Identification and typing of free-living Acanthamoeba spp. by MALDI-TOF MS Biotyper.
Over the years, the potential pathogenicity of Acanthamoeba for humans and animals has gained increasing attention from the scientific community. More than 24 species belong to this genus, however only some of them are causative agents of keratitis and encephalitis in humans. Due to technical difficulties in diagnosis, these infections are likely to be under-detected. The introduction of 18S rDNA amplification for the identification of Acanthamoeba has dramatically enhanced diagnosis performances, but the attestation of genotyping requires supplementary sequencing-based procedures. In this study, 15 Acanthamoeba strains were collected and grown on nutrient agar media. Each strain was genotyped by end-point PCR assay for the amplification of the 18S rDNA gene and the genotype was assigned by sequencing analysis through neighbor joining phylogenetic tree. In order to optimize standardization of the MALDI-TOF MS assay, we established the collection time point at the cystic phase. Two strains of each genotype were randomly chosen to customize the biotyper database. For all strains, 24 spectral measurements were acquired and submitted to identification and cluster analysis of spectra. The obtained results highlighted the correct identification of Acanthamoeba strains and the overlapping of spectra dendrogram clusters to the 18S genotype assignations. In conclusion, the MALDI-TOF MS Biotyper revealed the capability to identify and genotype the Acanthamoeba strains, providing a new frontier in the diagnostic identification of amaebae and in taxonomic and phylogenetic studies.
Human Microbiome Unit, Bambino Gesù Children's Hospital and Research Institute, IRCCS, Rome, Italy. Electronic address: federica.delchierico@opbg.net.
Federica
F
Del Chierico
Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy.
David
D
Di Cave
Human Microbiome Unit, Bambino Gesù Children's Hospital and Research Institute, IRCCS, Rome, Italy.
Cristel
C
Accardi
Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy.
Maristella
M
Santoro
Gene Expression-Microarrays Laboratory, Bambino Gesù Children's Hospital and Research Institute, IRCCS, Rome, Italy.
Andrea
A
Masotti
Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy.
Rossella
R
D'Alfonso
Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy.
Federica
F
Berrilli
Internal Medicine Department, University of Rome Tor Vergata, Rome, Italy; Fondazione Santa Lucia, Rome, Italy.
Andrea
A
Urbani
Human Microbiome Unit, Bambino Gesù Children's Hospital and Research Institute, IRCCS, Rome, Italy; Parasitology Unit, Bambino Gesù Children's Hospital and Research Institute, IRCCS, Rome, Italy.
Lorenza
L
Putignani
D000048:Acanthamoeba / Q000145:classification* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D015823:Acanthamoeba Keratitis / Q000469:parasitology*; D016000:Cluster Analysis; D016054:DNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000235:genetics; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods / Q000592:standards; D006801:Humans; D010641:Phenotype; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D040901:Proteomics / Q000379:methods; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S
Acanthamoeba genotyping; MALDI-TOF MS - based analytical identification assay; MALDI-TOF MS Biotyper