Animal could have adult worms that die and be left with circulating microfilariae
Microfilariae could be Dipetalonema reconditum, a parasite which does not produce the immitis antigen that our test detects.
Young dogs born to heartworm-positive mothers could be infected transplacently and be born with circulating microfilariae, but not have adult immitis, therefore no detectable D. immitis antigen.
Occasionally, microfilariae will be present before detectable levels of antigen have accumulated.