Bacterial PCR reflex NGS

Detection and identification of Bacterial DNA (16S rRNA target)

Sequencing of specific gene(s) provides a rapid and accurate identification of clinically significant organism. Judicious primer selection allows amplification of sequences at the species level or at the phylogenetic level. Raw sequences are assembled then classified by comparison to public sequence databases and the UWMC Clinical Microbiology Sequence Database in order to provide organism identification. With more than 20,000 sequences available in public databases (e.g. NCBI), 16S ribosomal RNA gene (rRNA) sequencing is considered by current taxonomists to be the gold standard in bacterial identification and classification. 16S rRNA gene contains conserved regions useful for the design of broad-range PCR primers that can amplify various fragments of the 16S rRNA gene from pathogenic and nonpathogenic bacteria. These fragments include hypervariable regions containing species-specific signature sequences useful for bacterial identification to species level.

In many specimens bacteria can be seen by microscopy of tissue sections but are very difficult to grow due to their fastidious nature, or are not viable as a result of antimicrobial therapy. Some specimens may never reveal the presence of a bacterial pathogen because of low abundance and/or lack of viability. The use of PCR to detect this DNA extracted directly from clinical specimens facilitates the identification of these pathogens. Selection of this test will reflex to Bacterial detection by 16S NGS [NGS16S] when there is evidence of multiple bacterial templates present.

Reflexive Testing
When suspected pathogenic microorganisms are detected, identification procedures are performed, as appropriate for the organism and specimen.

• Bartonella DNA detection by PCR [BRTDNA]
• Enterobacterales DNA Detection [ENBDNA]
• Legionella DNA detection by PCR [LEGDNA]
• Mycobacterium avium complex DNA Detection [MAVDNA]
• Mycoplasma and Ureaplasma DNA Detection by PCR [MPLDNA]
• M. tuberculosis complex DNA Detection [TBCDNA]
• Treponema pallidum DNA detection by NAAT [TPLDNA]
• Tropheryma whipplei DNA detection by PCR [TWHDNA]

Multiple Template Reflexes

• Bacterial detection by 16S NGS [NGS16S]

Acceptable specimens are listed below. Please see Molecular Microbiology Specimen Submission for complete specimen collection and handling instructions.

Shipping/Handling

• Fresh frozen tissue/fluid specimens should be collected into a DNA free container labeled with at least two identifiers and be submitted and maintained on dry ice.
• Formalin Fixed Paraffin-embedded tissues (FFPE, PET) can be sent ambient or with ice packs during warmer summer months to prevent melting.

Acceptable Specimens

• Fresh frozen tissue
• Fresh frozen fluid: any body fluid is acceptable if it is not listed under Unacceptable Specimens.
  • Common examples include: cerebrospinal fluid, pleural fluid, pericardial fluid, urine, bronchial lavage, joint fluid, bone marrow, vitreous fluid, etc.
• Formalin Fixed Paraffin-embedded tissues (FFPE, PET): blocks, scrolls, and unstained slides. Note that fresh tissue is considered the optimal specimen of choice, as it reduces the chance of introducing exogenous DNA templates or microorganisms during embedding/fixation, and because formalin fixation dramatically reduces the sensitivity of molecular assays due to reduced template yield and quality.
• Sputum: acceptable - except for Bacterial PCR reflex NGS [BCTDNA]/Bacterial DNA Detection by PCR (without reflex to NGS) [NRBDNA], Fungal PCR reflex NGS [FUNDNA]/Fungal DNA Detection by PCR (without reflex to NGS) [NRFDNA], and Nontuberculous Mycobacteria DNA Detection [NTMDNA]* assays
• eSwabs**, UTM (universal transport media), body fluid/bone marrow in EDTA (not including blood)
• Sodium polyanethol sulfonate (SPS, Wampole Isolator Tubes) acceptable with disclaimer

*Mycobacterium avium complex DNA Detection [MAVDNA] can be ordered on sputum

**Fungal PCR reflex NGS [FUNDNA] and Fungal DNA Detection by PCR (without reflex to NGS) [NRFDNA] may have interference due to some lots of eSwabs which have been found to contain Saccharomyces cerevisiae DNA, resulting in false positive detection. Clinical correlation and/or retesting with a different collection method is advised. The detection of S. cerevisiae from eSwab specimens can interfere with our ability to rule out other fungal DNA.

Unacceptable Specimens

• Blood, serum, plasma, stool/rectal swabs
• No citrated or heparinized solutions
• Tissues floating in formalin
• Swab/fluid collected in tube containing agar

Optimal Quantity:

• Fresh Tissue: 0.3-1.0 cm^3
• Fluid: 0.2-1 mL
• Formalin Fixed Paraffin-embedded Tissue (FFPE/PET): blocks are preferred over scrolls/unstained slides and will be sent back to client upon completion of testing
• Scrolls/unstained slides: cross-sectional area >1cm^2 send 10 sections of 10µm thickness, if <1cm^2 send 20 sections if available

Please note: We do not need a separate specimen aliquot for each test ordered. Only a single specimen aliquot or block of optimal quantity is necessary for performing multiple tests. If multiple aliquots or blocks of optimal quantity are sent, up to 2 will be pooled.

Fresh tissue is the optimal specimen of choice, as it reduces the chance of introducing exogenous DNA templates or microorganisms during embedding/fixation. Formalin fixation dramatically reduces the sensitivity of the assays due to reduced template yield and quality.