Assessment of the circulation of Dirofilaria immitis in dogs from northern Portugal through combined analysis of antigens, DNA and parasite forms in blood.

Dirofilariasis is a vector-borne disease frequent in many countries. Not only infected dogs, but also cats and wild canids (including wolves and foxes), represent important sources of infection for mosquitoes, which are the pathogen vectors. The disease is endemic in Mediterranean countries with increasing incidence in Italy, France, Greece and Spain, but limited epidemiological data is available from Portugal regarding its distribution and impact. Aiming to clarify this, canine whole blood samples (n = 244) from the north of Portugal were tested for Dirofilaria spp. antigens by use of a commercial rapid immunomigration test. Polymerase chain reaction (PCR) and the modified Knott test were also used to assess the presence of microfilariae. Results were also compared to assess the performance of each test used. Of the 244 animals tested, 118 (48.4%) were positive for Dirofilaria immitis (heartworm) in the serological adult worm rapid antigen detection test, and 36 (14.8%) had circulating microfilariae, identified as D. immitis. A combined positivity of 51.6% (126/244) was found. Results indicate that the risk of exposure to D. immitis in dogs is high in this region of Portugal, and that prophylaxis against the parasite is advisable to decrease the occurrence of canine infection and disease. The present study highlights the diagnostic value of serological and molecular tests in determining the prevalence of D. immitis.

Feasibility and comparative analysis of Dirofilaria immitis microfilaria freezing and fixation for student instruction and assessment of clinical parasitology skills.

BACKGROUND: Detection of D. immitis microfilaria (mf) is an important diagnostic skill in veterinary medicine and is critical to Day 1 veterinarians and technicians. Finding a supply of blood containing mf to teach the technique and formalin's adverse environmental effects used in the diagnostic microscopic tests present a challenge. RESULTS: This study evaluated the use of cryopreserved and recently drawn mf-infected blood along with two fixative reagents, acetic acid or formalin for mf detection. The specific aims included determining if veterinary students could 1) detect cryopreserved mf added to fresh blood using routine diagnostic testing and 2) detect morphological differences in the mf. The 236 students were kept blind from the sample status. The ability of the students to identify mf and the mf morphology were compared for the samples and fixatives evaluated. The results demonstrate using a combination of cryopreservation and acetic acid for teaching microfilaria diagnostic techniques is fleasible; however, the quality of the mf morphology is less than optimal when compared to freshly acquired mf containing blood. Compared to reference values, the mf demonstrated a decrease in size with each additional variable evaluated. CONCLUSION: A majority (98.3%) of the 236 students correctly identified the presence of mf. Teaching laboratories could utilize cryopreserved mf-spiked donor blood in lieu of freshly collected mf-containing blood from a naturally or experimentally infected dog. Substitution of less hazardous chemicals for the fixative can be used. Finally, the change in size measurements provides a mechanism to ensure students can correctly measure mf as students are required to do verifiable measurements and cannot copy reference values from a text book since the cryopreservation and fixation methods cause the mf to measure smaller than textbook reference values.

Cross-sectional survey of health management and prevalence of vector-borne diseases, endoparasites and ectoparasites in Samoan dogs.

OBJECTIVE: To determine the prevalence of selected canine vector-borne diseases (Leishmania infantum, Anaplasma spp., Ehrlichia canis, Borrelia burgdorferi and Dirofilaria immitis) and endo- and ectoparasites in Samoan dogs presenting for surgical sterilisation and to report on the general health management of the dogs. METHODS: This study was a prospective serological cross-sectional survey. Management data were obtained for 242 dogs by interview with their owners. Blood samples were collected from 237 dogs and screened for the canine vector-borne diseases using point-of-care qualitative ELISA assays. Anaplasma spp. positive samples were screened by PCR and sequenced for species identification. Rectal faecal samples were collected from 204 dogs for faecal flotation and immunofluorescent antibody tests were performed for Giardia and Cryptosporidium spp. on a subset of 93 faecal samples. The skin and coat of 221 dogs were examined for presence of ectoparasites. RESULTS: The D. immitis antigen was detected in 46.8% (111/237) of dogs. Seroprevalence of Anaplasma spp. was 8.4% (20/237); A. platys was confirmed by PCR. Prevalence of hookworm was 92.6% (185/205) and Giardia was 29.0% (27/93). Ectoparasites were detected on 210/221 (95.0%) of dogs examined and 228/242 dogs (94.2%) had previously never received any preventative medication. CONCLUSIONS: There was a very high prevalence of D. immitis, hookworm and external parasites in Samoan dogs, and prophylactic medication is rarely administered. This is the first report confirming A. platys in Samoa and the South Pacific islands. The public health implications of poor management of the dogs should be considered and investigated further.

GIS modeling for canine dirofilariosis risk assessment in central Italy.

A survey was conducted in an area of central Italy in order to study the prevalence of Dirofilaria immitis and D. repens in dogs. Blood samples were collected from 283 dogs and examined using a modified Knott's technique. In addition, in order to detect D. immitis occult infection, 203 serum samples were also analysed for D. immitis antigen detection. The results were analyzed in order to evaluate the behavioural and attitudinal risk factors. A geographical information system (GIS) for the study area was constructed, utilizing the following data layers: administrative boundaries, elevation, temperature, rainfall and humidity. Microfilariae were detected in 32 of the 283 dogs surveyed, constituting a total Dirofilaria prevalence of 11.3%. In particular, 20 dogs (7.1%) were positive for D. immitis and 12 dogs (4.2%) for D. repens microfilariae. One case of D. immitis occult infection was also detected. Choroplethic municipal maps were drawn within the GIS in order to display the distribution of each Dirofilaria species in the study area. Statistical analysis showed a significant association between Dirofilaria infection and animal attitude (hunting/truffle dogs showed a higher prevalence compared to guard/pet dogs). A higher prevalence was also recorded in 2 to 5-years old dogs. Furthermore a GIS-based modelling of climatic data, collected from 5 meteorological stations in the study area, was performed to estimate the yearly number of D. immitis generations in the mosquito vector. The results of the model as depicted by GIS analysis was highly concordant with the territorial distribution of positive dogs and showed that D. immitis spreading is markedly influenced by season. The potential transmission period in the study area was found to be confined to summer months with a peak in July and August, as expected for a temperate region where summer season is the most favourable period for the parasite.

Decision analysis: dealing with uncertainty in diagnostic testing.

Decision analysis is a process for systematically analyzing complex choices by considering all pertinent information. In this paper, we discuss how uncertainty associated with diagnostic testing can be included in a decision analysis using pay-off tables and decision trees (decision-flow diagrams). Variables associated with diagnostic test interpretation (such as pre-test and post-test probability of disease; test sensitivity, specificity and predictive values; fixed cut-offs versus continuous measurement scales; test dependence associated with the use of multiple tests) are considered. Several decision criteria and output measures are discussed (including MAXIMIN and MAXIMAX criteria, opportunity costs, expected monetary values, expected utility, sensitivity and risk-profile analysis, and threshold analysis). The application of decision analysis to diagnostic testing for Johne's disease and traumatic reticuloperitonitis of cattle, and for canine heartworm disease are used to illustrate both population- and patient-oriented applications and criteria for ranking the desirability of different outcomes.

A preliminary assessment of the recombinant antigen PLA2 in the diagnosis of human dirofilariosis.

Two recombinant antigens (P22U and PLA2), cloned in a L4 Dirofilaria immitis cDNA library, were analyzed by Western-blot and ELISA to investigate their characteristics for the diagnosis of human dirofilariosis. P22U seems related to a Di22 native antigen useful for the diagnosis of pulmonary dirofilariosis, but it is unspecifically recognized by sera from patients with different parasitic and non parasitic pulmonary diseases. PLA2 is not related to Di22 but specifically reacts in Western-Blot and ELISA with sera from patients with subcutaneous dirofilariosis.

Decision analysis in the evaluation of diagnostic tests.

Decision analysis was used to evaluate the clinical usefulness of 2 diagnostic tests: one for canine heartworm disease and the other for bovine traumatic reticulo-peritonitis. Several clinically relevant measures of test performance are introduced, including expected utility, risk profile, testing band, threshold analysis, and the relative cost of misdiagnosis. One of the principal benefits of decision analysis of diagnostic tests is that the technique can be used to determine how changes in underlying assumptions will affect clinical decisions. If clinicians can identify and assign values to relevant variables, then decision analysis can provide clinically meaningful guidelines for interpreting the results of diagnostic tests. To take advantage of these techniques, clinicians must become comfortable with quantitative expressions for test performance, risk, and prognosis.