Heartworm Screening methods in dogs, cats and human - Metode de realizare a Screening-ului in cazul dirofilariozei la caini, pisici si oameni

HEARTWORM SCREENING METHODS IN DOGS, CATS AND HUMAN

 

METODE DE REALIZARE A SCREENING-ULUI ÃŽN CAZUL DIROFILARIOZEI LA CÂINI, PISICI ŞI OAMENI

 

F. GUŞTERE,  Sânziana RĂDULESCU,  D. COBZARIU, S.

 BĂRĂITAREANU

*University of Agronomical Science and Veterinary Medicine
Splaiul Independentei 105, Sector 5, Bucharest,

e-mail: floringustere@yahoo.com

 

Key words: heartworm disease, dirofilaria immitis, rapid immunochromatographic tests, heartworm screening methods, microfilariae

 

SUMMARY

 

Heartworm disease ( a zoonosis disease which must be discovered and treated early enough in order to prevent its spreading ) represents a real challange for veterinary practitioners becouse of its common clinical signs and most of all because of its cronical evolution. The fact that mosquito’s bite is the most frequent cause of heartworm infection makes that the spreding area of the disease to be a large one ( we can find dirofilariosa in every area where temperature and humidity are high) . The purpose of this paper is not only to raise awareness about the importance of heartworm diagnosis but to describe one of the most efficient diagnostic tool. Diagnostic methods should detect both microfilariae and antigen.

 

Dirofilariosis is a zoonosis described in domestic and wild animals like dogs, cats, wolves and foxes. Dirofilaria immitis (fig.1) and Dirofilaria repens are the two most common filarial nematode worms responsable with producing the infection. The most frequent route of infection spreading is by female mosquito’s bite (the male is not able to develop the life cycle of Dirofilaria immitis). Important to know is  that hemotransfusions can also cause the desease transmision if the donor’s blood is contaminated. Dirofilariosis can be prevented with profilactic medication. On the other hand, the absence of a post–infection  treatment or innadequate drug administration can lead to death. In the last few years, concurrent with international trade development, the incidence of dirofilariosis in Romania has increased. The intermediate host of Dirofilaria Immitis (the mosquito) finds in Romania proper development conditions: temperatures of over 14 degrees and high humidity.

Fig1.Adult heartworm in Infectious Diseases Department.Veterinary Faculty Bucharest- original

 

In most of the human cases of dirofilariosis, Dirofilaria repens is the etiological agent that causes cutaneous changes. Literature does not mention any case of human dirofilariosis in our country, as opposed to Hungary, where the first case is mentioned in 1879. It seems that the number of cases gradually increased lately if we consult  the latest research. The diagnosis in these cases was made following a detailed case history and after a careful examination of the parasites found in different areas: subconjunctival space and the subcutaneous tissue of the thigh, forearm.[3]

The most important elements that contribute to the increasing cases of dirofilariosis in Romania consist of: the climatic conditions, international trade development and the fact that people travel along with their pets, bringing in contaminated animals from countries with a high  dirofilariosis prevalence. There is little information available about the incidence of dirofilariosis in our country. However, infections in dogs ranges from 2-17% in Bulgaria, Greece and Turkey up to 65% in Romania and some areas considered to be endemic[4]. Spiru Haret University  published some studies about the heartworm incidence in Romania: 12 out of 52 examined dogs were detected as beeing contaminated[4]. The lack of information has led to the creation of National Epidemiological Surveillance Network of Infectious and Parasitic Disease in Pets – PetEpiNetVet -, a network that provides assistance to owners and veterinarians.

The clinical signs that follow the heartworm infection consist of: anorexia, ascites, edema, tachycardia, tachypnea, cahexie, weight loss, syncope, cough, cough with blood streak, epistaxis, paresis - paralysis, cutaneous nodules, right heart failure - in case of massive contamination with filaria- , flu pipe. All these clinical signs should lead us to a confirmation diagnosis that can be done by at least two ways.

Heartworm treatement includes two aspects: heartworm prophylaxis that consists of drugs which belonging to macrocyclic lactones or macrolides class (ivermectin, selamectin, moxidectin) and adulticide therapy (milbemycin, ivermectin)[2].

If any specific signs are found during clinical examination, the animal needs further testing in order to see if a heartworm infection occured. If the test is positive there is no need for another diagnosis tool. On the other hand, if the test is negative but the animal continues to present specific simptoms an immunologic diagnosis is necessary. Microfilariae detection can be done by: fresh blood examination, concentration methods (Knott test and the filter test) and histochemical exam. Dirofilaria immitis microfilariae indentification based on morphology is considered a definitive proof of infection (specificity 100 %).

 

Fig.2 Microfilariae of Dirofilaria Immitis

 

For many years, the modified Knott test was used as a surveillance method[5,6]. Yet it turned out to be ineffective, being unable to detect sterile and unisexual infections. Taking that into consideration, nowadays rapid tests and serological tests[7] (for specific FILARCHEK antibodies or antigens) are preffered. Recent research show that antigen detection tests are more reliable than the ones that detect microfilariae. For detection of heartworm  specific circulating antigen, serological tests and immunochromatographic rapid tests are used. Although the two methods present a high sensitivity (the difference is insignificant), there are very few cases when they give false negative results.[8]

There are some of the serological tests used for heartworm surveillance/diagnostic:

            ELISA

1.      detecting adult forms antibodies (AB-ELISA);

2.      detecting adult forms  antigens (AG-ELISA);

            IMMUNOFLUORESCENCE

1.      detecting microfilariae - specific  antibodies (MF-IFA);

2.      detecting  specific adult forms antigens (AG-IFA);[8]

 

The efficacity of HEARTWORM IC – BIOPRONIX – AGROLABO was demonstrated by Agrolabo specialists in several clinical studies. Also, our unpublished studies revealed their practical value and that they are a very helpful diagnosis tool for field veterinarians. It is a simple and fast method; the results can be interpretated in 10-15 minutes; it doesn’t require special equipment and can-be made at any veterinary clinic or field;it has a sensitivity of 97.98%.

 

 

 

 

 

 

 

 

 

 

 

Fig.4 Recommended protocol for diagnosing and treating heartworm

disease (Charles Courtney - University of Florida)

 

TEST PRINCIPLE

 

Heartworm IC is a rapid test that employs the immunochromatographic sandwich technique using monoclonal antibodies against Dirofilaria Immitis antigen. A monoclonal antibody conjugated with colloidal gold and a further monoclonal antibody are immobilised on the test membrane. Samples containing Dirofilaria immitis antigen will bind to the colloidal gold conjugated antibody forming an antigen-antibody complex. The complex migrates along the membrane and is then captured by the second monoclonal antibody that is immobilised on the membrane at the level of window number two, forming a red line. Other capturing antibodies are immobilised at window three. These will bind with the fluid that continues to migrate along the membrane, forming a second red line which indicates that the test has been carried out correctly. This line should appear whether the test result is negative or positive.

 

TEST PROCEDURE

 

1.                  Using the appropriate pipette, dispense one drop of whole blood, serum or plasma sample into window no. one of the device.

2.                  Add two drops of diluent to window no. one of the device.

3.                  After 10 minutes read the result, do not read the result after more than 15 minutes

4.                  Result interpretation.

 

RESULT INTERPRETATION

If the test is negative, a red line will only appear in window number (internal control line).        
If the test is positive, two red lines will appear: one in window number two (test line) and one in window number three (internal control line).   
The test is considered invalid if no line appears in window number 3  of the device (internal control line), even if a line appears in window number two (test line). (fig. 3)

 

Fig 3. Result interpretation

 

For a complet screening veterinarians should use two diagnostic methods: one for microfilarae and the other for specific antigen detection. Moreover, for an efficient diagnosis it is important to choose high sensitivity and specificity tests.

Important to note is that currently, the easiest and most suitable method for screening  is the immunochromatographic test - it is fast (results in 10 minutes) and easy to use.

 

REFERENCES

 

1)       Baldock C.: - Clinical epidemiology.Interpretation of test results in heartworm diagnosis. Heartworm Symposium, 1988

2)       Clarke Atkins- Canine Heartworm Disease: Current Treatment and Prevention Approsches             Waltham/OSU Symposium, Small Animal Cardiology, 2002

3)       Claudio Genchi, Laura Rinaldi, Giuseppe Cringoli, Dirofilaria immitis and D. Repens in                dog and cat and human infections, 2007

4)       Fernando Simon, Claudio Genchi -Heartworm infections in humans and animals, , Editura              Salamanca, 2001

5)       Georgi JR, Georgi ME: Heartworms and other filarids. In: Canine clinical parasitology,   1992

6)       Greene RT, Bennett RA, Woody D, Troy GC- Evaluation of a microfilter technique and two             serological tests used in the diagnosis of canine, 1986

7)       Lois Roth, Lynne Brown- Comparison of three diagnostic tests for Dirofilaria immitis in a              low-incidence area,1993

8)       Thilsted JP, Whorton J -Comparasion of  four serotests for detection of Dirofilaria immitis              infection in dogs., AM J Vet 1987

9)       Societatea americana a dirofilariei http://www.heartwormsociety.org

10)    Diagnosticul dirofilariozei http://www.cyf-medical-distribution.ro

11)    Diagnostic for pet animals http://www.agrolabo.it