The buffy coat method: A tool for detection of blood parasites without staining procedures

Background: Blood parasites belonging to the Apicomplexa, Trypanosomatidae and Filarioidea are widespread in birds and have been studied extensively. Microscopical examination (ME) of stained blood films remains the gold standard method for the detection of these infections in birds, particularly because co-infections predominate in wildlife. None of the available molecular tools can detect all co-infections at the same time, but ME provides opportunities for this to be achieved. However, fixation, drying and staining of blood films as well as their ME are relatively time-consuming. This limits the detection of infected hosts during fieldwork when captured animals should be released soon after sampling. It is an obstacle for quick selection of donor hosts for parasite experimental, histological and other investigations in the field. This study modified, tested and described the buffy coat method (BCM) for quick diagnostics (~ 20 min/sample) of avian blood parasites. Methods: Blood of 345 birds belonging to 42 species was collected, and each sample was examined using ME of stained blood films and the buffy coat, which was examined after centrifugation in capillary tubes and after being transferred to objective glass slides. Parasite detection using these methods was compared using sensitivity, specificity, positive and negative predictive values and Cohen's kappa index. Results: Haemoproteus, Leucocytozoon, Plasmodium, microfilariae, Trypanosoma and Lankesterella parasites were detected. BCM had a high sensitivity (> 90%) and specificity (> 90%) for detection of Haemoproteus and microfilariae infections. It was of moderate sensitivity (57%) and high specificity (> 90%) for Lankesterella infections, but of low sensitivity (20%) and high specificity (> 90%) for Leucocytozoon infections. Trypanosoma and Plasmodium parasites were detected only by BCM and ME, respectively. According to Cohen's kappa index, the agreement between two diagnostic tools was substantial for Haemoproteus (0.80), moderate for Lankesterella (0.46) and fair for microfilariae and Leucocytozoon (0.28) infections. Conclusions: BCM is sensitive and recommended as a quick and reliable tool to detect Haemoproteus, Trypanosoma and microfilariae parasites during fieldwork. However, it is not suitable for detection of species of Leucocytozoon and Plasmodium. BCM is a useful tool for diagnostics of blood parasite co-infections. Its application might be extended to studies of blood parasites in other vertebrates during field studies.[Figure not available: see fulltext.