ABSTRACT: We compared information obtained by both microscopy and nested mitochondrial cytochrome b PCR in determining
prevalence of haemosporidian infections in naturally infected birds. Blood samples from 472 birds of 11 species belonging to 7
families and 4 orders were collected in Europe, Africa, and North America. Skilled investigators investigated them using the
PCR-based screening and microscopic examination of stained blood films. The overall prevalence of haemosporidian infections,
which was determined by combining results of both these methods, was 60%. Both methods slightly underestimated the overall
prevalence of infection, which was 54.2% after the PCR diagnostics and 53.6% after microscopic examination. Importantly, both
these tools showed similar prevalence for Haemoproteus spp. (21% by PCR and 22% by microscopy), Plasmodium spp. (17%
and 22%), and Leucocytozoon spp. (30% and 25%), verifying that microscopy is a reliable tool in determining patterns of
distribution of blood haemosporidian parasites in naturally infected birds. We encourage using optical microscopy in studies of
blood parasites in parallel to the now widely employed molecular methods. Microscopy is unlikely to result in false positives,
which is a major concern in large-scale PCR studies. Moreover, it is relatively inexpensive and provides valuable information
regarding the ways in which molecular methods can be further improved and most effectively applied, especially in the field
studies of parasites. Importantly, blood films, which are used for microscopic examination, should be of good quality; they should
be examined properly by skilled investigators. In spite of the substantial time investments associated with microscopy, such
examination provides opportunities for simultaneous determination and verification of taxonomically different parasites. Presently,
different PCR protocols must be used for the detection of parasites belonging to different genera; this is expensive and time
consuming.