Dirofilaria  ghid de diagnostic 3
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Dirofilaria ghid de diagnostic 3

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Simpozion Medical CYF Medical Diagnosticul Dirofilariozei

Simpozion Medical CYF Medical Diagnosticul Dirofilariozei
cyfmedical@gmail.com , pentru diagnostic metode de diagnostic si teste sunati la 0723318308.

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    Dirofilaria  ghid de diagnostic 3 Dirofilaria ghid de diagnostic 3 Presentation Transcript

    • Dirofilaria infections in animals and humans Guidelines for the diagnosis of Dirofilaria infection in dogs and cats  Claudio Genchi, Med Vet, PhD, EVPC Dipl Dept of Veterinary Science and Public Health, Università degli Studi di Milano Bucharest, June 15th, 2013
    • 1. Laboratory and in-clinic serological and blood diagnosis in dogs and cats 2. Radiography and echocardiography 3. Dog: staging, prognosis and choice of treatment 4. Practical guidelines for HW diagnosis in dogs and cats
    • 1. Circulating microfilariae: the intensity of microfilaraemia does not correlate with adult worm burden 2. Adult female circulating antigens [Ag]: partially correlate with adult worm burden Circulating antibodies [Ab]. Not useful in dogs DOG
    • … furthemore thoracic xR and in some case echocardiography are useful for staging the disease, prognosis and choice of the therapy
    • Fresh blood smears: not suitable, insufficient sensitivity and specificity; no species differentiation Concentration methods: 1. Filtration 2. Knott test: much more sensitive [about 2 ml of blood] and specific; species identification Acid-Phosphatase stain: 1.D. immitis: 2 activity spots around the anal and the excretory pores 2.D. repens: 1 spot around the anal pore PCR Mf examinations must always be performed [in dogs, mf/aemia is usually long lasting: sensitivity: 50-70%]
    • Knott test 2 ml of whole blood lysis with 0.2% formalin water solution centrifugation discard the surnatant, stain with ethylene blue or a drop of eosin
    • Species Length µm Width µm Features Dirofilaria immitis 290-330 5-7 No sheath, cephalic end pointed, tail straight with the end pointed. APh-S: two activity spots located around the anal and the excretory pores D. Repens 300-370 6-8 No sheath, cephalic end obtuse, tail sharp and filiform often ending like an umbrella handle. APh-S: one spot around the anal pore Acanthocheilonema reconditum 260-283 4 No sheath, cephalic end obtuse with a prominent cephalic hook, tail button hooked and curved. APh-S: activity throughout the body A. Dracunculoides 190-247 4-6.5 Sheath, cephalic end obtuse, caudal end sharp and extended. APh-S: three spots which include an additional spot in the medium body 1Microfilariae measured after concentration by the Knott test; when using the Difil® test, lengths are shorter. APh-S: acid phosphatase stain Morphological features of blood microfilariae from filarial worms of dogs and cats ESCCAP GL5, VBD
    • D. immitis/D. repens overlap in many regions and emphasizes the need of a correct differential diagnosis. Length µm Cephalic end Caudal end D. repens D. immitis 350-385 A-Ph 290-330 A-Ph tapered 2 spots rounded 1 spot hook-shaped pointed Diagnosis
    • CAT: microfilaemia Often unisexual infection or only pre-adult worms [sensitivity < 2%] When present, microfilaemia is short lasting A negative Knott test is not sufficient to rule out the infection in cats
    • Serological and whole blood test for heartworm diagnosis Antigens: Circulating Ag from adult female worms: In dogs positive after 6-7 months from infection [after 7-8 mths in cats] Most test kits very specific and sensitive in case of >2 adult female worms Commercially available both in-clinic and laboratory test kits
    • To note that the increased sensitivity of some test kits has probably decreased their specificity. Recently false positive results have been observed in dogs experimentally infected with Angiostrongylus vasorum. In case, serological results for D. immitis should be confirmed or excluded by additional diagnostic tests (Knott’s test for D. immitis microfilariae, serology or Baerman for L1 of A. vasorum in faeces) or Rx imaging frequently delivering pathognomonic findings for heart dirofilariosis or angiostrongylosis.
    • A. vasorum: dense alveolar patterns D. immitis: increase vascular patterns
    • Serological and whole blood test for heartworm diagnosis Antibodies [cats]: Circulating Ab from worms [L4-adult parasites-death parasites]; positive reactions from 2 months after infection and after several months [years] after the death of worms Not to be used in dogs In cats more suitable for assessing the infection risk than to confirm a clinical suspicion Positive results are not sufficient to role out a diagnosis of feline HW infection; it must be confirmed by an Ag test examination or other diagnostic approach
    • ELISAs for lab procedure [OD value] and in-clinic Immunomigration, in- clinic: easy and very rapid [5-10 min] whole blood, serum, plasma
    • Occult infections • Male worms or female worms only: very unusual in dogs/quite frequent in cats • Iatrogenic origin: macrocyclic lactones at high dosage • Immunological origin: old/very old female worms no more able to produce Mfs
    • Microfilarie Tick smear: drop of venous blood on a microscopic slide covered with a coverslip Modified Knott test [2 ml of blood] Filter test Mf immunohistochemical staining PCR on mf and adult worms Not sensitive, not specific Easy, sensitive, quite specific Easy, sensitive, quite specific Very specific and sensitive Very specific and sensitive Adult worms Morphology Not easy particularly in case of female worms Serology Ag/Ab ELISA WSP Ab ELISA Commercial test available Commercial test not available Histology Hematoxylin-eosin [morphology] WSP/immunohistochemical staining Not easy Very specific and sensitive Dirofilaria immitis
    • Microfilarie Tick smear: drop of venous blood on a microscopic slide covered with a coverslip Modified Knott test [2 ml of blood] Filter test Mf histochemical staining PCR on mf and adult worms Not sensitive, not specific Easy, sensitive, quite specific Easy, sensitive, quite specific Very specific and sensitive Very specific and sensitive Adult worms Morphology Not easy particularly in case of female worms Serology Ag/Ab ELISA WSP Ab ELISA Commercial test not available Commercial test not available Histology Hematoxylin-eosin [morphology] WSP/immunohistochemical staining Not easy, very thin cuticula indentation Very specific and sensitive Dirofilaria repens
    • whole blood smear one drop of venous blood on a microscopic slide covered with a coverslip Advantages Disadvantages Rapid and inexpensive Very low sensitivity, frequent false negative, no species diagnosis (it is not possible to differentiate microfilarie) Not useful in cats To note that intensity of microfilaremia is not correlated to the adult worm burden: in general, high microfilaremic dogs harbour few worms.
    • Modified Knott test Advantage Disadvantages Sensitive in dogs and specific: microfilarie belonging to different species can be differentiate Time consuming, need of a centrifuge and a skill operator with good knowledge of Mf morphology Specific but of low sensitivity in cats
    • Advantages Disadvantages Rapid and sensitive in dogs No need for a centrifuge apparatus Expensive (tests are sold as kit, Difil Test Evsco); difficulties in distinguish morphology; the lysate solution shrinks the Mf and new measurement standards are required to differentiate species Low sensitivity in cats Filter tests
    • Histology and histochemistry Advantages Disadvantages HISTO: very specific HISTOCHM: very specific and sensitive, suitable in case of “bad” specimens Costly, time consuming, need for a skilled laboratory technician
    • Histology: adult worm longitudinal ridges Courteously Laura H. Kramer, University of Parma D. repens D. immitis
    • WSP/immunohistochemical staining
    • Advantages Disadvantages Very specific and sensitive for HW diagnosis [when positive, the test is the definitive prove of heartworm infection in dogs and cats] Able to diagnose an actual infection [7-8 month-post infection/7-8 months after the death of worms] Costly, not available for other filarial infections Does not work in case of infections caused by male worms only Adult female HW circulating antigen
    • Advantages Disadvantages Very sensitive Able to detect the cat exposure to heartworm infection Suitable to asses the infection risk in cats and for epidemiological survey Costly Not fully specific Difficult to be interpreted Antibody test
    • Advantages Disadvantages Very sensitive, specific and accurate Able to discriminate all the filarial species Costly, time consuming Need for specialized laboratory and skilled technicians PCR
    • PCR Species-specific PCR amplifications of Dirofilaria immitis and D. repens 12S rDNA and of Acanthocheilonema reconditum coxI. a) PCR amplification using D. immitis 12S rDNA specific primers on D. immitis DNA (lanes 2-4), D. repens (lanes 5-6), A. reconditum (lanes 7- 8) and mixed DNAs (lanes 9-10); MW 1 2 3 4 5 6 D. immitis D. repens A. recon. 7 8 9 10 Mix 3 DNANeg a) c) MW 1 2 3 4 5 6 A. recon. D. immitis D. repens 7 8 9 10 Mix 3 DNANeg b) MW 1 2 3 4 5 6 D. repens D. immitis A. recon. 7 8 9 10 Mix 3 DNANeg
    • Differentiation of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood by real time PCR coupled to High Resolution Melting Analysis
    • Evaluation of thomboembolic risk • Lung arterial vessel conditions • Worm burden xR
    • PROGNOSIS Risk of developing post-adulticide treatment thromboembolism • Low risk of thromboembolic complications (low worm burden and no parenchyma and/ or pulmonary vascular lesions) Dogs included in this group must satisfy all this conditions • No symptoms • Normal thoracic radiographs • Low level of circulating antigens or a negative antigen test with circulating microfilariae • No worms visualized by echocardiography • No concurrent diseases • Permission of exercise restriction • High risk of thromboembolic complications In this group should be included all the dog that do not satisfy one or more of these conditions • Symptoms related to the disease (coughing, lipotimias, swelling of the abdomen) • Abnormal thoracic radiographs • High level of circulating antigens • Worms visualized by echocardiography • Concurrent diseases • No permission of exercise restriction
    • PROGNOSIS Risk of developing post-adulticide treatment thromboembolism Other factors to be considered: Age: low risk < 4 year-old; risk > 4 year-old Residency: low vs high prevalence in dog population
    • DOG:thoracicradiography
    • Echocardiography ECH detects echogenic walls of the immature or mature heartworm residing in the lumen of the pulmonary arterial tree, if within the visual window of the ultrasound. The adult parasite is echogenic, producing images of two, short parallel lines. The pulmonary arteries, right ventricle or rarely the right atria must be examined carefully because infections with one or only a few parasites could be overlooked.
    • DOG:echocardiography
    • Echo Doppler Tricuspid overflowing showing atrium-ventricular high rate pressure Systolic overflowing trough tricuspid valve and pulmonary diastolic overflowing
    • Cat: thoracic radiography
    • Feline HW Aelurostrongylus abstrusus
    • Aelurostrongylus abstrusus: nematode Metastrongiloidea, parasite of terminal bronchioles and alveolar ducts of cats Diagnosis: larvae in faeces throughout Berman apparatus
    • Echocardiography in cats is high specific and sensitive! Because the length of worms in cats is the same than in dogs [M: 12-18 cm; F: 25-30 cm] it is quite unlikely that worms could not be visualized by echo. In red the areas possible to be explored by echo
    • Cat: echocardiography
    • Mf Knott Ag test Interpretation Comment Positive Positive Definitive diagnosis of HW ThRx can help to manage the disease Clinical signs and the results of semiquantitative ELISA tests can help in discriminating between low and high risk of thromboembolism Positive Negative Definitive diagnosis of HW: very low HW burden if D. immitis Mf are present Filarial infection caused by other species than D. immitis Normal ThRx patterns Low/very low risk of thromboembolic complications Histochemical stain or PCR can be used to differentiate Mf Negative Positive Definitive diagnosis of HW occult infection Dogs were previously treated incorrectly with preventive drugs or with macrocyclic lactone injectable formulations ThRx and ECHO can help to manage the disease Clinical signs and the results of semiquantitative ELISA tests can help in discriminating between low and high risk of thromboembolism DOG
    • Mf Knott Ag test Ab test Interpretation Comment Positive Positive Positive Definitive diagnosis of HW ThR and ECHO can help to manage the disease Positive Negative Positive Definitive diagnosis of patent HW if D. immitis if Mf are present ThR and ECHO can help to manage the disease Positive Negative Negative Filarial infection due to other species than D. immitis Histochemical stain or PCR for differentiate Mf and give specific diagnosis Negative Positive Positive Definitive diagnosis of HW ThR and ECHO can help to manage the disease Negative Negative Positive Low adult female worm burden Immature worms Aborted infection Immune response to previous patent infection, but warms are already died ThR and ECHO are useful to confirm the suspicion of HW infection Re-testing after 4-8 months can help to confirm the suspicion Cat