Objectives: New technologies, particularly in the field of clinical metagenomics, have opened up new avenues to explore microbial involvement in illnesses where disease initiation is poorly understood. Here we present a series of six patients suffering from diseases with potential microbial involvement: systemic lupus erythematosus, chronic fatigue syndrome, fibromyalgia, multiple sclerosis, amyotrophic lateral sclerosis, and arteriosclerosis.

Material and methods: Peripheral blood (first five cases) and suction debris from carotid percutaneous angioplasty (arteriosclerosis) were refrigerated and transported to the site of analysis. DNA was extracted using the standard QIAamp DNA Blood Mini and Protozoal Extraction kits. Bacterial-specific DNA sequences were amplified and subsequently barcoded for DNA sequencing with 16S rRNA-directed primers flanking the variable regions 1 and 2 and variable regions 4 and 5, respectively. Protozoal-specific DNA sequences were amplified and subsequently barcoded for DNA sequencing using low stringency conditions and primers directed to variable regions in the 18S rRNA gene.

Results: We found previously unreported mixed bacterial and protozoal biofilm communities in all samples. In all six disease types we revealed protozoa, being close relatives to known aquatic-based protozoa, such as: Oblongichytrium, Chrysocapsa vernalis, Ochromonas, and Perkinsus qugwadi. In a blood sample from the patient with lupus erythematosus DNA stains and culture studies revealed an unknown protozoan exhibiting profound biofilm-forming capability: candidatus Protomyxzoa rheumatica.

Conclusions: Discovery of these potentially pathogenic protozoa in human blood may be of great clinical relevance. However, our preliminary results should be confirmed by independent researchers, and the role of biofilm-forming protozoa in human disease should be elucidated.