Serologic diagnosis of zoonotic pulmonary dirofilariasis

CLINICAL STUDIES

Serologic Diagnosis of Zoonotic Pulmonary Dirofilariasis

LAWRENCE Philadelphia,

ROBERT Madison,

T. GLICKMAN, Pennsylvania

B. GRIEVE,

Ph.D.

Wisconsin

PETER M. SCHANTZ, Atlanta,

V.M.D., Dr.P.H.

V.M.D.,

Ph.D.

Georgia

From the Dc”“~~“-+ nf rrrnical Studies, University of Penn Sylvania, MediSchoc )I of Veterinary tine, Philadelphia, Pennsyl mania. The work was supported in part by Nation 21 Institutes of Health Grant Al 18249. Requests for reprints should be addressed to Dr. Lawrence T. Glickman, Section of Epidemiology, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce Street, Philadelohia, Pennsvlvania 19104. Manuscript accepted February 2-1, 1985. q.s”‘LIII~III

“I

“II

Four symptomatic and four asymptomatic patients with histologically confirmed zoonotic pulmonary dirofilariasis caused by Dirofilaria immitis (dog heartworm) were evaluated serologically. Five patients had diagnostic indirect hemagglutination titers to D. immitis and six had positive findings by enzyme-linked immunosorbent assay. Of the two patients that had nondiagnostic titers by both enzyme-linked immunosorbent assay and indirect hemagglutination, one had an encapsulated necrotic adult worm that appeared to have been dead for some time, and the serum specimen for the second patient had been obtained five months following surgical removal of the granuloma. These findings suggest good sensitivity for these serologic methods in active cases, but declining antibody titers and decreased sensitivity following worm death. In general, cross-reactivity of the enzyme-linked immunosorbent assay with serum from patients with other nonfilarial parasitic infections or neoplasms was not observed. Dirofilaria species are nematodes that parasitize many mammals, including dogs and raccoons. They are transmitted by mosquitoes and are capable of infecting humans. Zoonotic dirofilariasis typically takes one of two clinical forms, namely subcutaneous or pulmonary [ 1,2]. Subcutaneous dirofilariasis is characterized by a lump anywhere on the bcdy. The most common cause of this condition in the United States is the raccoon parasite D. tenuis, and most cases have been acquired in the southeastern states, principally Florida and Louisiana. Human cases of ocular dirofilariasis caused by D. repens or D. conjunctivae have been reported infrequently from the Mediterranean area where D. repens is a common subcutaneous parasite of dogs [3]. D. immitis is mosquito-borne and its adult forms usually inhabit the right ventricle and pulmonary arteries of dogs; accordingly, it is commonly called the dog heartworm. Zoonotic pulmonary dirofilariasis caused by D. immitis was first described in 1954 [4], but it was not recognized as such until tissue specimens were reviewed in 1964 [l]. Ciferri [5] recently reviewed 60 cases of human pulmonary dirofilariasis indigenous to the United States. Of the 57 oatients for whom clinical information was available, 23 had symptoms usually consisting of chest discomfort, fever, cough, malaise, and occasionally hemoptysis. The pulmonary nodule(s) in the 34 asymptomatic patients had been discovered by radiographic chest examination. Laboratory results including eosinophil counts were not helpful in the diaanosis. Microfilariae were not demonstrated in the blood samples of fivepatients that were examined. Results of serologic tests were positive in only three of nine patients. The correct preoperative diagnosis was not made for any of the cases reported. Human pulmonary dirofilariasis has also been recognized as benign lung nodules

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ZOONOTIC PULMONARY DIROFILARIASIS-GLICKMAN ET AL

TABLE

I

Demographic,

Pathologic,

and Serologic

Findings

in Patients

with Zoonotic

Pulmonary

Dirofilariasis Tiler+

Patient

Age (years) and Sex

Residence

PreoperativeSymptoms/Diagnosis

1

72F

Iowa

2

73F

Oklahoma

3

44F

New York

53M

Florida

Pleuritic pain, inflammatory lesion or neoplasm Pleuritic pain, neoplasm

70F

New Hampshire

Pulmonary coin lesion

76F

Florida

Pulmonary coin lesion

67F

Virginia

Pulmonary coin lesion

42M

Missouri

Pulmonary coin lesion

Cough, pulmonary granuloma or neoplasm Pleuritic pain, pulmonary coin lesion

PathologicFinding

IHA

ELBA

Young male D. immitis worm Circumscribed yellow nodule with a D. immitis worm--“dead for some time” D. immitis

1:128

1:32

1:32

1:2

1:128

1:512

D. immitis granuloma D. immitis granuloma D. immitis granuloma D. immitis granuloma D. immitis granuloma

1:32

1:16

1:256

1:64

1:4,096

1:128

112,048

1:64

1:64

1:2

* IHA = indirect hemagglutination; a diagnostic titer is 1:128; ELISA = enzyme-linked immunosorbent assay; a diagnostic titer is 1:16. ‘Serum specimen obtained five months after surgery.

mimicking metastatic malignant neoplasms in a patient with transitional cell carcinoma [6]. Human dirofilariasis is typically a surgical diagnosis in contrast to the disease in dogs, in which circulating microfilariae are often evident. Serologic methods including enzyme-linked immunosorbent assay [7] and indirect immunofluorescence [8] are routinely used by veterinarians to diagnose amicrofilaremic (occult) canine heartworm infection, whereas indirect hemagglutination is used for humans with suspected pulmonary dirofilariasis [9]. However, indirect hemagglutination test results may be difficult to interpret because of cross-reactions with a wide variety of human helminth parasites [9]. This report describes the demographic and serologic findings in eight patients in the United States with histologically confirmed pulmonary dirofilariasis and serologic results in patients with other parasitic infections, or malignancies that could mimic this condition. Preliminary findings are presented on the sensitivity and specificity of two serologic tests for pulmonary dirofilariasis. PATIENTS

AND

nary nodules in whom a diagnosis of pulmonary dirofilariaA specimen

Division, Centers for Disease Control, Atlanta, Georgia. These included specimens from 10 patients with each of the following infections: toxocariasis, trichinosis, strongyloidiasis, toxoplasmosis, schistosomiasis, cysticercosis, echinococcosis, and nonendemic (e.g., acquired outside the United States) filariasis. Serum samples were also available from two patients with biopsy-proved subcutaneous dirofilariasis caused by D. tenuis and from seven patients with pulmonary or hepatic neoplasms. Serologic Studies. Antibodies to D. immitis were measured by indirect hemagglutination [lo] using a crude antigen prepared from adult D. immitis and by an enzymelinked immunosorbent assay using semipurified adult D. immitis antigen. The enzyme-linked immunosorbent assay previously described for canine serologic testing [ 1 I] was modified by substituting horseradish peroxidase-conjugated rabbit anti-human IgG for rabbit anti-canine IgG, and serum

samples were not preabsorbed with Toxocara canis antigens. Diagnostic titers for pulmonary dirofilariasis by indirect hemagglutination and enzyme-linked immunosorbent assay are 1: 128 [IO] and 1: 16, respectively. The later

cutoff titer was based on studies of naturally infected dogs

METHODS

Subjects. Preoperative serum specimens were obtained from seven patients with radiographically evident pulmosis was suspected.

had been serologically confirmed by the Parasitic Diseases

from an eighth patient was

in an endemic heartworm area [12] and on preliminary observations in humans (unpublished data). Indirect hemagglutination was performed at the Centers for Disease Control and enzyme-linked immunosorbent assay at the University of Pennsylvania, School of Veterinary Medicine.

obtained five months following surgery, at which time a D. immitis worm was identified histologically in surgically excised tissue. For all eight patients, the final pathologic diagnosis was pulmonary dirofilariasis caused by D. immitis. Serum samples were obtained from patients with other suspected parasitic diseases. The diagnosis in each case

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RESULTS

Of the eight patients with pulmonary dirofilariasis, Six were women and two were men; the age range was 42 to 76 years (Table I). These patients lived in seven different states and none had traveled outside the United States 60

ZOONOTIC

TABLE II

Antibody

Titer to Dirofilaria

immitis

in Patients

with Various

PULMONARY

Parasitic

Indirect Hemagglutination”

Condition

Echinococcosis Filariasis Schistosomiasis Strongyloidiasis Subcutaneous dirofilariasis (D. tenuis) Toxocariasis Toxoplasmosis Neoplasmst:

2

3 0

within the past five years. Four of the patients were symptomatic; three had pleuritic pain and one had a persistent cough. For the other four patients, the only evidence of disease was a solitary pulmonary coin lesion that was detected by radiographic chest examination. Results of enzyme-linked immunosorbent assay for antibody titer to D. immitis were positive in six patients, whereas results of indirect hemagglutination testing were positive in five. Of the two patients who had negative results of both enzyme-linked immunosorbent assay and indirect hemagglutination for D. immitis, one (Patient 2) had a necrotic adult D. immitis worm contained within a granuloma; the pathologist considered the worm to have been “dead for some time.” For the second patient with negative test results (Patient 8) the serum specimen had been obtained five months following surgical removal of a granuloma containing a D. immitis adult worm. Microfilariae were not observed in the peripheral blood of any of the eight patients, but specific procedures for the concentration of microfilariae had not been performed. Results of D. immitis serologic testing in patients with other parasitic infections or neoplasms are shown in Table II. Both patients with subcutaneous dirofilariasis caused by D. tenuis had positive results of indirect hemagglutination and enzyme-linked immunosorbent assay for D. immitis. Similarly, all eight patients with filarial infections acquired outside the United States had positive results of indirect hemagglutination for D. immitis whereas six had positive results of enzyme-linked immunosorbent assay. COMMENTS

Zoonotic dirofilariasis is being recognized with increased frequency among persons with no recent travel history outside of the United States and no intimate association with pet dogs, However, the geographic distribution of February

Assayt Number with Positive Resuli

8 10 7

-

one mesothelioma);

lmmunosorbenl

6 6 8 14 6 2

a 1

8 0 7

ET AL

or Neoplasms

Number Tested

2 1

* A diagnostic titer is 1: 128 or greater. + A diagnostic titer is 1:16 or greater. t Three pulmonary (two squamous cell carcinoma,

Infections Enzyme-Linked

Number with Positive Result

Number Tested

DIROFILARIASIS-GLICKMAN

four hepatic

(one angiosarcoma,

2 0 0

three

carcinoma).

human pulmonary dirofilariasis in the United States follows closely that of high-prevalence canine dirofilariasis [5]. Zoonotic transmission of D. immitis probably results from several factors. These are: (1) an estimated 41 million dogs living in one third to one half of all households, (2) canine heartworm infection rates that exceed 50 percent in some areas, and (3) isolation of D. immitis from many different mosquito vectors in the United States, some of which are serious human pests [ 131. The primary threat to human health from pulmonary dirofilariasis is not caused by the parasite, but rather by invasive procedures that are required to obtain a definitive diagnosis. These procedures, including bronchoscopy, bronchial biopsy, transthoracic needle aspiration, and thoracotomy, may pose a greater risk of complications than the parasite itself. Sensitive and specific serologic procedures for D. immitis could be helpful in the diagnostic process, although they cannot, by themselves, be used to rule out a diagnosis of pulmonary neoplasm. Serologic results must be considered and evaluated in conjunction with the patient’s demographic profile, travel and smoking history, physical and laboratory findings, and the presence of associated diseases. The enzyme-linked immunosorbent assay titers for D. immitis were diagnostic for six of the eight patients with pulmonary dirofilariasis; the indirect hemagglutination titers were diagnostic for five patients. In the two patients with nondiagnostic enzyme-linked immunosorbent and indirect hemagglutination results, the worm had either been surgically removed five months prior to obtainment of the serum specimen or was thought to have been dead for some time. The negative serologic findings therefore may reflect diminished antigen stimulation with declining antibody titers. It has been noted that enzyme-linked immunosorbent assay titers in dogs were infected with D. immitis and treated with an adulticide/microfilaricide drug 1986

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ZOONOTIC

PULMONARY

DIROFILARIASIS-GLICKMAN

ET AL

regimen decreased significantly within several months following successful treatment [ 14,151. Regardless of why serologic tests fail to detect significant antibody titers to D. immitis in patients with pulmonary dirofilariasis and radiographic lesions, the sensitivity of such serologic methods must be improved if they are to have an important role in the diagnosis of this infection. The specificity of enzyme-linked immunosorbent assay for D. immitis appeared adequate; only six of 55 patients with nonfilarial parasitic infections had diagnostic D. immitis titers by enzyme-linked immunosorbent assay. Indirect hemagglutination was less specific (x2 = 4.6, p = 0.003) with 10 of the 3 1 nonfilarial serum samples showing significant cross-reactivity with D. immitis. However, for these patients it was not possible to determine whether they might have been concurrently infected with D. immitis, since additional medical information was not available. Given widespread infection of dogs with D. immitis and the potential for zoonotic transmission, many persons might have subclinical D. immitis infection. In a recent survey, 12 (5.2 percent) of 230 human samples selected at random from a clinical laboratory in northern Illinois* were found to be serologically positive for D. immitis [ 161. Further evidence for the specificity of enzyme-linked immunosorbent assay or indirect hemagglutination for D. immitis is that patients with neoplastic diseases that could manifest as primary or metastatic pulmonary lesions and could be confused radiographically with dirofilariasis were all serologically negative. Both patients with subcutaneous dirofilariasis caused by D. tenuis had significant antibody titers to D. immitis by enzyme-linked immunosorbent assay and indirect hemag-

glutination, as did almost all patients with filarial infections acquired outside the United States. This was not unexpected, since cross-reactivity with D. immitis antigens has been observed in dogs naturally infected with Dipetalonema reconditum, a subcutaneous canine filarial parasite [12]. This lack of species-specific filarial reactivity is probably not critical for the serodiagnosis of human pulmonary dirofilariasis in the United States if the test is limited to persons with no travel history to other geographic areas in which human filariases are endemic. Such was the situation in the eight patients we included in this study. In conclusion, serologic methods for measuring D. immitis antibodies may be useful in patients with pulmonary lesions suggestive of dirofilariasis. They cannot by themselves be used to rule out a diagnosis of neoplastic or other pulmonary diseases, but they could possibly be utilized to monitor antibody titers as an indicator of treatment efficacy [ 151 when anthelmintic drugs are used for conservative therapy of pulmonary coin lesions suspected to be caused by D. immitis. The broad application of serologic methods for the diagnosis of human pulmonary dirofilariasis, however, must await additional data on the sensitivity and specificity of these methods in the clinical setting. ACKNOWLEDGMENT

We are grateful for the technical assistance of Marcia Mika-Grieve, Debra Jones, and Susanne Walqhuist. We ‘are indebted to Drs. J. Martin Johnson, William P. Illig, and Francis J. McMahon for sharing information on their patients.

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