Supportive Data

Fifty-two positive patient specimens were available for accuracy studies and correlated with results of organism-specific polymerase chain reaction (PCR). In addition, 20 negative samples from previous PCR testing were evaluated in verification. Using criteria established in verification, overall sensitivity of the assay is 82.7%, and specificity is 100% compared to single-analyte PCR tests. Sensitivity was lower due to the suboptimal recovery of Borrelia burgdorferi and increased to 97.4% without B burgdorferi included. A comment is added to reports that will reflect the limitations of detecting this organism.

The limit of detection was determined using four different organisms (Enterococcus gallinarum, Pseudomonas aeruginosa, Leptospira interrogans, and Anaplasma phagocytophilum) with an average sensitivity of 27 colony forming units per mL of whole blood for E gallinarum and P aeruginosa, and 63 copies per mL of whole blood for L interrogans and A phagocytophilum.

Specificity was tested using a panel of 10 nucleic acid extracts from viral, fungal, and parasitic organisms. No cross-reactivity to these organisms was observed.

Inclusivity studies were performed by sequencing 56 samples representing diverse types of bacteria (including tickborne bacteria). All bacteria were detected and correctly identified by next-generation sequencing.