COVID-19 increased in Italian children in the autumn and winter 2021-2022 period when Omicron was the dominant variant.

AIM: We examined the prevalence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in children during the autumn and winter season from 1 September 2021 to 30 January 2022 and compared it with the same period in 2020-2021. METHODS: This study was carried out int the paediatric emergency department (PED) of a tertiary Italian hospital. We compared the clinical and demographical features of all children who presented during the two study periods and tested positive for SARS-CoV-2. RESULTS: During the 2021-2022 autumn and winter season 5813 children presented to the PED, 19.0% were tested for SARS-CoV-2 and 133 (12.0%) of those tested positive. In 2020-2021, 2914 presented to the PED, 12.3% were tested, and 30 (8.3%) of those tested positive. There were no statistically significant differences in clinical severity during the two study periods, despite a higher percentage of neurological symptoms in 2020-2021. Of the SARS-CoV-2-positive cases, 29/133 (21.8%) were hospitalised during the 2021-2022 season and 10/30 (33.3%) during the previous one. Only 3/163 children required intensive care. CONCLUSION: The greater spread of SARS-CoV-2 was probably due to the greater transmissibility of the Omicron variant, but the symptoms were mild and only 3 children required intensive care.

Evaluation of three commercial assays for SARS-CoV-2 molecular detection in upper respiratory tract samples.

The increasing COVID-19 widespread has created the necessity to assess the diagnostic accuracy of newly introduced (RT-PCR based) assays for SARS-CoV-2 RNA detection in respiratory tract samples. We compared the results of the Allplex™ 2019-nCoV assay with those of the Simplexa™ COVID-19 Direct assay and the Quanty COVID-19 assay, respectively, all performed on 125 nasal/oropharyngeal swab samples of patients with COVID-19 suspicion. Fifty-four samples were positive, and 71 were negative with the Allplex™ assay, whereas 47 of 54 samples were also positive with the Simplexa™ assay. The Quanty assay detected 55 positive samples, including the 54 positive samples with the Allplex™ assay and 1 sample that was Allplex™ negative but Simplexa™ positive. Using a consensus result criterion as the reference standard allowed to resolve the eight samples with discordant results (one Allplex™ negative and seven Simplexa™ negative) as truly false negative. Interestingly, a Spearman's negative association was found between the viral RNA loads quantified by the Quanty assay and the CT values of RT PCRs performed with either the Allplex™ assay or the Simplexa™ assay. However, the strength of this association was higher for the Allplex™ assay (N gene, ρ = - 0.92; RdRP gene, ρ = - 0.91) than for the Simplexa™ assay (ORF1ab gene, ρ = - 0.65; S gene, ρ = - 0.80). The Allplex™ 2019-nCoV, the Simplexa™ COVID-19 Direct, and the Quanty COVID-19 assays yielded comparable results. However, the role these assays might play in future clinical practice warrants larger comparison studies.

Lumipulse G SARS-CoV-2 Ag assay evaluation using clinical samples from different testing groups.

OBJECTIVES: Compared to RT-PCR, lower performance of antigen detection assays, including the Lumipulse G SARS-CoV-2 Ag assay, may depend on specific testing scenarios. METHODS: We tested 594 nasopharyngeal swab samples from individuals with COVID-19 (RT-PCR cycle threshold [Ct] values ≤ 40) or non-COVID-19 (Ct values >40) diagnoses. RT-PCR positive samples were assigned to diagnostic, screening, or monitoring groups of testing. RESULTS: With a limit of detection of 1.2 × 104 SARS-CoV-2 RNA copies/mL, Lumipulse showed positive percent agreement (PPA) of 79.9% (155/194) and negative percent agreement of 99.3% (397/400), whereas PPAs were 100% for samples with Ct values of <18 or 18-<25 and 92.5% for samples with Ct values of 25-<30. By three groups, Lumipulse showed PPA of 87.0% (60/69), 81.1% (43/53), or 72.2% (52/72), respectively, whereas PPA was 100% for samples with Ct values of <18 or 18-<25, and was 94.4, 80.0, or 100% for samples with Ct values of 25-<30, respectively. Additional testing of RT-PCR positive samples for SARS-CoV-2 subgenomic RNA showed that, by three groups, PPA was 63.8% (44/69), 62.3% (33/53), or 33.3% (24/72), respectively. PPAs dropped to 55.6, 20.0, or 41.7% for samples with Ct values of 25-<30, respectively. All 101 samples with a subgenomic RNA positive result had a Lumipulse assay's antigen positive result, whereas only 54 (58.1%) of remaining 93 samples had a Lumipulse assay's antigen positive result. CONCLUSIONS: Lumipulse assay was highly sensitive in samples with low RT-PCR Ct values, implying repeated testing to reduce consequences of false-negative results.

Implementation of the eazyplex® CSF direct panel assay for rapid laboratory diagnosis of bacterial meningitis: 32-month experience at a tertiary care university hospital.

We aimed to report a 32-month laboratory experience with the eazyplex® CSF direct panel assay for the rapid diagnosis of meningitis due to six most common bacterial species (Escherichia coli, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, and Streptococcus pneumoniae). We included all cerebrospinal fluid (CSF) samples from patients admitted with a clinical suspicion of meningitis/encephalitis between May 2016 and December 2018 at our hospital. In addition to the eazyplex® assay, both Gram stain microscopy and culture were performed, and results were confirmed with 16S rRNA PCR/sequencing. Patients' demographics and relevant clinical information were collected. Of 135 studied patients, 44 (32.6%) had a microbiologically documented diagnosis of meningitis. Overall, we identified 21 S. pneumoniae, 10 N. meningitidis, 6 L. monocytogenes, 3 E. coli, 2 Streptococcus pyogenes, 1 S. agalactiae, and 1 Citrobacter koseri as aetiological agents. The eazyplex® assay allowed identification in 40 (90.9%) cases, with four not identified cases due to microorganisms not included in the panel at the time of testing. Thirty-two (72.7%) cases had positive culture results, whereas 28 (63.6%) cases had positive Gram stain results. Notably, combining Gram stain and eazyplex® assay allowed identification in 100% of cases. After notification of rapid results, physicians modified the empiric antibiotic therapy, which became appropriate in three patients (all with L. monocytogenes meningitis). The eazyplex® CSF panel assay worked better than culture in detecting the most common agents of bacterial meningitis and accelerated the diagnosis leading to timely initiation or continuation of appropriate antibiotic therapy.

Efficient Inactivation of Clinically Relevant Antimicrobial Drug Concentrations by BacT/Alert or Bactec Resin-Containing Media in Simulated Adult Blood Cultures.

We assessed the antimicrobial-inactivation capability of BacT/Alert (FA Plus and FN Plus) or Bactec (Plus Aerobic/F and Plus Anaerobic/F) media for 40 antibiotic-bacterium combinations in simulated adult blood cultures. Aside from high recovery rates (93.2% and 88.4%, respectively), we showed that at the lowest but clinically relevant antibiotic concentrations, both BacT/Alert and Bactec media recovered all the organisms tested with drugs except for Escherichia coli, which was tested in the presence of meropenem. Delayed recoveries were mainly associated with vancomycin.

Simplified Testing Method for Direct Detection of Carbapenemase-Producing Organisms from Positive Blood Cultures Using the NG-Test Carba 5 Assay.

We directly tested 484 organisms from clinical (n = 310) and simulated (n = 174) positive blood cultures using the NG-Test Carba 5 assay for carbapenemase-producing Enterobacterales detection. The assay identified all but 4 of the KPC (170/171), OXA-48-like (22/22), VIM (19/21), and NDM (14/15) producers with no false positives. Among the clinical Klebsiella pneumoniae organisms tested, 122 of 123 KPC, 1 of 1 OXA-48-like, and 1 of 2 VIM producers were detected by the assay. Some VIM and NDM producers yielded scant but still-readable bands with the assay. No organisms produced the IMPs that the assay was designed to detect.

Antimicrobial susceptibility testing of pathogens isolated from blood culture: a performance comparison of Accelerate Pheno™ and VITEK® 2 systems with the broth microdilution method.

Objectives: To compare the performance of the Accelerate Pheno™ system with that of the conventional phenotypic VITEK® 2 system for rapid antimicrobial susceptibility testing (AST) of bacterial pathogens from positive blood culture (PBC) samples, based on the reference broth microdilution (BMD) method. Methods: Prospectively collected PBCs that represented patient-unique bloodstream infection episodes were included. For PBC samples showing monomicrobial growth (n = 86), AST was performed using both Accelerate Pheno™ and VITEK® 2 systems directly from PBC broth. Colony isolates derived from subculture of PBC broth were then used for BMD testing. AST results were interpreted according to 2017 EUCAST breakpoints. Results: The overall categorical agreement between Accelerate Pheno™ system and BMD was 92.7% (467/504) for Gram-negative organisms and 99.0% (95/96) for Gram-positive organisms, with rates for very major errors of 3.6% (6/166), major errors 2.2% (9/416) and minor errors 3.8% (23/600). The overall categorical agreement between the VITEK® 2 system and BMD was 91.7% (463/505) for Gram-negative organisms and 99.0% (97/98) for Gram-positive organisms, with rates of very major errors of 2.4% (4/169), major errors 1.0% (4/416) and minor errors 5.8% (35/603). Importantly, unlike the VITEK® 2 system, no false-susceptible results occurred with two colistin-resistant organism-growing PBCs tested using the Accelerate Pheno™ system. Conclusions: Based on these findings, the Accelerate Pheno™ system can be a valid alternative for the rapid AST of Gram-negative and Gram-positive bacteria in bloodstream infections.

T2Bacteria magnetic resonance assay for the rapid detection of ESKAPEc pathogens directly in whole blood.

Objectives: To evaluate the magnetic resonance-based T2Bacteria Panel assay for direct detection of ESKAPEc (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli) pathogens in blood samples of patients with suspected bloodstream infection (BSI). Patients and methods: Adult patients admitted to the Emergency Medicine Department, Infectious Diseases Unit and ICU of a large tertiary-care hospital were included if they had a blood culture (BC) ordered concomitantly with a whole-blood sample for T2Bacteria testing. Results were compared with those of BC and other clinically relevant information. Results: A total of 140 samples from 129 BSI patients were studied. Single bacteria were detected in 15.7% (22/140) and 12.1% (17/140), and multiple bacteria in 2.9% (4/140) and 1.4% (2/140), of samples tested by T2Bacteria and BC, respectively. With respect to the six target (ESKAPEc) species, overall sensitivity and specificity of T2Bacteria across all detection channels in comparison with BC were 83.3% and 97.6%, respectively; these values increased to 89.5% and 98.4%, respectively, when a true-infection criterion (i.e. the same microorganism detected only by T2Bacteria was cultured from another sample type reflecting the source of infection) was used as the comparator. There were 808 T2Bacteria detection results across 112 samples, with concordant negative results, yielding a negative predictive value of 99.8%. The mean time to negative result was 6.1 ±âŸ1.5 h, whereas the mean time to detection/species identification was 5.5 ±âŸ1.4 h. Conclusions: The T2Bacteria Panel assay has the potential to provide accurate and timely diagnosis of ESKAPEc bacteraemia, which might support the direct therapeutic management of BSI patients.

Incidence and antimicrobial resistance trends in bloodstream infections caused by ESKAPE and Escherichia coli at a large teaching hospital in Rome, a 9-year analysis (2007-2015).

The proportion of antimicrobial resistance (AMR) among the ESKAPE and Escherichia coli (ESKAPEEc) pathogens causing bloodstream infection (BSI) increased worldwide. We described longitudinal trends in ESKAPEEc BSI and AMR over 9 years (2007-2015) at a large teaching hospital in Italy. Of 9720 unique BSI episodes, 6002 (61.7%) were caused by ESKAPEEc pathogens. The majority of these episodes (4374; 72.9%) were hospital-onset infections. The most frequent pathogen was E. coli (32.8%), followed by Staphylococcus aureus (20.6%), Klebsiella pneumoniae (16.1%), and Pseudomonas aeruginosa (11.6%). There was a significant increase of hospital-onset K. pneumoniae (from 2.3 to 5.0 per 10,000 patient-days; P = 0.001) and community-onset E. coli (from 3.3 to 9. 1 per 10,000 emergency admissions; P = 0.04) BSIs. Among hospital-onset BSIs, increases of extended-spectrum ß-lactamase (ESBL)-producing E. coli (from 25.4 to 35.2%, P = 0.006), carbapenemase-producing K. pneumoniae (from 4.2 to 51.6%, P < 0.001), and methicillin-resistant S. aureus (from 33.9 to 44.4%, P < 0.001) BSIs were observed between the 2007-2009 and 2010-2012 study periods. In contrast, a decrease of BSIs caused by P. aeruginosa resistant to ceftazidime (from 45.5 to 28.2%, P < 0.001), ciprofloxacin (from 46 to 36.3%, P = 0.05), and meropenem (from 55 to 39.9%, P = 0.03) was observed through all 9 years of the study period. Among community-onset BSIs, increases of BSIs caused by ESBL-producing E. coli (from 28.6 to 42.2%, P = 0.002) and carbapenemase-producing K. pneumoniae (from 0 to 17.6%) were observed between the 2007-2009 and 2010-2012 study periods. Our findings show increased rates of BSI and relative AMR for specific pathogen-health care setting combinations, and call for continued active surveillance and infection control policies.

Early assessment of blood culture negativity as a potential support tool for antimicrobial stewardship.

Objective: To assess whether 48-h negative blood culture (BC) bottles are still negative at the classic 120-h incubation endpoint and whether 48 h might be the time to make antimicrobial therapy decisions. Methods: Data from the first collected bottles from bloodstream infection (BSI) episodes of single patients were retrospectively analyzed. Probabilities of bottles being negative at the classic endpoint were calculated from 0 to 120 h of incubation. Results: Among BC-negative episodes (4018/4901 [82.0%]), most (2097/4018 (52.2%) occurred in medicine patients. At 48 h, probability was 100.0% (95% CI, 99.9-100.0) for all 4018 patients. Of these, 1244 (31.0%) patients remained on antibiotics until 120 h. Excluding 401 (32.2%) patients who received antibiotics for another (non-bloodstream) infection, 843 (67.8%) of 1244 patients could have merited early (48-h) discontinuation of antibiotics. Stopping treatment in these patients would have led to saving 5201 days of access (943 [18.1%] days), watch (3624 [69.7%] days), or reserve (634 [12.2%]) AWaRe groups' antibiotics, which correspond to 65.6% (5201/7928) of days of administered antibiotics in all 1244 patients. Conclusion: As an early indicator of BC negativity, the 48-h endpoint could reliably support antimicrobial stewardship, but the clinical judgment remains imperative especially when BSI is highly suspected.

In-depth characterization of multidrug-resistant NDM-1 and KPC-3 co-producing Klebsiella pneumoniae bloodstream isolates from Italian hospital patients.

Bloodstream infection (BSI) caused by carbapenem-resistant Klebsiella pneumoniae (KP) poses significant challenges, particularly when the infecting isolate carries multiple antimicrobial resistance (AMR) genes/determinants. This study, employing short- and long-read whole-genome sequencing, characterizes six New Delhi metallo-ß-lactamase (NDM) 1 and KP carbapenemase (KPC) 3 co-producing KP isolates, the largest cohort investigated in Europe to date. Five [sequence type (ST) 512] and one (ST11) isolates were recovered from patients who developed BSI from February to August 2022 or February 2023 at two different hospitals in Rome, Italy. Phylogenetic analysis revealed two distinct clusters among ST512 isolates and a separate cluster for the ST11 isolate. Beyond blaNDM-1 and blaKPC-3, various AMR genes, indicative of a multidrug resistance phenotype, including colistin resistance, were found. Each cluster-representative ST512 isolate harbored a blaNDM-1 plasmid (IncC) and a blaKPC-3 plasmid [IncFIB(pQil)/IncFII(K)], while the ST11 isolate harbored a blaNDM-1 plasmid [IncFII(pKPX1)] and a blaKPC-3 plasmid [IncFIB(K)/IncFII(K)]. The blaNDM-1 plasmids carried genes conferring resistance to clinically relevant antimicrobial agents, and the aminoglycoside resistance gene aac(6')-Ib was found on different plasmids. Colistin resistance-associated mgrB/pmrB gene mutations were present in all isolates, and the yersiniabactin-encoding ybt gene was unique to the ST11 isolate. In conclusion, our findings provide insights into the genomic context of blaNDM-1/blaKPC-3 carbapenemase-producing KP isolates.IMPORTANCEThis study underscores the critical role of genomic surveillance as a proactive measure to restrict the spread of carbapenemase-producing KP isolates, especially when key antimicrobial resistance genes, such as blaNDM-1/blaKPC-3, are plasmid borne. In-depth characterization of these isolates may help identify plasmid similarities contributing to their intra-hospital/inter-hospital adaptation and transmission. Despite the lack of data on patient movements, it is possible that carbapenem-resistant isolates were selected to co-produce KP carbapenemase and New Delhi metallo-ß-lactamase via plasmid acquisition. Studies employing long-read whole-genome sequencing should be encouraged to address the emergence of KP clones with converging phenotypes of virulence and resistance to last-resort antimicrobial agents.

Pilot study on cultural and metagenomic analysis of bile and biliary stentslead to unveiling the key players in stent occlusion.

Endoscopic Retrograde Cholangio-Pancreatography (ERCP) with biliary stenting is a minimally invasive medical procedure employed to address both malignant and benign obstructions within the biliary tract. Benign biliary strictures (BBSs), typically arising from surgical interventions such as liver transplants and cholecystectomy, as well as chronic inflammatory conditions, present a common clinical challenge. The current gold standard for treating BBSs involves the periodic insertion of plastic stents at intervals of 3-4 months, spanning a course of approximately one year. Unfortunately, stent occlusion emerges as a prevalent issue within this treatment paradigm, leading to the recurrence of symptoms and necessitating repeated ERCPs. In response to this clinical concern, we initiated a pilot study, delving into the microbial composition present in bile and on the inner surfaces of plastic stents. This investigation encompassed 22 patients afflicted by BBSs who had previously undergone ERCP with plastic stent placement. Our preliminary findings offered promising insights into the microbial culprits behind stent occlusion, with Enterobacter and Lactobacillus spp. standing out as prominent bacterial species known for their biofilm-forming tendencies on stent surfaces. These revelations hold promise for potential interventions, including targeted antimicrobial therapies aimed at curtailing bacterial growth on stents and the development of advanced stent materials boasting anti-biofilm properties.

Chip-Based Molecular Evaluation of a DNA Extraction Protocol for Candida Species from Positive Blood Cultures.

The diagnosis of Candida bloodstream infection (BSI) may rely on a PCR-based analysis of a positive blood culture (PBC) obtained from the patient at the time of BSI. In this study, a yeast DNA extraction protocol for use on PBCs was developed and evaluated with the molecular mouse (MM) yeast blood (YBL) chip-based PCR assay, which allowed us to detect nine medically relevant Candida species. We studied 125 simulated or clinical PBCs for Candida species. A positive correlation between the DNA concentration and colony-forming unit count was found for simulated (Spearman's ρ = 0.58; p < 0.0001) and clinical (Spearman's ρ = 0.23, p = 0.09) PBCs. The extracted DNA yielded positive results with the MM YBL chip assay that agreed with the Candida species-level identification results for 63 (100%) of 63 isolates from simulated PBCs and 66 (99.5%) of 67 isolates from clinical PBCs. The false-negative result was for one C. tropicalis isolate that grew together with C. albicans in PBC. None of the 30 (Candida)-negative clinical BCs included as negative controls yielded a positive result with the MM YBL chip assay. Our DNA extraction protocol for the Candida species couples efficiency and simplicity together. Nevertheless, further studies are needed before it can be adopted for use with the MM YBL chip assay.

Efficient Recovery of Candida auris and Five Other Medically Important Candida Species from Blood Cultures Containing Clinically Relevant Concentrations of Antifungal Agents.

Candida auris and other Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei) are important causes of bloodstream infection. Early or prolonged treatment with antifungal agents is often required. The inhibitory effect of antifungal agents in the patients' bloodstream may compromise the sensitivity of blood culture (BC) to diagnose and/or monitor patients with candidemia. Using a clinical BC simulation model, we compared antimicrobial drug-neutralizing BC media in BacT/Alert FA PLUS (FAP) or Bactec Plus Aerobic/F (PAF) bottles with non-neutralizing BC media in Bactec Mycosis IC/F (MICF) bottles to allow Candida growth in the presence of 100%, 50%, or 25% peak serum level (PSL) antifungal concentrations. In total, 117 organism/antifungal combinations were studied, and Candida growth was detected after incubating bottles into BacT/Alert VIRTUO or Bactec FX BC systems. Compared to control (without antifungal) bottles, both FAP and PAF bottles with 100% PSL antifungal concentrations allowed 100% recovery for C. auris, C. glabrata, and C. parapsilosis, whereas recovery was below 100% for C. albicans, C. krusei, and C. tropicalis. MICF bottles were less efficient at 100%, 50%, or 25% PSL antifungal concentrations, for all Candida species, except for C. auris. While azoles and amphotericin B did not hinder Candida growth in FAP or PAF bottles, echinocandins allowed C. auris, C. glabrata, and C. parapsilosis to grow in FAP, PAF, or MICF bottles. Overall, the maximum time to detection was 4.6 days. Taken together, our findings emphasize the reliability of BCs in patients undergoing antifungal treatment for candidemia. IMPORTANCE While echinocandins remain the preferred antifungal therapy for candidemia, bloodstream infections caused by C. auris, C. glabrata, or, at a lesser extent, C. parapsilosis may be difficult to treat with these antifungal agents. This is in view of the high propensity of the above-mentioned species to develop antifungal resistance or tolerance during treatment. Azoles and amphotericin B are possible alternatives. Thus, optimizing the recovery of Candida from BCs is important to exclude the likelihood of negative BCs for Candida species, owing to the inhibitory effect of antifungal agents present in the blood sample with which BCs are inoculated. Consistently, our results about the recovery of medically important Candida species (including C. auris) from simulated BCs in BacT/Alert FAP, Bactec PAF, or Bactec MICF bottles containing clinically relevant antifungal concentrations add support to this research topic, as well as to the use of BCs for monitoring the clinical and therapeutic course of candidemia.

Update of European Society of Clinical Microbiology and Infectious Diseases coronavirus disease 2019 guidelines: diagnostic testing for severe acute respiratory syndrome coronavirus 2.

SCOPE: Since the onset of COVID-19, several assays have been deployed for the diagnosis of SARS-CoV-2. The European Society of Clinical Microbiology and Infectious Diseases (ESCMID) published the first set of guidelines on SARS-CoV-2 in vitro diagnosis in February 2022. Because the COVID-19 landscape is rapidly evolving, the relevant ESCMID guidelines panel releases an update of the previously published recommendations on diagnostic testing for SARS-CoV-2. This update aims to delineate the best diagnostic approach for SARS-CoV-2 in different populations based on current evidence. METHODS: An ESCMID COVID-19 guidelines task force was established by the ESCMID Executive Committee. A small group was established, half appointed by the chair, and the remaining selected with an open call. The panel met virtually once a week. For all decisions, a simple majority vote was used. A list of clinical questions using the population, intervention, comparison, and outcome (PICO) format was developed at the beginning of the process. For each PICO, 2 panel members performed a literature search focusing on systematic reviews with a third panellist involved in case of inconsistent results. The panel reassessed the PICOs previously defined as priority in the first set of guidelines and decided to address 49 PICO questions, because 6 of them were discarded as outdated/non-clinically relevant. The 'Grading of Recommendations Assessment, Development and Evaluation (GRADE)-adoption, adaptation, and de novo development of recommendations (ADOLOPMENT)' evidence-to-decision framework was used to produce the guidelines. QUESTIONS ADDRESSED BY THE GUIDELINES AND RECOMMENDATIONS: After literature search, we updated 16 PICO questions; these PICOs address the use of antigen-based assays among symptomatic and asymptomatic patients with different ages, COVID-19 severity status or risk for severe COVID-19, time since the onset of symptoms/contact with an infectious case, and finally, types of biomaterials used.

SARS-CoV-2 Antigen Test Results to Infer Active or Non-Active Virus Replication Status in COVID-19 Patients.

We used nasopharyngeal swab samples of patients with a symptomatic (n = 82) or asymptomatic (n = 20) coronavirus disease 2019 (COVID-19) diagnosis to assess the ability of antigen detection tests to infer active (potentially transmissible) or inactive (potentially non-transmissible) infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using the subgenomic RNA (sgRNA) as an active replication marker of SARS-CoV-2, 48 (76.2%), 56 (88.9%), and 63 (100%) of 63 samples with sgRNA positive results tested positive with the SD BIOSENSOR STANDARD Q COVID-19 Ag (Standard Q), the SD BIOSENSOR STANDARD F COVID-19 Ag FIA (Standard F), or the Fujirebio LUMIPULSE G SARS-CoV-2 Ag (Lumipulse) assay, respectively. Conversely, 37 (94.9%), 29 (74.4%), and 7 (17.9%) of 39 samples with sgRNA negative results tested negative with Standard Q, Standard F, or Lumipulse, respectively. Stratifying results by the number of days of symptoms before testing revealed that most antigen positive/sgRNA positive results were among samples tested at 2-7 days regardless of the assay used. Conversely, most antigen negative/sgRNA negative results were among samples tested at 16-30 days only when Standard Q or Standard F were used. In conclusion, based on our findings, a negative antigen test, especially with the Lumipulse assay, or a positive antigen test, especially with the Standard F assay, may suggest, respectively, the absence or presence of replication-competent SARS-CoV-2.

Resistance and virulence features of hypermucoviscous Klebsiella pneumoniae from bloodstream infections: Results of a nationwide Italian surveillance study.

Among Enterobacterales, Klebsiella pneumoniae (Kp) is one of the major opportunistic pathogens causing hospital-acquired infections. The most problematic phenomenon linked to Kp is related to the dissemination of multi-drug resistant (MDR) clones producing carbapenem-hydrolyzing enzymes, representing a clinical and public health threat at a global scale. Over the past decades, high-risk MDR clones (e.g., ST512, ST307, ST101 producing bla KPC-type carbepenemases) have become endemic in several countries, including Italy. Concurrently, the spread of highly virulent Kp lineages (e.g., ST23, ST86) able to cause severe, community-acquired, pyogenic infections with metastatic dissemination in immunocompetent subjects has started to be documented. These clones, designated as hypervirulent Kp (hvKp), produce an extensive array of virulence factors and are highly virulent in previously validated animal models. While the prevalence and distribution of MDR Kp has been previously assessed at local and national level knowledge about dissemination of hvKp remains scarce. In this work, we studied the phenotypic and genotypic features of hypermucoviscous (HMV, as possible marker of increased virulence) Kp isolates from bloodstream infections (BSI), obtained in 2016-17 from 43 Italian Laboratories. Antimicrobial susceptibility testing, whole genome sequencing and the use of two animal models (G. mellonella and murine) were employed to characterize collected isolates. Over 1502 BSI recorded in the study period, a total of 19 Kp were selected for further investigation based on their HMV phenotype. Results showed that hvKp isolates (ST5, ST8, ST11, ST25) are circulating in Italy, although with a low prevalence and in absence of a clonal expansion; convergence of virulence (yersiniabactin and/or salmochelin, aerobactin, regulators of mucoid phenotype) and antimicrobial-resistance (extended-spectrum beta-lactamases) features was observed in some cases. Conventional MDR Kp clones (ST307, ST512) may exhibit an HMV phenotype, but with a low virulence potential in the animal models. To the best of our knowledge, this work represents the first systematic survey on HMV and hvKp in Italy, employing a functional characterization of collected isolates. Future surveillance programs are warranted to monitor the threatening convergence of virulence and resistance among MDR Kp and the spread of hvKp.