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

The effect of COVID-19 on the frequency of Pneumocystis jirovecii pneumonia: a monocentric, retrospective, and observational study

Background: Pneumocystis jirovecii pneumonia (PCP) and SARS-CoV2 share some similarities in their effects on the respiratory system, clinical presentation, and management. The COVID-19 pandemic required rapid action to curb transmission and mitigate its lethiferous impact. Non-pharmaceutical interventions (NPIs) were globally adopted. We hypothesized that these measures reduced the transmission and acquisition of P. jirovecii in both hospital and community settings. Methods: We conducted a retrospective observational study on 2950 respiratory specimens from patients with suspected pulmonary infection, analyzed at the Laboratory of Parasitology Unit of the Policlinico Tor Vergata of Rome, Italy, from January 2014 to December 2022. Results: We show a significant reduction in the frequency of PCP in the COVID-19 pandemic era compared to the previous period. Among the four sequence types of P. jirovecii identified, genotype 1 was the most prevalent (37%). We observed a non-significant trend of decreasing cases with genotype 1 and increasing cases with genotype 3 over the study period. Conclusions: The nationwide implementation of NPIs against COVID-19 may have changed the microbiological landscape of exposure, thereby decreasing the exposure to P. jirovecii and consequently reducing the incidence of PCP

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

Single and Synergistic Effects of Fenbendazole and Metronidazole Against Subclinical Infection by Giardia duodenalis in Non-Human Primates in a Zoological Garden in Southern Italy

The aim of this study was to assess the single and synergistic effects of fenbendazole (Fenb) and metronidazole (Metro) for the treatment of Giardia duodenalis infection in different species of non-human primates (NHPs) housed in a zoological garden of southern Italy. Moreover, the study also aimed to better define the circulation of G. duodenalis zoonotic assemblages in NHP and the potential occurrence of zoonotic transmission between the staff from the zoo and NHP. Briefly, six species that belonged to four families (Lemuridae, Cercopithecidae, Atelidae, and Hylobatidae) of NHP and housed in six cages (CG) were identified as Giardia positive and divided into two groups. Group F (N = 16 animals) was treated with Fenb (50 mg/kg, every 24 h for 5 consecutive days) and Group M (N = 7 animals) was treated with Metro (25 mg/kg, two times a day for 5 consecutive days). After the first round of therapy, all the animals were retreated for 5 days by inverting the drugs in each group. On each sampling day [study days (SDs) 3-24], the samples were tested for the presence of Giardia cysts using the FLOTAC technique. Multiple fecal tests for the antigen detection of Giardia, such as rapid ELISA and direct immunofluorescence (IFA), were performed at each sampling point only on samples that resulted in positive for Giardia cysts with FLOTAC. The efficacy of Fenb ranged from 30 to 67% and for Metro ranged from 82 to 96%. The results showed the synergistic effects of Metro and Fenb (98-100%) over the combination of Fenb and Metro (52-90%) against the infection by Giardia in NHPs. The overall k agreement between FLOTAC and IFA was reached 0.858 (p = 0.0001). In contrast, all the samples had a negative antigen result when using ELISA. At molecular analysis, six samples were confirmed positive for Giardia by nested PCR. Only two positive samples were successfully sequenced that showed 100% of identity with assemblage B. All the samples from the humans included in the study resulted in negative for Giardia cysts. Overall, the study emphasizes the need for regular monitoring of Giardia infections in NHP housed in zoos by traditional diagnostic tools combined with molecular characterization of the parasite

Prevalence, Risk Factors and Genotypes of Giardia duodenalis in Sheltered Dogs in Tuscany (Central Italy)

: In sheltered dogs, the prevalence of Giardia duodenalis is frequently high and may include potential zoonotic genotypes. The prevalence, genotypes and potential risk factors of G. duodenalis were assessed in 168 dogs from four kennels (Pistoia, Prato, Florence, Valdarno) in Tuscany, central Italy and compared with data from previous Italian studies. The prevalence of other intestinal parasites was also investigated. Individual dog faecal samples collected from each kennel were examined by parasitological techniques and a rapid immunoassay for the detection of G. duodenalis and Cryptosporidium faecal antigens. On Giardia-positive samples, molecular analysis was performed for genotype identification. Overall, 69 dogs scored positive for G. duodenalis (41%), but significant differences (p ≤ 0.05) were found among the four kennels and sampling seasons. The potentially zoonotic assemblages A and B and the canine-specific assemblage C (Pistoia: A-AII, B, C; Prato: A-AII, B; Florence: A-AII; Valdarno: A and C) were identified. Toxocara canis (8.9%), Trichuris vulpis (3.6%), hookworms (1.19%) and Cryptosporidium sp. (0.6%) were also identified. The high prevalence of G. duodenalis and the identification of potentially zoonotic genotypes in all examined kennels underline the need to improve routine parasite monitoring and control measures and to provide insights into the zoonotic potential of G. duodenalis

Giardia duodenalis assemblages and Entamoeba species infecting non-human primates in an Italian zoological garden: zoonotic potential and management traits

<p>Abstract</p> <p>Background</p> <p><it>Giardia duodenalis </it>and <it>Entamoeba </it>spp. are among the most common intestinal human protozoan parasites worldwide and they are frequently reported in captive non-human primates (NHP). From a public health point of view, infected animals in zoos constitute a risk for animal caretakers and visitors. In this study we carried out the molecular identification of <it>G. duodenalis </it>and <it>Entamoeba </it>spp. from nine species of primates housed in the zoological garden of Rome, to better ascertain their occurrence and zoonotic potential.</p> <p>Results</p> <p><it>G. duodenalis </it>was found only in <it>Lemur catta </it>(47.0%). <it>Entamoeba </it>spp. were detected in all species studied, with the exception of <it>Eulemur macaco </it>and <it>Varecia rubra</it>. The number of positive pools ranged from 5.9% in <it>L. catta </it>to 81.2% in <it>Mandrillus sphinx; </it>in <it>Pan troglodytes </it>the observed prevalence was 53.6%. A mixed <it>Entamoeba</it>-<it>Giardia </it>infection was recorded only in one sample of <it>L. catta</it>. All <it>G. duodenalis </it>isolates belonged to the zoonotic assemblage B, sub assemblage BIV. Three <it>Entamoeba </it>species were identified: <it>E. hartmanni</it>, <it>E. coli </it>and <it>E. dispar</it>.</p> <p>Conclusions</p> <p>Our results highlight the importance of regularly testing animals kept in zoos for the diagnosis of zoonotic parasites, in order to evaluate their pathogenic role in the housed animals and the zoonotic risk linked to their presence. A quick detection of the arrival of pathogens into the enclosures could also be a prerequisite to limit their spread into the structure via the introduction of specific control strategies. The need for molecular identification of some parasite species/genotype in order to better define the zoonotic risk is also highlighted.</p

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

Zoonotic Giardia duodenalis Genotypes and Other Gastrointestinal Parasites in a Badger Population Living in an Anthropized Area of Central Italy

The Eurasian badger (Meles meles) is widespread in Italy and occupies different habitats. The occurrence and species of gastrointestinal parasites were evaluated in a free-ranging badger population living in a highly anthropic area in central Italy. A total of 43 fecal samples were examined using the flotation test, the Mini-FLOTAC and Baermann techniques, and a rapid immunoassay for the detection of Giardia duodenalis and Cryptosporidium spp. fecal antigens. Molecular investigations were also performed that aimed at identifying Giardia genotypes. Overall, 37/43 samples (86%) were found positive. Specifically, 48.8% (21 samples) were positive for G. duodenalis, 23.2% (10/43) for Cryptosporidium spp., and 7% (3/43) for coccidian oocysts. Strongyloides sp. nematode larvae were detected in 3/43 samples (7%). Ascarid (1/43, 2.3%), capillariid (1/43, 2.3%), and strongyle-type eggs (76.7%, 33/43) were also identified. Among the 11 readable sequences of samples that were positive for G. duodenalis by end-point PCR (18/21), the zoonotic assemblage A sub-assemblage AII and mixed assemblage A and B were identified. This is the first report of zoonotic G. duodenalis genotypes in the Eurasian badger. Moreover, most of identified parasites have zoonotic potential and/or potential impact on the population health of wild badgers and other wild and domestic animals

Intestinal parasites in Rupicapra spp. populations: study in the framework of the relevant italian project (PRIN)

Intestinal parasites can seriously threaten the performances and well-being of wild ungulates. In this study, we investigated the occurrence and parasitic burden of protozoans and gastro-intestinal helminths (GIH) in Rupicapra spp. From September 2013 to January 2016, 352 fresh fecal samples were collected from Rupicapra rupicapra rupicapra in the Alps (N=262) and from Rupicapra pyrenaica ornata in the Apennines (N=90). Samples were examined using standard copro-parasitological methods for Eimeria and GIH and an immunofluorescence test for Cryptosporidium and Giardia duodenalis. Parts of gp60 and ssRNA/gdh/βgiardin genes were used to identify these protozoa species/genotypes. In R.r.rupicapra and in R.p.ornata, 7 and 6 parasite taxa were identified, respectively, with a mean number of 1.7 species/host (min-max 0-5) and 2.05 (min-max: 0-4), respectively. Overall, 85.3% (95%, C.I.=81.5-89.1) of the animals investigated scored microscopically positive to Eimeria spp. with a mean intensity of emission (m.i.e.) of up to 776 o.p.g.; 5.4% (95%, C.I.=3.07.7) were positive to G. duodenalis and 82% (95%, C.I. 77.91-86.15) to GIH with a m.i.e. of up to 147 e.p.g. Prevalence in R.r.rupicapra was 81.2% with a m.i.e. of 380 o.p.g. for Eimeria, 6.87% for Giardia, and 77.45% for GIH with a m.i.e. of 142 e.p.g. Prevalence in R.p.ornata was 94.4% with m.i.e. of 1,093 o.p.g. for Eimeria, 1.1% for G.duodenalis, and 94.4% for GIH with a m.i.e. of 151 e.p.g. Assemblages A/AI and E were identified in R.r.rupicapra and assemblage A/AIII in R.p.ornata. None of the animals tested positive for Cryptosporidium. The results show that the prevalence of Eimeria, G.duodenalis and GIH in both host species is nonnegligible, with a significantly higher parasitic burden in R.p.ornata. The detection of G. duodenalis in Rupicapra spp. is noteworthy. This study updates the data on parasitic fauna of these wild bovids. The impact of these parasites on chamois population dynamics will be inferred from the results/variables obtained throughout the entire interdisciplinary project

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. (c) 2006 Elsevier Ireland Ltd. All rights reserved