Supportive Data

The following validation data supports the use of this assay for clinical testing.

Accuracy/Diagnostic Sensitivity and Specificity:

Results from this real-time polymerase chain reaction (RT-PCR) assay on the LightCycle were compared to those generated using conventional PCR assay for Anaplasma phagocytophilum on 127 unique, archived whole blood specimens (26 positive and 99 negative specimens by conventional PCR). Using the conventional PCR as the gold standard, the diagnostic sensitivity and specificity for detection of Anaplasma phagocytophilum were 100%. In addition, 12 known Ehrlichia chaffeensis isolates and 2 Ehrlichia ewingii isolates (reference strains) were tested by the LC PCR and were positive.

Supplemental Data (Spiking Studies):

To supplement the above data, 30 negative whole blood samples were spiked with Anaplasma phagocytophilum positive control plasmid at the limit of detection (LOD) (10 copies/mcL). The 30 spiked specimens were run in a blinded manner along with 30 negative (non-spiked) specimens. 100% of the spiked specimens were positive, and 100% of the non-spiked specimens were negative.

Analytical Sensitivity/ LOD:

The lower LOD of this assay for each of the species in EDTA blood is as follows:

-Anaplasma phagocytophilum=approximately 10 targets per microliter

-Ehrlichia chaffeensis=approximately 5 targets per microliter

-Ehrlichia muris eauclairensis =approximately 100 targets per microliter

-Ehrlichia ewingii/canis=approximately 10 targets per microliter

Analytical Specificity:

No PCR signal was obtained from extracts of the following organisms: herpes simplex virus, Epstein-Barr virus, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, Bartonella henselas, Bartonella quintana, Ricketssia typhi, Ricketssia rickettsii, Toxoplasma gondii, Babesia microti MN, Babesia microti ATCC 53899, Borellia burgdorferi ATCC 51990, Ehrlichia risticii ATCC VR-986, and Anaplasma marginale. Positive results were obtained from nucleic extracts of 2 Ehrlichia canis strains (patient strain and ATCC CRL-10390 strain), with a melting temperature (Tm) of 49.5° C (indistinguishable from Ehrlichia ewingii). A positive melting peak was also noted with Ehrlichia muris (ATCC VR-1411), but the Tm (55.24° C) was easily distinguished from the Tm of the target organisms.

Precision:

Interassay precision was 97%, and intra-assay precision was 96%.

Reference Range:

Fifty whole blood specimens from normal donors were tested and found to be negative for targeted or detectable Ehrlichia and Anaplasma species.

Reportable Range:

This is a qualitative assay, and results are reported as either negative or positive for targeted Ehrlichia/Anaplasma species (positive for Anaplasma phagocytophilum, Ehrlichia chaffeensis, Ehrlichia muris eauclairensis, or Ehrlichia ewingii).