Persistence of SARS-CoV-2 infection and viral intra- and inter-host evolution in COVID-19 hospitalized patients.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) persistence in COVID-19 patients could play a key role in the emergence of variants of concern. The rapid intra-host evolution of SARS-CoV-2 may result in an increased transmissibility, immune and therapeutic escape which could be a direct consequence of COVID-19 epidemic currents. In this context, a longitudinal retrospective study on eight consecutive COVID-19 patients with persistent SARS-CoV-2 infection, from January 2022 to March 2023, was conducted. To characterize the intra- and inter-host viral evolution, whole genome sequencing and phylogenetic analysis were performed on nasopharyngeal samples collected at different time points. Phylogenetic reconstruction revealed an accelerated SARS-CoV-2 intra-host evolution and emergence of antigenically divergent variants. The Bayesian inference and principal coordinate analysis analysis showed a host-based genomic structuring among antigenically divergent variants, that might reflect the positive effect of containment practices, within the critical hospital area. All longitudinal antigenically divergent isolates shared a wide range of amino acidic (aa) changes, particularly in the Spike (S) glycoprotein, that increased viral transmissibility (K417N, S477N, N501Y and Q498R), enhanced infectivity (R346T, S373P, R408S, T478K, Q498R, Y505H, D614G, H655Y, N679K and P681H), caused host immune escape (S371L, S375F, T376A, K417N, and K444T/R) and displayed partial or complete resistance to treatments (G339D, R346K/T, S371F/L, S375F, T376A, D405N, N440K, G446S, N460K, E484A, F486V, Q493R, G496S and Q498R). These results suggest that multiple novel variants which emerge in the patient during persistent infection, might spread to another individual and continue to evolve. A pro-active genomic surveillance of persistent SARS-CoV-2 infected patients is recommended to identify genetically divergent lineages before their diffusion.

Low temperature bacterial expression of the neutral amino acid transporters SLC1A5 (ASCT2), and SLC6A19 (B0AT1).

It is well established that Escherichia coli represents a powerful tool for the over-expression of human proteins for structure/function studies. In many cases, such as for membrane transporters, the bacterial toxicity or the aggregation of the target protein hamper the expression limiting the application of this tool. The aim of this study was finding the appropriate conditions for the expression of reluctant proteins that is the human neutral amino acid transporters ASCT2 and B0AT1, that have great relevance to human health in cancer therapy and in COVID-19 research, respectively. The cDNAs coding for the proteins of interest were cloned in the pCOLD I vector and different E. coli strains (BL21 codon plus RIL, and RosettaGami2) were cultured in absence or in presence of glucose (0.5-1%), at low temperature (15 °C), and low inducer concentrations (10-100 µM). Cell growth and protein production were monitored by optical density measurements and western blotting assay, respectively. Even though in different conditions, the expression of both amino acid transporters was obtained.Reducing the growth rate of specific E. coli strains by lowering the temperature and the IPTG concentration, together with the addition of glucose, two reluctant human neutral amino acid transporters have been expressed in E. coli. The results have a potentially great interest in drug discovery since ASCT2 is an acknowledged target of anticancer therapy, and B0AT1 together with ACE2 is part of a receptor for the SARS-Cov-2 RBD proteins.

Clostridioides difficile Infection: Use of Inflammatory Biomarkers and Hemogram-Derived Ratios to Predict Mortality Risk in Hospitalized Patients.

BACKGROUND: Clostridioides difficile infection (CDI) is a significant cause of mortality, especially in healthcare environments. Reliable biomarkers that can accurately predict mortality in CDI patients are yet to be evaluated. Our study aims to evaluate the accuracy of several inflammatory biomarkers and hemogram-derived ratios in predicting mortality in CDI patients, such as the neutrophil-to-lymphocyte ratio (NLR), the systemic immune-inflammation index (SII), the platelet-to-neutrophil ratio (PNR), the derived neutrophil-to-lymphocyte ratio (dNLR), C-reactive protein (CRP), the platelet-to-lymphocyte ratio (PLR), and procalcitonin (PCT). RESULTS: NLR showed a sensitivity of 72.5% and a specificity of 58.42% with an area under curve (AUC) = 0.652. SII had a sensitivity of 77.5%, a specificity of 54.74%, and an AUC = 0.64. PNR, neutrophils, dNLR, and lymphocytes had lower AUCs which ranged from 0.595 to 0.616, with varied sensitivity and specificity. CRP, leukocytes, and platelets showed modest predictive values with AUCs below 0.6. PCT had a sensitivity of 100%, a low specificity of 7.41%, and an AUC = 0.528. METHODS: We conducted a retrospective analysis of CDI patients from two different hospital settings in Italy and Romania during the COVID-19 pandemic, from 1 January 2020 to 5 May 2023. Statistical analyses included t-tests, Wilcoxon rank-sum tests, χ2 tests, and multivariate logistic regression to identify predictors of mortality. ROC analysis assessed the accuracy of biomarkers and hemogram-derived ratios. A p value < 0.05 was considered significant. CONCLUSIONS: Neutrophils, dNLR, NLR, SII, and PNR are valuable biomarkers for predicting mortality in CDI patients. Understanding these predictors can improve risk stratification and clinical outcomes for CDI patients.

Prevalence of Enteric Pathogens and Antibiotic Resistance: Results of a Six-Year Active Surveillance Study on Patients Admitted to a Teaching Hospital.

BACKGROUND: Acute Infectious Diarrhea (AID) and the short- and long-term complications associated with it are major causes of hospitalization worldwide. In Italy, due to a lack of robust surveillance programs, only limited data has been collected on their prevalence and circulation. This study aims to evaluate the resistance pattern of enteric pathogens and their epidemiological trends over a six-year period. METHODS: This cross-sectional retrospective study was conducted from January 2018 to December 2023. Stool samples were analyzed during routine diagnosis with culture methods, syndromic molecular tests, and enzyme immunoassay. RESULTS: Bacteria were the most isolated enteric pathogens (62.2%), followed by fungi (29.0%), viruses (8.2%), and parasites (0.6%). Most bacteria were isolated from outpatients (29.5%) and from patients in the Oncology ward (26.2%). The most prevalent target was EPEC (11.1%), followed by C. difficile toxin A/B-producing strains (8.3%), C. jejuni (2.5%), and S. enterica, (1%.). Norovirus and Candida spp. were the most prevalent in pediatric patients (6.5% and 39.6%, respectively). In the last years, enteric pathogens have been a frequent cause of infections characterized by a problematic resistance to common antimicrobials. In our study, S. enterica showed resistance to amikacin, gentamicin, ampicillin, levofloxacin, and ciprofloxacin. C. jejuni was susceptible to all tested drugs. CONCLUSION: Timely notification of gastroenteric infections is crucial in identifying potential outbreak sources and ensuring strict adherence to food safety and hygiene practices, so as to protect the most vulnerable populations. The present study offers insights into the epidemiological characteristics and the antibiotic susceptibility of the main enteric AID pathogens in order to implement infection control measures in health care settings.

Orthopedic Device-Related Infections Due to Emerging Pathogens Diagnosed by a Combination of Microbiological Approaches: Case Series and Literature Review.

Orthopedic and trauma device-related infections (ODRI) due to high virulence microorganisms are a devastating complication after orthopedic surgery. Coagulase-negative Staphylococci (CoNS) are mainly involved but commensal bacteria, located in human mucous membranes, are emerging pathogens in ODRI. Currently, bacterial culture is the gold standard for ODRI but the diagnostic process remains time consuming and laborious. We evaluated a combination of microbiological approaches in the diagnosis of emerging pathogens involved in ODRI. We analyzed two synovial fluids, five tissue samples and five surgical wound swabs from two different patients with ODRI, attending the Department of Orthopedic and Trauma Surgery of Mater Domini Teaching Hospital, Catanzaro, Italy. Identification was carried out with a combination of microbiological approaches (culture, mass spectrometry and 16s rRNA gene sequencing). We demonstrated the importance of a combination of microbiological approaches for the diagnosis of emerging pathogens in ODRI, because the low number of cases in the literature makes it very difficult to formulate guidelines for the management of patients.