First Case of VIM-1-like-Producing Pseudomonas putida Bacteremia in an Oncohematological Pediatric Patient in Italy.

Bacterial infections caused by multidrug-resistant (MDR) Gram-negatives are of great concern worldwide, as they are frequently associated with high mortality and morbidity rates. To date, two cases of VIM-2 metallo-ß-lactamase (MBL)-producing Pseudomonas putida bacteremia have been ever reported in France and Spain between 2004 and 2010. Here, we present the first case of VIM-1-like-producing P. putida isolated in blood culture collected from an oncohematological pediatric patient admitted to Bambino Gesù Children's Hospital (IRCCS) in Rome, Italy.

Clonal Spread of Hospital-Acquired NDM-1-Producing Klebsiella pneumoniae and Escherichia coli in an Italian Neonatal Surgery Unit: A Retrospective Study.

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.

"CORE" a new assay for rapid identification of Klebsiella pneumoniae COlistin REsistant strains by MALDI-TOF MS in positive-ion mode.

Due to the global spread of pan resistant organisms, colistin is actually considered as one of the last resort antibiotics against MDR and XDR bacterial infections. The emergence of colistin resistant strains has been observed worldwide in Gram-negative bacteria, such as Enterobacteriaceae and especially in K. pneumoniae, in association with increased morbidity and mortality. This landscape implies the exploration of novel assays able to target colistin resistant strains rapidly. In this study, we developed and evaluated a new MALDI-TOF MS assay in positive-ion mode that allows quantitative or qualitative discrimination between colistin susceptible (18) or resistant (32) K. pneumoniae strains in 3 h by using the "Autof MS 1000" mass spectrometer. The proposed assay, if integrated in the diagnostic workflow, may be of help for the antimicrobial stewardship and the control of the spread of K. pneumoniae colistin resistant isolates in hospital settings.

Effective Rapid Diagnosis of Bacterial and Fungal Bloodstream Infections by T2 Magnetic Resonance Technology in the Pediatric Population.

Children are prone to bloodstream infections (BSIs), the rapid and accurate diagnosis of which is an unmet clinical need. The T2MR technology is a direct molecular assay for identification of BSI pathogens, which can help to overcome the limits of blood culture (BC) such as diagnostic accuracy, blood volumes required, and turnaround time. We analyzed results obtained with the T2Bacteria (648) and T2Candida (106) panels in pediatric patients of the Bambino Gesù Children's Hospital between May 2018 and September 2020 in order to evaluate the performance of the T2Dx instrument with respect to BC. T2Bacteria and T2Candida panels showed 84.2% and 100% sensitivity with 85.9% and 94.1% specificity, respectively. The sensitivity and specificity of the T2Bacteria panel increased to 94.9% and 98.7%, respectively, when BC was negative but other laboratory data supported the molecular result. T2Bacteria sensitivity was 100% with blood volumes <2 mL in neonates and infants. T2Bacteria and T2Candida provided definitive microorganism identification in a mean time of 4.4 and 3.7 h, respectively, versus 65.7 and 125.5 h for BCs (P < 0.001). T2 panels rapidly and accurately enable a diagnosis of a pediatric BSI, even in children under 1 year of age and for very small blood volumes. These findings support their clinical use in life-threatening pediatric infections, where the time to diagnosis is of utmost importance, in order to improve survival and minimize the long-term sequalae of sepsis. The T2 technology could be further developed to include more bacteria and fungi species that are involved in the etiology of sepsis.

Evidence of pediatric sepsis caused by a drug resistant Lactococcus garvieae contaminated platelet concentrate.

Owing to an increasing number of infections in adults, Lactococcus (L.) garvieae has gained recognition as an emerging human pathogen, causing bacteraemia and septicaemia. In September 2020, four paediatric onco-hematologic patients received a platelet concentrate from the same adult donor at Bambino Gesù Children's Hospital IRCCS, Rome. Three of four patients experienced L. garvieae sepsis one day after transfusion. The L. garvieae pediatric isolates and the donor's platelet concentrates were retrospectively collected for whole-genome sequencing and shot-gun metagenomics, respectively (Illumina HiSeq). By de novo assembly of the L. garvieae genomes, we found that all three pediatric isolates shared a 99.9% identity and were characterized by 440 common SNPs. Plasmid pUC11C (conferring virulence properties) and the temperate prophage Plg-Tb25 were detected in all three strains. Core SNP genome-based maximum likelihood and Bayesian trees confirmed their phylogenetic common origin and revealed their relationship with L. garvieae strains affecting cows and humans (bootstrap values >100 and posterior probabilities = 1.00). Bacterial reads obtained by the donor's platelet concentrate have been profiled with MetaPhlAn2 (v.2.7.5); among these, 29.9% belonged to Firmicutes, and 5.16% to Streptococcaceae (>97% identity with L. garvieae), confirming the presence of L. garvieae in the platelet concentrate transfusion. These data showed three episodes of sepsis for the first time due to a transfusion-associated transmission of L. garvieae in three pediatric hospitalized hematology patients. This highlights the importance to implement the screening of platelet components with new human-defined pathogens for ensuring the safety of blood supply, and more broadly, for the surveillance of emerging pathogens.

Prevalence and Molecular Typing of Carbapenemase-Producing Enterobacterales among Newborn Patients in Italy.

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.

Case Report: Trichosporon japonicum Fungemia in a Pediatric Patient With Refractory Acute B Cell Lymphoblastic Leukemia.

Trichosporon japonicum is a very rare opportunistic yeast causing fungal disease in humans, especially in immunocompromised hosts. Here, we describe a new case of T. japonicum isolated from the blood of a pyrexial pediatric patient with refractory acute B cell lymphoblastic leukemia and acute respiratory distress. Prompt diagnosis through early clinical suspicion and appropriate molecular microbiology analysis allowed the yeast to be accurately identified at species level. Subsequent drug susceptibility testing and focused antifungal treatment with voriconazole and amphotericin B led to a complete clinical and mycological resolution of the infection, which represents the second successful case of T. japonicum bloodstream infection described in literature to date.

Diagnosis of COVID-19 in children guided by lack of fever and exposure to SARS-CoV-2.

BACKGROUND: The objective of this study is to test how certain signs and symptoms related to COVID-19 in children predict the positivity or negativity of the SARS-CoV-2 nasopharyngeal swab in children. METHODS: We review the data of children who were tested for SARS-CoV-2 for a suspected infection. We compared the clinical characteristics of the subjects who tested positive and negative, including the sensibility, positive and negative predictive value of different combination of signs and symptoms. RESULTS: Of all the suspected infected, 2596 tested negative (96.2%) and 103 tested positive (3.8%). The median age was 7.0 and 5.3 years for the positive and negative ones, respectively. The female to male ratio was ~1:1.3. Fever and respiratory symptoms were mostly reported. Most positive children had a prior exposure to SARS-CoV-2-infected subjects (59.2%). A total of 99.3% of patients without fever nor exposure to the virus proved negative to the SARS-CoV-2 test. CONCLUSIONS: Our study suggests that a child without fever or contact with infected subjects is SARS-CoV-2 negative. If this were to be confirmed, many resources would be spared, with improved care of both COVID-19 and not COVID-19-affected children. IMPACT: Key message: lack of fever and exposure to SARS-CoV-2-infected people highly predicts a negative results of the SARS-CoV-2 nasopharyngeal swab in the paediatric population. Added value to the current literature: this is the first article to prove this point. IMPACT: reduction of emergency department accesses of children with suspected SARS-CoV-2 infection; increased outpatient management of children with cough or other common respiratory symptoms of infancy; sparing of many human and material health resources.

Highly Specific Memory B Cells Generation after the 2nd Dose of BNT162b2 Vaccine Compensate for the Decline of Serum Antibodies and Absence of Mucosal IgA.

Specific memory B cells and antibodies are a reliable read-out of vaccine efficacy. We analysed these biomarkers after one and two doses of BNT162b2 vaccine. The second dose significantly increases the level of highly specific memory B cells and antibodies. Two months after the second dose, specific antibody levels decline, but highly specific memory B cells continue to increase, thus predicting a sustained protection from COVID-19. We show that although mucosal IgA is not induced by the vaccination, memory B cells migrate in response to inflammation and secrete IgA at mucosal sites. We show that the first vaccine dose may lead to an insufficient number of highly specific memory B cells and low concentration of serum antibodies, thus leaving vaccinees without the immune robustness needed to ensure viral elimination and herd immunity. We also clarify that the reduction of serum antibodies does not diminish the force and duration of the immune protection induced by vaccination. The vaccine does not induce sterilizing immunity. Infection after vaccination may be caused by the lack of local preventive immunity because of the absence of mucosal IgA.

High Resolution Melting-Typing (HRMT) of methicillin-resistant Staphylococcus aureus (MRSA): The new frontier to replace multi-locus sequence typing (MLST) for epidemiological surveillance studies.

We report an implemented molecular-typing-method based on HRMA to detect SNPs within MLST loci, characterizing 100 clinical MRSA and 11 control strains, representative of Italian clones. The results provide solid evidence that HRMT could be a fast, cost-effective and reliable alternative to MLST, for MRSA molecular epidemiology.