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  • Comparative analysis of Parasitological and Molecular methods in detecting blood microfilaremia
  • Negar Hemmati,1 Fateme Jalousian,2,* Negar Najafifard,3
    1. DVM student, Faculty of Veterinary Medicine, Tehran University
    2. Parasitology Department, Faculty of Veterinary Medicine, University of Tehran
    3. DVM student, Faculty of Veterinary Medicine, Tehran University


  • Introduction: Bloodworms, particularly Dirofilaria immitis, are significant parasites in both veterinary and human health contexts. These parasites are primarily transmitted through arthropod vectors and are endemic in Iran, where various veterinary filarial worms, including Dirofilaria immitis, Dirofilaria repens, and Acanthocylonma reconditum, are found. Diagnosis of filariasis involves examining parasites in peripheral blood using the modified Knott method. However, molecular methods have emerged as more accurate and specific tools for detecting filarial parasites. The main objective of this study is to investigate the modified Knott method and molecular PCR method for detecting Dirofilaria Immitis in dog blood. The secondary goal of this study is to compare the effectiveness of these two methods in diagnosing Dirofilariasis.
  • Methods: In this study, a total of 66 blood samples were collected from dogs residing in Mazandaran, Gilan, and Qazvin provinces. Each blood sample, comprising 1 milliliter, was mixed with 9 milliliters of 2% formalin. We employed the modified Knott method to examine the presence of microfilaria. Additionally, DNA extraction was carried out using a commercial kit(MBST, Iran), followed by amplification and sequencing of a partial sequence of the Cytochrome oxidase subunit I(COXI) gene through PCR.
  • Results: The results obtained from the modified Knott method demonstrated the presence of Dirofilaria immitis microfilaria in 6 cases (9.1%) of the blood samples. The average length of the observed microfilaria was calculated to be 311.8 ± 9.8 micrometers. On the other hand, the PCR method successfully detected the presence of the Dirofilaria immitis DNA genome in 33 (50%) of the blood samples. To establish a positive control for the study, we acquired a Dirofilaria immitis sample from the heart of a dog that unfortunately died in an accident in Gilan province. This particular sample was generously provided by the Parasitology Museum and was utilized in our research. The modified Knott method exhibited sensitivity, specificity, and efficiency rates of 55% (with a confidence interval of 67.88%-41.61%), 100%, and 77.5%, respectively. For the PCR method, the sensitivity, specificity, and efficiency were all reported as 100%. To assess the agreement between the two methods, we calculated the Kappa coefficient, which resulted in a value of 0.4. This suggests a relative agreement between the modified Knott method and PCR method employed in this study.
  • Conclusion: The modified Knott method is an affordable and reasonably specific option for diagnosing Dirofilaria immitis microfilariae. However, for diagnosing dirofilariasis in dogs, molecular methods are more sensitive and efficient. Molecular methods can effectively detect the presence of the disease even when microfilariae are not visible in the peripheral blood. It is important to consider that while molecular methods offer better efficiency, they require advanced laboratory equipment and are more expensive to perform. As a result, the modified Knott method can be used as a cost-effective and relatively quick initial diagnostic approach. However, in areas where dirofilariasis is prevalent or hyper-endemic, like the northern and surrounding regions of the Caspian Sea and East Azerbaijan province, it is crucial for laboratories to be equipped with the necessary instruments for molecular diagnosis. Additionally, the use of rapid serology detection kits in these areas can greatly expedite the process of diagnosing infections. It is worth emphasizing that accurate diagnosis of contamination is fundamental in developing effective and efficient programs for controlling and preventing infectious diseases. Furthermore, the implementation of a one-health approach, which considers the health of animals alongside human welfare, is essential for mitigating risks posed by emerging diseases and enhancing overall well-being. A comprehensive approach that addresses both animal and human health is vital to achieve successful outcomes in disease prevention.
  • Keywords: Dirofilariasis, Microscopic examination, Mitochondrial gene, vector-borne transmission, zoonotic