Invasive group A streptococci infections are increasing worldwide, mainly due to the emm1 lineage M1UK emergence. Although this variant has recently been described in adult Italian patients, its circulation in children has not yet been established. Characterizing by whole genome sequencing 6 invasive group A streptococci strains isolated between 2022 and 2023, we highlighted M1UK lineage circulation in pediatric patients in Italy.

This article reports a rapid and unexpected spread of colonization cases of NDM-1 carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in a neonatal surgical unit (NSU) at Bambino Gesù Children's Hospital in Rome, Italy. Between the 16th of November 2020 and the 18th of January 2021, a total of 20 NDM-1 carbapenemase-producing K. pneumoniae (n = 8) and E. coli (n = 12) were isolated from 17 out of 230 stool samples collected from neonates admitted in the aforementioned ward and time period by an active surveillance culture program routinely in place to monitor the prevalence of colonization/infection with multidrug-resistant Gram-negative microorganisms. All strains were characterized by antimicrobial susceptibility testing, detection of resistance determinants, PCR-based replicon typing (PBRT) and multilocus-sequence typing (MLST). All isolates were highly resistant to most of the tested antibiotics, and molecular characterization revealed that all of them harbored the blaNDM-1 gene. Overall, IncA/C was the most common Inc group (n = 20/20), followed by IncFIA (n = 17/20), IncFIIK (n = 14/20) and IncFII (n = 11/20). MLST analysis was performed on all 20 carbapenemase-producing Enterobacterales (CPE) strains, revealing three different Sequence Types (STs) among E. coli isolates, with the prevalence of ST131 (n = 10/12; 83%). Additionally, among the 8 K. pneumoniae strains we found 2 STs with the prevalence of ST37 (n = 7/8; 87.5%). Although patient results were positive for CPE colonization during their hospital stay, infection control interventions prevented their dissemination in the ward and no cases of infection were recorded in the same time period.

Background: It is well known that the best nutritional option for infants is human milk, and that when breastfeeding is not possible, human milk banks are a possible alternative. However, in the case of infants with fat transport disorder like chylothorax, defatting of human milk is mandatory. Research Aim: The aim of the study was to reduce milk fat content without reducing other nutrients, increasing oxidative stress, or introducing harmful microorganisms. Methods: In this prospective, cross-sectional, observational study, we examined the influence of defatting and pasteurization of 50 donor samples on fat, macro- and micronutrients, as well as on oxidative stress markers. Results: Low-temperature centrifugation proved to be very efficient in defatting, reducing the concentration of triglycerides by 85% and cholesterol by 50%. The macronutrients (proteins, albumin, and Immunoglobulin A) did not undergo significant changes due to defatting and pasteurization procedures, while iron decreased by 36%. However, as the majority of iron is retained, this result does not remarkably change the milk composition. Furthermore, oxidative stress markers and antioxidant levels were unchanged, and the milk result was microbiologically safe. Conclusions: Cold milk centrifugation proved to be an effective technique that allows the reduction of human milk lipids. The determination of triglycerides and cholesterol can be used as an indicator of skimming. This procedure is not accompanied by substantial modifications of other components present in the milk.

The spread of carbapenemase-producing Enterobacterales (CPE), especially Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli), is a serious public health threat in pediatric hospitals. The associated risk in newborns is due to their underdeveloped immune system and limited treatment options. The aim was to estimate the prevalence and circulation of CPE among the neonatal intensive units of a major pediatric hospital in Italy and to investigate their molecular features. A total of 124 CPE were isolated from rectal swabs of 99 newborn patients at Bambino Gesù Children’s Hospital between July 2016 and December 2019. All strains were characterized by antimicrobial susceptibility testing, detection of resistance genes, and PCR-based replicon typing (PBRT). One strain for each PBRT profile of K. pneumoniae or E. coli was characterized by multilocus-sequence typing (MLST). Interestingly, the majority of strains were multidrug-resistant and carried the blaNDM gene. A large part was characterized by a multireplicon status, and FII, A/C, FIA (15%) was the predominant. Despite the limited size of collection, MLST analysis revealed a high number of Sequence Types (STs): 14 STs among 28 K. pneumoniae and 8 STs among 11 E. coli, with the prevalence of the well-known clones ST307 and ST131, respectively. This issue indicated that some strains shared the same circulating clone. We identified a novel, so far never described, ST named ST10555, found in one E. coli strain. Our investigation showed a high heterogeneity of CPE circulating among neonatal units, confirming the need to monitor their dissemination in the hospital also through molecular methods.

In 2014, the Italian Working Group for Infections in Critically Ill Patient of the Italian Association of Clinical Microbiologists updated the recommendations for the diagnostic workflow for bloodstream infections (BSI). Two years after publication, a nationwide survey was conducted to assess the compliance with the updated recommendations by clinical microbiology laboratories. A total of 168 microbiologists from 168 laboratories, serving 204 acute care hospitals and postacute care facilities, were interviewed during the period January–October 2016 using a questionnaire consisting of nineteen questions which assessed the level of adherence to various recommendations. The most critical issues were as follows: (a) The number of sets of blood cultures (BC) per 1,000 hospitalization days was acceptable in only 11% of laboratories; (b) the minority of laboratories (42%) was able to monitor whether BCs were over or under‐inoculated; (c) among the laboratories monitoring BC contamination (80%), the rate of contaminated samples was acceptable in only 12% of cases;(d) the Gram‐staining results were reported within 1 hr since BC positivity in less than 50% of laboratories. By contrast, most laboratories received vials within 2–4 hr from withdrawal (65%) and incubated vials as soon as they were received in the laboratory (95%). The study revealed that compliance with the recommendations is still partial. Further surveys will be needed to monitor the situation in the future.

BACKGROUND:Holder pasteurization (HoP) is the recommended method of pasteurization for donor human milk (DHM). The aim of the present study was to compare nutritional and microbiological impact on DHM of a new technique of pasteurization based on technical changes of HoP.METHODS:We analyzed milk samples from 25 donors. Each sample, derived from one breast milk expression, was subdivided into three aliquots according to pasteurization: The first was not pasteurized, the second pasteurized by HoP, and the third was pasteurized by modified HoP (MHoP). Each aliquot was assessed as to its microbiological and nutritional profile. Nutritional profile included calcium and triglycerides concentrations detected by spectrophotometry and amino acid levels assessed by high-performance liquid chromatography (HPLC).RESULTS:Triglycerides were significantly lower in pasteurized, by both methods, than in not pasteurized aliquots, while calcium and amino acids concentration were similar. Microbiological profile did not differ between HoP and MHoP aliquots.CONCLUSIONS:HoP and MHoP seem to have similar efficacy in preserving some nutritional characteristics of DHM and to confer similar microbiological safety. MHoP is time-saving and potentially costs-effective when compared to HoP, and it is; therefore, potentially of more interest from a practical point of view. Further studies are needed to confirm these findings.

Background and aims: Blood culture (BC) results are essential to guide antimicrobial chemotherapy for patients with sepsis. However, BC is a time-consuming exam, which can take several days. Reducing BCs turn around time (TAT) could impact on multiple outcome parameters and TAT monitoring is an important tool for measurement of microbiology laboratory performance. The aim of this study was to provide an overview of BC TATs among Italian microbiology laboratories. Materials and methods: Five laboratories collected and recorded, for a month period, date and time of the BC processing events. Cumulative TATs were analysed using the GraphPad software. Results: Participating laboratories reported data from 302 sepsis episodes. The median time from when the BC system produced a positive signal until Gram-stain results were reported was 7.6 hours. A rapid molecular identification and antimicrobial susceptibility testing (AST) was performed in 26.5% of BCs. Mean TAT for identification report was significantly lower when a molecular approach was adopted (12 vs. 28.7 hours, P<0.001). Similarly, results of the molecular AST were obtained more than 24 hours in advance compared with phenotypic AST (mean 13.2 vs. 47.6, P<0.001). TATs from BC positivity of laboratories opened 7 days/week were not significantly lower than those of laboratories opened 6 days/week. Conclusions: BC is a time-consuming exam, however, molecular identification and AST methods can drastically reduce time to results. The lack of difference between TATs observed for laboratories working 7 days/week and 6 days/week, coupled with a high rate of BCs turning positive during the night enable to conclude that the most urgent measure to reduce TATs is the expansion of laboratory regular duty hours.

We evaluated a multiplex PCR assay, the Seeplex Diarrhoea ACE detection, that simultaneously detects 15 enteric pathogens, including Salmonella spp., Shigella spp., Vibrio spp., toxin B producer Clostridium difficile, Campylobacter spp., Clostridium perfringens, Yersinia enterocolitica, Aeromonas spp., Escherichia coli O157:H7, verocytotoxin-producing Escherichia coli, adenovirus, Group A rotavirus, norovirus GI and GII, and astrovirus. We compared this assay with clinical methods routinely used in our laboratory, for detecting enteropathogens in stool samples collected from 245 paediatric patients with suspected infectious gastroenteritis. We recovered 61 bacterial pathogens and 121 enteric viruses with our laboratory assays, while we detected 78 bacteria and 167 viruses with the molecular assay. We calculated specificity and sensitivity for both methods after analysis of discordant results and demonstrated greater sensitivity for multiplex PCR than for our routine methods, with the exception of Salmonella spp. and toxigenic C. difficile detection. The multiplex PCR assay proved to be a reliable tool to directly detect the most common enteropathogens in stool samples but with some limitations.

OBJECTIVE:To investigate the accuracy of procalcitonin (PCT) as a diagnostic marker of nosocomial sepsis (NS) and define the most accurate cut-off to distinguish infected from uninfected neonates.SETTING:Six neonatal intensive care units (NICUs).PATIENTS:762 neonates admitted to six NICUs during a 28-month observational study for whom at least one serum sample was taken on admission.MAIN OUTCOME MEASURES:Positive and negative predictive values at different PCT cut-off levels.RESULTS:The overall probability of an NS was doubled or more if PCT was >0.5 ng/ml. In very-low-birth-weight (VLBW) infants, a cut-off of >2.4 ng/ml gave a positive predictive value of NS near to 50% with a probability of a false-positive diagnosis of NS in about 10% of the patients.CONCLUSIONS:In VLBW neonates, a serum PCT value >2.4 ng/ml prompts early empirical antibiotic therapy, while in normal-birth-weight infants, a PCT value ≤2.4 ng/ml carries a low risk of missing an NS.

Background: The need for reducing unnecessary antibiotic treatment is being emphasized in the management of urinary tract infections (UTI), a disease frequent in childhood. An ideal test should provide early diagnosis without the waiting times of urine culture, but even a simple test of exclusion could significantly improve patient management.Methods: We evaluated the sensitivity, specificity, negative and positive predictive value of automated microscopy IRIS iQ200 combined with the dipstick analyses in children with suspected UTI. Multivariable logistic regression analysis was used to identify the set of variables that best predict positive culture results and develop a numerical risk score.Results: Of 474 consecutive urine samples retrospectively analyzed, 69 were positive at urine culture with prevalence of infection of 14.6%. Parameters significantly associated with the presence of infection in multivariable analysis were age <1 year (p<0.001). leukocyte esterase >= 15 x 10(boolean AND)6/L (p<0.001). number of small particles (ASP) >= 5500x 10(boolean AND)6/L (p<0.001) and bacteria >= 3 x 10(boolean AND)6/L (p = 0.01). The derived score ranged from 0 to 10, with higher values indicating higher risk of UTI. The area under the score ROC curve was 79% (95% CI 0.72-0.85), and was better than those of the individual urinary chemical and microscopic analyses.Conclusions: This routine method could improve the management of UTI in children by early identifying patients with low probability of infection, for whom antibiotic treatment can be withheld until the results of urine culture become available. (C) 2011 Elsevier B.V. All rights reserved.

Proteomics is particularly suitable for characterising human pathogens with high life cycle complexity, such as fungi. Protein content and expression levels may be affected by growth states and life cycle morphs and correlate to species and strain variation. Identification and typing of fungi by conventional methods are often difficult, time-consuming and frequently, for unusual species, inconclusive. Proteomic phenotypes from MALDI-TOF MS were employed as analytical and typing expression profiling of yeast, yeast-like species and strain variants in order to achieve a microbial proteomics population study. Spectra from 303 clinical isolates were generated and processed by standard pattern matching with a MALDI-TOF Biotyper (MT). Identifications (IDs) were compared to a reference biochemical-based system (Vitek-2) and, when discordant, MT IDs were verified with genotyping IDs, obtained by sequencing the 25-28S rRNA hypervariable D2 region. Spectra were converted into virtual gel-like formats, and hierarchical clustering analysis was performed for 274 Candida profiles to investigate species and strain typing correlation. MT provided 257/303 IDs consistent with Vitek-2 ones. However, amongst 26/303 discordant MT IDs, only 5 appeared "true''. No MT identification was achieved for 20/303 isolates for incompleteness of database species variants. Candida spectra clustering agreed with identified species and topology of Candida albicans and Candida parapsilosis specific dendrograms. MT IDs show a high analytical performance and profiling heterogeneity which seems to complement or even outclass existing typing tools. This variability reflects the high biological complexity of yeasts and may be properly exploited to provide epidemiological tracing and infection dispersion patterns.

Introduction. The bacteriology processes almost all the clinical specimens through manually performed cultures and therefore needs to evolve toward automated systems.We report our experience on the benefits of combination between liquid-based flocked swab transport systems (Copan Innovation) and platforms of plating and automatic streaking of agar plates.The main innovation is that, for the first time, the automation can be performed not only for urine and other few liquids materials, but also to most of other biological samples. Methods. Our considerations are based on more than one year of experience on the instrument InocuLAB® (Dynacon Inc.) in two Italian Hospitals, supplemented by theoretical knowledge of other marketed in Italy instruments.The three instruments currently available in Italy (A: InocuLAB® Copan, B:WASP® Copan and C: Previ-Isola bioMérieux®) can inoculate and streak liquid or eluated specimens from liquidbased flocked swab transport systems on any agar plate.The main differences are: streaking of already deposited material on plate (only A and B); choice among various protocols of streak and inoculate (only A and B); automatic opening and recapping of the specimen containers (only A and B); automatic choice of the plates (B: 9 columns and C: 5 columns); partition of the streaked plates in groups (B: up to 4 group s and C: up to 3); forced use of disposables devices (only instrument C). No instrument present problems of carryover and the quality of plated specimens is excellent. Conclusions. Although significantly different, all the platforms combined with flocked swabs represent a pre-analytical workstation for microbiology automation (inoculating, streaking, and bar-coding of liquid specimens). The main advantages can be seen in the operator’s production time, in safety and in quality of streak.The almost complete elution of the biological sample in liquid-based flocked swab transport systems is a standard base which allows to perform many diagnostic tests, including quantitative streaking for quantitative evaluation and multiple culture for additional investigation.

Salmonella enterica est couramment mise en cause dans les maladies gastro-intestinales en Italie. S. Typhimurium represente environ 40 % des isolats, dont la plupart des souches appartiennent au type DT104. Nous decrivons ici les investigations menees lors de l’apparition de S. Typhimurium DT104A, un sous-type jamais observe auparavant en Italie, a Rome au cours du printemps 2004.

Las salmonelas entericas son una causa frecuente de infeccion gastrointestinal en Italia. S. typhimurium representa aproximadamente el 40% de las cepas aisladas y la mayoria de ellas pertenecen al fago tipo DT104. Describimos la investigacion de un brote de S. typhimurium DT104A, un subtipo nunca observado hasta ahora en Italia, que se produjo en Roma durante la primavera de 2004.

Resistance to antibiotics frequently used to treat Helicobacter pylori infection is one of the most important factors in treatment failure. The aims of this study were to determine the accuracy of culture and other invasive and non-invasive tests for diagnosis of H. pylori infection in children, using histological examination as the gold standard, and to evaluate rates of resistance to metronidazole, clarithromycin and amoxicillin. Between March 2003 and December 2004, gastric biopsies from 215 children were tested by histological examination, a rapid urease test and culture. In addition, stool samples were obtained from all children for H. pylori antigen testing. Sixty-four (30%) patients were positive for H. pylori by histological examination. The H. pylori fecal antigen test revealed a sensitivity of 57.8%, a specificity of 93.4%, a positive predictive value of 78.7% and a negative predictive value of 83.9%; rapid urease test had a sensitivity of 70.3%, a specificity of 94.7%, and positive and negative predictive values of 83.3% and 88.2%; culture had a sensitivity of 90.6%, a specificity of 100%, and positive and negative predictive values of 100% and 96.2%. All positive cultures were tested for susceptibility to amoxicillin, metronidazole and clarithromycin by the E-test method. Resistance rates to clarithromycin and metronidazole were 20.7% and 27.6% respectively; no resistance to amoxicillin was observed. This study confirms the place of histological examination as the definitive test for H. pylori, and shows the importance of H. pylori culture in allowing evaluation of antibiotic resistance profiles in pediatric patients.