• Client-owned dogs presented to the University Veterinary Small Animal Teaching Hospital of Liège, Belgium, between March 2013–April 2017 were retrospectively selected.
• Dogs were of various breeds, aged from 9 months–10 years.
• Inclusion criteria were respiratory clinical signs (cough, respiratory distress or exercise intolerance) and recorded diagnostic results for natural A. vasorum infection (Baermann coprology, Angio Detect™, and enzyme linked immunosorbent assays [ELISA] both for antigens and specific antibodies).
• A. vasorum infection was suspected based on compatible respiratory signs (as above), radiographic findings, bronchoscopy, and bronchoalveolar lavage (BAL) cytology examination.
• Owner-reported duration of respiratory clinical signs varied from 2 weeks–2 months.
• Angiostrongylosis was confirmed in all seven dogs by:
  • positive quantitative (q) PCR from BAL samples and clinical recovery after administration of either 50 mg/kg q24h fenbendazole PO for 3 week treatment duration or 2.5 mg/kg moxidectin repeated after 2–4 weeks.

• At the time of diagnosis one or two serum samples were obtained and frozen prior to analysis.
• One sample per dog was thawed for Angio Detect™ diagnostic testing.
• The second serum sample underwent A. vasorum antigen and antibody sandwich-ELISAs; both analysed at the Institute of Parasitology, Vetsuisse Faculty, University of Zurich, Switzerland.
• Faecal samples were obtained from each dog over 3 consecutive days for Baermann coprology. All coprology was undertaken in the Laboratory of Parasitology of the Faculty of Veterinary Medicine, University of Liège, Belgium.

• Positive or negative results from Angio Detect™, A. vasorum antigen sandwich-ELISA, A. vasorum antibody sandwich-ELISA and Baermann coprology.
  • Positive Baermann coprology result was determined by identifying L1 larvae of A. vasorum in the faeces. A. vasorum L1 larvae distinguished from Crenosoma vulpis (C. vulpis) L1 larvae due to their characteristic tail notch.

• Of the seven angiostrongylosis cases, two were positive on Angio Detect™ and three were positive on Baermann coprology.
• In one dog, positive on A. vasorum antigen sandwich-ELISA, the Angio Detect™ assay was repeated twice as invalid results were obtained (the control line was not visible).
• Compared to Baermann coprology, Angio Detect™ had a specificity of 100% (95% CI 39.8–100%; n = 4/4) and sensitivity of 66.7% (95% CI 9.4–99.2%; n = 2/3). Not all diagnostic test results were available for each dog; however, all PICO relevant data were available.

• Small sample size.
• The title is suggestive of angiostrongylosis infection with negative in-house diagnostic results, which was not reflective of all seven cases.
• Results of all diagnostic tests available for each dog were not reported.
• The spectrum of clinical signs and severity of the target disease (angiostrongylosis) found within the university hospital setting may introduce referral bias. The population used may not be representative of all clinical contexts.
• It was unclear whether the indeterminate index result (the invalid Angio Detect™ reading obtained twice for dog 3) was classified as a negative or positive result and therefore could introduce information and interpretive bias to the results. No postulated reason for indeterminate Angio Detect™ results was provided, which could aid evaluation of the usefulness of this diagnostic test within a clinical setting.
• No measure of diagnostic accuracy was reported. Evaluation of measures of diagnostic accuracy may have aided the analysis of superiority or non-inferiority among the index diagnostic tests used.

• Faecal samples were obtained from all dogs for Baermann coprology.
• Blood samples were taken from all dogs and placed into ethylenediaminetetraacetic acid (EDTA) sample pots. Plasma from each blood sample underwent Angio Detect™ diagnostic testing.

Day 0

• Dogs that had positive test results on day -15 testing (n = 3) were treated with topical Advocate^®; Bayer (10% imidacloprid and 2.5% moxidectin).

Days 14 and 28

• The three treated dogs had faecal samples collected for Baermann coprology and phlebotomy for Angio Detect™ (methodology as above for both diagnostic tests).

• Positive or negative results from Angio Detect™ and Baermann coprology:
  • Positive Baermann coprology result was determined by identifying L1 larvae of A. vasorum. The dog was considered negative if A. vasorum larvae were not identified.

• Dog 4 tested positive with Baermann coprology but negative with Angio Detect™.
• Dogs 13 and 14 tested positive with both Baermann coprology and Angio Detect™.
• All three of these dogs were negative with both diagnostic tests on days 14 and 28.
• Compared to Baermann coprology, Angio Detect™ had a specificity of 100% (95% CI 73.5–100%; n = 12/12) and sensitivity of 66.7% (95% CI 9.4–99.2%; n = 2/3).

• Small sample size.
• It was assumed eligibly was limited only to residing in the kennel as eligibility criteria were not reported.
• The population was asymptomatic leading to challenges in comparing data obtained to patients exhibiting clinical signs.
• The prophylactic anthelmintic treatment history of each individual dog prior to enrolling in the study was not reported, which could potentially introduce bias in the results.
• It was unclear if the population for this study had been infected with A. vasorum before during the kennel’s previous history of A. vasorum infection and if this would impact on the results.
• It was not clear if a veterinarian(s) conducted the clinical examinations of the dogs and what aspects of physical examination were being assessed by the individual(s).
• Single, rather than three consecutive day, faecal samples were obtained for Baermann coprology; potentially reducing Baermann sensitivity and introducing measurement bias.

• Client-owned dogs were enrolled from one of 17 volunteer small animal practices based in Wallonia, Belgium from November 2014–February 2016.
• The study population included case dogs showing clinical signs suggestive of A. vasorum and control dogs presented to their registered clinic for conditions unrelated to A. vasorum.
• The clinical signs group contained dogs that presented with clinical signs suggestive of A. vasorum, including; dyspnoea, cough, exercise intolerance, bleeding abnormalities and neurological disorders.
• The control group contained dogs that presented to the practices for routine healthcare or unrelated conditions. Eligibility for enrolment was not restricted by age, breed or sex.
• Dogs were excluded if travel outside Belgium had occurred within 3 months prior to entering the study.

• 757 dogs in a control group;
• 222 dogs in a clinical signs group.

• Angio Detect™ test results were performed following manufacturer’s instructions with all tests undertaken within a strictly adhered 15-minute time frame.
• All staff undertaking the Angio Detect™ test were trained and observed by the first author with positive results recorded by intensity (+, ++, or +++).
• Faecal samples were obtained for Baermann coprology from 41 seropositive dogs (24 controls and 17 symptomatic dogs) and six seronegative dogs with strong suspicion of angiostrongylosis (one control and five symptomatic dogs).
  • recently passed faeces were collected on 3 consecutive days, or one or two samples per dog where this was not possible.
• Owners completed a questionnaire detailing the patient’s location, breed, sex, age, recent antiparasitic prophylactic protocol, and lifestyle.

• Positive or negative results from Angio Detect™ and Baermann coprology:
  • Positive Baermann coprology result was determined by identifying L1 larvae of A. vasorum (using their characteristic tail notch to identify them compared to other parasitic larvae). The dog was considered negative if A. vasorum larvae were not identified.
• Prevalence of A. vasorum infection in both dogs displaying consistent clinical signs and controls.

• Prevalence of Angio Detect™ positive cases was 3.6% (n = 27/757).
• 3/27 Angio Detect™ positive cases did not have Baermann coprology performed. 17/24 (70.8%) Angio Detect™ positive dogs had negative Baermann coprology.

Clinical Signs Group

• Prevalence of Angio Detect™ positive cases was 8.6% (n = 19/222).
• 2/19 Angio Detect™ positive cases did not have Baermann coprology performed. 8/17 (47.1%) Angio Detect™ positive dogs had negative Baermann coprology.
• 1/17 positive Angio Detect™ dogs had only C. vulpis larvae found on Baermann coprology.
• 1/5 Angio Detect™ negative dog was positive on Baermann coprology (mixed parasite burden found).
• Of the 19 Angio Detect™ positive dogs:
  • 6 were coughing
  • 3 were dyspnoeic
  • 3 had exercise intolerance
  • 5 had coughing with exercise intolerance
  • 1 had a bleeding disorder
  • 1 had a neurological disorder

• Volunteer bias could have been introduced due to the selection process of small animal practices. There was variability in faecal sample collection and the frequency with <3 consecutive daily samples collected was not reported. Both of these factors introduce measurement bias to Baermann coprology.
• It is unclear what time period was considered recent for faecal collection. The method of faecal storage prior to Baermann coprology was also not explained. There may have been inconsistencies and variation, introducing Baermann coprology measurement bias.
• Angio Detect™ positive readings were recorded as +, ++, or +++, however the relevance and results of this classification system were not explained.
• Unable to calculate diagnostic accuracy for Angio Detect™ compared to Baermann coprology, as only seropositive dogs were sampled.

• Client-owned dogs were enrolled from one of 26 volunteer small animal practices based throughout Belgium and The Netherlands from February–June 2017.
• The dogs had non-parasitic conditions (examples provided include neutering, trauma, and vaccination).
• Dogs were > 6 months, of either sex and any breed.
• Dogs were excluded if travel outside Belgium or The Netherlands occurred within 3 months or if anthelmintic treatment had been administered within 2 months of entering the study.

• Faeces, collected by the owner, underwent Baermann coprology.
• Dogs underwent phlebotomy at the veterinary clinic. Blood was collected in heparinised or plain tubes, centrifuged and Angio Detect™ conducted using manufacturer’s instructions.

• Positive or negative results from Angio Detect™ and Baermann coprology:
  • Positive Baermann coprology was determined by identifying L1 larvae of A. vasorum. The dog was considered negative if A. vasorum larvae were not identified.
• Prevalence of A. vasorum in dogs not presenting for parasitic conditions.

• 3/216 (1.4%) faecal samples were positive for A. vasorum on Baermann coprology.
• 1/237 (0.4%) serum samples were positive for A. vasorum on Angio Detect™. This dog was also one of the 3 Baermann-positive dogs.
• Compared to Baermann coprology, Angio Detect™ had a specificity of 100% (95% CI 98.3–100%; n = 213/213) and sensitivity of 33.3% (95% CI 0.84–90.6%; n = 1/3).
• None of the dogs that tested positive by either test method had signs of gastrointestinal parasitism.

• Volunteer bias could have been introduced due to the selection process of small animal practices.
• Recall bias is possible regarding the owner’s recollection of whether and/or when anthelmintics had been administered, which could have affected sample selection since this was an exclusion criterion.
• It is unclear what time period was considered recent for faecal collection. The method of faecal storage prior to Baermann coprology was also not explained. There may have been inconsistencies and variation, introducing measurement bias.
• No explanation for missing diagnostic test results is provided (Baermann 23 dogs and Angio Detect™ two dogs).
• A. vasorum-positive dogs were reported not to have clinical signs of gastrointestinal parasitism, but information regarding other clinical signs was not reported.

• Client-owned dogs that were suspected of clinical A. vasorum infection were selected by their registered veterinarian (based within the United Kingdom [UK]) during June 2012–April 2013.
• Inclusion criteria for suspected clinical A. vasorum infection were at the discretion of the patient’s veterinarian.
• Dogs that had been historically diagnosed (via Baermann coprology performed by two authors based in either Zurich, Switzerland or Copenhagen, Denmark) were also included in the study.
• A control group contained samples from dogs that were A. vasorum negative but positive for other parasite infections.

• 195 dogs suspected of being infected with A. vasorum volunteered from the UK.
• 19 dogs diagnosed with A. vasorum by Baermann coprology via archive.

An additional 89 negative control samples were obtained:

• 79 dogs negative for A. vasorum based on antigen ELISA and positive for Dirofilaria immitis (via post-mortem).
• Three dogs negative for A. vasorum and positive for Crenosoma vulpis based on Baermann coprology.
• Four dogs experimentally infected with Dirofilaria repens.
• Three dogs experimentally infected with whipworms, hookworms, and ascarids.

• Faecal samples obtained from the suspected A. vasorum UK cases underwent Baermann coprology at IDEXX Reference commercial Laboratories.
• Serum or plasma obtained from the suspected A. vasorum UK cases were frozen at -20°C (due to time delay between collection and Angio Detect™ testing).
• Serum samples were obtained (from archive) for the historically A. vasorum diagnosed dogs.
• All serum samples (from UK dogs, archive and controlled) underwent randomized blind labelling prior to Angio Detect™ diagnostic testing undertaken by blinded personnel.
• In cases where Baermann coprology results did not match Angio Detect™ results the patient’s sample underwent A. vasorum antigen ELISA testing.

• Positive or negative results from Angio Detect™ and Baermann coprology, and A. vasorum antigen ELISA (where applicable):
  • Positive Baermann coprology was determined by identifying L1 larvae of A. vasorum (using their characteristic tail notch to identify them compared to other parasitic larvae). The dog was considered negative if A. vasorum larvae were not identified.

Dogs with suspected clinical A. vasorum infection from the UK:

• 16/195 of dogs (8.2%) had A. vasorum positive Baermann coprology results.
• 15/16 dogs had a positive Angio Detect™ test result.
• The remaining dog of these 16 (i.e. with a positive Baermann coprology result and a negative Angio Detect™ test result) tested negative with A. vasorum antigen ELISA.
• 2/179 dogs with negative A. vasorum larvae Baermann coprology result tested positive with Angio Detect™. These two samples also tested positive with A. vasorum antigen ELISA.

Archive dog samples:

• All 19 samples were positive on Angio Detect™ testing. To be eligible for this group all 19 had previously tested positive for  A. vasorum larvae on Baermann coprology.

Compared to Baermann coprology, Angio Detect™ had a reported specificity of 98.9% (95% CI 96.0–99.9%) and sensitivity of 97.1% (95% CI 85.1–99.9%).

• The process of archive sample selection was not described. This could influence the eligibility of this population group, may introduce selection bias and therefore reduce the strength of the evidence obtained.
• The eligibility criteria for clinical A. vasorum signs were not described and were left to the submitting veterinarian’s opinion, with veterinarians possibly differing in the methods used to identify eligible cases. This could introduce misclassification bias and result in this group not being a true representation of dogs with clinical A. vasorum. It could also affect the accuracy of diagnostic data obtained.
• The clinical settings (e.g. first opinion, referral) of UK practices where faecal and serum samples were obtained were not noted in the methods section. This could introduce spectrum and/or referral bias as the spectrum of clinical signs and disease severity could vary in different clinical settings.
• The method(s) used for faecal sample transport was not described and it is unclear if they were consistent. If faecal storage was not adequate it could have resulted in false-negative results obtained by Baermann coprology.
• The process of thawing the serum/plasma samples and duration of sample freezing was not described and it is therefore uncertain if these could have altered Angio Detect™ testing and introduced measurement bias.
• It is unknown how many faecal samples were obtained over multiple days for a single patient compared to single faecal samples submitted, which could introduce measurement bias for Baermann coprology.

• Dogs that resided at a kennel in Italy.
• Two breeds were present – Belgian Shepherds (n = 3, all males) and Cavalier King Charles Spaniels ((CKCS) n = 17; 13 females and four males).

Retrospective A. vasorum case series:

• These were dogs that had been diagnosed with A. vasorum infection (by post-mortem, Angio Detect™, BAL, and/or Baermann coprology) and were recorded on archive at the Veterinary Pathology Laboratories, Veterinary School of Milan University, Italy, between 1998–2016.
• Breeds included Jack Russell terrier (n = 1), Cao de Agua Portuguese (n = 1), Maltese (n = 1), Springer Spaniel (n = 1), Italian Hound (n=1), Lurcher (n = 1), Rhodesian Ridgeback (n = 1) and mixed-breed (n = 4).
• Ages ranged from 8 months to 144 months (but not all age records were complete).
• Both male (n = 5) and female (n = 6) dogs were represented.

• Two clinical angiostrongylosis cases (both CKCS).
• 18 dogs resident at same kennels.

Retrospective case series: 11 dogs.

• Faecal samples were obtained from each dog for Baermann coprology.
• Each dog was tested with Angio Detect™.
• All of the above tests were not performed on the dog that was the second clinical case.

Retrospective case series:

• Data recorded on cases within the Veterinary Pathology Laboratories archives were examined for positive A. vasorum results.

• Positive or negative results from Angio Detect™ and Baermann coprology:
  • Positive Baermann coprology was determined by identifying L1 larvae of A. vasorum. The dog was considered negative if A. vasorum larvae were not identified.

Retrospective case series:

• Clinical signs, Angio Detect™ results, results obtained for A. vasorum larvae identification, post-mortem findings and clinical outcome of all cases that were recorded positive for A. vasorum in the archive records.

• 1 dog had positive Baermann coprology and was also Angio Detect™ positive.
• Compared to Baermann coprology, Angio Detect™ had a specificity of 100% (95% CI 81.5-100%; n = 18/18) and sensitivity of 100% (95% CI 2.5-100%; n = 1/1).

Retrospective case series:

• Four dogs had both Baermann coprology and Angio Detect™ performed.
  • Of these, all four were positive on Angio Detect™ and only one was positive on Baermann coprology.

• Small sample size.
• In the cross-sectional study, the origin of each dog was recorded but the relevance of this information was not explained. Additional information such as how long the dog had been in the kennel along with history of when and where any additional boarding may have occurred could have made this information more relevant.
• The search strategy of the retrospective case series was not included and bias may have occurred in retrieving the data due to record keeping potentially not being standardised.
• It is unknown how many different clinicians treated the archive cases and they may not have had standardisation of record keeping. This could introduce selection bias or could have resulted in lower numbers within this study than were truly eligible.
• Single, rather than three consecutive day, faecal samples were obtained for Baermann coprology for the cross-sectional study. Similarly, frequency of faecal collection was not clear in the retrospective case series. Inconsistencies potentially reduced Baermann sensitivity and introduced measurement bias.

• Naturally infected A. vasorum-positive dogs which included:
  • A. vasorum Baermann positive dogs from Switzerland.
  • A. vasorum-positive dogs (confirmed by larval analysis) from Germany that had been enrolled on previous lungworm studies.
• The population also included experimentally infected A. vasorum-positive dogs, some of which were treated with imidacloprid/moxidectin topical medication.
• There was also a control group consisting of dogs both experimentally and naturally infected with parasitic infections other than A. vasorum as well as negative Baermann coprology. These dogs came from Germany, Switzerland, Czech Republic and Italy.

• The group with natural A. vasorum infection included 39 dogs

The group of 38 dogs with experimental A. vasorum infection included:

• 10 dogs who received imidacloprid/moxidectin topical treatment.

The control group contained 133 dogs.

Natural A. vasorum infection group:

• Serum was obtained from all dogs.

Experimental A. vasorum infected group:

• For 28 of these dogs that were experimentally infected with a known A. vasorum worm burden, serum samples were obtained before inoculation and also:
  • at days 21, 35, 49, and 62 (±1) after inoculation for all 28 dogs.
  • at days 21, 35, 49, 62, and 76 (±1) after inoculation for 20 of these 28 dogs.
  • at days 21, 35, 49, 62, 76, and 91 (±1) after inoculation for 15 of these 28 dogs.
  • at days 21, 35, 49, 62, 76, 91, and 97 (±1) after inoculation for four of these 28 dogs.
  • two of these 28 dogs received serum samples up until 286 days after inoculation.
  • Baermann coprology results were available for these 28 dogs.
• An additional 10 dogs had serum samples obtained before and after experimental inoculation of A. vasorum. They had Advocate^® (10 mg/kg imidacloprid / 2.5 mg/kg moxidectin. This is the equivalent of 0.1 ml/kg Advocate^® product application) topically administered between days 81–110 after inoculation.
  • Baermann coprology results were available for these 10 dogs.

Control group:

• This group was enrolled for cross-reactivity testing.
• Experienced laboratory personnel analysed all serum samples with Angio Detect™ (following instructions from manufacturer). Serum samples were positive or negative for Angio Detect™ with positive bar readings being subclassified into:
  • + meaning a visible but slight colour intensity positive;
  • ++ meaning a good visible colour intensity positive;
  • +++ meaning an intensely coloured positive.
• The serum samples also underwent A. vasorum antigen ELISA testing. Serum samples were either positive or negative for A. vasorum antigen ELISA testing with optical density (OD) readings being subclassified into:
  • Negative being OD < 0.159;
  • + being OD 0.159–0.350;
  • ++ being OD 0.351–0.800;
  • +++ being OD > 0.800.

• Positive or negative results from Angio Detect™ and Baermann coprology:
  • Positive Baermann coprology was determined by identifying L1 larvae of A. vasorum. The dog was considered negative if A. vasorum larvae were not identified.
• A. vasorum antigen ELISA was also used to compare/confirm a positive or negative A. vasorum infection.

• All 39 dogs had positive Baermann coprology.
• 33/39 dogs tested positive with Angio Detect™.
• Compared to Baermann coprology, Angio Detect™ had a reported sensitivity of 84.6% (95% Cl 69.5–94.1%).

Experimental A. vasorum infected group:

• The 28 A. vasorum inoculated dogs:
  • Within 47–55 days post inoculation patency was achieved in all 28 dogs with Baermann coprology.
• The 10 dogs treated with Advocate^®:
  • One dog (which had a worm burden on post-mortem examination) was positive for Angio Detect™ but produced a negative result on Baermann coprology.
  • Baermann coprology was negative 9–20 days after treatment, at 3 weeks after treatment three dogs had negative Angio Detect™ results.

Control group:

• Positive Angio Detect™ results were obtained for both dogs that had A. vasorum and C. vulpis combined infections.
• Compared to Baermann coprology, Angio Detect™ had a reported specificity of 100% (95% CI 74.1–100%).

• The method of selecting the eligible population is not reported and there is the potential for selection bias.
• Sensitivity reportedly based on 39 serum samples from naturally infected dogs; however methods state these comprised samples from 28 dogs from Switzerland and 13 serum samples from Germany from previous lungworm studies, with no explanation provided for this discrepancy.
• The severity and spectrum of clinical signs of the population are not reported. This makes extrapolation of the tests’ diagnostic accuracy to a clinical setting difficult.
• There was no explanation why not all 28 dogs within the experimentally inoculated group were tested for the same duration with both Angio Detect™ and antigen ELISA tests. Flow of experimentally inoculated participants throughout the study was also not clear. As a result, interpretation of the results should be cautious.

• Client-owned dogs presenting with respiratory distress to one of three Italian clinics from November 2016–January 2020.
• Eligible ages were 4 months–2 years (median age 10 months).

• All dogs underwent conscious thoracic ultrasonography (B-Mode and M-Mode) in standing position.
• Faecal samples were obtained from all dogs (except one) for Baermann coprology.
• Faecal sample from thermometer underwent microscopic faecal smear.
• All dogs were tested with Angio Detect™.

• Positive Baermann coprology was determined by identifying L1 larvae of A. vasorum. The dog was considered negative if A. vasorum larvae were not identified.

• A. vasorum-positive Baermann coprology in 12 of 25 cases.
• Angio Detect™ positive in 11 of 26 cases (9 were also Baermann positive).
• Compared to Baermann coprology, Angio Detect™ had a specificity of 92.3% (95% CI 64.0–99.8%; n = 12/13) and sensitivity of 75.0% (95% CI 42.8–94.5%; n = 9/12).

• Small sample size.
• High risk of spectrum bias, since the study population comprised solely of young dogs presented for respiratory distress, and as such is not representative of the population of dogs undergoing diagnostic testing for A. vasorum infection.
• 25/26 dogs were reported by their owners to be receiving monthly ivermectin-based chemoprophylaxis at the time of presentation, but time since last administration was not reported.
• Single, rather than three consecutive day, faecal samples were obtained for Baermann coprology, potentially reducing Baermann sensitivity and introducing measurement bias.