Naegleria Infection Workup

The diagnosis of primary amebic meningoencephalitis (PAM) is always parasitic and is based on detection and identification of N fowleri trophozoites in the CSF or brain biopsy samples.

Specimens

The CSF is the specimen of choice for demonstration of the amebae.

Direct wet-mount microscopy

The CSF is centrifuged at 150 xg for 5 minutes. The supernatant is aspirated, and the sediment is suspended in the remaining fluid. A drop of sediment suspension is kept on a slide and mounted with a coverslip and is examined with compound light microscopy using 10X and 40X objectives. The specimen is best examined with phase contrast microscopy. This may show trophozoites with lobopodia extension and retraction.

The amebae are detected based on their active directional movements. Close observation is important because PAM can be diagnosed based on the observation of trophozoites; however, these have been confused with WBCs in reported cases. Cyst and flagellated stages are not found in CSF samples; if both cysts and trophozoites are found in CSF, it is highly suggestive of Acanthamoeba infection, ruling out Naegleria PAM.

Examination of stained CSF smear

CSF Gram stain findings are usually negative. RBCs are present. Wright-Giemsa–stained CSF may show trophozoites with large karyosome and may show a contractile vacuole. Direct fluorescent antibody staining of CSF smears is useful for demonstrating N fowleri in the CSF.

Culture

Naegleria species can be readily cultivated on either nonnutrient agar or agar media containing low concentrations of nutrients (eg, peptone 0.05%, yeast extract 0.05%, glucose 0.1%) in the presence of living or killed bacteria. In general, the bacteria of choice include nonmucoid strains of Klebsiella pneumoniae, Enterobacter species (Enterobacter aerogenes and Enterobacter cloacae), and Escherichia coli. After several days, the plate is microscopically inspected; Naegleria cysts are identified by trails left by migrating amebae in the lawn of the bacteria. Various molecular methods can be used for final confirmation of the identity of the species.

Serodiagnosis

Serologic testing has no role in the diagnosis of acute PAM, since little time is available from onset to death to mount an antibody response. In one survivor, detectable antibody persisted for more than 4 years.

Molecular diagnosis

PCR is available at some research sites using numerous primers. Molecular characterization of strains is also useful in tracking infections to a source and in recognizing potential risks for swimmers or bathers in particular locales. A species-specific DNA probe is available to identify N fowleri in environmental samples, followed by restriction fragment length polymorphism (RFLP) analyses of whole-cell DNA for confirmation. Epidemiologic typing of N fowleri was used in an analysis of the 5.8S rRNA gene and the internal transcribed spacer (ITS) of clinical isolates. In a study performed in the United States, a rapid, sensitive, and specific assay for the detection of N fowleri was developed using Mp2C15 probe in a nested PCR assay format.^[20] A nested PCR assay has also been applied to detect the presence of the parasite in domestic water sources.^[21]

Recently, flow cytometry has been used for the diagnosis of N fowleri infection.^[22] Flores et al evaluated flow cytometry and monoclonal antibodies in differentiating Naegleria fowleri from Acanthamoeba species.^[23]

Lately, a real-time PCR using hybridization fluorescent-labelled probes, targeting the N fowleri Mp2Cl5 gene sequence, has been developed. The reaction detection limit in their study was 1 copy of the Mp2Cl5 DNA sequence.^[24]

Visvesvara has reported the development of a multiplex real-time PCR that could simultaneously look for Naegleria, Acanthamoeba, and Balamuthia species in a single specimen, thus reducing the time for diagnosis. This is especially useful as infection with any of the 3 amoebae could have clinical presentations indistinguishable from each other.^[25]

Histology

Both immunofluorescence and immunoperoxidase methods are useful for demonstrating N fowleri trophozoites in the histologic sections of the brain biopsy samples.

Head CT scanning yields nonspecific findings, showing a loss of the subarachnoid space and diffuse gray material enhancement.

CSF studies show the following:

• Sanguinopurulent or bloody CSF, showing a nonspecific polymorphonuclear (PMN) neutrophil – predominant neutrophilia
• Increased opening pressure
• PMN pleocytosis
• Elevated RBC count or frank hemorrhagic CSF
• Normal-to-low CSF glucose level
• Elevated protein level

Lumbar puncture: Wet-mount examination of CSF is the main diagnostic tool in PAN.

N fowleri infection produces lesions mainly in the base of the brain, brain stem, and cerebellum. The olfactory mucosa and bulbs are the most commonly affected areas. The lesions consist of an acute necrotizing meningoencephalitis associated with moderately purulent exudates. Only trophozoites are found in the CNS lesions, not cysts