Test Catalog

Test Id : ACARP

Acanthamoeba species Molecular Detection, PCR, Ocular

Useful For
Suggests clinical disorders or settings where the test may be helpful

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


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.

Method Name
A short description of the method used to perform the test

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

NY State Available
Indicates the status of NY State approval and if the test is orderable for NY State clients.


Reporting Name
Lists a shorter or abbreviated version of the Published Name for a test

Acanthamoeba species Detection, PCR

Specimen Type
Describes the specimen type validated for testing


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.


Question ID Description Answers
SRCAS Specimen Source

Specimen Required
Defines the optimal specimen required to perform the test and the preferred volume to complete testing

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.


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

Specimen Minimum Volume
Defines the amount of sample necessary to provide a clinically relevant result as determined by the Testing Laboratory

Tissue: 5 mm biopsy

Scrapings: 0.5 mL

Contact Lens Solution in contact lens cases: 1 mL

Reject Due To
Identifies specimen types and conditions that may cause the specimen to be rejected

Calcium alginate-tipped swab Wood swab Transport swab containing gel Specimens containing scalpel blades Unstained slides Reject

Specimen Stability Information
Provides a description of the temperatures required to transport a specimen to the performing laboratory, alternate acceptable temperatures are also included

Specimen Type Temperature Time Special Container
Varies Refrigerated (preferred) 7 days
Frozen 7 days

Useful For
Suggests clinical disorders or settings where the test may be helpful

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

Clinical Information
Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test

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.

Reference Values
Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.


Provides information to assist in interpretation of the test results

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.

Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances

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.

Supportive Data

The following assay verification data supports the use of this assay for clinical testing.


Species Inclusivity:

The Acanthamoeba polymerase chain reaction (PCR) assay detects the 20 different strains of Acanthamoeba, including the genotypes that cause human disease.


Accuracy/Diagnostic Sensitivity and Specificity-Fresh Specimens:

Results from this PCR assay detecting the 18S rRNA gene of Acanthamoeba species were compared to culture results on 112 contact/ocular specimens. Of the 12 specimens that were positive by culture, 11 were detected by PCR (sensitivity 92%). PCR also detected an additional 2 positive specimens, which were both from the same patient with a clinical diagnosis of amebic keratitis (AK), while 98 specimens were negative by both culture and PCR (specificity 98%).


Accuracy/Diagnostic Sensitivity and Specificity-Formalin-Fixed Paraffin-Embedded Specimens:

Twenty-four formalin-fixed paraffin embedded (FFPE) archived tissue blocks were tested by the Acanthamoeba species PCR assay and results were compared to histopathologic (light microscopic) diagnosis. Fourteen of the tissues had a morphologic diagnosis of acanthamebic keratitis; of these, 12 were positive by PCR (sensitivity 86%). Ten specimens were negative by both histopathology and PCR (specificity 100%).


Supplemental Accuracy Data:

Spiking studies were performed using ocular material in transport media (contact lens fluid, minimal essential media), fresh tissue, and FFPE tissue spiked with Acanthamoeba genomic DNA at an approximate concentration of 50 targets/mcL. All samples were then extracted and tested in a blinded fashion. At 50 targets/mcL, 100% of the ocular material, the fresh, and the FFPE tissue were positive by PCR.


Analytical Sensitivity/Limit of Detection:

-The limit of detection (LOD) determined with serial dilution of cultured Acanthamoeba cysts (counted using a hemocytometer) was considered to be 1 cyst per processed sample.

-The LOD established using genomic DNA spiked into contact lens solution/minimal essential transport media   is 1.26 target copies/mcL.

-The LOD established using genomic DNA spiked into fresh tissue matrix is 6.5 target copies/mL.

-The LOD established using genomic DNA spiked into FFPE tissue matrix is 5.7 target copies/mL.


Analytical Specificity:

No PCR signal was obtained from the extracts of 47 bacterial, viral, parasitic, and fungal isolates from similar organisms or from organisms commonly found in the specimens tested.



Qualitative inter- and intra-assay precision was 100%. All crossing point values were within 2 cycles of the mean.


Reference Range:

The reference range is "Negative" for this assay. PCR and culture performed on 291 contact lenses from asymptomatic individuals failed to detect Acanthamoeba DNA or growth.


However, PCR may detect Acanthamoeba species colonization due to the widespread distribution of this free-living ameba in the environment, and PCR testing should only be used for patients with a clinical history and ocular symptoms consistent with AK.


Reportable Range:

This is a qualitative assay, and the results are reported as negative or positive for targeted Acanthamoeba species.

Clinical Reference
Recommendations for in-depth reading of a clinical nature

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

Method Description
Describes how the test is performed and provides a method-specific reference

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)

PDF Report
Indicates whether the report includes an additional document with charts, images or other enriched information


Day(s) Performed
Outlines the days the test is performed. This field reflects the day that the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time before the test is performed. Some tests are listed as continuously performed, which means that assays are performed multiple times during the day.

Monday through Saturday

Report Available
The interval of time (receipt of sample at Mayo Clinic Laboratories to results available) taking into account standard setup days and weekends. The first day is the time that it typically takes for a result to be available. The last day is the time it might take, accounting for any necessary repeated testing.

2 to 3 days

Specimen Retention Time
Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded

7 Days

Performing Laboratory Location
Indicates the location of the laboratory that performs the test


Several factors determine the fee charged to perform a test. Contact your U.S. or International Regional Manager for information about establishing a fee schedule or to learn more about resources to optimize test selection.

  • Authorized users can sign in to Test Prices for detailed fee information.
  • Clients without access to Test Prices can contact Customer Service 24 hours a day, seven days a week.
  • Prospective clients should contact their Regional Manager. For assistance, contact Customer Service.

Test Classification
Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR) product.

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
Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Clinic Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.

CPT codes are provided by the performing laboratory.


LOINC® Information
Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the order and results codes of this test. LOINC values are provided by the performing laboratory.

Test Id Test Order Name Order LOINC Value
ACARP Acanthamoeba species Detection, PCR 41429-2
Result Id Test Result Name Result LOINC Value
Applies only to results expressed in units of measure originally reported by the performing laboratory. These values do not apply to results that are converted to other units of measure.
SRCAS Specimen Source 31208-2
38058 Acanthamoeba species PCR 41429-2

Test Setup Resources

Setup Files
Test setup information contains test file definition details to support order and result interfacing between Mayo Clinic Laboratories and your Laboratory Information System.

Excel | Pdf

Sample Reports
Normal and Abnormal sample reports are provided as references for report appearance.

Normal Reports | Abnormal Reports

SI Sample Reports
International System (SI) of Unit reports are provided for a limited number of tests. These reports are intended for international account use and are only available through MayoLINK accounts that have been defined to receive them.

SI Normal Reports | SI Abnormal Reports