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Test Code BRBST Tickborne Bacterial, PCR and Sequencing, Blood


Necessary Information


Specimen source is required.



Specimen Required


Container/Tube:

Preferred: Lavender top (EDTA)

Acceptable: Royal blue top (EDTA), pink top (EDTA), or sterile vial containing EDTA-derived aliquot

Specimen Volume: 1 mL

Collection Instructions: If not submitting in original vial, mix well before transferring to a sterile vial.


Useful For

Detecting and identifying pathogenic tickborne bacteria infecting normally sterile whole blood

 

Potential detection of bacteria that cause similar illnesses to tickborne infections

 

This test should not be used as first tier test. It should only be used when routine testing is negative.

 

This test is not recommended as a test of cure because nucleic acids may persist for long periods of time after successful treatment.

Reflex Tests

Test ID Reporting Name Available Separately Always Performed
SPID2 Specimen Identification by PCR No, (Bill Only) No

Testing Algorithm

For information see Acute Tick-borne Disease Algorithm.

Reporting Name

Tickborne Bacterial PCR+Sequence, B

Specimen Type

Whole Blood EDTA

Specimen Minimum Volume

0.5 mL

Specimen Stability Information

Specimen Type Temperature Time
Whole Blood EDTA Refrigerated (preferred) 14 days
  Frozen  14 days

Reject Due To

  All specimens will be evaluated at Mayo Clinic Laboratories for test suitability.

Reference Values

No tickborne bacterial DNA detected

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.

Method Description

The test utilizes DNA extraction from EDTA whole blood and polymerase chain reaction (PCR) of a highly variable fragment of the 16S ribosomal RNA (rRNA) gene. If present, the amplified DNA is sequenced to obtain identification of the source organism from the patient sample. Specimens undergo total nucleic acid extraction, and the purified eluate is produced by the MP96 (Roche Diagnostics). The DNA eluate is concentrated two-fold from the specimen input volume. PCR is performed on a LightCycler 480II instrument (Roche Diagnostics) to amplify approximately 530 base pairs (bp) of the bacterial 16S rRNA gene (V1-V3, exact length varies by species) with SYBR Green DNA detection.

 

Amplification inhibition is detected with a second PCR reaction performed using the extracted specimen spiked with a low concentration of positive control DNA. Due to high crossing point values reflecting the low abundance of pathogens, all patient samples for this test will undergo next-generation sequencing (NGS).

 

Samples are sequenced using NGS on a MiSeq sequencing platform (Illumina). The NGS process generates 500 bases of sequencing reads (250 bp in the forward and reverse directions). Due to the size of amplicon generated, there is a gap in coverage between forward and reverse sequences. Verification results and in silico analysis have demonstrated that forward sequence results (≥210 bp) offer the same bacterial identity as full-length amplicon sequence for all organisms that can be reported by this assay. Quality filtering and results generation from NGS files is performed with Pathogenomix cloud-based software (RipSeq NGS).

 

High-quality sequence (Q ≥30) of 210 bp or more in length are sorted into clusters based on the observed sequence and used for identification. Positive and negative controls are used throughout all processes to ensure assay performance. Forward and reverse sequences are analyzed to determine bacterial identity using the cloud-based analysis program RipSeq NGS (Pathogenomix).(Rodino KG, Wolf MJ, Sheldon S, et al. Detection of tick-borne bacteria from whole blood using 16S ribosomal RNA gene PCR followed by next-generation sequencing. J Clin Microbiol. 2021;59(5):e03129-20. doi:10.1128/JCM.03129-20)

Day(s) Performed

Monday through Friday

Report Available

14 to 28 days

Specimen Retention Time

7 days

Performing Laboratory

Mayo Clinic Laboratories in Rochester

CPT Code Information

87801-Broad Range Bacterial PCR and Sequencing

87798-Specimen Identification by PCR (if appropriate)

LOINC Code Information

Test ID Test Order Name Order LOINC Value
BRBST Tickborne Bacterial PCR+Sequence, B 76575-0

 

Result ID Test Result Name Result LOINC Value
BRBST Tickborne Bacterial PCR+Sequence, B 76575-0