Dirofilariasis Workup

Updated: Oct 05, 2015
  • Author: Alena Klochko, MD; Chief Editor: Pranatharthi Haran Chandrasekar, MBBS, MD  more...
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Workup

Approach Considerations

Studies used in the diagnosis and evaluation of dirofilariasis include the following:

  • Complete blood cell (CBC) count - Using a CBC, eosinophilia may be detected in up to 20% of cases of human pulmonary dirofilariasis (HPD)
  • Overall blood eosinophilia and elevated serum immunoglobulin E levels are rarely observed and are of limited value in screening for dirofilariasis
  • Sputum cytology - The presence of eosinophils may support a diagnosis of HPD in patients with a coin lesion observed on radiography, although the test lacks enough specificity for an accurate diagnosis
  • Serologic studies - Using ELISA, serologic studies may yield positive results in 75% of patients with HPD
  • Polymerase chain reaction (PCR) assay - Has been successful in the diagnosis of D immitis and D repens infections
  • Imaging studies - Including chest radiography, computed tomography (CT) scanning, magnetic resonance imaging (MRI), and ultrasonography
  • Pulsed-field gel electrophoresis
  • Biopsy - Including surgical biopsy and fine-needle aspiration
  • Histology - Diagnosing dirofilariasis based purely on histopathology has its pitfalls, especially when the morphology of the nematode is altered owing to inflammatory response or surgical artifact
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Serology

Experimental complement fixation tests, indirect hemagglutination, and ELISAs using whole-body or somatic antigens (SA) have been employed in the diagnosis of dirofilariasis. Serologic studies using ELISA may yield positive results in 75% of patients with HPD. While these are useful tools in epidemiologic surveys in known endemic areas, they have low specificity in the general population and are not routinely available.

Findings may be false-positive in as many as 30% of patients being tested for dirofilariasis, because of cross-reactions with other nematode antigens (eg, Toxocara canis). This means that the antigens chosen for such tests in humans must be shown not to react with antibodies against T canis, Ascaris lumbricoides, Strongyloides stercoralis, Ancylostoma duodenale, or Necator americanus. All of these nematodes have a migratory pathway that potentially exposes humans to antigens, enabling them to stimulate an antibody response. [69] In regions outside the United States, antibody to taxonomically more distantly related helminths (eg, Taenia solium, Clonorchis sinensis) may have to be shown to be non–cross-reactive with the D immitis antigen.

An improved specificity with ELISA has been demonstrated if a 22-kd protein (Di22) from adult D immitis or recombinant antigens (ie, a 35-kd fusion protein, P22U, phospholipase A2 [PLA2]) are used for antibody capture. P22U and PLA2 are larval excretory/secretory proteins of D immitis. P22U is probably related to Di22 and is not recognized in Western blot tests of sera from patients with other parasitic and nonparasitic pulmonary diseases. PLA2 is not related to Di22 but reacts specifically with sera from patients with subcutaneous dirofilariasis.

Nevertheless, because of the low pretest probability of HPD, a positive ELISA result using Di22 or other antigens must be supported by radiologic characteristics, presentation, patient residence and travel history, and other data so that the clinician can decide whether invasive techniques are needed to reach a definitive diagnosis.

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PCR Assay

Recently, a duplex real-time PCR has been assessed for the discrimination between D immitis and D repens and their quantification in blood samples and mosquitoes. [70]

A multiplex PCR based on the amplification of a mitochondrial gene of blood-circulating microfilariae of D immitis and D repens has also been shown to be useful for their molecular detection and differentiation in blood and skin samples. [71]

PCR assay using paraffin samples can be used for diagnosis when identification of the species is not possible with conventional morphology owing to poor conservation of the worm. [72] D immitis and D repens infections have been successfully diagnosed with amplification of genomic DNA extracted from single worms isolated in patients with clinical dirofilariasis.

The biopsy material or worms intended for PCR assay should be not be stored in formalin, because formalin fragments genomic DNA and interferes with Taq polymerase.

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Imaging Studies

Chest radiography

The incidental finding of a pulmonary lesion on chest radiography is the usual presentation of HPD. The lesion is usually a well-circumscribed, peripheral coin lesion or nodule. In as many as 90% of cases, there is a solitary nodule. The lesions are usually smaller than 30 mm [73] and are typically subpleural (68%) and on the right side (76%), showing a particular preference for the right lower lobe (46% of all cases). Multiple lesions can be observed in either the same lobe or multiple lobes. (See the image below.)

Plain chest radiographic appearance of pulmonary c Plain chest radiographic appearance of pulmonary coin lesion secondary to Dirofilaria immitis infection in a man.

Pulmonary lesions can be transitory. The coin lesion is an end-stage result of the parasite's death in the vascular bed of the lungs and the stimulation of a pneumonitis followed by granuloma formation. This pneumonitis phase of HPD often goes unrecognized by the radiologist, because the developing nodule is obscured by the lung inflammation. [69] The shortest time reported between a normal finding on chest radiography and the presence of a coin lesion due to D immitis infection was 5 months. Miyoshi et al noted pleural effusions in 13% of patients with HPD. [73]

The diagnosis in children may underappreciated, as dirofilarial lesions may be may be mistakenly labeled as Ghon foci secondary to pulmonary tuberculosis and, as such, not followed or treated.

CT scanning

Chest or abdominal CT scanning may be performed for further evaluation of pulmonary or abdominal lesions and for assessment of mediastinal lymphadenopathy. It may also be used to guide fine-needle aspiration and biopsy of suspicious lesions

MRI

MRI may be useful in differentiating subcutaneous dirofilarial lesions of the head and eyes from lesions of other etiologies (eg, histiocytosis, neuroblastoma).

Ultrasonography

Ultrasonography of abdominal or cutaneous lesions assists in the etiologic diagnosis of dirofilariasis and may guide fine-needle aspiration biopsy of the lesions.

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Pulsed-Field Gel Electrophoresis

Multilocus electrophoresis analysis of DNA from worms recovered from pulmonary or subcutaneous nodules may be useful in identifying specific Dirofilaria species, especially in areas where both D repens and D immitis are present. (Histologic and phenotypic features of recovered worms may be damaged during removal or they may degenerate, preventing accurate identification.)

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Biopsy

Surgical biopsy of subcutaneous nodules

D repens usually presents as a subcutaneous nodule in the body sites discussed earlier. The nodule almost always is excised for histopathologic diagnosis and confirmation of infection. The size of the nodules can vary from 0.5-2.5 cm in diameter.

Fine-needle aspiration of peripheral pulmonary lesions

Transthoracic needle aspiration (which can be performed with or without CT guidance) has been employed successfully in the diagnosis of HPD, having been used to identify morphologic features of D immitis and prevent invasive surgery.

Bronchoscopy with cytology and transbronchial biopsy

Bronchoscopy has been attempted in cases of HPD, with little success in positively identifying the cause of the pulmonary lesion. [74]

Results of cytology of bronchoalveolar lavage specimens and brushings should be interpreted with caution because the bronchial mucosa in patients with HPD may be focally metaplastic and mimic invasive squamous cell carcinoma.

A predominance of eosinophils or the presence of eosinophilic pneumonia may be a clue to the diagnosis of HPD.

Open lung biopsy with wedge resection of affected lung segment

Upon removal, most lesions are well-circumscribed, spherical, and gray-yellow in appearance. Wedge-shaped lesions may be observed in 32% of cases.

Fine-needle aspiration of retroperitoneal dirofilarial masses

The successful use of ultrasonographically guided fine-needle aspiration to obtain a biopsy of a pseudotumorous para-aortic mass has been described. [75]

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Histologic Findings

Differentiating D repens from D immitis in microscopic sections is based on the diameter of the parasites, the thickness of the cuticle, the number and distribution of the fibers in the muscular layer, and other morphologic details, including the presence or absence of external cuticular ridges (found in D repens but not in D immitis). [76]

Diagnosing dirofilariasis based purely on histopathology has its pitfalls, however, especially when the morphology of the nematode is altered owing to inflammatory response or surgical artifact. (See the images below.) [77]

Transverse section through an immature adult Dirof Transverse section through an immature adult Dirofilaria immitis removed from the right chest wall of an 18-month-old child in Sydney, Australia. The large lateral chords and multilayered cuticle are typical of Dirofilaria. The smooth cuticle is a feature of D immitis.
Low-power view of a bulbar conjunctival biopsy spe Low-power view of a bulbar conjunctival biopsy specimen obtained from a 72-year-old man from Queensland, Australia, showing degenerate pieces of an immature Dirofilaria immitis worm in cross-sections and longitudinal sections with Masson stain.
Midpower view of bulbar conjunctival biopsy specim Midpower view of bulbar conjunctival biopsy specimen showing degenerate pieces of an immature Dirofilaria immitis worm in cross-section and longitudinal section view with Masson stain.
High-power view of bulbar conjunctival biopsy spec High-power view of bulbar conjunctival biopsy specimen showing a cross-section of immature Dirofilaria immitis. The thin, smooth cuticle with internal, lateral, longitudinal ridges; the thin hypodermis; the large lateral cords; the well-developed coelomyarial muscles; and the ill-defined reproductive organs are diagnostic of an immature D immitis (hematoxylin and eosin stain).

HPD with D immitis infection

On microscopy, lesions have a necrotic center of lung tissue around a centrally thrombosed artery, with fragments of a nonviable, immature worm. This core is surrounded by a granulomatous zone of epithelial cells, plasma cells, lymphocytes, and a fibrous capsule rich in eosinophils (66% of cases). The overlying pleura is usually inflamed and fibrotic (75%). Calcification and caseous necrosis may be present (41%). Charcot-Leyden crystals may be detected in the nodules (27%).

The adjacent lung may show a desquamative, interstitial, pneumonia–like reaction (66%); follicular bronchiolitis (46%); and patchy organizing pneumonia (34%). A focal vasculitis involving the muscular arteries and capillaries is recognized in 51% of cases.

Anatomic features of the worm are better appreciated when histologic sections are stained with Movat pentachrome, Gomori methenamine-silver (GMS), or periodic acid-Schiff (PAS) stains. The ability to identify internal features of the worm (ie, digestive system, genital tract) is common. Nonspecific fluorescent whitener stains have also been used to rapidly identify worms in pulmonary specimens. Worms have been identified on frozen section biopsy specimens in 2 cases.

D repens infection

In most cases, the worm contained in the lesion shows varying grades of degradation. Some areas of the worm show more degeneration than others (ie, myoid fibers, lateral chords). Other features, such as the cuticle (outer covering), walls of the digestive tract, and sexual tubules, offer a certain resistance to attack. The external ridges of the cuticle are the feature that most easily distinguishes D repens from D immitis in such lesions.

The worm is surrounded by a large number of eosinophils and other inflammatory cells of varying degrees. An epithelioid, granulomatous reaction with multinucleate giant cells is observed in as many as 33% of cases and may resemble mycobacterial or fungal infections. Lymphocytes may form large aggregates and follicles with germinal centers that can extend into the connective tissue, mimicking lymphoma.

A fibrotic capsule and calcification may be observed in 6% of cases. The subcutaneous fat shows a septal and lobular inflammation mediated by eosinophils and lymphocytes. The absence of fat necrosis in the presence of inflammation with such cells indicates a diagnosis of dirofilariasis. However, sparganosis, a parasitic zoonosis caused by the larval stage of Spirometra species, may cause a similar lesion. (See the images below.)

Transverse section through a mature female Dirofil Transverse section through a mature female Dirofilaria repens removed from the superomedial orbital rim of a 67-year-old man. The patient was infected in Corfu, Greece, 6 months prior to diagnosis in Sydney, Australia. Features characteristic of Dirofilaria are the arrangement of the longitudinal muscles and the multilayered cuticle, which is expanded in the region of the large lateral chords.
Higher-magnification image of the Dirofilaria repe Higher-magnification image of the Dirofilaria repens displayed in the previous image. The nematode was removed from the eye of a 67-year-old man. The features characteristic of D repens are the longitudinal ridges on the cuticle; the ridges are 6-7 micrometers wide, spaced at 11- to 12-micrometer intervals.
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