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Test ID FLARP Free-Living Amebae, Molecular Detection, PCR, Varies

Useful For

Aids in the diagnosis of primary amebic meningoencephalitis and granulomatous amebic encephalitis in spinal fluid and tissue in conjunction with clinical and radiologic findings

 

This test should not be used to screen asymptomatic patients.

Highlights

This assay is intended as an aid in the diagnosis of primary amebic meningoencephalitis and granulomatous amebic encephalitis in conjunction with clinical and radiologic findings.

 

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

Method Name

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

Reporting Name

Free-living Amebae Detection, PCR

Specimen Type

Varies


Necessary Information


Specimen source is required.



Specimen Required


Submit only 1 of the following specimens:

 

Specimen Type: Spinal fluid

Container/Tube: Sterile container

Specimen Volume: 0.5 mL

Collection Instructions: Send vial number 2.

 

Specimen Type: Tissue: Fresh

Sources: Brain, skin, lung

Container/Tube: Sterile container

Specimen Volume: 5 to 10 mm

Collection Instructions: Submit tissue in a sterile container with 1 mL of sterile saline or minimal essential media (MEM).

 

Preferred: Paraffin-embedded tissue block:

Supplies: Tissue Block Container (T553)

Specimen Type: Formalin-fixed, paraffin-embedded tissue block (FFPE)

Sources: Brain, skin, lung

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: Brain, skin, lung

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 Minimum Volume

Spinal Fluid: 0.3 mL; Tissue: 5 mm biopsy

Specimen Stability Information

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

Reject Due To

All specimens will be evaluated at Mayo Clinic Laboratories for test suitability.  

Clinical Information

Several free-living amebae can infect the central nervous system (CNS) and cause devastating, usually fatal, disease. The route of entry and clinical course of infection varies with the type of ameba involved. Naegleria fowleri typically causes rapidly progressive primary amebic meningoencephalitis (PAM) in previously healthy children or adults. Infection is acquired during contact with contaminated water, including swimming and diving in warm stagnant freshwater lakes and by nasal irrigation with nonsterile water. During contact, the amebae enter the nasal sinuses and travel along the olfactory nerve through the cribriform plate of the skull and into the CNS. PAM is almost uniformly fatal within several days of exposure. Because of the rarity of the infection and difficulty in initial detection, about 75% of diagnoses are made after the death of the patient. In contrast, Acanthamoeba species and Balamuthia mandrillaris usually cause a subacute CNS illness, usually in adults who are immunocompromised, called granulomatous amebic encephalitis (GAE). The presentation of GAE can mimic a brain abscess, aseptic or chronic meningitis, or CNS malignancy. The amebae usually disseminate to the CNS from the lungs or a primary skin lesion.

 

These amebae are usually identified by microscopic examination of cerebrospinal fluid or brain tissue and agar culture. Culture is more sensitive than microscopy alone but takes up to 7 days to produce a positive result. Also, B mandrillaris will not grow in routine culture. Real-time polymerase chain reaction assays offer a rapid and sensitive alternative to microscopy and culture.

Reference Values

Negative

Interpretation

A positive result indicates the presence of free-living amoeba DNA and is consistent with active or recent infection. While positive results are highly specific indicators of disease, they should be correlated with symptoms and clinical findings of primary amebic meningoencephalitis and granulomatous amebic encephalitis.

Cautions

Primary amebic meningoencephalitis due to Naegleria fowleri is a rapidly fatal disease, and rapid detection may improve the likelihood of survival. The fastest way to make a diagnosis is through examination of spinal fluid for characteristic trophozoites. This should be performed in addition to polymerase chain reaction testing.

 

While this assay is designed to detect symptomatic infection with free-living amoeba species, the widespread distribution of these microscopic amebae in the environment may contaminate inanimate objects. Thus, testing should be reserved for patients with a clinical history and symptoms consistent with granulomatous amebic encephalitis and primary amebic meningoencephalitis.

 

Inadequate specimen collection or improper storage may invalidate test results.

 

Free-living amoeba DNA may be detectable for an unknown period of time after adequate treatment.

Clinical Reference

1. Cope JR, Ali KM, Visvesvara GS: Pathogenic and opportunistic free-living amebae. In: Carroll KC, Pfaller MA, Landry ML, et al, eds. Manual of Clinical Microbiology. 12th Ed. ASM Press; 2019:chap142

2. Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases (NCEZID), Division of Foodborne, Waterborne, and Environmental Diseases (DFWED): Parasites - Acanthamoeba - Granulomatous Amebic Encephalitis (GAE); Keratitis. CDC; Updated December 29, 2021. Accessed March 28, 2023. Available at www.cdc.gov/parasites/acanthamoeba/health_professionals/acanthamoeba_keratitis_hcp.html

3 Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases (NCEZID), Division of Foodborne, Waterborne, and Environmental Diseases (DFWED): Naegleria fowleri - Primary Amebic Meningoencephalitis (PAM) - Amebic Encephalitis. Updated July 7, 2022. Accessed February 19, 2023. Available at www.cdc.gov/parasites/naegleria/health_professionals.html

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 (TPS). The LC 480 II instrument is an automated instrument that amplifies and monitors the development of target nucleic acid (amplicon) after each polymerase chain reaction (PCR) cycle.

 

The DNA target for this PCR assay is a gene encoding the nuclear small subunit ribosomal 18S ribosomal RNA (rRNA).

 

The PCR is run as 2 separate assays: primers and probes for Acanthamoeba species and an internal control will comprise 1 reaction, while primes and probes for Balamuthia mandrillaris and Naegleria fowleri are contained in the second reaction. The forward and reverse primers are for template amplification and the TaqMan probes (FAM and CY5) are for detection. The FAM and CY5 probes contain 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 ameba 18S rDNA, it is subsequently degraded by a DNA polymerase with 5'-3' exonuclease activity, resulting in release of the fluorophore and production of signal. Presence of the specific organism nucleic acid is confirmed by the presence of an amplification curve, which is used to determine if a specimen is positive or negative.(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 Nov 4;152[6]:799-807)

Day(s) Performed

Monday through Saturday

Report Available

2 to 3 days

Test Classification

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

CPT Code Information

87798 x 3

87999 (if appropriate for government payers)

NY State Approved

Yes

Forms

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

Testing Algorithm

For more information see Meningitis/Encephalitis Panel Algorithm.