for this infection is done by blood tests. Antigen testing is the most sensitive method to detect an infection with adult (female) worms. Microscopic examination of a blood smear or Knott’s test can be used to detect circulating microfilariae, the infective larvae. To increase the sensitivity of the antigen test by decreasing the false negative test results, heating of the blood sample has been recommended in recent guidelines. Heating is believed to remove blocking immune-complexes. Circulating microfilariae are not specific findings for heartworm infection, as other nematodes (among others, Acanthocheilonema dracunculoides ) can also result in microfilaremia. Although the type of microfilariae cannot be determined by microscopy alone, real-time PCR can reliably identify the infecting nematode species. Correct identification of the parasite is of major importance, as an infection with D. immitis requires antiparasitic therapy, whereas A. dracunculoides is thought to be a clinically irrelevant coincidental finding. The present case report describes a microfilaremic dog where the initial antigen test for D. immitis turned positive after heat treatment, whereas real-time PCR revealed that the microfilariae were A. dracunculoides (syn. Dipetalonema dracunculoides ). Results A circa 5-year old, asymptomatic Spanish mastiff dog was referred for heartworm therapy because microfilariae were found via a screening blood test. The dog was recently imported to the Netherlands from Spain, where it had been a stray dog. Antigen tests on a plasma sample for D. immitis were performed with three different test kits, which all turned out to be negative. However, heat treatment of two of these samples were carried out and both of them led to a positive antigen test result. Real-time PCR showed that the circulating microfilariae belonged to A. dracunculoides species. Three administrations of moxidectin spot-on at monthly intervals resulted in a negative antigen and a negative Knott’s tests one month after the last treatment. Conclusions We conclude that heat treatment of initially negative blood samples for D. immitis could lead to false positive antigen test results if the dog is infected with A. dracunculoides.

Background Canine heartworm ( Dirofilaria immitis ) is a life-threatening infection of dogs with a global distribution. Information on the prevalence of D . immitis and associated risk factors for canine heartworm antigen positivity—and thus disease—in Australia is scarce or outdated. The current reference method for D . immitis diagnosis in dogs is via the detection of heartworm antigen in blood using commercially available microwell-based enzyme-linked immunosorbent assays (ELISAs). Heat treatment of canine plasma prior to testing has been suggested to increase test sensitivity. The aim of the current study was to estimate the prevalence of D . immitis in dogs confined to shelters in Queensland, Australia. The impact of heat treatment on antigen test results was also assessed. Methods Blood samples ( n  = 166) were collected directly from dogs in seven shelters across Queensland (latitudinal span of approx. 1700 km) into EDTA blood collection tubes. A commercially available ELISA (DiroCHEK®) was used to detect canine heartworm antigen in untreated and heat-treated plasma. Whole blood was concurrently tested for the presence of microfilariae and D . immitis DNA using a modified Knott’s test and real-time PCR, respectively. Risk factors (age, gender, source, location) associated with the odds of positivity for canine heartworm were assessed using binary logistic regression models. Results A total of 16 dogs (9.6%; 95% confidence interval [CI]: 5.9–15.2%) were positive for canine heartworm based on combined test results. Heat treatment did not impact on the positivity of D . immitis antigen within samples (Cohen’s kappa = 0.98), but the optical density was significantly increased in paired plasma samples for D . immitis antigen-positive samples (Wilcoxon matched-pairs signed rank test, two-tailed P  < 0.01). Location of the dog in a shelter in northern Queensland was the only risk factor significantly associated with the odds of a dog being more likely to be D . immitis antigen positive (odds ratio: 4.39; 95% CI: 1.26–13.51). All samples positive for the modified Knott’s test were also positive for D . immitis DNA by PCR. Conclusions This study demonstrated the presence of heartworm-positive dogs in shelters in Queensland, with positive animals significantly more likely to occur in northern Queensland than southern Queensland. Sustained testing for the presence of D . immitis microfilariae and antigen remain important diagnostic tools in areas with known and re-emerging canine heartworm activity. Graphical Abstract

Background: Heartworm disease (HWD) is a vector-borne disease caused by the filarial nematode Dirofilaria immitis. Low antigen levels caused by immune complex formation preclude HWD diagnosis. Heat treatment is an immune complex dissociation technique used to enhance antigen detection. Only a few studies have reported the benefits of heat treatment in nationwide surveys. Methods: To investigate the impact of heat treatment on the seroprevalence of HWD in companion dogs in the USA, serum samples (n = 3253) were analyzed for D. immitis antigen (DiroCHEK®, Zoetis) without and with heat treatment of the samples. Results: Compared to sera without heat treatment, heat treatment significantly increased overall prevalence from 3.8% (123/3253) to 7.3% (237/3253) (p < 10−4), expanding antigen detection from 32 to 39 of the 48 states and Washington District of Columbia included in this study. Conclusions: This study represents the largest nationwide survey of HW antigen detection in dogs in the US applying heat treatment to canine sera. The heat treatment used herein has the advantage of requiring a low volume of serum, making it optimal for use in routine diagnosis. Heat treatment should be used routinely by reference laboratories and veterinary clinics in patients with a negative initial test.