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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

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. PCR 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.

Negative

Interpretation 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.

Cautions 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 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 (FFPE) Specimens:

Twenty-four 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, MEM), 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 (LoD):

-The 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/MEM 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.

 

Precision:

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;60:399-405

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