Novel data from Italian Vermamoeba vermiformis isolates from multiple sources add to genetic diversity within the genus.

Vermamoeba vermiformis represents one of the most common free-living amoebae identified in worldwide environmental surveys. We analyzed 56 water samples with varying characteristics, including temperature and the particular settings in which humans may be exposed to water, plus one corneal scraping from a keratitis patient, with the following aims: (i) to investigate the presence of V. vermiformis; (ii) to identify the isolate subtypes; (iii) to place the Italian isolates in the broader picture of the genetic diversity within V. vermiformis. Twenty-two isolates were identified upon culturing and sequencing of > 600 bp in the 18S ribosomal RNA (rRNA) gene sequence, bringing to 27 the number of sequences recovered from Italian sources. By adding deposited sequences, we assembled a dataset of 74 isolates. Three of our isolates were characterized by allelic code 7-5-1-1, never reported before, and two showed 100% identity with an uncultured eukaryote and carried the 719T>C variant. We show that the variable segments E5, E3, F, and G convey most of the information on diversity, enabling the clustering of the isolates in a replicable fashion. The presence of different strains in natural thermal waters and in distribution systems indicated heterogeneity of the amoebic populations. Also, ours and the only other sequence from human infection were mapped in different clades. Overall, we enlarged the repertoire of single nucleotide and indel variants and the list of allelic codes, proceeding one step further in the description of the diversity within the genus.

Molecular identification of Mesocestoides sp. metacestodes in a captive gold-handed tamarin (Saguinus midas).

Mesocestoides spp. are tapeworms harbored in the intestine of many domestic and wild carnivores, birds of prey and, rarely, of humans. The life cycle is assumed to involve three hosts and the metacestode juvenile stage (tetrathyridium) may reproduce asexually, provoking severe systemic infections. In the present study, a case of a peritoneal infection by Mesocestoides sp. occurred in a captive gold-handed tamarin (Saguinus midas) is described. Phylogenetic positions based on CO1 and 12S loci were analysed to describe in details this unusual finding of Mesocestoides in a callitrichid monkey. The phylogenetic analyses has not clearly resolve relationships between the metacestode forms from the tamarin and any of the described Mesocestoides species available so far, including those described from non-human primates, thus supporting the hypothesis of a new taxon within the genus.

Identification and phylogenetic position of Naegleria spp. from geothermal springs in Italy.

Naegleria spp. are free-living amoebae belonging to the family Vahlkampfiidae, in the class Heterolobosea. Among the recognized species, Naegleria fowleri causes primary amoebic meningoencephalitis (PAM), while two other species, Naegleria australiensis and Naegleria italica, have been reported as pathogenic in experimental animals. Due to the thermotolerance properties of some species, geothermal water sources including hot springs represent suitable habitats for their proliferation. The main aim of this study was a year-round sampling in two geothermal springs in Central Italy, to investigate the presence of Naegleria spp. using PCR/DNA sequencing based methods. The affinities between the sequences generated here and others reported in the literature were explored by using POY, which implements the concept of dynamic homology. Naegleria australiensis, Naegleria italica, and Naegleria lovaniensis, plus an unassigned Naegleria spp. were detected. Indels in the rDNA ITS1 and ITS2 turned out to be critical to distinguish the three species and confirmed their phylogenetic relationships. This is the first molecular report on the Naegleria spp. occurrence in geothermal waters in Central Italy, coupled with a fine genetic characterization.

Blastocystis in Côte d'Ivoire: molecular identification and epidemiological data.

Blastocystis is an enteric protozoan infecting humans and animals in both developed and developing countries at all latitudes. Despite this, data on Blastocystis infection are not available for several geographical areas, including many African countries. In this study, a survey was conducted on Blastocystis among humans and domestic animals in rural and urban localities in Côte d'Ivoire, in order to investigate the prevalence, the subtype distribution, and the zoonotic potential in association with sociodemographic factors, seasonality, symptoms, and co-infections. A total of 110 fecal samples were collected from patients living in four localities. Molecular and phylogenetic analyses were conducted for Blastocystis detection and subtyping. Positive samples from symptomatic patients were tested by Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) to evidence the presence of other common intestinal pathogens. Overall, a prevalence of 58.2% was observed in humans and subtypes ST1(50.0%), ST2 (22.0%) and ST3 (28.1%) were identified. The prevalence values varied significantly among the sites but not in relation to the subtype. The seasonal rains significantly increase the infection rate in all localities. No significant differences in the ST distribution between asymptomatic and symptomatic subjects were observed. As regard the zoonotic transmission, an additional sampling was conducted in another village where fecal samples were simultaneously collected from humans and animals. Blastocystis STs 1-3 and ST7 were identified in eight humans and four chickens, respectively. This study provides the first exhaustive data on the prevalence and molecular epidemiology of Blastocystis in Côte d'Ivoire.

Social determinants associated with Giardia duodenalis infection in southern Côte d'Ivoire.

The purpose of this investigation was to analyze the association between different social determinants of health at the local scale and Giardia duodenalis infection in diverse settlements in southern Côte d'Ivoire. Stool samples from 306 individuals aged 1-16 years were collected from six rural villages and a small town. Five variables were categorized to classify the increasing risk levels of acquiring intestinal parasites. Giardia prevalences (%) and odds ratios (ORs) were evaluated. Correlation and regression analyses were conducted to determine the correlation coefficients and to propose predictive models based on social determinants to forecast the risk of giardiasis. The overall observed prevalence of Giardia was 21.6 %. When the analysis was conducted at the local level, the percentage of infected people varied from a minimum of 12.7 up to 36.4 %. A significant association (p < 0.001) was found between the selected social determinants and G. duodenalis prevalence in the different localities. Correlation and regression analyses allowed us to describe two predictive models to estimate the OR of Giardia transmission. This study helps to clarify the possible impact of different social determinants of health on the risk of giardiasis at the local scale. Both predictive models could be suitable in order to assess the likelihood of the transmission of intestinal parasitic infection in developing countries.

Genetic characterization of Giardia duodenalis by sequence analysis in humans and animals in Pemba Island, Tanzania.

Giardia duodenalis represents one of the most widespread human enteric parasites: about 200million people in Asia, Africa and Latin America are infected. Giardia exerts a deep impact on public health because of high prevalence and possible effects on growth and cognitive functions in infected children. The major aim of this study was to detect and genetically characterize G. duodenalis in both human and animal fecal samples collected in Pemba Island, in the archipelago of Zanzibar (Tanzania), in order to deepen the knowledge of genotypes of Giardia in this area. Between October 2009 and October 2010, we collected 45 human fecal samples from children from 2 primary schools and 60 animal fecal samples: 19 from zebus (Bos primigenius indicus) and 41 from goats (Capra hircus). Detection and genetic identification were performed by multilocus analysis of ssu-rDNA and gdh genes. In humans we found a higher prevalence of assemblage B (sub-assemblage BIV), in goats of assemblage E and in zebus of assemblage A. Our study represents an important contribution to the epidemiological knowledge of G. duodenalis in this area of Tanzania.

Molecular analysis based on mtLSU-rRNA and DHPS sequences of Pneumocystis jirovecii from immunocompromised and immunocompetent patients in Italy.

Pneumocystis jirovecii is an opportunistic fungus predominantly reported in immunocompromised individuals, who develop severe interstitial pneumonia (PcP). However, it is known that asymptomatic or mild pulmonary infections, defined as colonization, are widely observed in the general adult population. So far, genetic and epidemiological data of P. jirovecii infections in Italy are rather scarce and limited to defined geographical regions, mainly regarding isolates from HIV-infected patients. The aim of this study was to evaluate the polymorphisms at the mtLSU-rRNA and the DHPS loci by the screening and genotyping of a cohort of patients from two major hospitals in Rome (Italy). The study included 263 patients divided into two groups, all enrolled consecutively from January 2006 to December 2010: (i) 38 immunocompromised subjects including 25 HIV-infected; (ii) 225 immunocompetent patients. Sixty-seven out of 263 patients (25.5%) were found positive after PCR amplification of the mtLSU-rRNA gene. Overall, genotyping at mtLSU-rRNA locus revealed that the genotype 2 was the most frequent. Sequences of the DHPS gene were obtained from 21 patients, 9 from immunocompromised patients (6 from HIV infected individuals), 12 from immunocompetent ones. Considering the most common DHPS mutations usually detected at amino acid positions 55 and 57 and potentially related to drug resistance, all samples analyzed showed the wild-type signatures. These are the first data in Italy on prevalence and genotypes of P. jirovecii regarding colonized immunocompetent adults. Further multicenter analyses on P. jirovecii infection will be necessary to better define the specific epidemiology of the disease in the Italian populations.

Investigation on Opisthorchis felineus occurrence and life cycle in Italy.

Opisthorchiasis is a fish borne parasitic infection caused by helminths of the genus Opisthorchis (Digenea, Opisthorchiidae), affecting humans and other fish-eating mammals. Despite Opisthorchis felineus was first described in Italy in 1884, no cases of human opisthorchiasis were reported in this country until 2004; from then on, 4 outbreaks due to this species have been recorded in Central Italy. Following the more relevant of these outbreaks, involving 34 people in August 2007, snails, fishes and fecal samples collected from the Bolsena and Bracciano lakes (Central Italy) were analyzed in order to define the cycle of O. felineus in the area and investigate its prevalence in the different hosts. Pools of 20-40 snails each (4983 specimens altogether) of the genus Bithynia were analyzed by PCR for parasite DNA detection. Eight hundred and ninety-four fish belonging to 12 species were collected from the two lakes and tested for metacercariae both by muscle compression and digestion techniques. Eighty-seven fecal samples of 5 putative definitive host species were collected very close to the two lakes and tested for parasite eggs detection by formalin-ethyl acetate concentration technique. Identification at the species level of metacercariae and eggs, respectively, from fish and stool was confirmed by PCR analysis and sequencing. O. felineus DNA was detected in 0.08% (overall minimum infection rate) of snails of the genus Bithynia from the two lakes. The tench, Tinca tinca, was the only fish found infested in both lakes (prevalence 88.5%). O. felineus eggs were found only in cat feces (prevalence 46.4%). The tench represents the only threat for the human consumption in the study area while Coregonus sp., the most economically important species for the local fishery and frequently consumed raw marinated, resulted to be not infected. The high prevalence recorded both in fish and in definitive host suggests a widespread and massive presence of the parasite in the area. Further studies are needed to better investigate the possible role of some cyprinids species as intermediate hosts, in order to check their safety for human consumption.

Investigation of Toxoplasma gondii presence in farmed shellfish by nested-PCR and real-time PCR fluorescent amplicon generation assay (FLAG).

To evaluate the presence of Toxoplasma gondii in edible farmed shellfish, 1734 shellfish specimens i.e., 109 Crassostrea gigas (6 pools), 660 Mytilus galloprovincialis (22 pools), 804 Tapes decussatus (28 pools) and 161 Tapes philippinarum (6 pools), were collected from the Varano Lagoon (Apulia, Italy). Shellfish from 62 pools were subjected to two molecular techniques: a nested-PCR assay, and a fluorescent amplicon generation (FLAG) real-time PCR assay, both based on the multi-copy B1 target, were performed. One pooled sample of gills from C. gigas and one pooled sample of haemolymphs from T. decussatus were assessed as positive for T. gondii DNA by both techniques. The results demonstrated the presence of T. gondii in edible farmed C. gigas and T. decussatus and indicate that there may be a considerable health threat involved in eating contaminated raw shellfish.

Genotyping of Giardia duodenalis among children and dogs in a closed socially deprived community from Italy.

Molecular characterization of Giardia duodenalis cysts from humans and animals living in well-defined contexts is useful to study the circulation of isolates and represents a tool to evaluate zoonotic infection risk. The presence of giardiasis in children living in a disadvantaged and socially deprived small Rom community, as well in dogs roaming freely in the same context was carried out by microscopic analysis and beta-giardin gene amplification. Five out of 14 children were found positive at microscopic examination for G. duodenalis and six positive at PCR, while eight out of 14 dogs tested both microscopically and molecularly positive for G. duodenalis. Moreover, most of the children and dogs were symptomatic. Molecular characterization of Giardia positive samples from children and dogs showed 99.5% identity with Giardia Assemblage A1. The dog-specific genotypes C and D were not found. The findings of this survey provide the first European evidence to support the possible role of dogs in zoonotic transmission involving children and stray dogs in a closed context with very low standards of hygiene (i.e. Rom community), and these results show the need to monitor the health of marginal populations to safeguard ethnic minority groups.

Acanthamoeba T4 and T15 genotypes associated with keratitis infections in Italy.

Thus far there is little data available concerning Acanthamoeba associated amoebic keratitis (AK) from Italy. In order to understand the incidence of Acanthamoeba in patients with ocular infections and to characterize the isolates at the molecular level, ocular specimens and contact lenses or lens case solutions from 140 patients were analysed by culture and by an 18S rRNA (Rns) gene-based PCR method. Nineteen (13.6%) patients showed Acanthamoeba culture positive samples. Eleven out of the 14 genetically characterized isolates were assigned to the T4 genotype. Three isolates, two of them from patients with keratitis responding to specific anti-Acanthamoeba therapy, were identified as belonging to the T15 genotype. This finding represents the first association between the T15 genotype and human amoebic keratitis. PCR amplification of the 18S ribosomal DNA proved to be a sensitive method, potentially able to detect Acanthamoeba without the need of long culture incubation, and thus considerably useful for clinical applications.

Giardia cysts and Cryptosporidium oocysts in membrane-filtered municipal wastewater used for irrigation.

A wastewater tertiary treatment system based on membrane ultrafiltration and fed with secondary-treated municipal wastewater was evaluated for its Giardia cyst and Cryptosporidium oocyst removal efficiency. Giardia duodenalis (assemblages A and B) and Cryptosporidium parvum were identified in feed water but were found in filtered water only during occasional failure of the filtration system.

Prevalence and genotyping of human isolates of Giardia duodenalis from Albania.

Microscopical and PCR-based techniques were performed in order to investigate the prevalence of infection and the genotypes of Giardia duodenalis from 125 stool samples collected from children living in the urban and the rural areas of Tirana (Albania) and hospitalized with acute gastroenteritis. 7 out of 125 samples resulted positive for Giardia at the microscopic examination (5.6%). In 50 selected samples including the 7 samples positive for Giardia by microscopy, 3 and 15 additional positive samples were detected by immunofluorescence and PCR, respectively. Seasonality appeared as an important parameter to be evaluated in order to better understand the prevalence of infection. Sequence analysis revealed both human Assemblage A and B. This result represents the first data on G. duodenalis genotypes in Albania.

Ribosomal and mitochondrial DNA sequence variation in Sarcoptes mites from different hosts and geographical regions.

In order to investigate the extent of the genetic variation in the DNA sequences of Sarcoptes scabiei, mite populations collected on Alpine chamois ( Rupicapra rupicapra), Pyrenean chamois ( Rupicapra pyrenaica) and red fox ( Vulpes vulpes) from different localities of Italy and Spain were studied. Sequence analyses were carried out on the second internal transcribed spacer of the nuclear ribosomal DNA and on the 16S mitochondrial rRNA gene. ITS-2 sequences showed a higher degree of genetic polymorphism, mostly randomly distributed in the isolates from different hosts and localities, although both genomic regions are characterised by fixed nucleotide substitutions which were able to discriminate the mites collected on Pyrenean chamois from north-western Spain and on foxes from north-eastern Spain and from north-western Italy with respect to the other isolates. These results suggest the existence of a limitation to free gene exchange between the studied populations, probably related to the genetic structuring of local populations rather than to a differential adaptation to host species.

Helminth communities in eels Anguilla anguilla from Adriatic coastal lagoons in Italy.

The composition and diversity of the total and intestinal component and infra-communities were determined in eels Anguilla anguilla from three shallow lagoons on the Adriatic coast of Italy to determine whether the helminth communities would differ in composition and structure from those in eels from lagoons on the Tyrrhenian coast. The lagoons differed in respect of their management regimes and the extent of freshwater influx. Both freshwater and marine species of helminths were found in the eels in all three lagoons, but the freshwater component was richer in Valle Figheri. A suite of three digenean eel specialist species occurred in all three lagoons, of which any two members dominated each community. This conferred a high degree of similarity between the communities of the three lagoons. The same three species also dominated helminth communities in eels in lagoons along the Tyrrhenian coast of Italy, and compositional similarity levels were similar within and between western and eastern groups. Species richness was higher in the component communities of the eels of the Adriatic lagoons when compared to the Tyrrhenian ones, but diversity and dominance indices were of a similar order of magnitude and range. Intestinal helminth communities were richer and more diverse in two of the Adriatic lagoons because the proportion of eels with zero or one helminth species was, unusually, in the minority. It was nevertheless concluded that infracommunity structure was similar in eels from both western and eastern lagoons and that the hypothesis that it would differ in Adriatic lagoons could not be supported. The findings provide further evidence of the similarity in composition and structure of helminth communities in eels from coastal lagoons throughout Europe.

Composition and diversity of helminth communities in eels anguilla anguilla in the river tiber: long-term changes and comparison with insular europe

Most studies of helminth communities in the European eel Anguilla anguilla have been undertaken in the British Isles, and there are very few analyses of community composition and structure from continental Europe. To fill this gap and test the hypothesis that helminth communities in freshwater eels in the British Isles are not typical of those of continental Europe, helminth communities of eels in the River Tiber below Rome were analysed by season using data collected in 1980 and new data from 1996. The intestinal helminth communities in the Tiber eels were species poor and characterized by low diversity. Most eels harboured one or no parasite species and communities were heavily dominated by the acanthocephalan Acanthocephalus clavula. Intestinal helminth infracommunity richness and diversity did not differ between seasons within a year or between the same seasons in 1980 and 1996, although some changes in composition were apparent. Intestinal infracommunities from Tiber eels were very similar in characteristics to those analysed from the British Isles, and their temporal changes also showed close similarities to those reported from rivers in the UK. It seems likely therefore that conclusions derived from British studies can be applied to helminth communities of eels on the continent.

Composition and structure of helminth communities in eels Anguilla anguilla from Italian coastal lagoons.

The composition and diversity of total and intestinal helminth component and infracommunities were determined in eels Anguilla anguilla from four shallow coastal lagoons near Rome. The lagoons differed principally in respect of their salinity. Only Lake Burano still received an input of freshwater, and both freshwater and marine helminth species were found in eels. In the other three lagoons all helminth species were marine or euryhaline and communities were dominated by digeneans. At component community level these three lagoons were far more similar to each other than to Lake Burano, but this distinction almost disappeared at infracommunity level. Species richness and diversity declined with increasing salinity at component community level but not at infracommunity level. Despite the changes and differences in helminth community composition, intestinal helminth infracommunity structure was very similar in all four lagoons and to that in eels from freshwater localities in Britain. The findings thus provide further and unexpected support for the view that some factor(s) other than supply side ones must be operating to produce the fundamental structural similarity in helminth communities in eels.