Comparative evaluation of UNEX-based DNA extraction for molecular detection of Cyclospora cayetanensis, Toxoplasma gondii, and Cryptosporidium parvum as contaminants of berries

Abstract The potential public health impact of foodborne parasites transmitted via contaminated fresh produces indicates the necessity for robust and reliable laboratory methods for their detection and identification on this infection vehicle. Standardization of methods for detection of common FBP in fresh produce is to be expected and ensuring that the DNA extraction approach is most appropriate for the FBP of interest and for the matrix being analyzed is also important. Therefore, the aim of the present study was to compare the efficacy of two commercially available DNA extraction procedures, the UNEX-based method and DNeasy PowerSoil kit in the detection of three protozoan parasites, C. cayetanensis, C. parvum, and T. gondii, on contaminated berries. Oocysts of each parasite were spiked into the pellets of raspberry and blueberry washes. The spiked pellets were then randomly assigned to DNA extraction using either the PowerSoil or UNEX method, with DNA extraction with both methods performed by two independent analysts. The detection rate when berry washes were spiked with 20 oocysts of C. cayetanensis, T. gondii, and C. parvum was 95%, 85%, and 40%, respectively, when using the PowerSoil kit; whereas the equivalent results using the UNEX method were 55%, 60%, and 5%, respectively. In addition, significantly lower Cq values were achieved for each parasite in the samples spiked with 500 oocysts when the PowerSoil kit was used. Possible reasons for these results are discussed, and include the composition of both the beads and the buffers in each method

Accidental Nasal Myiasis Caused by Megaselia rufipes (Diptera: Phoridae) in a Child

A case of a nasal myiasis in a 3-yr-old Italian girl who was referred to Bambino Gesù Hospital in Rome, Italy, is reported. Larvae discharged with the nasal mucus were microscopically identified as Megaselia spp.; DNA barcoding analysis showed that they belonged to the 'scuttle fly' species Megaselia rufipes (Meigen). Based on the patient's history, she became infected when she played outside. This is the first report of myiasis in humans due to M. rufipes (Diptera: Phoridae)

Leishmania infantum Seroprevalence in Cats From Touristic Areas of Italy and Greece

Leishmaniosis by Leishmania infantum is a major zoonotic Vector-Borne Disease (VBD) in terms of geographic distribution, pathogenicity and zoonotic potential. While dogs are the main reservoir of L. infantum, the infection in cats is poorly understood although increasingly reported from enzootic and non-enzootic areas. The Mediterranean basin is a key area for leishmaniosis and includes touristic spots that require continuous surveillance for VBDs in consideration of the growing tendency of tourists to travel with their pets. This study evaluated L. infantum seroprevalence in cats living in selected touristic localities of Italy and Greece. A total of 269 cat serum samples from three Sites i.e., 76, 40, and 153 from Adriatic Coast of Abruzzo, Italy (Site A), Giglio Island, Tuscany, Italy (Site B), and Mykonos Island, Greece (Site C), respectively, were included in the survey. Sera samples were subjected to an indirect immunofluorescence antibody assay for the detection of anti-L. infantum specific IgG. Associations between possible risk factors and seropositivity to L. infantum were statistically evaluated. Antibodies against L. infantum were detected in eight out of 269 (3.0%) cats tested i.e., 4/76 (5.3%), 1/40 (2.5%), and 3/153 (2.0%), from sites A, B, and C, respectively. A statistical association between anti-L. infantum antibodies and cohabitation with dogs was shown. This study indicates that feline populations living in the examined Italian and Greek touristic areas are exposed to L. infantum and that they may contribute to the circulation of L. infantum, enhancing the risk of infection for dogs and humans

What's in a child's ear? A case of otomyiasis by Sarcophaga argyrostoma (Diptera, Sarcophagidae)

A clinical report of otomyiasis in a 1-year-old girl is reported. A III instar larva of Sarcophaga sp. was microscopically identified and Sarcophaga (Liopygia) argyrostoma (Diptera, Sarcophagidae) was suspected. A molecular method targeting a fragment of the cox1 gene was used to confirm the identity of the specimen. Although myiases are not frequent manifestations in otolaryngology, they should arouse the attention of doctors, social workers and parents dealing with disabled people, the elderly and children. This contribution also highlights the need of combining microscopy and molecular tools to achieve a correct and reliable identification of the specimen/s

Zoonotic intestinal helminthes diagnosed in a 6-year period (2015–2020) in privately owned dogs of sub-urban and urban areas of Italy

Intestinal helminthes of dogs have a relevant role for health and welfare of canine populations and many of them are zoonotic. Thus, a constant surveillance is pivotal to safeguard companion dogs and human health. Data from faecal examinations of pet dogs obtained in the last six years during the routine activity of the laboratory of Parasitology and Parasitic diseases of the University Teaching Veterinary Hospital of the University of Teramo, Italy, are here reported. From 2015 to 2020 a total of 1083 individual faecal samples from privately owned dogs were examined using conventional copro-microscopic techniques. Endoparasite elements were found in 278 (25.7%) dogs, and among them, eggs of intestinal zoonotic helminthes were detected in 184 (17%) samples. The most common zoonotic parasites were Toxocara canis and hookworms (8.9% positivity rate for both). Most dogs (88%) were positive for a single zoonotic parasite while 12% of the positive animals harbored two or more zoonotic helminthes. A statistical analysis showed a significant association between dogs with less than one year of age and positivity to at least one intestinal zoonotic helminth, and that infections by T. canis are significantly more prevalent in dogs under one year of age. Despite being well known in canine clinical practice, these results indicate that zoonotic helminthes are still frequent in owned dogs. Thus, increased awareness of veterinary practitioners and pet owners is necessary to implement current diagnostic and control strategies to minimize the risk of zoonotic transmission of intestinal helminthes

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

Contamination of fresh produce sold on the Italian market with Cyclospora cayetanensis and Echinococcus multilocularis

To investigate the presence of Cyclospora cayetanensis, Toxoplasma gondii and Echinococcus spp. in fresh produce sold in Italy, 324 locally produced ‘ready-to-eat’ (RTE) mixed-salad packages belonging to three brands and 324 berries packages (blueberries and blackberries imported from Peru and Mexico, respectively, and raspberries grown in Italy) were purchased at retail. Nine individual packages from each of the six types of fresh produce were collected monthly for one year, and with the same produce pooled, this resulted in a total of 72 pools for the whole year. Using microscopy (FLOTAC), a Cyclospora-like oocyst was detected in a blueberry sample and a taeniid egg was detected in a RTE-salad sample. Molecular tools confirmed these to be C. cayetanensis and Echinococcus multilocularis, respectively. Toxoplasma gondii was not detected in any of the samples. This study shows for the first time in Europe that imported berries on the Italian market may be contaminated with C. cayetanensis and RTE salads grown in Italy with E. multilocularis. The results indicate a new epidemiological scenario and highlight that current management of fresh produce, locally produced or imported, does not ensure products are free from parasite contamination

Ready-to-eat salads and berry fruits purchased in Italy contaminated by Cryptosporidium spp., Giardia duodenalis, and Entamoeba histolytica

Ready-to-eat (RTE) salads and berries are increasingly consumed in industrialized countries. These products can be contaminated by pathogenic parasites that have been responsible for foodborne outbreaks worldwide. In Italy, there are few data on contamination of RTE salads and berries with parasite transmission stages and this requires more-in-depth investigations. To estimate the prevalence of contamination with Cryptosporidium spp. and Giardia duodenalis in these fresh products, a total of 324 packages of local RTE mixed salads - belonging to three different industrial brands - and 324 packages of berries - blueberries from Peru, blackberries from Mexico, raspberries from Italy - were bought from supermarkets located in the Provinces of Bari and Foggia, Apulia, Italy. A pool size of nine packages was chosen and a total of 72 pools were processed in the whole year. After washing, the pellets were examined by microscopy (FLOTAC) and tested using conventional simplex PCR, targeting Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp., and sequencing. Several Cryptosporidium species and Giardia duodenalis assemblages, some of which are of potential zoonotic relevance, as well as Entamoeba spp., were identified in both matrices. By microscopy, Giardia-like cysts in local raspberries and Entamoeba-like cysts in imported blueberries were detected. Giardia duodenalis (Assemblages A, B and E) and Entamoeba histolytica were molecularly confirmed with overall prevalences of 4.6% (95% C.I. 3.0-6.8) and 1% (95% C.I. 0.3-2.1), respectively. Molecular methods identified Cryptosporidium ryanae, Cryptosporidium bovis, Cryptosporidium xiaoi, and Cryptosporidium ubiquitum in both matrices, with a prevalence of 5.1% (95% C.I. 3.3-7.3). A distinct seasonality in prevalence was observed for G. duodenalis, with most positives occurring in spring, whereas Cryptosporidium showed no significant seasonal variations. These results highlight that inadequate management of fresh produce, both locally produced and imported, along the food chain may have the potential for consequences on human health

Ready-to-eat salads and berry fruits purchased in Italy contaminated by Cryptosporidium spp., Giardia duodenalis, and Entamoeba histolytica

Ready-to-eat (RTE) salads and berries are increasingly consumed in industrialized countries. These products can be contaminated by pathogenic parasites that have been responsible for foodborne outbreaks worldwide. In Italy, there are few data on contamination of RTE salads and berries with parasite transmission stages and this requires more-in-depth investigations. To estimate the prevalence of contamination with Cryptosporidium spp. and Giardia duodenalis in these fresh products, a total of 324 packages of local RTE mixed salads - belonging to three different industrial brands – and 324 packages of berries - blueberries from Peru, blackberries from Mexico, raspberries from Italy - were bought from supermarkets located in the Provinces of Bari and Foggia, Apulia, Italy. A pool size of nine packages was chosen and a total of 72 pools were processed in the whole year. After washing, the pellets were examined by microscopy (FLOTAC) and tested using conventional simplex PCR, targeting Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp., and sequencing. Several Cryptosporidium species and Giardia duodenalis assemblages, some of which are of potential zoonotic relevance, as well as Entamoeba spp., were identified in both matrices. By microscopy, Giardia-like cysts in local raspberries and Entamoeba-like cysts in imported blueberries were detected. Giardia duodenalis (Assemblages A, B and E) and Entamoeba histolytica were molecularly confirmed with overall prevalences of 4.6% (95% C.I. 3.0–6.8) and 1% (95% C.I. 0.3–2.1), respectively. Molecular methods identified Cryptosporidium ryanae, Cryptosporidium bovis, Cryptosporidium xiaoi, and Cryptosporidium ubiquitum in both matrices, with a prevalence of 5.1% (95% C.I. 3.3–7.3). A distinct seasonality in prevalence was observed for G. duodenalis, with most positives occurring in spring, whereas Cryptosporidium showed no significant seasonal variations. These results highlight that inadequate management of fresh produce, both locally produced and imported, along the food chain may have the potential for consequences on human health

A Comparison of Copromicroscopic and Molecular Methods for the Diagnosis of Cat Aelurostrongylosis

The gold standard method for the diagnosis of cat aelurostrongylosis is the detection of Aelurostrongylus abstrusus first stage larvae with the Baermann’s examination. Nevertheless, molecular assays have shown higher diagnostic performances compared to copromicroscopy. This study evaluated the usefulness of an A. abstrusus species-specific PCR on different biological samples collected in clinical settings from 100 privately-owned cats in Italy (n. 60) and Greece (n. 40). A fecal sample was collected from each animal and a pharyngeal swab was also obtained for cats from Italy. All stool samples were subjected to flotation and Baermann’s test. The cats were categorized in three groups based on the results of copromicroscopy, i.e., Group A (n. 50 cats with A. abstrusus infection regardless of positivity for other helminths), Group B (n. 25 cats negative for A. abstrusus but positive for at least one of any other helminth), Group C (n. 25 cats negative for any helminth). DNA was extracted from individual aliquots of feces, flotation supernatant, Baermann’s sediment and the pharyngeal swab and then subjected to a PCR specific for A. abstrusus. At least one fecal aliquot or the pharyngeal swab scored positive by the A. abstrusus-specific PCR for 48/50 (96%) cats enrolled in Group A; in particular, 38/50 (76%), 35/50 (70%), 41/50 (82%) and 21/25 (84%) DNA extracts from feces, flotation supernatant, Baermann’s sediment and pharyngeal swabs were positive by PCR. These results confirm that molecular tools are highly sensitive and specific and indicate that pharyngeal swabs are the most suitable sample for molecular analysis in clinical settings