Acetic acid as an alternative reagent in the modified Knott test

Abstract

The suitability of acetic acid as a safer alternative to formalin in the modified Knott test was evaluated for the diagnosis of canine heartworm (Dirofilaria immitis). Microfilaria concentration was measured by both methods and found to agree within reasonable limits (−5.84 % bias; −88.1–76.4 % limits of agreement). The level of agreement was lower when samples were prepared with a 24 h delay, but this was due to the formalin method tending to yield lower counts (−20.1 % bias; −90.5–50.2 % limits of agreement). Clearing the sample of hemoglobin improves readability and is a key feature of the modified Knott test. Hemolysis was significantly lower in the acetic acid method than the formalin method as measured by red blood cell count (6.83 × 106 and 8.79 × 106 cells/ml, respectively; p = 0.015) and absorbance at 415 nm (33.20 and 34.75, respectively; p < 0.001). Visual assessment, however, revealed little practical difference in readability. Finally, lengths of microfilariae were measured to ensure the validity of species identification by the acetic acid method; mean length was significantly shorter after acetic acid treatment (273 μm) than formalin treatment (316 μm; p < 0.001). Length reduction was also observed in acetic acid-treated Acanthocheilonema reconditum (254 μm versus 262 μm; p = 0.035), though these samples were stored prior to testing and are not directly comparable. We conclude that, while the readability of samples is similar for both methods, species differentiation must still be accomplished by other means. For most clinical purposes in determining the presence or absence of blood circulating microfilariae, however, acetic acid appears to be a suitable alternative to formalin in the modified Knott test.