TEST CATALOG ORDERING & RESULTS SPECIMEN HANDLING CUSTOMER SERVICE EDUCATION & INSIGHTS
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 (CSF); and both fresh and formalin-fixed paraffin-embedded (FFPE) 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 positive, then reflex sequencing will be performed at an additional charge.

 

If PCR is negative, no sequencing will be performed.

 

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 (PCR) amplification of a portion of the 16S ribosomal RNA 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 (NGS) 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(s) 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.

Supportive Data

One hundred and 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 -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 (CFU)/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 (FFPE) 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