| Summary: | Apicomplexan parasites of the genus <i>Babesia</i> cause babesiosis in humans and animals worldwide. Human babesiosis is a predominantly zoonotic disease transmitted by hard ticks that is of increasing health concern in the USA and many other countries. Microscopic examination of stained blood smears, detection of serum antibodies by immunoassays and identification of parasite nucleic acid in blood by qPCR and fluorescence in situ hybridization (FISH) are some methods available for diagnosing babesiosis. This study investigated the use of a <i>Babesia</i> genus-specific FISH test for detecting <i>Babesia</i> parasites in blood smears and immunofluorescence assay (IFA) for detecting serum antibodies to <i>Babesia duncani</i> and <i>Babesia microti</i>, two common species that cause human babesiosis in the USA. The findings with clinical samples originating from USA, Australia, Europe and elsewhere demonstrate that the parallel use of <i>Babesia</i> genus-specific FISH and IFA tests for <i>B. duncani</i> and <i>B. microti</i> provides more useful diagnostic information in babesiosis and that <i>B. duncani</i> infections are more widespread globally than presently recognized.
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