Aspergillus fumigatus DNA detection by PCR

General Information

Lab Name

Lab Code

ASPDNA

Epic Name

Aspergillus fumigatus DNA detection by PCR

Description

Detection of Aspergillus fumigatus DNA

Aspergillus fumigatus is considered the most prevalent airborne fungal pathogen that causes severe and often fatal invasive aspergillosis (IA) among immunocompromised individuals such as patients with leukemia or those who have undergone organ transplantation. Aspergillus is a ubiquitous environmental hyaline mold that is responsible for a spectrum of disease. Clinical syndromes range from non-invasive colonization such as aspergilloma (fungal ball) and allergic responses such as allergic bronchopulmonary aspergillosis (ABPA), to semi-invasive and invasive disease including necrotizing pneumonia, invasive pulmonary aspergillosis, and disseminated invasive aspergillosis involving tissue. Aspergillus fumigatus is the most frequently isolated species and is most commonly associated with invasive infection. Diagnosing these infections relies upon accurately recognizing a constellation of clinical signs and symptoms, as well as imaging and laboratory studies. These diagnoses are difficult, expensive, and time consuming. In the laboratory, culture of invasive fungal pathogens is slow and can be complicated by the presence of contaminating molds in the environment, and Aspergillus spp. are rarely if ever isolated from blood culture. Further, in many specimens from patients, fungal organisms can be seen by microscopy of tissue sections or body fluids, but are not viable. Some specimens may never reveal the presence of a fungal pathogen because of low abundance and/or lack of viability.

A rapid test with high sensitivity and specificity for detecting A. fumigatus DNA in clinical specimens is required for successful antifungal therapy.

Internal Transcribed Spacer sequence exhibit species-specific polymorphisms suitable for distinguishing A. fumigatus from other fungi including genetically closely related species such as Neosartorya ficheri. A nested PCR protocol with A. fumigatus specific FRET probes is employed to achieve high sensitivity and specificity.

The Aspergillus fumigatus DNA detection by PCR [ASPDNA] allows the rapid detection of Aspergillus fumigatus using primers and Aspergillus fumigatus sequence-specific probes designed to bind to the internal transcribed spacer of the rRNA operon. Fluorescence detection utilizes a FRET hybridization probe and a TaqMan hydrolysis probe specific for Aspergillus fumigatus. Real-time PCR is used to detect DNA extracted directly from clinical specimens facilitating more rapid detection of fungal elements.

Forms & Requisitions

Molecular Microbiology Order Form

Synonyms

Aspergillosis PCR, Aspergillus fumigatus detection, Aspergillus fumigatus identification, Aspergillus fumigatus PCR, Aspergillus identification, Aspergillus PCR, molecular Aspergillus, universal PCR

Components

Code	Name
ASPRS	A fumigatus PCR: Detection, nested ITS
ASPID	A fumigatus PCR: Identification, nested ITS
ASPSI	A fumigatus PCR: Specimen Description
ASPSPI	A fumigatus PCR: External Identifier
ASPSR	A fumigatus PCR: Special Requests
ASPSC	A fumigatus PCR: Specimen Comments
ASPNAE	A fumigatus PCR: Specimen DNA Extraction
ASPREV	A fumigatus PCR: Pathologist Review
ASPME	A fumigatus PCR: Method Note

Interpretation

Guidelines

Method

DNA extraction, nucleic acid purification, polymerase chain reaction (PCR), sequencing

Reference Range

See individual components

Ordering & Collection

Specimen Type

Tissue (Fresh frozen or paraffin-embedded), Fluid (see Acceptable Specimens for details)

Collection

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.

Forms & Requisitions

Molecular Microbiology Order Form

Handling Instructions

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

Quantity

Requested: See "Collection" for Optimal Quantity
Minimum: Specimens below optimal quantity are acceptable for testing, however, diagnostic yield is generally proportional to specimen size.

Processing

Receiving Instructions: 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.

Misc Sendout

Performance

Lab Department

Micro Molecular Diag(MMD)

Frequency

Fresh frozen tissues/fluids result in 2-3 business days after receipt of specimen. Formalin Fixed Paraffin-embedded tissues result in 3-4 business days after receipt of specimen.

Available STAT?

Performing Location(s)

UW-MT	Microbiology, Molecular Diagnostics 206-520-4600 ---------------------------------------- Shipping Address Attn: Molecular Microbiology UW CLSPS 1601 Lind Ave SW Room 117 Renton, WA 98057 Phone: 206-520-4600 Alternate phone: 206-598-6147 Performing Lab Address Clinical Microbiology Lab, NW177 University of Washington Medical Center 1959 NE Pacific Street Seattle, WA 98195 Phone: 206-598-5735 Alternate phone: 206-598-6147	Contact Information Please e-mail us with any questions or comments you may have. Your inquiry will be answered as soon as possible. email: molmicdx@uw.edu The Molecular Microbiology lab is open from Monday-Friday, 7am-4pm PDT. Billing inquiries and requests for faxed reports can be made to our Client Services Department at (206) 520-4600 or (800) 713-5198. For results or other inquiries, we can be reached by phone at the following numbers: Phone: (206) 598-5735 Alternate phone: (206) 598-6147 FAX: (206) 520-4903 For assistance during weekends, holidays and after hours, please contact Lab Medicine Resident at (206) 598-6190

Billing & Coding

CPT Codes: 87798
LOINC: 62478-3
Interfaced Order Code: UOW4404