Test Catalog

Test Id : BRBPS

Broad Range Bacterial PCR and Sequencing, Varies

Useful For
Suggests clinical disorders or settings where the test may be helpful

Detecting and identifying bacteria (including mycobacteria) from normally sterile sources, including synovial fluid; body fluids such as pleural, peritoneal, and pericardial fluids, cerebrospinal fluid; and both fresh and formalin-fixed paraffin-embedded  tissues

 

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

Highlights

This test is used for detection and identification of bacteria (including mycobacteria) in normally sterile specimens.

 

This test is optimal for situations in which bacteria (including mycobacteria) are visualized in the specimen but other laboratory methods have failed to yield a diagnosis.

Reflex Tests
Lists tests that may or may not be performed, at an additional charge, depending on the result and interpretation of the initial tests.

Test Id Reporting Name Available Separately Always Performed
ISBA Bacterial Ident by Sequencing No, Bill Only No
ISNGS Ident by Next Generation Sequencing No, Bill Only No
SPID2 Specimen Identification by PCR No, Bill Only No
CSFME Meningitis Encephalitis Panel, PCR Yes No

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

If polymerase chain reaction (PCR) testing is negative, no sequencing is performed, and the test is resulted as negative.

 

If PCR testing is positive, sequencing is performed. Strong positive results are first submitted to Sanger sequencing, which can yield results in as few as 4 days. Weak positive results, or Sanger sequencing results that are mixed, are submitted to next-generation sequencing (ie, targeted metagenomics testing).

 

The following algorithms are available in Special Instructions:

-Infective Endocarditis: Diagnostic Testing for Identification of Microbiological Etiology

-Meningitis/Encephalitis Panel Algorithm

Special Instructions
Library of PDFs including pertinent information and forms related to the test

Method Name
A short description of the method used to perform the test

16S Ribosomal RNA Gene Polymerase Chain Reaction (PCR) followed by Sequencing

NY State Available
Indicates the status of NY State approval and if the test is orderable for NY State clients.

Yes

Reporting Name
Lists a shorter or abbreviated version of the Published Name for a test

Broad Range Bacteria PCR+Sequencing

Aliases
Lists additional common names for a test, as an aid in searching

16S rRNA gene sequencing

Bacterial sequencing

Broad range

Broad-range

16S

Broad

Mycobacteria

Mycobacterial

Ribosomal RNA

rRNA

Sequencing

Next Generation Sequencing

NGS

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

If polymerase chain reaction (PCR) testing is negative, no sequencing is performed, and the test is resulted as negative.

 

If PCR testing is positive, sequencing is performed. Strong positive results are first submitted to Sanger sequencing, which can yield results in as few as 4 days. Weak positive results, or Sanger sequencing results that are mixed, are submitted to next-generation sequencing (ie, targeted metagenomics testing).

 

The following algorithms are available in Special Instructions:

-Infective Endocarditis: Diagnostic Testing for Identification of Microbiological Etiology

-Meningitis/Encephalitis Panel Algorithm

Specimen Type
Describes the specimen type validated for testing

Varies

Necessary Information

Specimen source is required.

Specimen Required
Defines the optimal specimen required to perform the test and the preferred volume to complete testing

Fresh tissue is preferred over formalin-fixed, paraffin-embedded tissue.

 

Submit only 1 of the following specimens:

 

Preferred Specimen Type:

Specimen Type: Fresh tissue or biopsy

Sources: Normally sterile tissue such as bone, lymph node, joint, heart valve, brain, viscera, organ, lung

Container/Tube: Sterile container

Specimen Volume: Entire collection or 5 mm(3)-approximately the size of a pencil eraser

Collection Instructions:

1. Collect fresh tissue specimen.

2. Submit tissue only, do not add fluid to tissue.

3. Freeze specimen.

Specimen Stability Information: Frozen <14 days (preferred)/Refrigerated <14 days

 

Alternate Specimen Type:

Preferred: Paraffin-embedded tissue block:

Supplies: Tissue Block Container (T553)

Specimen Type: Formalin-fixed, paraffin-embedded (FFPE) tissue block

Sources: Normally sterile or deep tissues such as bone, lymph node, joint, heart valve, brain, viscera, organ, lung

Container/Tube: Tissue block

Collection Instructions: Submit a formalin-fixed, paraffin-embedded tissue block to be cut and returned.

Specimen Stability Information: Ambient (preferred)/Refrigerated

 

Acceptable: Paraffin-embedded tissue block:

Specimen Type: Section (scrolls) of FFPE tissue block

Sources: Normally sterile or deep tissues such as bone, lymph node, joint, heart valve, brain, viscera, organ, lung

Container/Tube: Sterile container for each individual cut section (scroll)

Collection Instructions: Perform microtomy and prepare five separate 10-micron sections. Each section (scroll) must be placed in a separate sterile container for submission.

Specimen Stability Information: Ambient (preferred)/Refrigerated

 

Specimen Type: Fluid

Sources: Normally sterile body fluids such as cerebrospinal, vitreous humor, pleural, abdominal, peritoneal, ascites, pericardial, pelvic

Container/Tube: Screw-capped, sterile container

Specimen Volume: 1 mL

Collection Instructions:

1. Collect fresh fluid specimen.

2. Freeze specimen.

Specimen Stability Information: Frozen <14 days(preferred)/Refrigerated <14 days

 

Specimen Type: Synovial fluid

Container/Tube:

Preferred: Red top or sterile container

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

Specimen Volume: 1 mL

Collection Instructions: Send specimen in original tube (preferred).

Specimen Stability Information: Frozen <14 days (preferred)/ Refrigerated <14 days

Special Instructions
Library of PDFs including pertinent information and forms related to the test

Forms

If not ordering electronically, complete, print, and send a Microbiology Test Request (T244) with the specimen.

Specimen Minimum Volume
Defines the amount of sample necessary to provide a clinically relevant result as determined by the Testing Laboratory

Fluid: 0.5 mL

Fresh tissue or biopsy: 5 mm(3)

Paraffin-embedded tissue block: two 10-micron sections

Reject Due To
Identifies specimen types and conditions that may cause the specimen to be rejected

Tissue in formalin, formaldehyde, acetone, or any other fluid Bone marrow Decalcified bone Slides Skin biopsy Colon biopsy Reject

Specimen Stability Information
Provides a description of the temperatures required to transport a specimen to the performing laboratory, alternate acceptable temperatures are also included

Specimen Type Temperature Time Special Container
Varies Varies (preferred)

Useful For
Suggests clinical disorders or settings where the test may be helpful

Detecting and identifying bacteria (including mycobacteria) from normally sterile sources, including synovial fluid; body fluids such as pleural, peritoneal, and pericardial fluids, cerebrospinal fluid; and both fresh and formalin-fixed paraffin-embedded  tissues

 

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

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

If polymerase chain reaction (PCR) testing is negative, no sequencing is performed, and the test is resulted as negative.

 

If PCR testing is positive, sequencing is performed. Strong positive results are first submitted to Sanger sequencing, which can yield results in as few as 4 days. Weak positive results, or Sanger sequencing results that are mixed, are submitted to next-generation sequencing (ie, targeted metagenomics testing).

 

The following algorithms are available in Special Instructions:

-Infective Endocarditis: Diagnostic Testing for Identification of Microbiological Etiology

-Meningitis/Encephalitis Panel Algorithm

Clinical Information
Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test

Cultures from patients with suspected bacterial infection involving normally sterile sites may fail to provide bacterial (including mycobacterial) growth for identification due to the presence of fastidious or slow-growing bacteria or as a result of antecedent antimicrobial chemotherapy. Polymerase chain reaction amplification of a portion of the 16S ribosomal RNA (rRNA) gene followed by sequencing of the amplified product can be used to detect bacterial (including mycobacterial) nucleic acids in such situations, enabling a diagnosis. Sterile sources accepted for testing may have more than one bacterial species present or the presence of copy variants of the 16S rRNA gene within a single bacterial species, confounding Sanger sequencing analysis. Next-generation sequencing can be useful in such cases. Ideal specimens are those in which bacteria (includes mycobacteria) are visualized by microscopy. Heart valves from patients with endocarditis with positive Gram stains are, for example, especially suitable.

Reference Values
Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.

No bacterial DNA detected

Interpretation
Provides information to assist in interpretation of the test results

A positive broad-range polymerase chain reaction (PCR)/sequencing result indicates that bacterial nucleic acid of the specified organism was detected, which may be due to bacterial infection or environmental or contaminating nucleic acids in the specimen.

 

A negative broad-range PCR/sequencing result indicates the absence of detectable bacterial (including mycobacterial) nucleic acids in the specimen but does not rule-out false-negative results that may occur due to sampling error, sequence variability underlying the primers, the presence of bacterial nucleic acids in quantities less than the limit of detection of the assay, or inhibition of PCR. If PCR testing appears to be negative but there is evidence of PCR inhibition, testing will be repeated. If inhibition is again detected, the result will be reported as "PCR inhibition present."

Cautions
Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances

This test does not detect nonbacterial organisms (eg, viruses, fungi, helminths, protozoa), but does detect mycobacteria.

 

False-positive results are theoretically possible if patient specimens are contaminated with bacterial nucleic acids either from the environment or from patient microbiota (eg, skin microbiota contamination).

 

This test is validated for normally sterile sources.

 

In extenuating circumstances, sequencing, especially next-generation sequencing, may be associated with an extended turnaround time, approaching or possibly exceeding the published maximum laboratory time (28 days).

Supportive Data

One hundred thirty positive patient specimens were available for accuracy studies and correlated with results of culture, organism-specific polymerase chain reaction (PCR), or previous broad-range bacterial PCR and sequencing. In addition, 63 negative samples from previous Sanger sequence-based testing were used in verification. All samples were tested with both Sanger and next-generation sequencing (NGS) technologies enabling resolution of poor-quality Sanger results and identifying polybacterial presence in some samples. Using criteria established in verification, sensitivity of the assay is 99% and specificity is 97%. Some samples were spiked with gram-negative or gram-positive bacteria due to the scarcity of clinically positive samples. Testing demonstrated 100% correlation with expected results from spiked material.

 

The limit of detection was less than 65 colony forming units per PCR reaction for all sources as determined by spiking Streptococcus gallolyticus and Escherichia coli into PCR-negative fresh tissue, synovial fluid, formalin-fixed, paraffin-embedded  tissue, sonicate fluid, body fluid, and cerebrospinal fluid.

 

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 amplifying 42 genomic DNA samples representing diverse types of bacteria (including mycobacteria) expected to be present in the specimen types acceptable for this assay. All bacteria and mycobacteria were detected and correctly identified by both Sanger and NGS.

 

An additional study of 15 specimens previously characterized only as polybacterial revealed the ability of NGS to detect and differentiate multiple bacteria for reporting. The laboratory section director is responsible for reporting of polybacterial results.

Clinical Reference
Recommendations for in-depth reading of a clinical nature

1. Oyvind K, Simmon K, Karaca D, Langeland N, Wiker HG: Dual priming oligonucleotides for broad-range amplification of the bacterial 16S rRNA gene directly from human clinical specimens. J Clin Microbiol. 2012;50(4):1289-1294. doi: 10.1128/JCM.06269-11

2. Gomez E, Cazanave C, Cunningham SA, et al: Prosthetic joint infection diagnosis using broad-range PCR of biofilms dislodged from knee and hip arthroplasty surfaces using sonication. J Clin Microbiol. 2012;50:3501-3508

3. Virk A, Pritt B, Patel R, et al: Mycobacterium lepromatosis Lepromatous leprosy in US citizen who traveled to disease-endemic areas. Emerg Infect Dis. 2017 Nov; 23(11):1864-1866. doi: 10.3201/eid2311.171104

4. Liesman RM, Pritt BS, Maleszewski JJ, Patel R: Laboratory diagnosis of infective endocarditis. J Clin Microbiol. 2017 Sep;55(9):2599-2608. doi: 10.1128/JCM.00635-17

5. Ramakrishna JM, Libertin CR, Yang JN, Diaz MA, Nengue AL, Patel R: 16S rRNA Gene PCR/sequencing of cerebrospinal fluid in the diagnosis of post-operative meningitis. Access Microbiology. 2020 Feb 3;2. doi: 10.1099/acmi.0.000100

Special Instructions
Library of PDFs including pertinent information and forms related to the test

Method Description
Describes how the test is performed and provides a method-specific reference

This test utilizes specimen processing, DNA extraction, and polymerase chain reaction (PCR) of a highly variable fragment of the 16S ribosomal RNA (rRNA) gene. The variability of the targeted V1-V3 region allows for taxonomically specific reporting. If positive by PCR based on signal strength, the amplified DNA is sequenced to obtain identification of the source organism. If PCR is negative, no sequencing is performed. PCR inhibition is detected with a second PCR reaction and amplification is performed on a LightCycler. Only high-quality consensus sequence of 400 base pairs or more (usable data for both forward and reverse direction) is used for Sanger sequencing identification. If sequence data is not interpretable using Sanger sequencing, next-generation sequencing (NGS) is performed. Quality filtering is performed for NGS and only results with 100X coverage are used in analysis. Positive and negative controls are used throughout all processes to ensure assay performance. Sequence quality (specimen score) and data analysis for organism identification is accomplished with Pathogenomix RipSeq software.(Unpublished Mayo method)

PDF Report
Indicates whether the report includes an additional document with charts, images or other enriched information

No

Day(s) Performed
Outlines the days the test is performed. This field reflects the day that the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time before the test is performed. Some tests are listed as continuously performed, which means that assays are performed multiple times during the day.

Monday through Friday

Report Available
The interval of time (receipt of sample at Mayo Clinic Laboratories to results available) taking into account standard setup days and weekends. The first day is the time that it typically takes for a result to be available. The last day is the time it might take, accounting for any necessary repeated testing.

14 to 28 days

Specimen Retention Time
Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded

1 week

Performing Laboratory Location
Indicates the location of the laboratory that performs the test

Rochester

Fees
Several factors determine the fee charged to perform a test. Contact your U.S. or International Regional Manager for information about establishing a fee schedule or to learn more about resources to optimize test selection.

  • Authorized users can sign in to Test Prices for detailed fee information.
  • Clients without access to Test Prices can contact Customer Service 24 hours a day, seven days a week.
  • Prospective clients should contact their Regional Manager. For assistance, contact Customer Service.

Test Classification
Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR) product.

This test was developed, and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.

CPT Code Information
Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Clinic Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.

CPT codes are provided by the performing laboratory.

87801-Broad Range Bacterial PCR and Sequencing

87798-Bacterial Ident by Sequencing (if appropriate)

87798-Specimen Identification by PCR (if appropriate)

87798-Ident by Next Generation Sequencing (if appropriate)

87483-Meningitis Encephalitis Panel, PCR (if appropriate)

Test Setup Resources

Setup Files
Test setup information contains test file definition details to support order and result interfacing between Mayo Clinic Laboratories and your Laboratory Information System.

Excel | Pdf

Sample Reports
Normal and Abnormal sample reports are provided as references for report appearance.

Normal Reports | Abnormal Reports

SI Sample Reports
International System (SI) of Unit reports are provided for a limited number of tests. These reports are intended for international account use and are only available through MayoLINK accounts that have been defined to receive them.

SI Normal Reports | SI Abnormal Reports