Diagnosis of Infections Caused by Pathogenic Free-Living Amoebae

Abstract

Naegleria fowleri, Acanthamoeba spp., Balamuthia mandrillaris, and Sappinia sp. are pathogenic free-living amoebae. N. fowleri causes Primary Amoebic Meningoencephalitis, a rapidly fatal disease of the central nervous system, while Acanthamoeba spp. and B. mandrillaris cause chronic granulomatous encephalitis. Acanthamoeba spp. also can cause cutaneous lesions and Amoebic Keratitis, a sight-threatening infection of the cornea that is associated with contact lens use or corneal trauma. Sappinia pedata has been identified as the cause of a nonlethal case of amoebic encephalitis. In view of the potential health consequences due to infection with these amoebae, rapid diagnosis is critical for early treatment. Microscopic examination and culture of biopsy specimens, cerebral spinal fluid (CSF), and corneal scrapings have been used in the clinical laboratory. For amoebic keratitis, confocal microscopy has been used to successfully identify amoebae in corneal tissue. More recently, conventional and real-time PCR assays have been developed that are sensitive and specific for the amoebae. In addition, multiplex PCR assays are available for the rapid identification of these pathogens in biopsy tissue, CSF, and corneal specimens.

Rapid detection and simultaneous molecular profile characterization of Acanthamoeba infections

“…HRM allowed corneal samples to be readily assayed after DNA extraction without interference from DNA sequences that may be found in the specimen. For the series of 20 corneal scrapings tested here, HRM detecting capacities and specificity were higher than culture and all other nucleic-acid amplification techniques (Behets et al, 2006;Da Rocha-Azevedo et al, 2009;Rivière et al, 2006). The loop-mediated isothermal amplification (LAMP) is a method that does not require thermocyclers and DNA purification procedures.…”

“…Great improvement was obtained using TaqMan rtPCR tests but they are unable to detect strains from genotype T5 (Qvarnstrom et al, 2006) and A. astronyxis (Da Rocha-Azevedo et al, 2009;Qvarnstrom et al, 2006). Moreover, the cost for the numerous TaqMan probes and for the postamplification procedures required for characterizing Acanthamoeba is high (Gatti et al, 2010;Goldschmidt et al, 2009b;Khairnar et al, 2011;Rivera and Adao, 2009;Schroeder et al, 2001;Yera et al, 2007).…”

“…The use of labeled TaqMan® probes (Qvarnstrom et al, 2006;Yera et al, 2007) instead of SYBRgreen improved rtPCR specificity but did not detect all the genotypes. In addition, TaqMan rtPCRs were unable to provide group or genotype data (Da Rocha-Azevedo et al, 2009;Qvarnstrom et al, 2006). We reported a broad-spectrum rtPCR performed directly from clinical or environmental samples (targeting a highly conserved mitochondrial gene) that made possible the detection of all the available American Type Culture Collection (ATCC) strains (0.1 cyst/μL of sample).…”

“…In this study, we developed a new molecular strategy based on HRM that should a) detect in 1 run the equivalent of 1 cyst of Acanthamoeba or less per sample; b) produce results without molecular probes based on the dye-melting derivative curve analysis with no need for gel electrophoresis, hybridizations, or immunoenzymatic assays; and c) draft different profiles for different species according to the guanine cytosine content and to the size of the amplicons neosynthesised during the nucleic-acid amplification process 30867], and A. hatchetti). Acanthamoeba were cultured and maintained according to the procedures indicated by the ATCC for each strain (Da Rocha-Azevedo et al, 2009;Marciano Cabral, 2003).…”

“…After rinsing fluorescein and topical anesthetics from the eye surface, ophthalmologists performed deep scraping of the cornea with sterile stainless steel blades in order to obtain materials from patients presenting with corneal ulcers and requiring microbiological diagnosis (Goldschmidt et al, 2006(Goldschmidt et al, , 2008 Aliquots of each scraping were discharged into flasks with 5 mL of 0.9% sodium chloride and Escherichia coli suspensions and incubated up to 30 days before being discarded as culture negative (Da Rocha-Azevedo et al, 2009;Marciano Cabral, 2003;Yera et al, 2007); c) DNA extraction for diagnosis of Herpesviridae by real-time PCR (Goldschmidt et al, 2006;Van Doornum et al, 2003) and of Acanthamoeba (Behets et al, 2006;Goldschmidt et al, 2008).…”