Regional spread of contact lens-related Acanthamoeba keratitis in Italy.

Acanthamoeba ocular infections, known as Acanthamoeba keratitis, are an emerging problem among contact lens wearers. Infections mediated by Acanthamoeba are uncommon, but they can be underestimated due to poor awareness and delayed diagnosis. The routine use of rapid and cost-effective molecular methods like Real Time PCR for the diagnosis of this important pathogen could improve diagnosis and therapy outcome. This report describes the detection by Real Time PCR assay of six T4 and one T3 Acanthamoeba infections, as the first reported cases in Tuscany, Italy.

Update on Acanthamoeba jacobsi genotype T15, including full-length 18S rDNA molecular phylogeny.

Free-living amoebae of the genus Acanthamoeba are worldwide present in natural and artificial environments, and are also clinically important, as causative agents of diseases in humans and other animals. Acanthamoeba comprises several species, historically assigned to one of the three groups based on their cyst morphology, but presently recognized as at least 20 genotypes (T1-T20) on the basis of their nuclear 18S ribosomal RNA (rRNA) gene (18S rDNA) sequences. While strain identification may usually be achieved targeting short (<500 bp) 18S ribosomal DNA (rDNA) fragments, the use of full-length gene sequences (>2200 bp) is necessary for correct genotype description and reliable molecular phylogenetic inference. The genotype T15, corresponding to Acanthamoeba jacobsi, is the only genotype described on the basis of partial sequences (~1500 bp). While this feature does not prevent the correct identification of the strains, having only partial sequences renders the genotype T15 not completely defined and may furthermore affect its position in the Acanthamoeba molecular tree. Here, we complete this gap, by obtaining full-length 18S rDNA sequences from eight A. jacobsi strains, genotype T15. Morphologies and physiological features of isolated strains are reported. Molecular phylogeny based on full 18S rDNA confirms some previous suggestions for a genetic link between T15 and T13, T16, and T19, with T19 as sister-group to T15.

Detection and genotyping of Dientamoeba fragilis from symptomatic patients: New insights from Italy into a little-known gastrointestinal protozoan.

Dientamoeba fragilis (D. fragilis) represents a common protozoan in both high and low income countries. Despite this, epidemiological data on dientamoebiasis are still limited, and it is possible that the actual prevalence rates of D. fragilis have been underestimated due to the challenges in its detection and identification. In the present study, symptomatic patients from Rome (Central Italy) were surveyed for two years to determine D. fragilis percentage of infection and genotypes. Stool samples collection was performed over 864 patients, DNA extracted, and RT-PCR performed by the SeeGene Allplex™ Gastrointestinal Parasite Panel Assays. Seventy-nine resulted positive for D. fragilis (9.1%). Co-infections were detected in 22 isolates: 21 displayed Blastocystis sp. + D. fragilis (27.8%). Based on the sequence of a central fragment of the SSU rRNA gene, only genotype 1 was identified. These findings are among the few available data regarding genetic diversity of D. fragilis in Italy. Large-scale human and animal research are required to enhance our knowledge of prevalence, host range, genetic variability and zoonotic transmission of this little-known intestinal protozoan.

Acanthamoeba Sequence Types and Allelic Variations in Isolates from Clinical and Different Environmental Sources in Italy.

The genus Acanthamoeba comprises free-living amoebae distributed in a wide variety of environments. These amoebae are clinically significant, causing opportunistic infections in humans and other animals. Despite this, limited data on Acanthamoeba sequence types and alleles are available in Italy. In the present study, we analyzed all Acanthamoeba sequences deposited from Italy with new positive Acanthamoeba clinical samples from symptomatic AK cases, to provide an overview of the genetic variants' spatial patterns from different sources within the Italian context. A total of 137 Acanthamoeba sequences were obtained. Six sequence types were identified: T2/6, T3, T4, T11, T13, and T15. Only T4 and T15 were found in both sources. The Acanthamoeba T4 sequence type was found to be the most prevalent in all regions, accounting for 73% (100/137) of the Italian samples analyzed. The T4 sequence type demonstrated significant allelic diversity, with 30 distinct alleles from clinical and/or environmental samples. These outcomes enabled a better understanding of the distribution of Acanthamoeba isolates throughout Italy, reaffirming its well-recognized ubiquity. Acanthamoeba isolates analysis from keratitis, together with the environmental strains monitoring, might provide important information on different genotypes spreading. This might be useful to define the transmission pathways of human keratitis across different epidemiological scales.

Intestinal parasites infecting captive non-human primates in Italy.

Non-human primates (NHPs) living in captive conditions are susceptible to intestinal parasites that can contribute to mortality and morbidity, and cause zoonotic infections. Thus, parasite surveys on NHP populations under human care are relevant as part of the evaluation of NHPs welfare and in the zoonotic disease risk assessment, as well as in the exploration of parasite transmission pathways, according to the One-Health concept. This study aimed to identify intestinal parasites infecting NHPs living in two wildlife recovery centers and in a zoological garden, in Italy. Ninety-three fecal samples from Macaca tonkeana, Macaca fascicularis, Sapajus apella, Chlorocebus aethiops, Macaca fuscata, Macaca sylvanus, and Cebus capucinus were collected at Piano dell'Abatino Park (Lazio), and fecal smears and flotation were performed in order to identify parasites according to morphological keys. Additionally, one carcass of M. fuscata from the Bioparco Zoological Garden of Rome (Lazio) and one of M. fascicularis from the Center for the Recovery of Exotic and Maremma Wild Animals (Tuscany) were necropsied and intestinal adult nematodes were collected and characterized at morphological and molecular level, using the mitochondrial cox1 and rrnL markers. Protozoans (Entamoeba coli, Iodamoeba bütschlii, Dientamoeba fragilis-like, Giardia sp.), chromists (Balantidium/Buxtonella sp.) and nematodes (Capillaria sp., Trichuris sp., strongyliform larvae and Oesophagostomum sp.) were found through fecal smears and flotation. The collected adult nematodes from dead NHPs were morphologically identified as whipworms (genus Trichuris). Phylogenetic analyses grouped Trichuris specimens into the Trichuris trichiura complex of species, with specimens from M. fuscata clustering into a host-specific branch, and whipworms from M. fascicularis clustering within a clade formed by Trichuris infecting several primate species, including humans. The results here collected revealed the presence of potentially zoonotic parasites circulating in captive primates in Italy, providing useful information for the formulation of management and care plans for captive NHPs, and for the elaboration of safety measures for visitors and animal keepers.

Giardia duodenalis in Wildlife: Exploring Genotype Diversity in Italy and across Europe.

Fragmented data are so far available on genotype diversity of G. duodenalis in wildlife in different countries in Europe, in particular, in Italy. In the present study, G. duodenalis sequences obtained from different Italian wild animals [12 porcupines (Hystrix cristata), 4 wild boars (Sus scrofa), 1 wolf (Canis lupus italicus), 6 Alpine chamois (Rupicapra rupicapra rupicapra)] were compared with those available from wild host species in Europe to add new data on the geographic distribution of Giardia assemblages/sub-assemblages and their transmission patterns among natural hosts. Thirty-eight sequences were obtained by MLG analysis (SSU-rRNA, bg, gdh, and tpi genes) and subsequently compared by phylogenetic and network analyses with those from wild species monitored in the last decades in Europe. The results revealed the presence of potentially zoonotic (A-AI, A-AII from wild boar; B from porcupine) and host-adapted (D from wolf; E, A-AIII from chamois) assemblages and sub-assemblages and represent the first report for Italian wild boar. The analysis did not find any evidence of spatial or host segregation for specific genetic variants, mostly shared between different hosts from different European countries. However, conflicting evidence was found in genotypic assignment, advocating for data improvement and new genomic approaches.

Exploring the nature of interaction between shiga toxin producing Escherichia coli (STEC) and free-living amoeba - Acanthamoeba sp.

Free-living amoebae (FLA) are widely distributed protozoa in nature, known to cause severe eye infections and central nervous system disorders. There is growing attention to the potential role that these protozoa could act as reservoirs of pathogenic bacteria and, consequently, to the possibility that, the persistence and spread of the latter may be facilitated, by exploiting internalization into amoebae. Shiga toxin-producing strains of Escherichia coli (STEC) are zoonotic agents capable of causing serious diseases, such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Cattle represent the main natural reservoir of STEC, which are frequently found also in other domestic and wild ruminants, often without causing any evident symptoms of disease. The aspects related to the ecology of STEC strains in animal reservoirs and the environment are poorly known, including the persistence of these microorganisms within niches unfavorable to survival, such as soils or waters. In this study we investigated the interaction between STEC strains of serotype O157: H7 with different virulence gene profiles, and a genus of a wild free-living amoeba, Acanthamoeba sp. Our results confirm the ability of STEC strains to survive up to 20 days within a wild Acanthamoeba sp., in a quiescent state persisting in a non-cultivable form, until they reactivate following some stimulus of an unknown nature. Furthermore, our findings show that during their internalization, the E. coli O157 kept the set of the main virulence genes intact, preserving their pathogenetic potential. These observations suggest that the internalization in free-living amoebae may represent a means for STEC to resist in environments with non-permissive growth conditions. Moreover, by staying within the protozoa, STEC could escape their detection in the vehicles of infections and resist to the treatments used for the disinfection of the livestock environment.

Population Analysis of O26 Shiga Toxin-Producing Escherichia coli Causing Hemolytic Uremic Syndrome in Italy, 1989-2020, Through Whole Genome Sequencing.

Shiga toxin-producing Escherichia coli (STEC) belonging to the O26 serogroup represent an important cause of Hemolitic Uremic Syndrome (HUS) in children worldwide. The localization of STEC virulence genes on mobile genetic elements allowed the emergence of clones showing different assets of this accessory genomic fraction. A novel O26 STEC clone belonging to Sequence Type (ST) 29 and harboring stx2a, ehxA and etpD plasmid-borne genes has emerged and spread in Europe since the mid-1990s, while another ST29 clone positive for stx2d and lacking plasmid-borne virulence genes was recently described as emerging in France. In Italy, O26 has been the most frequently detected STEC serogroup from HUS cases since the late 1990s. In this study we describe the genomic characterization and population structure of 144 O26 STEC strains isolated from human sources in Italy in the period 1989-2020. A total of 89 strains belonged to ST21, 52 to ST29, two to ST396 and one to ST4944. ST29 strains started to be isolated from 1999. 24 strains were shown to harbour stx1a, alone (n=20) or in combination with stx2a (n=4). The majority of the strains (n=118) harbored stx2a genes only and the two ST396 strains harbored stx2d. A Hierarchical Clustering on Principal Components (HCPC) analysis, based on the detection of accessory virulence genes, antimicrobial resistance (AMR) genes and plasmid replicons, classified the strains in seven clusters identified with numbers from 1 to 7, containing two, 13, 39, 63, 16, 10 and one strain, respectively. The majority of the genetic features defining the clusters corresponded to plasmid-borne virulence genes, AMR genes and plasmid replicons, highlighting specific assets of plasmid-borne features associated with different clusters. Core genome Multi Locus Sequence Typing grouped ST21 and ST29 strains in three clades each, with each ST29 clade exactly corresponding to one HCPC cluster. Our results showed high conservation of either the core or the accessory genomic fraction in populations of ST29 O26 STEC, differently from what observed in ST21 strains, suggesting that a different selective pressure could drive the evolution of different populations of these pathogens possibly involving different ecological niches.

Giardia duodenalis in colony stray cats from Italy.

Giardia duodenalis is the most common intestinal protozoan in humans and animals worldwide, including eight morphologically identical assemblages, infecting pets, livestock, wildlife and human beings. Assemblages A and B are those with the higher zoonotic potential, and they have been detected in several mammals other than humans; the others (C to H) show a higher host specificity. Cats can harbour both the specific Assemblage F and the zoonotic ones A and B. Several studies have been carried out on G. duodenalis genotypes in cats; however, the role of this species in the epidemiology of giardiasis is still poorly understood. In this scenario, the present study carried out the detection and genetic characterization at sub-assemblage level of G. duodenalis from colony stray cats in central Italy. In the period 2018-2019, 133 cat faecal samples were analysed for the presence of G. duodenalis cysts by a direct immunofluorescence assay. Positive samples were subsequently subjected to molecular analyses for assemblage/sub-assemblage identification. Forty-seven samples (35.3%) were positive for G. duodenalis cysts by immunofluorescence. G. duodenalis DNA was amplified at SSU-rDNA locus from 39 isolates: 37 were positive for zoonotic Assemblage A and 2 showed a mixed infection (A + B). Positive results for the ß-giardin gene were achieved for 25 isolates. Sequence analysis revealed 16 isolates belonging to Sub-assemblage AII and 8 to Sub-assemblage AIII. One isolate resulted as ambiguous AI/AIII. Large sequence variability at the sub-assemblage level was detected, with several double peaks and mutations, making complex a proper isolate allocation. When compared with previous studies, the 35.3% prevalence of G. duodenalis in cats reported in the present article was surprisingly high. Moreover, all positive cats resulted to be infected with zoonotic assemblages/sub-assemblages, thus indicating stray cats as a possible source of human giardiasis and highlighting the sanitary relevance of cat colonies in the study area.

PCR-based identification of thermotolerant free-living amoebae in Italian hot springs.

Several thermal areas, also used for leisure purposes, may represent suitable habitats for free-living amoebae (FLAs), but few studies have been carried out in search for these organisms. The aim of this study was to assess the presence and distribution of FLAs by culture detection and molecular identification, over a one year-round sampling of two sites in Central Italy. Two geothermal springs (Site A and Site B) were investigated for a total of 36 water samples. Four sets of primers were used to amplify FLA DNA from all cultures positive for amoebic growth at both 37 °C and 45 °C. Overall, 33 (91.6%) water samples produced PCR amplification. Eleven taxa were identified. The array of identified species varied over the sampling period, and differed between the two hot springs, Site A harbouring 11 taxa compared to 5 of site B. However, both sites were characterized by the most common species Vermamoeba vermiformis and Naegleria australiensis. Acanthamoeba genotypes T4 and T15 were found at low frequency. Differences in the composition between the two sites could reflect environmental changes in biotic and chemical/physical parameters. From a public health perspective, the detection of potentially pathogenic amoebae could unveil a potential risk for humans.

Giardia microti in pet Microtus guentheri: Evidence of a parasite never detected in Italy.

The genus Giardia includes several species distinguished by morphological, biological and molecular features. Currently, eight species within the genus are retained as valid. In Italy no identification of Giardia species other than Giardia duodenalis has been so far reported. Fecal samples were collected from two Günther's Voles (Microtus guentheri) positive to Giardia cysts by microscopic investigation and immunofluorescence. The voles were born in Milan (Northern Italy) from two gravid females imported from the Netherlands and kept for sale in a pet shop in Varese (Northern Italy). Positive feces were subjected to a nested PCR to amplify a 18S rRNA fragment for molecular characterization. A phylogenetic analysis was conducted to compare the obtained sequence with those of all other Giardia species available in GenBank for the 18S locus, using the Maximum Likelihood (ML) method by R software. Sequence analyses unambiguously identified the isolates as belonging to G. microti, showing 99% of identity with those of its isolates available in GenBank. A well-defined cluster, supported by significant bootstrap values and corresponding to the G. microti cluster, including sequences obtained from M. guentheri, was evidenced in the ML tree, confirming species assignment. The present finding represents the first report of G. microti from pet animals in Italy.

Nuclear and Mitochondrial Data on Trichuris from Macaca fuscata Support Evidence of Host Specificity.

Whipworms are parasitic intestinal nematodes infecting mammals, and traditionally humans and other primates that have so far been considered infected by Trichuris trichiura. Recent molecular studies report a more complex scenario suggesting the presence of a species complex with several Trichuris taxa specifically infecting only one primate species as well as taxa able to infect a range of primate species. The systematics of the group is important for taxonomic inference, to estimate the relative zoonotic potential, and for conservation purposes. In fact, captive animals living in zoological gardens are usually infected by persistent monoxenous intestinal parasites. Here, two Japanese macaques living in the Bioparco Zoological Garden of Rome were found infected by Trichuris sp. Nematodes were characterized at the molecular level using nuclear (btub and 18S) and mitochondrial (16S and cytb) markers and then compared to Trichuris collected previously in the same location, and to other Trichuris infecting primates. Evidences from mitochondrial and nuclear markers allowed for the identification of Trichuris sp. specific to Macaca fuscata. Results obtained here also described a uniform taxonomic unit of Trichuris, separated but closely related to Trichuris trichiura, thus, emphasizing its zoonotic potential for workers and visitors.

Molecular characterization of Trichuris spp. from captive animals based on mitochondrial markers.

Monoxenous parasites may easily infect animals in captivity, and nematodes belonging to the genus Trichuris are commonly reported in zoological gardens worldwide. Infections in captive animals should be accurately monitored and the characterization of pathogens is highly advisable, as a tool to infer possible routes of intra- and interspecific transmission pathways and to assess the related zoonotic potential. Whipworms are usually identified on the basis of few morphological features of adults males and eggs and by an host-affiliation criterion. Given the strong morphological convergence of adaptive traits and the possible occurrence of hybridization and/or cross-infections events, the use of molecular methods is of great utility. Here, we analysed two partial mitochondrial loci, the cytochrome c oxidase I and the cytochrome b regions, in Trichuris spp. infecting four animal species hosted in the Bioparco Zoological Garden of Rome. Results from molecular systematics, compared to previous data, suggested that the five Trichuris taxa recovered were well separated, showing a significant degree of host affiliation (herbivorous, primates/swine and rodents/canids). The screw horn antelopes and the camels were infected with two variants of Trichuris ovis; Trichuris sp. similar to those observed in rodents from South America was infecting the Patagonian maras. Moreover, Trichuris from the ring-tailed lemur showed a great similarity to Trichuris infecting the Japanese macaque previously analysed from the same zoological garden, and clustering together with Trichuris trichiura, posing a potential zoonotic threat for visitors and workers.

Diversity of Eimeria Species in Wild Chamois Rupicapra spp.: A Statistical Approach in Morphological Taxonomy.

Wildlife is frequently infected by intestinal protozoa, which may threaten their fitness and health. A diverse community of Eimeria species is known to occur in the digestive tract of mountain-dwelling ungulates, including chamois (genus Rupicapra). However, available data on Eimeria diversity in these taxa is at times inconsistent and mostly dated. In the present study, we aimed to revisit the occurrence of Eimeria spp. in the Alpine subspecies of the Northern chamois (Rupicapra rupicapra rupicapra) and the Apennine subspecies of the Southern chamois (Rupicapra pyrenaica ornata) in Italy, using an integrated approach based on a hierarchical cluster analysis (HCPC) applied to oocyst morphology and morphometry. A total of 352 fecal samples were collected from R. r. rupicapra (n = 262) and R. p. ornata (n = 90). Overall, 85.3% (300/352) of the animals tested microscopically positive to Eimeria spp. Based on morphological analysis, we identified all the eimerian species described in chamois. Through the HCPC method, five clusters were generated, corresponding to E. suppereri, E. yakimoffmatschoulskyi, E. riedmuelleri (two different clusters), and E. rupicaprae morphotypes. The well-defined clusters within E. riedmuelleri support the existence of two distinct morphological groups, possibly referable to different taxonomic units. This study suggests that combining a morphometrical approach with a powerful statistical method may be helpful to disentangle uncertainties in the morphology of Eimeria oocysts and to address taxonomic studies of eimeriid protozoa at a specific host taxon level.

Outbreak of swimmer's itch in Central Italy: Description, causative agent and preventive measures.

Swimmer's itch is caused by the penetration of free-swimming larvae of trematodes of the family Schistosomatidae in human skin. It is usually reported in people engaged in recreational water activities in freshwater bodies and in most of cases, it is provoked by bird schistosomes of the genus Trichobilharzia. In the summer 2017, many cases of dermatitis were recorded in people bathing in the waters of the Albano Lake (Rome, Italy) and a parasitological investigation was carried out in order to ascertain the causative agent of these cases. Snails of the family Lymnaeidae, natural intermediate hosts of bird schistosomes, were collected from lake shallow waters to detect the presence of trematodes of the genus Trichobilharzia. Pools of maximum 10 snails were placed in Petri dishes, and cercarial emergence was stimulated exposing snails to strong artificial light intensity at 25°C. Three hundred and thirty-seven snails were collected and screened for the shedding of cercariae. Furcocercariae of the family Schistosomatidae, with a morphology overlapping that of the genus Trichobilharzia, were detected in seven Petri dishes. Assuming that in each positive Petri dish just one snail was shedding furcocercariae, the minimum infectious rate was 2.1%. Molecular analysis of furcocercariae allowed ascribing them to the species Trichobilharzia franki. Snails of the species Radix auricularia were identified as intermediate hosts of the parasite. This is the second record of T. franki causing cercarial dermatitis in Central Italy, the third in Italy. The 2017 was in Italy exceptionally warm and dry. Trematodes are sensitive to changes in temperature, being cercarial production and emission rates temperature dependent. Small increases in water temperature would speed up parasite development and transmission, leading to a manifold increase in cercarial emergence. Moreover, high temperatures raise chances to acquire the infection, due to increased time spent in water by people.

Isolation and molecular characterization of free-living amoebae from different water sources in Italy.

Free-living amoebae (FLA) are protozoa ubiquitous in Nature, isolated from a variety of environments worldwide. In addition to their natural distribution, some species have been found to be pathogenic to humans. In the present study a survey was conducted in order to evaluate the presence and to characterize at molecular level the isolates of amoebic organisms collected from different water sources in Italy. A total of 160 water samples were analyzed by culture and microscopic examination. FLA were found in 46 (28.7%) of the investigated water samples. Groundwater, well waters, and ornamental fountain waters were the sources with higher prevalence rates (85.7%, 50.0%, and 45.9%, respectively). Identification of FLA species/genotypes, based on the 18S rDNA regions, allowed to identify 18 (39.1%) Acanthamoeba isolates (genotypes T4 and T15) and 21 (45.6%) Vermamoeba vermiformis isolates. Other FLA species, including Vahlkampfia sp. and Naegleria spp., previously reported in Italy, were not recovered. The occurrence of potentially pathogenic free-living amoebae in habitats related to human population, as reported in the present study, supports the relevance of FLA as a potential health threat to humans.