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Epic Code LAB1230707 Acanthamoeba species Molecular Detection, PCR, Ocular

Additional Codes

Mayo code: ACARP

Interface code: 1230707

Performing Laboratory

Mayo Clinic Laboratories in Rochester

Useful For

Aids in the diagnosis of amebic keratitis in conjunction with clinical findings

Specimen Type

Varies


Ordering Guidance


Although verification experiments did not detect Acanthamoeba species DNA in contact lenses from asymptomatic adults, it is possible that the polymerase chain reaction (PCR) 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: Formalin-fixed, paraffin-embedded tissue block (FFPE)

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, minimal essential media (MEM), or viral transport media.

3. Remove the collection device from the collection container before submitting to the lab.

4. Specimens containing scalpel blades will be canceled.

Additional Information: Swabs are not the preferred specimen for this test and may yield false-negative results. Specimens collected using wooden shafted swabs and calcium alginate-tipped swabs will be canceled.

 

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 minimal essential media (MEM).

2. Right and left lenses must be submitted individually using multiple sterile containers or in the original contact lens case. Multiple orders must be created.

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. Multiple orders must be created.

2. Indicate Right or Left in the specimen source.


Specimen Minimum Volume

Tissue: 5 mm biopsy
Scrapings: 0.5 mL
Contact Lens Solution in contact lens cases: 1 mL

Forms

If not ordering electronically, complete, print, and send a Microbiology Test Request (T244) with the specimen.

Highlights

This assay is intended as an aid in the diagnosis of amebic keratitis in conjunction with clinical findings.

 

This test has similar sensitivity and specificity to culture but provides a more rapid result.

 

Acanthamoeba species are free-living organisms and may be found widely in the environment.

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

Cautions

While this assay is designed to detect symptomatic infection with Acanthamoeba species, the widespread distribution of these free-living microscopic amebae in the environment may contaminate inanimate objects such as contact lenses and cases. Thus, it should be used for patients with a clinical history and ocular symptoms consistent with amebic keratitis.

 

Inadequate specimen collection or improper storage may invalidate test results.

 

Acanthamoeba species DNA may be detectable for an unknown period of time after adequate treatment.

Clinical Information

Acanthamoeba are ubiquitous, free-living, microscopic amebae that cause rare, but severe, infections of the eye, skin, lungs, and central nervous system (CNS). They are found worldwide in water and soil and may enter the body through inhalation, contamination of wounds, and contact lens use. As many as 24 species comprising 18 genotypes (T1-T18) have been described, although most human infections are due to genotype T4. Given their widespread distribution in the environment, many people will be exposed to Acanthamoeba during their lifetime, but very few will become sick from this exposure.

 

The most common form of Acanthamoeba infection is amebic keratitis (AK). Infection occurs primarily in contact lens wearers due to contamination of lenses, cleaning solutions, or storage cases. Amebae can also enter the cornea following trauma. AK is a painful, subacute corneal infection associated with extensive scarring and blindness if untreated. Cases generally respond to treatment but relapse is common. Compared to corneal infection, involvement of the CNS is rare and seen primarily in severely immunocompromised individuals such as organ transplant recipients and patients with AIDS. CNS infection may also be caused by a related ameba, Balamuthia mandrillaris.

 

AK is usually clinically suspected based on symptoms and confocal ophthalmologic examination. Confirmation of infection is classically identified by microscopic examination and culture of corneal tissue and contact lenses or equipment using tap water agar plate overlain with bacteria as a food source for the amebae. Unfortunately, it must be held and examined for 7 days for maximum sensitivity. A polymerase chain reaction assay provides a more rapid result with similar sensitivity to culture and is, therefore, the preferred method for confirming the clinical diagnosis of AK.

Method Name

Real-Time Polymerase Chain Reaction (PCR)/TaqMan DNA Probe Hybridization

Report Available

2 to 3 days

Reporting Name

Acanthamoeba species Detection, PCR

Reference Values

Negative

Interpretation

A positive result indicates the presence of Acanthamoeba species DNA and is consistent with active or recent infection. While positive results are highly specific indicators of disease, they should be correlated with symptoms, clinical findings, and confocal ophthalmologic examination.

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 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)

Day(s) Performed

Monday through Saturday

Clinical Reference

1. Trabelsi H, Dendana A, Sellami A, et al: Pathogenic free-living amoebae: epidemiology and clinical review. Pathol Biol (paris). 2012 Dec;60(6):399-405

2. Thompson PP, Kowalski RP, Shanks RMQ, Gordon YJ: Validation of real-time PCR for laboratory diagnosis of Acanthamoeba keratitis. J Clin Microbiol. 2008 Oct;46(10):3232-3236

Test Classification

This test was developed, and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.

CPT Code Information

87798

LOINC Code Information

Test ID Test Order Name Order LOINC Value
ACARP Acanthamoeba species Detection, PCR 41429-2

 

Result ID Test Result Name Result LOINC Value
SRCAS Specimen Source 31208-2
38058 Acanthamoeba species PCR 41429-2