Identification and typing of free-living Acanthamoeba spp. by MALDI-TOF MS Biotyper

Highlights

• Infections from pathogenic free-living Acanthamoeba are human rising diseases.

• MALDI-TOF MS platform identifies and types Acanthamoeba strains rapidly.

• Acanthamoeba genotypes are characterized by distinctive spectral profiles.

Abstract

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.

Introduction

Free-living amoebae belonging to the genus Acanthamoeba are protozoa widespread in the environment (e.g., soil, water, drinking water, air conditioning systems, marine sediments) (Visvesvara et al., 2007). Over the years, Acanthamoeba has gained increasing attention from the scientific community because of its potential pathogenicity, since these organisms are able to cause diseases in humans and animals (Trabelsi et al., 2012). More than 24 species belong to this genus, but only some of them are causative agents of keratitis and encephalitis in humans. The identification of the different species of Acanthamoeba only based on morphological characteristics is inadequate due to changes in the morphology of the amoebae (Smirnov et al., 2011). This difficulty in the assignment methodology prompted researchers to find a new type of classification. Recently, the genus Acanthamoeba has been divided into 20 different genotypes named from T1 to T20 on the basis of ribosomal RNA sequences (particularly the 18S gene) (Fuerst et al., 2015).

However, diagnostic or phylogenetic analyses based solely on short fragments might lead to unreliable results. In fact, recently, Corsaro et al. (2015) revised the wrong assignment of several partial sequences, ascribed to genotype T16 or T4, and assigned them to the proper genotypes (Corsaro et al., 2015).

The Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology is a rapid and consolidated method for automated and accurate microbial phenotypic identification (Clark et al., 2013). In the last few years, MALDI-TOF MS has also been applied as a potential complementary method to discriminate genus, species, and strains on the basis of characteristic patterns of proteins (biomarkers) among opportunistic free-living amoebae Acanthamoeba, Balamuthia and Naegleria fowleri (Visvesvara et al., 2007). Recently, Moura et al. (2015) developed new high-resolution MALDI-TOF MS processing methods to analyze Naegleria species and N. fowleri strains (Moura et al., 2015). In Italy, epidemiological surveys have reported the frequency of different Acanthamoeba genotypes in symptomatic individuals in several regions (Di Cave et al., 2014, Di Cave et al., 2009, Gatti et al., 2010). However, the relevance of Acanthamoeba keratitis is still grossly underestimated, not only at the level of the individual patient, but most of all as a public health concern, also due to the lack of standardized guidelines for the clinical diagnosis.

Further studies on amoebae would represent a new and useful frontier in their diagnostic identification as well as a further approach to perform taxonomic and phylogenetic studies on amoebae. In this study, we developed a MALDI-TOF MS-based assay to identify and genotype Acanthamoeba clinical strains. The reliability of this new method for parasite identification has been confirmed by overlapping the spectra profile dendrogram to the phylogenetic tree obtained with the gold-standard method based on PCR genotyping assay.