Supportive Data

Borrelia miyamotoi:

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

Accuracy/Diagnostic Sensitivity and Specificity: Clinical Samples:

Sixty-two clinical EDTA blood specimens received in the clinical laboratory for Ehrlichia/Anaplasma polymerase chain reaction (PCR) were tested using the Borrelia miyamotoi PCR assay. Results were compared to the MDH 16S ribosomal RNA TaqMan assay. In addition, 2 retrospectively identified B miyamotoi-positive specimens were confirmed by the B miyamotoi PCR assay and the MDH TaqMan assay.

Spiking studies:

To supplement the clinical specimens, negative whole blood and cerebrospinal fluid (CSF) specimens were spiked with genomic or plasmid DNA of B miyamotoi near the limit of detection and tested in a blinded fashion. The sensitivity of the PCR assay was 100%, and the specificity with spiked specimens was 100%. The samples were extracted and tested in a blinded fashion.

Species Inclusivity:

-Four strains of B miyamotoi were detected by the PCR assay.

-Three other Borrelia species that cause human tick-borne relapsing fever (Borrelia hermsii, Borrelia parkeri, and Borrelia turicatae) were also detected at a melting temperature (Tm) below that of B miyamotoi. Thus, related Borrelia species may be detected and differentiated with this assay.

-Sixteen different strains from the Borrelia burgdorferi sensu lato group were not detected with this PCR assay.

Analytical Sensitivity/Limit of Detection:

-The limit of detection (LOD) is 2800 target copies/mL of whole blood or CSF.

Analytical Specificity:

-No PCR signal was obtained from the extracts of 31 bacterial, viral, parasitic, and fungal isolates from either similar organisms or organisms commonly found in the specimens tested.

Precision:

Interassay and intra-assay precision were 100%.

Reference Range:

The reference range of this assay is negative. This assay is designed to detect only species of clinical significance and is to be used for patients with a clinical history and symptoms consistent with tick-borne relapsing fever.

Reportable Range:

This is a qualitative assay, and the results are reported as negative or positive for B miyamotoi DNA.

Ehrlichia/Anaplasma:

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

Accuracy/Diagnostic Sensitivity and Specificity:

Results from this real-time PCR assay on the LightCycler (LC PCR) 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 A 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 A phagocytophilum positive control plasmid at the 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:

-A phagocytophilum=approximately 10 targets per microliter

-E chaffeensis=approximately 5 targets per microliter

-Ehrlichia muris eauclairensis=approximately 100 targets per microliter

-E 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 henselae, Bartonella quintana, Rickettsia typhi, Rickettsia rickettsii, Toxoplasma gondii, Babesia microti MN, B microti ATCC 53899, B 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 Tm of 49.5° C (indistinguishable from E ewingii). A positive melting peak was also noted with E 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 A phagocytophilum, E chaffeensis, E muris eauclairensis, or E ewingii).

Babesia:

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

Accuracy/Diagnostic Sensitivity and Specificity:

Ninety-six whole blood specimens were tested by this real-time PCR assay and another real-time PCR assay. Concordance was 99%.

Analytical Sensitivity/LOD:

The LOD established using whole organism spiked into specimen matrix (whole blood) is as follows:

-Babesia microti, ATCC PRA 99: 2670 target copies/mL

-Babesia duncani ATCC PRA 302: 1540 target copies/mL

-Babesia MO-1 positive patient DNA: 10,700 target copies/mL

-Babesia divergens-positive patient DNA: 5270 target copies/mL

Serial 10-fold dilutions of microscopy-positive specimens were also tested in a blinded fashion using conventional thick and thin blood films and the Mayo Babesia species PCR. The PCR was able to consistently detect two 10-fold dilutions lower than using microscopy.

Analytical Specificity:

No cross-reactivity was noted using a panel of 34 bacteria, viruses, parasites, and fungi were detected by the Babesia species PCR.

Precision:

Interassay and intra-assay precision were 100%.

Reference Range:

The reference range is negative. This was confirmed by testing 93 blood specimens from asymptomatic individuals for the presence of Babesia species by the Babesia species PCR assay. All 93 specimens were negative.

Reportable Range:

This test is a qualitative assay, and results are reported as positive or negative for Babesia species (B microti, B duncani, B divergens, and B MO-1).