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