Acanthamoeba and Balamuthia species PCR

General Information

Lab Name

Lab Code

AMBPCR

Epic Name

Acanthamoeba and Balamuthia species PCR

Description

Detection and identification of Acanthamoeba and Balamuthia species DNA

Free living amoeba of the Acanthamoeba and Balamuthia families are opportunistic protozoans that are widely distributed in the environment and are well recognized to produce serious human infections, including cutaneous infections, keratitis, and encephalitis. Although these infections are relatively rare, they can incur significant morbidity and mortality. PCR has emerged as a key tool in diagnosing disease caused by these organisms, and has been shown to outperform conventional tests in terms of turnaround times, sensitivity, and specificity. Our broad range PCR assay offers highly sensitive molecular identification of Acanthamoeba and Balamuthia species, having a limit of detection of a single organism per reaction. Uniquely, our broad-range test provides clinical diagnosis of infections caused by either of those genera using a single test.

The 18S rRNA gene has been proposed as a generally useful target for identifying and classifying parasites. Broad range primer design is challenging, however, as conserved regions of 18S rRNA often remain emerged across eukaryotic species, including humans. In this assay, Acanthomoeba-specific primers described by Maubon et al, which do not cross-react with human DNA, were used as the basis for designing broader range primers with affinity for members of both Acanthamoeba and Balamuthia genera.

References

1. K. Khairnar, G.S. Tamber, F. Ralevski, D.R. Pillai Comparison of molecular diagnostic methods for the detection of Acanthamoeba spp. from clinical specimens submitted for keratitis.
Diagn Microbiol Infect Dis, 70 (2011), pp. 499-506

Forms & Requisitions

Molecular Microbiology Order Form

Synonyms

18S PCR, Acanthamoeba PCR, Amoeba PCR, Balamuthia PCR, parasite identification, protozoan identification

Components

Code	Name
AB18RS	Amoeba PCR: Detection, 18S rDNA
AB18ID	Amoeba PCR: Identification, 18S rDNA
AMBSI	Amoeba PCR: Specimen Description
AMBSPI	Amoeba PCR: External Identifier
AMBSR	Amoeba PCR: Special Requests
AMBSC	Amoeba PCR: Specimen Comments
AMBNAE	Amoeba PCR: Specimen DNA Extraction
AMBREV	Amoeba PCR: Pathologist Review
AMBME	Amoeba PCR: Method Note

Interpretation

Method: DNA extraction, nucleic acid purification, polymerase chain reaction (PCR), sequencing
Reference Range: See individual components
References: 1. K. Khairnar, G.S. Tamber, F. Ralevski, D.R. Pillai Comparison of molecular diagnostic methods for the detection of Acanthamoeba spp. from clinical specimens submitted for keratitis.
Diagn Microbiol Infect Dis, 70 (2011), pp. 499-506

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: 87801
LOINC: 18725-2
Interfaced Order Code: UOW4211