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HelpOrder Code LABACARP Acanthamoeba species Molecular Detection, PCR, Ocular
Useful For
Aids in the diagnosis of amebic keratitis in conjunction with clinical findings
Method Name
Real-Time Polymerase Chain Reaction (PCR)/ DNA Probe Hybridization
Reporting Name
Acanthamoeba species Detection, PCRSpecimen Type
VariesOrdering Guidance
Although verification experiments did not detect Acanthamoeba species DNA in contact lenses from asymptomatic adults, it is possible that the polymerase chain reaction may detect asymptomatic colonization/contamination and, therefore, testing should not be performed on asymptomatic individuals.
Necessary Information
Specimen source is required.
Specimen Required
The preferred specimen for this test is corneal scraping or biopsy.
Submit only 1 of the following specimens:
Specimen Type: Tissue, fresh
Sources: Ocular
Container/Tube: Sterile container
Specimen Volume: 5-10 mm
Collection Instructions: Submit tissue in a sterile container with 1 mL of sterile saline, minimal essential media (MEM), or viral transport media.
Preferred Paraffin-Embedded Tissue Block:
Supplies: Tissue Block Container (T553)
Specimen Type: Formalin-fixed, paraffin-embedded tissue block (FFPE)
Sources: Ocular
Container/Tube: Tissue block
Collection Instructions: Submit a FFPE tissue block to be cut and returned.
Acceptable Paraffin-Embedded Tissue Block:
Specimen Type: FFPE section
Sources: Ocular
Container/Tube: Sterile container for each individual cut section (scroll).
Collection Instructions: Perform microtomy and prepare five separate 10-micron sections. Each section (scroll) must be placed in a separate sterile container for submission.
Specimen Type: Scrapings, swabs
Sources: Eye, ocular, cornea
Container/Tube: Sterile container
Specimen Volume: 1 mL
Collection Instructions:
1. Collect corneal scrapings using a scalpel or other sharp device to remove the outer layer of cells from the eye.
2. Swish the collection device in 1 mL of sterile saline, MEM, or viral transport media.
3. Remove the scalpel blade or sharp device from the collection container before submitting to the lab.
4. Specimens containing scalpel blades will be canceled.
5. If collected via swab, swab must be placed into viral transport media and submitted with the specimen. Specimens received without swabs will be canceled.
Additional Information: Swabs are not the preferred specimen for this test and may yield false-negative results.
Specimen Type: Contact lenses
Container/Tube: Sterile container
Specimen Volume: Entire collection
Collection Instructions:
1. Place entire contact lens in a sterile container with 1 mL sterile saline, viral transport media, or MEM.
2. Right and left lenses must be submitted individually using multiple sterile containers or in the original contact lens case. A separate order must be created for each lens being tested.
3. Indicate Right or Left in the specimen source.
Specimen Type: Contact lens cases without lenses
Container/Tube: Sterile container
Specimen Volume: 1 mL solution or entire case
Additional Information:
1. Depending on the type of case submitted, it may be necessary to test right and left chambers individually. A separate order must be created for each chamber being tested.
2. Indicate Right or Left in the specimen source.
Specimen Minimum Volume
Scrapings: 0.5 mL; Other specimen types: See Specimen Required
Specimen Stability Information
| Specimen Type | Temperature | Time | Special Container |
|---|---|---|---|
| Varies | Refrigerated (preferred) | 7 days | |
| Frozen | 7 days |
Reject Due To
| Calcium alginate-tipped swab Wood swab Transport swab containing gel Specimens containing scalpel blades Unstained slides |
Reject |
Reference Values
Negative
Method Description
The assay is performed on the Roche LightCycler (LC) 480 II instrument following DNA extraction on the Roche MagNA Pure or the Siemens Tissue Preparation System. The LC 480 II instrument is an automated instrument that amplifies and monitors the development of target nucleic acid (amplicon) after each cycle of polymerase chain reaction (PCR).
The DNA target for this PCR assay is a gene encoding the nuclear small subunit ribosomal 18S ribosomal RNA (rRNA).
The PCR mix contains a forward and reverse primer specific for Acanthamoeba species template amplification and 1 TaqMan probe (CY5). The CY5 probe contains a fluorophore (5'-end) and a quencher (3'-end) in close proximity; the quencher inhibits the fluorescence signal from the fluorophore while the probe is intact. After the probe anneals to the targeted Acanthamoeba 18S rRNA, it is subsequently degraded by a DNA polymerase with 5'-3' exonuclease activity, resulting in release of the fluorophore and production of detectable fluorescent signal.(Qvarnstrom Y, Visvesvara GS, Sriram R, da Silva AJ: Multiplex real-time PCR assay for simultaneous detection of Acanthamoeba spp, Balamuthia mandrillaris, and Naegleria fowleri. J Clin Microbiol. 2006 Oct;44(10):3589-3595; Connelly L, Anijeet D, Alexander CL. A descriptive case of persistent Acanthamoeba keratitis: raising awareness of this complex ocular disease. Access Microbiol. 2019 Nov 28;2(3):acmi000084; Norgan AP, Sloan LM, Pritt BS: Detection of Naegleria fowleri, Acanthamoeba spp, and Balamuthia mandrillaris in formalin-fixed, paraffin-embedded tissues by real-time multiplex polymerase chain reaction. Am J Clin Pathol 2019;152:799-807)
Day(s) Performed
Monday through Sunday
Performing Laboratory
Mayo Clinic Laboratories in Rochester
CPT Code Information
87798
Forms
If not ordering electronically, complete, print, and send a Microbiology Test Request (T244) with the specimen.