Deer keds on wild ungulates in northern Italy, with a taxonomic key for the identification of Lipoptena spp. of Europe.

Deer keds (Lipoptena spp.) are blood-sucking ectoparasites of domestic and wild animals, and also accidentally of humans. In Europe, five Lipoptena spp. have been recorded, although the lack of specific taxonomic keys has often led to mistaken identification or to missing data. The present study aimed to develop an identification key of the European species and also to identify Lipoptena spp. found on wild ungulates in northern Italy. In total, 390 hippoboscids were collected from Rupicapra rupicapra, Capreolus capreolus, Cervus elaphus and Ovis aries musimon in an Alpine area of Italy. After morphological identification, 140 specimens were subjected to phylogenetic analysis based on mitochondrial (CO1) and nuclear (CAD) gene sequences. Despite the expected presence of slight morphological variations, all specimens examined were identified both microscopically and molecularly as Lipoptena cervi (100% identity for both CO1 and CAD genes). The massive increase in wild ungulate populations can favour the possibility of detecting other species of Lipoptena. The identification keys proposed in the present study may help with monitoring the presence of Lipoptena species, particularly in European countries where this ectoparasite is neglected and for which various data (from diffusion to control methods) are still missing.

Surveillance of healthcare acquired infections by "alert microorganisms": preliminary results.

BACKGROUND: We evaluated the trend of four years (2015 - 2018) of "alert organisms" surveillance carried out at the 450 bed teaching hospital S. Andrea in Rome. METHODS: All patients with an "Alert organism" isolation were screened. In accordance with definitions used by the Centers for Disease Control patients with an "alert organism" isolation were evaluated for infection or colonization, by an infection control team (ICT). RESULTS: Between April 2015 and December 2018 a total 4,762 specimens with "Alert organism" isolation were screened and 1,601 patients were surveyed and included in the study. Overall 780 (48.8%) patients developed an healthcare acquired infection (HAI) at our institution, whereas 311 (19.4%) entered with a community acquired infection, 254 (15.8%) with an infection acquired in another healthcare setting and 256 (16.0%) resulted simply colonized. The 780 patients who developed an HAI at our institution presented 878 infectious episodes and the isolation of 931 microorganisms. C. difficile infections were the most common (27.2%), followed by 21.3% respiratory tract infections, 16.9% urinary tract infections, 15.5% surgical site infections, 12.5% bloodstream infections, 3.6% ulcers and 3.0% others. Among HAI group Gram negatives (54.1%) were more frequent than Gram positives (45.9%), whereas in patients entering in the hospital already with a community infection Gram positives overpassed Gram negatives (58.7% vs. 41.3%; p<0.001). Most common pathogens responsible for HAI were C. difficile (25.6%), Klebsiella spp. (25.5%), MRSA (19.6%) and Acinetobacter spp. (15.3%). Notably 30.0% HAI at other institutions were represented by C. difficile. Impressively, >40% of community acquired infections were related to MRSA. CONCLUSIONS: The present study provided some useful insight into the major multi-resistant pathogens epidemiology at our institution. The Authors succeeded in organizing a multidisciplinary ICT that created a partnership feeling with the hospital personnel.

Cyclospora cayetanensis travels in tap water on Italian trains.

Tap water samples from the toilets of an Italian national railway train were collected over a period of 10 months and tested for the presence of Cyclospora cayetanensis (C. cayetanensis) using EvaGreen® real-time polymerase chain reaction (RT-PCR) assay coupled with high resolution melting (HRM) analysis for protozoan detection and oocyst quantification. C. cayetanensis positive samples were detected in March, April, and May 2013, with the number of oocysts of 4, 5, and 11 per liter, respectively. This is the first finding of C. cayetanensis in water samples in Italy. The findings call for an improvement of hygiene and water safety by the Italian national railway company.

Saccharomyces cerevisiae fungemia, a possible consequence of the treatment of Clostridium difficile colitis with a probioticum.

The yeast Saccharomyces boulardii is a biotherapeutic agent used for the prevention and treatment of several gastrointestinal diseases, such as diarrhoea caused by Clostridium difficile, in addition to the antibiotic therapy. In this study we report a case of Saccharomyces cerevisiae fungemia in a patient with Clostridium difficile-associated diarrhoea (CDAD) treated orally with S. boulardii in association with vancomycin. The identification of the S. cerevisiae was confirmed by molecular technique. Fungemia is a rare, but a serious complication to treatment with probiotics. We believe it is important to remind the clinicians of this risk when prescribing probiotics, especially to immunocompromised patients.

Molecular characterization and phylogenetic inferences of Dermanyssus gallinae isolates in Italy within an European framework.

In order to investigate the genetic relationships between Dermanyssus gallinae (Metastigmata: Dermanyssidae) (de Geer) isolates from poultry farms in Italy and other European countries, phylogenetic analysis was performed using a portion of the cytochrome c oxidase subunit 1 (cox1) gene of the mitochondrial DNA and the internal transcribed spacers (ITS1+5.8S+ITS2) of the ribosomal DNA. A total of 360 cox1 sequences and 360 ITS+ sequences were obtained from mites collected on 24 different poultry farms in 10 different regions of Northern and Southern Italy. Phylogenetic analysis of the cox1 sequences resulted in the clustering of two groups (A and B), whereas phylogenetic analysis of the ITS+ resulted in largely unresolved clusters. Knowledge of the genetic make-up of mite populations within countries, together with comparative analyses of D. gallinae isolates from different countries, will provide better understanding of the population dynamics of D. gallinae. This will also allow the identification of genetic markers of emerging acaricide resistance and the development of alternative strategies for the prevention and treatment of infestations.

Detection of Toxoplasma gondii and Cyclospora cayetanensis in Mytilus galloprovincialis from Izmir Province coast (Turkey) by Real Time PCR/High-Resolution Melting analysis (HRM).

To evaluate the presence of Toxoplasma gondii and Cyclospora cayetanensis in edible shellfish, a total of 795 specimens of Mytilus galloprovincialis from 8 different sites on the west coast of Turkey (Bays of Izmir and Mersin) were examined. Fifty-three pooled samples were created and subjected to EvaGreen(®) RealTime PCR assay and HRM analysis for protozoans detection and oocyst quantification. Seven out of 8 sites were contaminated with at least one protozoan species. Twenty-one out of 53 (39.6%) samples tested positive for T. gondii, C. cayetanensis or both pathogens: 26.4% samples positive for C. cayetanensis; 9.4% for T. gondii, and 3.8% for both protozoans. The number of oocysts in positive samples ranged from 6 to 30 for T. gondii and from 3 to 48 for C. cayetanensis. This is the first report of T. gondii Type 1, and C. cayetanensis in M. galloprovincialis, the most consumed shellfish species in Turkey.

Evidences of increasing risk of dirofilarioses in southern Italy.

Given the spread of Aedes albopictus from northern to southern Italy, and the lack of updated data on Dirofilaria infections, this study was carried out to assess the infection risk for dogs and cats in Apulia region. During a 2-year study, 175 A. albopictus female specimens and samples of blood from 427 dogs (309 privately owned dogs and 118 shelter dogs) and 12 cats were collected. All blood samples were subjected to a modified Knott method, to a test for the detection of circulating Dirofilaria immitis antigen, and to a Dirofilaria species-specific real-time PCR for the simultaneous detection of D. immitis and Dirofilaria repens, targeting on partial cytochrome oxidase subunit 1 and internal transcribed spacer-2, respectively. Two abdomen and one thorax pools from A. albopictus were positive for D. immitis, with minimum infection rates of 1.14 and 0.51, respectively, and a probability of a single positive specimen to be infected of P = 0.6 % (95 % confidence interval (CI) = 0.12-1.73). Out of 439 examined subjects, 22 (5.0 %) tested positive for Dirofilaria spp. in at least one diagnostic test. A specific D. immitis infestation rate of 3.5 % was found among the privately owned dogs, while shelter dogs tested positive only for D. repens with a prevalence of 3.4 %; one cat tested molecularly positive for D. immitis. There was a significantly higher rate of positivity among guard dogs for D. immitis (odds ratio, 6.24, 95 % CI, 1.26-25.28; P < 0.05). The increasing risk of D. immitis infection in southern Italy is supported by the noteworthy positivity of A. albopictus populations and the cat. Our data highlight the usefulness to include filarioid infestation in routine diagnosis.

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.

Evaluation of the poultry red mite, Dermanyssus gallinae (Acari: Dermanyssidae) susceptibility to some acaricides in field populations from Italy.

Red mite field populations from seven naturally infested Italian caged laying poultry farms were investigated for their susceptibility to acaricide formulations available on the market, containing amitraz, carbaryl and permethrin. A minimum of 3,000 mites of all stages were collected from each farm and were tested with five acaricide concentrations (5, 10, 20, 50, 100%) plus an untreated control (0%). Field red mite populations were found to be tolerant even with the highest concentrations with carbaryl and permethrin for six (86%) and three (42%) of the investigated farms, respectively (P < 0.05). Furthermore, six (86%) of the investigated farms showed a red mite population susceptible to amitraz at any concentration. Out of the seven field populations tested with amitraz, one population is becoming less tolerant whereas another was the most tolerant to carbaryl and permethrin at any concentration. Data show that the lack of effectiveness of some acaricides is spreading in Europe and call for the adoption of alternative management strategies to avoid development of resistance.

Phylogenetic relationship between Dermanyssus gallinae populations in European countries based on mitochondrial COI gene sequences.

Phylogenetic analysis of Dermanyssus gallinae mites originating from UK, France and Italy was performed using partial amplification of the mitochondrial COI gene. Results showed that UK samples reveal the greatest variation and diversity and are linked to one of the French populations highlighting North-South genetic transitions in European red mite populations. Intra-farm variations between mite samples highlighted the diversity between national populations and possibly its origin from the different chemical strategies used in each country.

Theileria sp. OT3 and other tick-borne pathogens in sheep and ticks in Italy: molecular characterization and phylogeny.

PCR Reverse Line Blot (RLB) hybridization and sequencing were used to determine the dynamics of infection with tick-borne pathogens in one hundred apparently healthy sheep in Italy. Blood samples were tested once prior to the onset of the grazing season (June 2010) and once after the end of the grazing season (August 2010). Ticks collected from sheep and from the vegetation were also tested by PCR/RLB. Before grazing, 56% of the sheep harbored several tick-borne pathogens: Anaplasma ovis was the most prevalent (41%), followed by A. ovis co-infected with Theileria sp. OT3 (14%). After grazing, 87% of sheep were positive for A. ovis alone (41%), co-infected with Theileria sp. OT3 (8%) or co-infected with Babesia motasi (5%). Other sheep were infected with Anaplasma phagocytophilum alone (20%), co-infected with B. motasi (7%) or with Theileria sp. OT3 (5%) (p<0.001). After grazing, sheep were significantly more infected with tick-borne pathogens than before grazing. Ticks collected were all Haemaphysalis punctata (n-89) and 36% were positive for A. ovis, Ehrlichia ovina and A. ovis combined with A. phagocytophilum. Phylogenetic analysis including isolates from countries in the Mediterranean Basin show circulation of the same variants of Theileria sp. OT3, whereas two different geographical origins for the isolates of A. ovis and A. phagocytophilum were identified. This is the first report from Italy of Theileria sp. OT3 in sheep, whereas the detection of Ehrlichia ovina in ticks is worth noting, and the presence of A. phagocytophilum in sheep and in ticks poses a potential public health risk.

Are elevated levels of soluble ICAM-1 a marker of chronic graft disease in heart transplant recipients?

Positivity for circulating intercellular adhesion molecule-1 (ICAM-1) in heart transplant recipients has been claimed to predict the development of coronary artery disease and risk of graft failure. Soluble ICAM-1 were evaluated in 32 heart transplant recipients. Five of these patients, who had undergone transplantation several years before, were positive for soluble ICAM-1 but did not present any clinical sign of graft rejection. Furthermore, although heart graft coronary disease was diagnosed in 15 of the 32 patients, they did not show significantly higher titres of soluble ICAM-1 compared to the remaining patients. These findings suggest that major caution is necessary when considering ICAM-1 positivity as a marker of graft disease.

High-level expression in insect cells and purification of secreted monomeric single-chain Fv antibodies.

We have constructed a recombinant baculovirus encoding an anti-(phenyl-oxazolone) single-chain Fv antibody (anti-phOx-scFv) fused to the baculovirus GP67 secretion signal sequence, 6 liters of Sf9 insect cells were infected with this virus at a multiplicity of infection of one and cultured in a bioreactor for 72 h. The dialyzed supernatant was subjected to cation exchange chromatography at pH 6.0 followed by size exclusion chromatography on a Sephadex G100 superfine matrix. This rapid protocol resulted in the isolation of monomeric scFv with a purity of greater than 98%. The final yield was 32 mg/l (10(9) cells/l). Partial amino-terminal sequencing revealed that the GP67 signal sequence was completely removed upon secretion. The dissociation constant of the scFv monomers is about 1 x 10(-4) M. By competitive ELISA scFv dimers yielded a half maximum inhibitory concentration of 3.4 x 10(-7 M which matches the earlier measured Kd for the anti-phOx-scFv (3.2-5.3 x 10-7 M. Marks et al. (1991) J. Mol. Biol. 222, 581-597: Marks et al. (1992) Bio/Technology 10, 779-783). This method is readily scaled up for the preparation of scFv antibodies in high yield and purity obviating any affinity chromatography and/or refolding steps by exploitation of insect cell expression as an efficient alternative to E. coli expression.

Role of group B streptococcal capsular polysaccharides in the induction of septic arthritis.

The ability of different serotypes of group B streptococci (GBS) to induce septic arthritis in mice was compared. Types II, III, IV, V, VI and VII GBS were investigated. A highly capsulate strain of type III GBS, COH1, and its mutants, COH1-11 (lacking capsular sialic acid) and COH1-13 (non-capsulate), obtained by transposon insertional mutagenesis, were used to assess the role of type-specific polysaccharide on the induction of arthritis. At an intravenous dose of 10(7) cfu/mouse, reference strains of types II, III, IV, VI and VII and type III strain COH1 induced arthritis with an incidence ranging from 70 to 90%. For type V and strain COH1-11, 10(8) cfu/mouse was required to obtain a 50% incidence of arthritis; lesions were not evident with strain COH1-13. The presence of the capsule played a major role in the induction of GBS septic arthritis. The presence and amount of sialic acid in capsular polysaccharide influenced the incidence of articular lesions. The bacterial dose affected the manifestations of arthritis; the less virulent strains of GBS also induced articular lesions when an adequate number of micro-organisms reached the joints.

A new serological technique, time-resolved fluoroimmunoassay (TRFIA), for the immunological diagnosis of urinary schistosomiasis.

The application of a new serological method, time-resolved fluoroimmunoassay (TRFIA), is described for the diagnosis of urinary schistosomiasis. A chelate of lanthanides (europium) with a long fluorescent life-time is used as label. The intensity of fluorescence is measured after a delay selected to eliminate almost completely the background fluorescence, which decays rapidly. TRFIA was compared with an established method, enzyme linked immunosorbent assay (ELISA). Using sera from proven cases of Schistosoma haematobium infection, 98.1% of the samples were positive by TRFIA and 86.5% by ELISA. Sera from patients infected with helminths other than schistosomes produced only 1.5% of false positives with TRFIA, compared with 12.3% by ELISA. TRFIA is more sensitive and specific than ELISA.

Expression modes of urinary N-acetyl-beta-D-glucosaminidase in patients with chronic renal insufficiency.

BACKGROUND: Urinary N-acetyl-beta-D-glucosaminidase (NAG) activity has emerged as potentially useful early marker of renal tubular injury. This activity is usually evaluated in random urine samples and is related to urinary creatinine concentration. Reports about the lack of correlation between NAG activity of 24-h urines and activity of random urine samples in some clinical and experimental situations led us to study the correlation existing between different procedures for expressing urinary NAG in patients with chronic renal insufficiency. METHODS: Thirty samples of 24-h urine and 30 random urine samples from chronic renal insufficiency patients were collected. The activity of urinary NAG was examined fluorimetrically. RESULTS: The following correlations were observed: (1) r = 0.431 (P = 0.017) for activity in random urine samples and total activity in 24-h urines); (2) r = 0.281 (P = 0.005) for activity in random samples and activity, expressed as U/l, in 24-h urines. CONCLUSIONS: The data show that collection of urine excreted over the whole day and evaluation of total daily excretion of NAG seems the method of choice, at least for patients with chronic renal insufficiency.

The relationship between glycated hemoglobin and polymorphonuclear leukocyte leukotriene B4 release in people with diabetes mellitus.

In order to evaluate polymorphonuclear leukocyte (PMN) activity in diabetes mellitus, leukotriene B4 (LTB4) levels were measured in sixty patients, 31 affected with Type 1 diabetes mellitus and 29 affected with Type 2 diabetes mellitus. The LTB4 levels (12.1+/-0.2 pg/100 microl) in diabetic patients were higher compared to those of the control group (7.9+/-0.1 pg/100 microl) (P < 0.001), and remained significantly higher (P < 0.001) (12.8+/-0.2 pg/100 microl) than in the control group (11.0+/-0.2 pg/100 microl) after stimulation with calcium ionophore. A significant and positive correlation between glycated hemoglobin and LTB4 was demonstrated (P < 0.001, r = 0.80). This study demonstrates that in diabetic patients there is a PMN activation and that this activation is correlated to glycated hemoglobin level.

Anti-neutrophil cell antibodies in newly diagnosed patients with type-1-diabetes.

Both anti neutrophil cell antibodies and anti endothelial cell antibodies were found in 7 out of 30 newly-diagnosed type-1 diabetic patients. This confirms the abnormal activation of the immunological system in the early stage of type-1 diabetes mellitus.

Rapid test on amniotic fluid for diagnosis of foetal pulmonary maturity.

The Authors have carried out a study on samples of amniotic fluid taken at various gestational ages. They compare the optic density at 650 nm and the Lecithin/Sphingomyelin ratio, evaluated from the quantitative point of view according to the method of Gluk and Coll. The results show a significant correlation between samples with optic density greater than or equal to 0.15 and those with L/S ratio greater than or equal to 2.0. Finally, the Authors analyze the possible usefulness of this method as a rapid test to assess foetal pulmonary maturity.

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.