dirofilaria ghid de diagnostic 3
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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
SpeciesLength
µm
Width
µmFeatures
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 smearone 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
DO
G: th
ora
cic
rad
iogra
phy
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.
DO
G: echo
card
iog
raph
y
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