Acetic acid as an alternative reagent in the modified Knott test

Area of Science:

• Veterinary Parasitology
• Diagnostic Microbiology
• Comparative Pathology

Background:

• The modified Knott test is a standard diagnostic method for canine heartworm (Dirofilaria immitis).
• Formalin, a common reagent, poses safety concerns due to its toxicity and carcinogenicity.
• Safer alternatives are sought to improve laboratory safety and reduce environmental impact.

Purpose of the Study:

• To evaluate the suitability of acetic acid as a safer alternative to formalin in the modified Knott test for Dirofilaria immitis diagnosis.
• To compare microfilaria concentration, hemolysis levels, and microfilarial length measurements between acetic acid and formalin methods.
• To assess the impact of delayed sample preparation on the accuracy of both methods.

Main Methods:

• Microfilaria concentration was determined using both acetic acid and formalin in the modified Knott test.
• Hemolysis was quantified by red blood cell count and spectrophotometric absorbance at 415 nm.
• Microfilarial lengths were measured to assess potential effects on species identification.

Main Results:

• Microfilaria concentrations showed reasonable agreement between acetic acid and formalin methods (bias: -5.84%).
• Acetic acid significantly reduced hemolysis compared to formalin (p < 0.015 for RBC count, p < 0.001 for absorbance).
• Mean microfilarial length was significantly shorter after acetic acid treatment (273 μm) versus formalin (316 μm; p < 0.001).

Conclusions:

• Acetic acid is a suitable alternative to formalin for the modified Knott test in detecting circulating microfilariae for most clinical purposes.
• While sample readability is similar, species differentiation requires methods beyond length measurement after acetic acid treatment.
• Further research is needed to validate species identification accuracy with acetic acid, especially for Acanthocheilonema reconditum.