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

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

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

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

High Diversity of Giardia duodenalis Assemblages and Sub-Assemblages in Asymptomatic School Children in Ibadan, Nigeria

Giardia duodenalis is a significant contributor to the burden of diarrheal disease in sub-Saharan Africa. This study assesses the occurrence and molecular diversity of G. duodenalis and other intestinal parasites in apparently healthy children (n = 311) in Ibadan, Nigeria. Microscopy was used as a screening method and PCR and Sanger sequencing as confirmatory and genotyping methods, respectively. Haplotype analyses were performed to examine associations between genetic variants and epidemiological variables. At microscopy examination, G. duodenalis was the most prevalent parasite found (29.3%, 91/311; 95% CI: 24.3-34.7), followed by Entamoeba spp. (18.7%, 58/311; 14.5-23.4), Ascaris lumbricoides (1.3%, 4/311; 0.4-3.3), and Taenia sp. (0.3%, 1/311; 0.01-1.8). qPCR confirmed the presence of G. duodenalis in 76.9% (70/91) of the microscopy-positive samples. Of them, 65.9% (60/91) were successfully genotyped. Assemblage B (68.3%, 41/60) was more prevalent than assemblage A (28.3%, 17/60). Mixed A + B infections were identified in two samples (3.3%, 2/60). These facts, together with the absence of animal-adapted assemblages, suggest that human transmission of giardiasis was primarily anthroponotic. Efforts to control G. duodenalis (and other fecal-orally transmitted pathogens) should focus on providing safe drinking water and improving sanitation and personal hygiene practices.This research was funded by the Health Institute Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness, grant number PI19CIII/00029.S

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 beta-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

Final destination: The Mediterranean Sea, a vulnerable sea. The long journey of Giardia duodenalis cysts

The Mediterranean Sea is considered a “litmus paper” of pollution risks for any parameter, including faecal contamination. Giardia duodenalis is one of the most important protozoan parasites responsible for diarrhoea in a wide range of hosts, including humans, domestic and wild animals, worldwide. The degree of contamination related to the protozoan's resistant forms on land, and the consequent transport through rivers from point sources to the sea are important aspects to better understand the processes involved in the microbiological pollution of aquatic ecosystems. However, land-sea transfer routes and the complex transmission patterns often remain neglected. This contribution deals with the contamination by G. duodenalis of the Mediterranean Sea through its inhabitants (shellfish, marine mammals, fishes), and provides data on the origin of such contamination on land from humans and animals to soil, fresh produce and waters; this scenario allows to understand the long journey of the protozoan following the drainage basins (i.e., natural watersheds) from the mainland towards the final destination. The Mediterranean Sea contamination is also explained in the light of the Giardia survival in water and the effects of climatic change with the related consequences. Addressing faecal contamination threats in the Mediterranean Sea is a difficult task, but a number of mitigation measures need to be implemented and/or in some countries even applied. Effective management must become a priority in the agenda of policy makers of all Mediterranean Countries for the implementation of successful measures and can only be applied in the perspective of the One Health approach

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