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Machine learning in embedded systems has become a reality, with the first tools for neural network firmware development already being made available for ARM microcontroller developers. This paper explores the use of one of such tools, namely the STM X-Cube-AI, on mainstream ARM Cortex-M microcontrollers, analyzing their performance, and comparing support and performance of other two common supervised ML algorithms, namely Support Vector Machines (SVM) and k-Nearest Neighbours (k-NN). Results on three datasets show that X-Cube-AI provides quite constant good performance even with the limitations of the embedded platform. The workflow is well integrated with mainstream desktop tools, such as Tensorflow and Keras.

Since 2001 the Istituto Superiore di Sanità established a quality assurance programme for molecular genetic testing that covers four pathologies: Cystic Fibrosis (CF), Beta Thalassemia (BT), Fragile X Syndrome (FX), and Familial Adenomatous Polyposis Coli (APC). Since 2009 this activity is an institutional activity and participation is open to both public and private laboratories. Seven rounds have been performed until now and the eighth is in progress. Laboratories receive 4 DNA samples with mock clinical indications. They analyze the samples using their routine procedures. A panel of assessors review the raw data and the reports; all data are managed through a web utility. In 2010 the number of participants was 43, 17, 15, 5 for CF, BT, FX, APC schemes respectively. Genotyping results were correct in 96%, 98.5%, 100%, and 100% of CF, BT, FX, and APC samples, respectively. Interpretation was correct in 74%, 91%, 88%, and 60% of CF, BT, FX, and APC reports, respectively; however in most of them it was not complete but a referral to genetic counseling was given. Reports were satisfactory in more than 60% of samples in all schemes. This work presents the 2010 results in detail comparing our data with those from other European schemes.

The Italian scheme of External Quality Assessment for beta-thalassemia started in 2001 as part of a project twice funded by the Italian Ministry of Health and coordinated by the Istituto Superiore di Sanità. To date, five trials have been performed (2001-2004 and 2006). The aim of the Italian scheme is to help public laboratories in improving and reaching a high standard of quality when performing a molecular test. Many laboratories took part in the 5-year project, and their participation was constant during the whole period. The aims of this paper are to describe the genotyping and reporting results as well as focusing on the techniques and the testing strategies adopted to detect mutations. Almost 99% of the alleles analyzed were correctly detected by laboratories, while 0.33% of the analyses gave a wrong result. Reverse dot blot was the most used technique, and it was always used in the strategies adopted by laboratories to detect mutations. The reports sent by laboratories showed incompleteness and heterogeneity; thus, a new model for written reports has been introduced since 2004. It will be interesting to monitor the effects of the reporting model and the output of this educational action in the future.

The Italian External Quality Assessment scheme for fragile X syndrome started in 2001 as an activity funded by the National Health System and coordinated by the National Institute of Public Health. The aim of this work is to present the data of 5 years (2001--2004 and 2006) of survey. The External Quality Assessment scheme was designed to cover the following points: (a) genotyping and (b) interpretation and reporting of results. Overall, the scheme covered about 65% of all Italian public laboratories. The average reporting of results was 91.6%, with an overall success rate of 76%. The rate of diagnostic errors observed was on average 5%. Inaccuracy in sizing of CGG repeats of normal and premutated alleles was reported. During the survey the proportion of laboratories using a Southern blotting, polymerase chain reaction, and ABI sizing kit in combination rose from 36.8% to 70.6%. The reports from laboratories showed incompleteness and considerable variations in expected outcomes. For this reason, in 2004 a model for written reports was introduced. In conclusion, these data underscore the need to participate in External Quality Assessment schemes as an educational resource to ensure quality in molecular genetic testing.

Salivary gland tumours are rare tumours characterized by histopathologic complexity and a wide variety of morphologic features. Studies on genetic changes in different histological subtypes of salivary gland tumours are important to better understand molecular pathogenetic mechanisms and to identify diagnostic and prognostic markers. Data are even more scanty dealing with unusual subtypes of these tumours. The aim of the present study was to analyse two high grade transformation adenoid cystic carcinomas (hgACC) and one hybrid tumour in order to identify, by mutational and microsatellite analysis, genetic alterations in TP53, CDKN2A/ARF, RAS, BRAF, PTEN, MAPK2 and EGFR genes. The two hgACCs showed snps missense in RAS genes and alterations with allelic instability in CDKN2A/ARF; moreover, a double mutation in TP53 was detected in one case. The hybrid tumour showed alterations in CDKN2A/ARF gene and snps missense in NRAS genes. Our data suggest that CDKN2A/ARF pathway might be involved in pathogenesis of the salivary gland tumours analysed. Further molecular analyses of these very rare tumours are necessary to better understand the role of other genetic alterations detected in our study.

Our study showed the detection of S. suis in DNA from tissue belonging to 4 patients out of 8 with salivary gland tumors. To our knowledge there are no prevalence data of S. suis in the region where the patients were recruited; however, over the past few years the number of reported S. suis infections in humans has increased significantly (5) and it is worthwhile to report this finding to accumulate significant data about possible risk factors for these rare tumors.

Background: The Italian external quality assessment scheme in classical cytogenetics was started in 2001 as an activity funded by the National Health System and coordinated by the Italian Public Institute of Health. Objectives: The aim of our work is to present data from the first 4 years of activity, 2001–2004. Methods: Italian cytogenetics public laboratories were enrolled on a voluntary basis, and this nationwide program covered prenatal, postnatal and oncological diagnosis. The scheme is annual and retrospective; a panel of experts reviewed the quality of images and reports in order to assess technical, analytical and interpretative performance. Results: Over the 4-year period, the number of participating laboratories increased: from 36 in 2001, 46 in 2002, 49 in 2003 to 51 in 2004. The overall technical performance was satisfactory. Inadequacy or lack of information in reporting was the most frequent analytical inaccuracy identified in all parts of the scheme. However, the percentage of complete reports increased significantly during the period: by 36% in postnatal diagnosis between 2001 and 2004 (p < 0.001) and by 42% in oncological diagnosis between 2002 and 2004 (p = 0.003). Conclusions: Our experience reveals that participation in external quality assessment programs has significant advantages, helping to standardize and to assure quality in cytogenetic testing.

Background: Despite the rapid transition into routine clinical practice of molecular techniques based on PCR, external quality assessment (EQA) is still not widely available. The European Union and European Communities Confederation of Clinical Chemistry have supported the EQUAL project as a series of 3 different EQA programs for the assessment of molecular methods independently from analytes. We present the results from the EQUAL-qual program designed to evaluate the analytical aspects of DNA analysis by means of a conventional qualitative PCR experiment. Methods: The EQUAL-qual program provided DNA, blood samples, and primer sets to participant laboratories to assess DNA extraction and PCR amplification. We have developed statistical procedures to identify laboratories performing poorly in DNA extraction (quality and quantity), PCR efficiency, and data interpretation after electrophoresis. Results: An application to participate was obtained from 213 laboratories (from 25 countries), and 175 (82%) of laboratories submitted results for assessment. Questionable results in terms of quality and/or quantity of DNA derived from blood extractions were returned by 27% of laboratories (46 of 166). PCR efficiency showed high variability, with 3% of laboratories (5 of 163) showing a consistently low rate of amplification and 10% (18 of 175) not reporting the expected number of bands of the amplified targets. Conclusions: The results showed considerable variability in all phases of the experiment. The approach confirms the validity of EQA as a method for evaluating analytical aspects of PCR-based tests.

The Italian External Quality Control Programme for cystic fibrosis molecular diagnosis started in 2001; public laboratories distributed throughout Italy participated on a voluntary basis.: The Italian Public Health Institute (Istituto Superiore di Sanità) sent six validated DNA samples to participating laboratories: technical and clinical information was provided for each sample. Laboratories were required to analyse all six samples. For each sample the laboratories had to provide the results (including raw data) and a report of molecular analysis within 2 months using current methods and nomenclature. Raw data and reports were evaluated by a Steering Committee and their comments were sent to each laboratory.: Genotyping results indicated a general good level of quality for all laboratories, i.e., ∼1% of alleles were incorrectly assigned each year due to analytical (45%) and misinterpretation (45%) errors. During the first 2 years, more than 70% of laboratories did not test for some regional Italian mutations. Commercial kits for reverse dot-blot and oligonucleotide ligation assay PCR were used to detect mutations by 52.8% and 29.5%, respectively, of the participating laboratories. Reporting of results was still inadequate; in 2004 a model for the written report was introduced, but not all laboratories used it.: Our data show that few genotyping errors were made by laboratories and were principally due to misinterpretation and analytical reasons. However, reports are still inadequate and it will be interesting to evaluate the introduction of the reporting model in future years.Clin Chem Lab Med 2007;45:254–60.

The epidemiological importance of tracing plasmids conferring drug resistance prompted us to develop a PCR method based on replicons (inc/rep PCR) of the major plasmid incompatibility groups among Enterobacteriaceae. Eighteen pairs of primers were designed to perform 5 multiplex- and 3 simplex-PCRs, recognizing FIA, FIB, FIC, HI1, HI2, I1-Igamma, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA. The specificity of the method was tested on a collection of 61 reference plasmids and on 20 Salmonella enterica strains of different serotypes isolated in Italy. Results indicated that the inc/rep PCR method demonstrates high specificity and sensitivity in detecting replicons on reference plasmids and also revealed the presence of recurrent and common plasmids in epidemiologically unrelated Salmonella isolates of different serotypes. These results suggest that the method is potentially applicable to a large number of strains to trace the diffusion of specific multi-drug resistance plasmids in different environments.

The present paper reports the characterization of HERV-E endogenous retroviral sequences in the human genome by using three complementary approaches. Firstly, we identified genomic clones containing HERV-E by BLAST screening of human DNA databases by using the entire sequence of a characterized HERV-E clone (M10976) as a query. The genomic structure and integration sites of the HERV-E elements were characterized. Secondly, new integration sites of HERV-E elements were revealed by a retroviral LTR-arbitrary primer-PCR (RELAP-PCR) technique. BLAST analysis of the PCR products identified a subgroup that shows low identity (75%) to the original clone M10976 and slightly higher identity (80%) to a closely related HERV-E (Ac. n. K02166). Finally, we performed FISH mapping, which revealed sites of integration of HERV-E not previously identified at the cytogenetic level. A preliminary analysis of genes mapping in the same bands as HERV-E integration sites was performed: several loci relevant to physiological and/or pathological processes were detected. Our findings may provide clues to identify HERV-E integration sites adjacent to genes with important biological roles.

The uropathogenic Escherichia coli strain J96 (04:K6) is able to produce four adherence factors [P-fimbriae (pap and prs), F1C-fimbriae (foc) and Type 1-fimbriae (fim)], two alpha-hemolysins (hlyI and II) and the cytotoxic necrotizing factor type 1 (cnf1). Using phenotypic test systems and genotypic analysis, it has been shown that the mutant strain J96-M1 has lost the hlyII, prs and cnf1 genes. The three virulence associated determinants are linked on one particular region on the chromosome, which is termed 'pathogenicity island II' (Pai II).

One hundred and sixty-eight isolates of Escherichia coli obtained in Italy from 112 children with diarrhoea and 56 age-matched controls were examined by the HEp-2 cell adhesion assay. Sixteen strains showed localised adherence (LA), 29 showed diffuse adherence (DA) and eight strains showed aggregative adherence (AA). No adhesion pattern was significantly associated with disease. Strains that showed LA or AA were further characterised by serotyping, fluorescent actin staining (FAS) test and hybridisation with the EPEC adherence factor (EAF), E. coli attaching and effacing (eae) and enteroaggregative (EAgg) DNA probes. Strains that showed poor LA were FAS-negative, did not belong to EPEC serotypes and did not hybridise with EPEC probes. Conversely, the two strains that showed a good LA pattern belonged to serotype O128:H2, were FAS positive and hybridised with the eae probe. No isolate hybridised with the EAF probe. Only three of the eight strains with the AA pattern hybridised with the EAgg probe. Probe positivity correlated with the ability to produce clumps at the surface of the liquid culture and to agglutinate rat erythrocytes. In two of these EAgg probe-positive strains, electronmicroscopy revealed the presence of fibrillar bundles which seem to mediate bacterial aggregation.

Escherichia coli strains isolated from 81 fecal samples from red deer (Cervus elaphus), roe deer (Capreoulus capreoulus), chamois (Rupicapra rupicapra) and alpine marmot (Marmota marmota) living in the Stelvio National Park, Italy, were examined for antimicrobial resistance and production of toxic factors. Direct plating of specimens on media containing antimicrobial drugs allowed us to isolate resistant strains of E. coli from 10 of 59 (17%) specimens examined by this technique. Nine of 31 specimens from red deer (29%) contained resistant strains. Different animals were likely colonized by the same resistant strain of E. coli. Conjugative R plasmids were found in four strains isolated from the marmot, roe deer and chamois. A strain from red deer produced heat-stable enterotoxin and another strain produced both hemolysin and cytotoxic necrotizing factor. A marmot isolate produced hemolysin alone. No strains were found to produce heat-labile enterotoxin or verotoxins.

Nine strains of ETEC isolated in Italy have been compared with 13 isolates from Somalia with respect to toxin production, serotype and antimicrobial resistance pattern. None of the strains isolated from Italy belonged to any serotype or serogroups found among the strains from Somalia. Remarkable differences between the two groups of isolates were also observed with regard to the susceptibility to antimicrobials and the presence of R-plasmids. These findings suggest that ETEC strains isolated in Italy are not related to the strains widespread in Somalia and, generally, in developing countries.

A purification procedure for a protein obtained from an pathogenic strain of E. coli is described. The protein — called CNF — is active in inhibiting the duplication of cultured mammalian cells. Since nuclei division is apparently normal, treatment of cultured cells with CNF leads to the formation of gigantic, polynucleated cells. The purified protein is chromatographically and electrophoretically homogeneous. A partial characterization of CNF protein is also given.

Pathogens associated with diarrheal disease were isolated in a 10% systematic sample of 1667 patients newborn-14 years in the Banaadir Pediatric Hospital Mogadishu Somalia in 1983-1984. While 33% of patients had prior undetermined treatment pathogens isolated were similar. 30% of the 50000 children attending the Banaadir Hospital in 1983-84 had diarrhea with rates proportional to the population in each district. 62% had acute illness; 20% were ill >2 weeks. 76% had fever 67% vomiting 93% abdominal pain 40% moderate-severe dehydration and 55% watery diarrhea. The most frequently isolated pathogens were rotavirus (25%) ETEC (11%) Shigella (9%) Giardia (8%) Clostridia jejuni (8%) V. cholerae non-O1 (6%) and A. hydrophila (9%). 61% of children screened had 1 or more and 15% had 2-5 pathogens. No Yersinia enterocolitica was found. Children 3 years had high rates of Giardia Shigella and V. Cholerae non-O1. Breast-fed infants had relatively lower rates of Shigella: their predominant pathogens were rotavirus ETEC Clostridia and Salmonella. At least 1 parasite was found in 811 children screened. Fever and mucus and blood in the stool were linked with Shigella. There were seasonal peaks in rotavirus in the dry and rainy season in ETEC at the end of the dry season and Shigella in the rainy season and in November.

The effect of the cytotoxic necrotizing factor of Escherichia coli on HEp-2 cells was studied by fluorescence and scanning electron microscopy. This cytotoxin, known for inducing the formation of giant multinucleated cells in several cell lines, caused changes in actin and tubulin organization. The presence of membrane ruffles at the cell border and of numerous thick bundles of actin crossing the cell body, suggests that the factor promotes cell spreading; this probably interferes with cytokinesis, ultimately leading to the formation of very large flattened multinucleated cells. Moreover, the nuclear segmentation observed in treated cells seems to be associated with a rearrangement of actin in the perinuclear region and with the presence of tubulin bundles in proximity to nuclear clefts. Although the primary target is still unknown, these findings suggest that the cytoskeleton is affected accounting for the multinucleation process induced by the factor.

Summary. A cell-culture assay was used to detect toxins directly in stools from sporadic cases of infantile diarrhoea. Cytotoxins were revealed in 11 out of 58 samples from children with diarrhoea, nine of whom had no common enteric pathogens in their stools. A preliminary characterisation of the cytotoxins was obtained by neutralisation tests with clostridial antitoxins.

SUMMARYTwo hundred and ninety-nine different strains ofEscherichia coli, isolated from 172 patients with diarrhoea and 113 healthy subjects, were examined for enterotoxin, cytotoxin and haemolysin (Hly) production and for mannose-resistant haemagglutination (MRHA) and invasive properties. Three strains proved enterotoxigenic, none enteroinvasive; cytotoxin and Hly production was shown in 25 strains from patients and in 3 from controls. Ten strains produced the cytotoxic necrotizing factor (CNF), 6 released other factors which kill cell cultures. Hly production was shown in 21 strains, 9 of which were also positive for CNF. MRHA was detected in 26% of strains from diarrhoca compared with 14% of strains from healthy people. A strong association between toxin production and MRHA was demonstrated. Serotyping results showed that the strains exhibiting virulence traits mostly belonged to serogroups commonly involved in extra-intestinal infections. The possible role of strains ofE. colishowing one or more virulence factors as opportunistic pathogens in diarrhoeal diseases is discussed.

SummaryThe susceptibility to antimicrobial agents of 569 salmonella isolates collected in 1977–8 from patients in hospitals in Rome was tested. Fifty-nine per cent of all isolates were resistant to one or more antimicrobials. Resistance was most common to sulphathiazole, tetracycline, streptomycin, whereas colistin, gentamicin, tobramycin, trimethoprim-sulphamethoxazole and nalidixic acid were the most activein vitro.Multiple resistance was most frequently found in strains ofSalmonella wienandS. typhimurium(94% and 38% respectively).A significant change in the resistance pattern ofS. wienwas observed between 1977 and 1978, with a significant increase of susceptibility to some antimicrobials in 1978.Twenty-one R-plasmids transmissible toE. coliK12 were derived from 46 resistant strains ofS. typhimurum.

Multidrug-resistant Vibrio cholerae O1 El Tor strains isolated during the 1994 outbreak of cholera in Albania and Italy were characterized for the molecular basis of antibiotic resistance. All strains were found to be resistant to tetracycline, streptomycin, spectinomycin, trimethoprim, sulfathiazole, and the vibriostatic compound O/129 (2,4-diamino-6,7-diisopropylteridine). Resistance genes were self-transferable by a conjugative plasmid of about 60 MDa, with the exception of spectinomycin resistance, which was conferred by the aadA1 gene cassette located in the bacterial chromosome within a class 1 integron. The resistance to trimethoprim and O/129 was conferred by the dfrA1 gene, which was present on the plasmid. Although the dfrA1 gene is known to be borne on an integron cassette, class 1, 2, or 3 intI genes were not detected as part of the plasmid DNA from the strains studied.

Two hundred and nineteen strains of Escherichia coli from extraintestinal infections and feces of healthy subjects were examined for hemolysin (Hly) and cytotoxic necrotizing factor (CNF) production and for mannose-resistant hemagglutination. Of 105 strains from extraintestinal infections, 42 (40.0%) were positive for production of both Hly and CNF, and 21 (20.0%) were positive for Hly alone; on the contrary, only 1 Hly- and CNF-positive strain and 2 Hly-positive strains were found among 114 strains from normal stools. CNF production was not found to occur among the nonhemolytic strains, confirming the close association existing between these toxic factors. Hemolytic strains positive for CNF showed mannose-resistant hemagglutination less frequently than did Hly-positive, CNF-negative strains (25.6 versus 82.6%), suggesting the existence of two distinct classes among hemolytic strains of E. coli.