Chlamydiales DNA Detection by PCR

Chlamydiales is an order of intracellular bacterial parasites that are responsible for a variety of human diseases. Such organisms can cause acute and chronic respiratory infections (including Chamyida pneumoniae, C. psittaci, and C. abortus), ocular infections (including C. pneumoniae, C. psittaci, C. felis, C. pecorum, C. caviae, C. suis, and C. muridarum), and sexually transmitted infections (including C. trachomatis). Depending on the species and disease entity involved, infection may occur through direct, zoonotic, or environmental exposure.

Although some species within order Chlamydiales are well-described and well-established human pathogens, others remain poorly characterized and are classified as emerging agents of human disease, some of which are being actively investigated through ongoing research efforts. Such emerging pathogens include Waddliaceae chondrophila in adverse pregnancy outcomes, and Parachlamydia acanthamoebae, P. naegleriophila and Simkaniaceae negevensis in respiratory disease.

Diagnosis of infection with bacteria from the order Chlamydiales can be challenging. Because these organisms only replicate within eukaryotic cells, they cannot be recovered or identified by standard microbiological culture. Moreover, the 16s rRNA genes of these bacteria encode an unusual sequence composition that prevents their molecular detection by standard broad-range bacterial PCR assays Bacterial PCR reflex NGS [BCTDNA] and Bacterial DNA Detection by PCR (without reflex to NGS) [NRBDNA]: dedicated molecular testing is required.

Broad-range Chlamydiales DNA detection is conducted using a hybridization probe-based real-time PCR assay to identify organisms from across that taxonomical order. Specimens testing positive for amplification by real-time PCR are subjected to DNA sequencing to determine the identity of the detected organism.

Please note that for the detection of Chlamydia trachomatis from specific specimen types (Endocervical or urethral, Liquid Pap, Vaginal swabs, Throat swabs, Anal or rectal swabs, or fresh urine), ordering Chlamydia trachomatis (CT) Amplified Nucleic Acid Detection [CHLAD] or GC (N. gonorrhoeae) and CT (Chlamydia trachomatis) Amplified Nucleic Acid Detection [GCCTAD] is recommended.

For more information on molecular microbiology testing, please visit Molecular Diagnosis Microbiology Section.

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.

UWMC/HMC: Store and send fresh tissue/fluid specimens refrigerated, if specimen storage and transport will exceed 8 hours, freeze at -20°C. Freeze all fresh tissue/fluid specimens at -20°C upon arrival in UW Molecular Microbiology.

Please see Molecular Microbiology Specimen Submission for complete specimen collection and handling instructions.