Comparative methodology for the detection and differentiation of circulating microfilariae of Dirofilaria immitis in the dog

The sensitivities of the Knott's test (four 20-μl sediment aliquots), quantitative buffy coat capillary tube method (QBC tube, 111 μl of whole blood) and direct blood smear (DBS, 20 μl of whole blood) were evaluated for the detection of microfilaraemia in dogs. Undiluted whole blood samples taken from 70 Dirofilaria immitis antigen-positive dogs and 10 serially diluted microfilaraemic blood samples at concentrations of 400, 200, 100, 50, 25 and 12 microfilariae (mff) ml−1 were examined. For filarial speciation, the buffy coat of QBC tubes was mixed with one drop of methylene blue–formalin solution and examined as a direct smear. In 52/70 microfilaraemic blood samples, the number of mff ranged from 12 to 321987 ml−1 (median: 3199 ml−1). The diagnostic sensitivity of the Knott's test, QBC tube method and DBS in undiluted blood samples attained the 100%, 98% and 92.3% levels, respectively. Eighteen dogs tested amicrofilaraemic by all three methods. At concentrations of 400 mff ml−1, a 100% sensitivity was found by all three methods, while at 200 mff ml−1 the Knott's test, QBC tube and DBS were 100%, 100% and 90% sensitive, respectively. The relevant figures at 100 mff ml−1 were 100%, 100% and 80%, at 50 mff ml−1 100%, 100% and 50%, at 25 mff ml−1 100%, 100% and 10% and at 12 mff ml−1 80%, 50% and 10%. At 50 and 25 mff ml−1, the DBS was less sensitive compared to the other two methods, while at 12 mff ml−1, only to the Knott's test. A significant correlation was found between the QBC tube method and Knott's test regarding mff speciation. Therefore, the QBC method may be considered a reliable alternative to the Knott's test for both the detection and speciation of mff in the dog.