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Background Low T3 syndrome is frequent in patients admitted to intensive care units for critical illness and pneumonia. It has been reported also in patients with COVID-19, Hodgkin disease and chronic lymphocytic leukemia. We analyzed the clinical relevance of Low T3 syndrome in COVID-19 patients and, in particular, in those with associated hematological malignancies. Methods Sixty-two consecutive patients, hospitalized during the first wave of SARS-CoV-2 outbreak in Sant’Andrea University Hospital in Rome, were subdivided in 38 patients (Group A), showing low levels of FT3, and in 24 patients (Group B), with normal FT3 serum values. During the acute phase of the disease, we measured serum, radiologic and clinical disease severity markers and scores, in search of possible correlations with FT3 serum values. In addition, in 6 COVID-19 patients, 4 with Low T3 syndrome, including 2 with a hematological malignancy, and 2 with normal FT3 values, we performed, high-dimensional single-cell analysis by mass cytometry, multiplex cytokine assay and gene expression profiling in peripheral blood mononuclear cells (PBMC). Results Low FT3 serum values were correlated with increased Absolute Neutrophil Count, NLR and dNLR ratios and with reduced total count of CD3+, CD4+ and CD8+ T cells. Low FT3 values correlated also with increased levels of inflammation, tissue damage and coagulation serum markers as well as with SOFA, LIPI and TSS scores. The CyTOF analysis demonstrated reduction of the effector memory and terminal effector subtypes of the CD4+ T lymphocytes. Multiplex cytokine assay indicates that mainly IL-6, IP-10 and MCAF changes are associated with FT3 serum levels, particularly in patients with coexistent hematological malignancies. Gene expression analysis using Nanostring identified four genes differently expressed involved in host immune response, namely CD38, CD79B, IFIT3 and NLRP3. Conclusions Our study demonstrates that low FT3 serum levels are associated with severe COVID-19. Our multi-omics approach suggests that T3 is involved in the immune response in COVID-19 and coexistent hematological malignancy and new possible T3 target genes in these patients have been identified.

Influenza is a relevant public health problem, also due to the risk of complications. The most effective measure to prevent influenza is vaccination; therefore, at present, there is consensus among European countries, regarding the need for routine seasonal influenza vaccination of elderly and individuals at increased risk of severe influenza. At the same time, influenza surveillance is necessary to understand the viruses circulating and effectiveness of vaccination strategies. The present study reports the results of two seasons influenza surveillance (2017/2018 and 2018/2019) conduced in an University Hospital in Rome among hospitalized patients aged ≥65 years.A prospective cohort study.The study consisted of systematic daily screening of all admissions among patients aged ≥65 years meeting a syndromic SARI case definition during two consecutive influenza seasons: 2017/2018 and 2018/2019. Characteristics of patients and their risk factors were collected by a standardized questionnaire and nose-pharyngeal swabs were performed to each patient. Influenza vaccine effectiveness (IVE), rates of vaccinated subjects and case fatality rate were also evaluated.Influenza was laboratory confirmed in 11 (9.9%) of the 111 and 11 (9.6%) of the 115 enrolled patients in seasons 2017/18 and 2018/19, respectively. Adjusted IVE against all influenza type, calculated for each season, was 88.5% (95% CI: 38.9 to 97.8) and 61.7% (95% CI: -59.9 to 90.9) for 2017/2018 and 2018/2019 seasons, respectively. Our analysis shows a Case Fatality Rate of 2.7% and 4.3% for the 2017/18 and 2018/19 seasons, respectively.The surveillance of SARI conduced in one hospital in Rome confirmed that influenza is an important cause of hospital admissions. Routine monitoring of infectious diseases and related aetiology associated with SARI, also at the local-level, is useful for targeting the right preventive measures.

Background Two methods for detecting synovial fluids alpha defensins are available: the enzyme-linked immunosorbent assay and the lateral flow test. For both, the proper role and accuracy remain uncertain. The purpose of this study was to assess the accuracy of the matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) for alpha defensin detection in synovial fluids of patients with total knee arthroplasty/total hip arthroplasty failures. The hypothesis was that the alpha defensin measurement through MALDI-TOF MS assay could be a high sensitive and specific test for periprosthetic joint infections (PJI) diagnosis as compared with Musculoskeletal Infection Society (MSIS) criteria. Methods The study included 138 patients. The 2018 MSIS criteria were used to diagnose PJIs. Synovial fluids were assessed for routinely synovial fluid tests and alpha defensin measurement through MALDI-TOF MS. Sensitivity, specificity, overall diagnostic accuracy, positive and negative predictive values, receiver operator curves, and area under the curve were calculated. Results As per the 2018 MSIS criteria, 59 PJIs (43%) and 79 aseptic failures (57%) were diagnosed. The MALDI-TOF MS assay showed an overall accuracy of 94.9%. The sensitivity was 93%, the specificity was 96%, the positive predictive value was 95%, and the negative predictive value was 95%. Receiver operator curves analysis demonstrates an area under the curve of 0.95 (P Conclusion The MALDI-TOF MS assay showed high sensitivity and specificity for alpha defensin detection in case of total knee arthroplasty/total hip arthroplasty failures. The advantages of the technology, such as the few milliliters of sample needed, the rapidity of obtaining results, and the cost-effectiveness of the procedure could make the MALDI-TOF MS alpha defensin assay a useful and widespread test in clinical practice.

SARS-CoV-2 environmental contamination occurs through droplets and biological fluids released in the surroundings from patients or asymptomatic carriers. Surfaces and objects contaminated by saliva or nose secretions represent a risk for indirect transmission of COVID-19. We assayed surfaces from hospital and living spaces to identify the presence of viral RNA and the spread of fomites in the environment. Anthropic contamination by droplets and biological fluids was monitored by detecting the microbiota signature using multiplex RT-PCR on selected species and massive sequencing on 16S-amplicons.A total of 92 samples (flocked swab) were collected from critical areas during the pandemic, including indoor (3 hospitals and 3 public buildings) and outdoor surfaces exposed to anthropic contamination (handles and handrails, playgrounds). Traces of biological fluids were frequently detected in spaces open to the public and on objects that are touched with the hands (>80%). However, viral RNA was not detected in hospital wards or other indoor and outdoor surfaces either in the air system of a COVID-hospital, but only in the surroundings of an infected patient, in consistent association with droplets traces and fomites. Handled objects accumulated the highest level of multiple contaminations by saliva, nose secretions and faecal traces, further supporting the priority role of handwashing in prevention.In conclusion, anthropic contamination by droplets and biological fluids is widespread in spaces open to the public and can be traced by RT-PCR. Monitoring fomites can support evaluation of indirect transmission risks for Coronavirus or other flu-like viruses in the environment.ImportanceSeveral studies searched for SARS-CoV-2 in the environment because saliva and nasopharyngeal droplets can land on objects and surfaces creating fomites. However, the ideal indicator would be the detection of the biofluid. This approach was not yet considered, but follows a traditional principle in hygiene, using indicators rather than pathogens. We searched for viral RNA but also for droplets on surfaces at risk. For the first time, we propose to monitor droplets thorugh their microbiota, by RT-PCR or NGS.Even if performed during the pandemic, SARS-CoV-2 wasn’t largely spread on surfaces, unless in proximity of an infectious patient. However, anthropic contamination was frequently at high level, suggesting a putative marker for indirect transmission and risk assessment. Moreover, all SARS-CoV-2-contaminated surfaces showed the droplets’ microbiota.Fomites detection may have an impact on public health, supporting prevention of indirect transmission also for other communicable diseases such as Flu and Flu-like infections.GRAPHICAL ABSTRACT

Immune dysregulation is a hallmark of patients infected by SARS-CoV2 and the balance between immune reactivity and tolerance is a key determinant of all stages of infection, including the excessive inflammatory state causing the acute respiratory distress syndrome. The kynurenine pathway (KP) of tryptophan (Trp) metabolism is activated by pro-inflammatory cytokines and drives mechanisms of immune tolerance. We examined the state of activation of the KP by measuring the Kyn:Trp ratio in the serum of healthy subjects (n = 239), and SARS-CoV2-negative (n = 305) and -positive patients (n = 89). Patients were recruited at the Emergency Room of St. Andrea Hospital (Rome, Italy). Kyn and Trp serum levels were assessed by HPLC/MS-MS. Compared to healthy controls, both SARS-CoV2-negative and -positive patients showed an increase in the Kyn:Trp ratio. The increase was larger in SARS-CoV2-positive patients, with a significant difference between SARS-CoV2-positive and -negative patients. In addition, the increase was more prominent in males, and positively correlated with age and severity of SARS-CoV2 infection, categorized as follows: 1 = no need for intensive care unit (ICU); 2 ≤ 3 weeks spent in ICU; 3 ≥ 3 weeks spent in ICU; and 4 = death. The highest Kyn:Trp values were found in SARS-CoV2-positive patients with severe lymphopenia. These findings suggest that the Kyn:Trp ratio reflects the level of inflammation associated with SARS-CoV2 infection, and, therefore, might represent a valuable biomarker for therapeutic intervention., Highlights • The Kynurenine:Tryptophan ratio is an activation biomarker of the kynurenine pathway. • The kynurenine pathway has a key role in the regulation of the immune system. • The Kyn:Trp ratio is significantly increased in SARS-CoV2-postive patients. • The increase is more prominent in males than in females' SARS-CoV2-positive patients. • Increased Kyn:Trp ratio is associated with lymphopenia and COVID-19 severity.

Rationale Detection of α-defensins in synovial fluid is gaining more and more interest in the field of correct diagnosis of periprosthetic joint infections (PJIs). At present, they can be assessed by a quantitative enzyme-linked immunosorbent assay which is expensive and time-consuming and by a qualitative lateral flow immunoassay which is rapid but quite expensive and whose clinical sensitivity is debated. Thus, developing an alternative rapid, accurate, and low-cost assay for α-defensins is important to make α-defensins actionable as novel key clinical markers. Methods Synovial fluid (SF) samples were obtained from 18 patients undergoing revision of primary joint arthroplasty. Of these, eight met the 2013 Musculoskeletal Infection Society (MSIS) criteria for PJIs, the remaining were classified as aseptic failure. Microbiological analysis and Synovasure assays were carried out on all samples. Sample preparation and the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) settings were adjusted to detect human neutrophil peptide (HNP)-1, -2 and -3 and to obtain optimal results in term of sensitivity and stability. Results MALDI-TOF MS was able to detect HNPs in SF from septic patients. No signals for HNPs were detected in SF from aseptic failure. The limits of detection (LOD) were 2.5 and 1.25 μg/mL for HNP-2 and HNP-1, respectively. The turnaround time of the analysis is 20 min, and SF samples are stable at -20°C for up to 3 days. Assay sensitivity, specificity, and positive and negative predictive values (PPV and NPV) were 100% for all parameters. On the same SF samples, the Synovasure assay showed lower sensitivity specificity, and PPV and NPV of 87.5%, 90%, 87.5% and 90%, respectively. Microbiological analysis of SF confirmed the presence of bacteria only in SF MSIS-positive patients. Conclusions The reported MALDI-TOF MS assay was able to detect and differentiate HNPs in SF samples and showed a slightly better diagnostic accuracy than the Synovasure assay.

In March 2020, the World Health Organization (WHO) declared that the COVID-19 outbreak recorded over the previous months could be characterized as a pandemic. The first known Italian SARS-CoV-2 positive case was reported on 21 February. In some countries, cases of suspected &ldquo, COVID-19-like pneumonia&rdquo, had been reported earlier than those officially accepted by health authorities. This has led many investigators to check preserved biological or environmental samples to see whether the virus was detectable on dates prior to those officially stated. With regard to Italy, the results of a microbiological screening in sewage samples collected between the end of February and the beginning of April 2020 from wastewaters in Milan (Northern Italy) and Rome (Central Italy) showed presence of SARS-CoV-2. In the present study, we evaluated, by means of a standardized diagnostic method, the SARS-CoV-2 infection prevalence amongst patients affected by severe acute respiratory syndrome (SARI) in an academic hospital located in Central Italy during the period of 1 November 2019&ndash, 1 March 2020. Overall, the number of emergency room (ER) visits during the investigated period was 13,843. Of these, 1208 had an influenza-like syndrome, but only 166 matched the definition of SARI as stated in the study protocol. A total of 52 SARI cases were laboratory confirmed as influenza: 26 as a type B virus, 25 as a type A, and 1 as both viruses. Although about 17% of the total sample had laboratory or radiological data compatible with COVID-19, all the nasopharyngeal swabs stored underwent SARS-CoV-2 RT-PCR and tested negative. Based on our result, it is confirmed that the COVID-19 pandemic spread did not start prior to the &ldquo, official&rdquo, onset in central Italy. Routine monitoring of SARI causative agents at the local level is critical for reporting epidemiologic and etiologic trends that may differ from one country to another and also among different influenza seasons. This has a practical impact on prevention and control strategies.

Background: Low T3 syndrome is a frequent finding in critical illness The prevalence and clinical relevance of the Low T3 syndrome in coronavirus infections ha

Measles continue to be a major public health issue worldwide with high morbidity and mortality rates. The disease is still endemic in Europe and during 2017 a vast outbreak was described in Italy, Romania and Hungary, which led to thousands of new cases and several deaths. In Italy, 3931 confirmed cases of measles were reported to the Italian national surveillance system from many Italian administrative regions; Lazio, in central Italy, exhibited the highest number of infected patients 1322 (33.63%) and as well as the highest incidence. In this study, we describe the results of a retrospective analysis, carried out during 2016 and 2017, concerning the measles antibody prevalence in patients and healthcare workers attending the Sant’Andrea Hospital of Rome (Lazio). A total of 94 patients (median 30 years of age) were screened in 2016, and 316 (median 40 years of age) during 2017, with an increase of 236% compared to previous year. During 2017, 41 confirmed cases of measles were reported while none in 2016 (P47 years of age) and only one confirmed measles infection was recorded in 2017. These results suggest that there is still an unvaccinated portion of the adult population, who sustain the endemic circulation of measles in Italy. In addition to reach herd immunization on children of 2 years old, catch-up vaccination campaign targeting adult population in Italy and other European countries needs to be implemented to prevent future measles outbreak.

IntroductionNickel exposure usually presents as Allergic Contact Dermatitis. However, Nickel not only causes dermatitis, but an excess of dietary Nickel is reported to be responsible for overweight, metabolic disorders and imbalance of gut microflora.ObjectiveThe aim of study is to expand a preliminary reported evidence of the presence of Nickel-resistant bacteria isolated in human microbiome and further evaluate their association with nickel allergy and overweight in females, the gender mostly affected by Nickel exposure.Materials and MethodsWe collected stool samples from 11 lean female with a nickel allergy (BMI 25). 11 subjects not allergic to nickel served as control group. Stool cultures were supplemented with increasing concentrations of nickel sulphate (NiSO4⋅6H2O) from 0.1mM up to 50 mM, in both aerobic and anaerobic conditions(culturomics-approach Lusi, 2017). Stool cultures not supplemented with nickel were used as controls. Identification of Nickel resistant bacteria was made by MALDI-TOF technology.ResultsIn control subjects, 5 mM NiSO4 was the cut off for microbial growth. Conversely, gut bacteria continued to grow at concentration higher than 5 mM in allergic subjects. In particular, Nickel resistant bacteria able to tolerate 32 mM of NiSO4 was detected in 10% of lean allergic and 29% of overweight allergic females. Gut microbes able to grow in at extremely high NiSO4 concentration (50mM) could only be detected in overweight patients with a severe nickel allergy. At increasing NiSO4 concentration, allergic females, especially those with increased BMI, showed a progressive decrease ofEnterobacteriaceaealong with an increased presence ofLactobacillaceae,BacillaceaeandClostridiaceaecompared to control subjects. Major changes in microbial composition were noted at 50 mM of NiSO4 in overweight allergic females.ConclusionOverweight females with a nickel allergy harbor gut microbes highly resistant to nickel and the role of these bacterial strains must be further elucidated.

In order to characterize newborn mouse gut microbiota phylotypes in very early-life stages, an original metaproteomic pipeline, based on LC–MS 2 -spectra and Mascot driven NCBI non-redundant repository database interrogation was developed. An original computational analysis assisted in the generation of a taxonomic gut architecture from protein hits to operational taxonomic units (OTUs) and related functional categories. Regardless of the mouse's genetic background, a prevalence of Firmicutes (Lactobacillaceae) and Proteobacteria (Enterobacteriaceae) was observed among the entire Eubacteria taxonomic node. However, a higher abundance of Firmicutes was retrieved for Balb/c gut microbiota compared to Rag2 ko mice, the latter was mainly characterized by a Proteobacteria enriched microbiota. The metaproteomic-obtained OTUs were supported, for the identification (ID) of the cultivable bacteria fraction, corroborated by axenic culture-based MALDI-TOF MS IDs. Particularly, functional analysis of Rag2 ko mice gut microbiota proteins revealed the presence of abundant glutathione, riboflavin metabolism and pentose phosphate pathway components, possibly related to genetic background. The metaproteomic pipeline herein presented may represent a useful tool to investigate the highly debated onset of the human gut microbiota in the first days of life, when the bacterial composition, despite its very low diversity (complexity), is still very far from an exhaustive description and other complex microbial consortia. Biological significance The manuscript deals with a “frontier” topic regarding the study of the gut microbiota and the application of a metaproteomic pipeline to unveil the complexity of this fascinating ecosystem at the very early stages of life. Indeed during these phases, its diversity is very low but the bacterial content is highly “instable”, and the relative balance between mucosal and fecal bacteria starts its dynamics of “fight” to get homeostasis. However, in the neonatal period, especially immediately after birth, a comprehensive description of this microbial eco-organ is still lacking, while it should be mandatory to highlight its first mechanisms of homeostasis and perturbation, while it co-develops with and within the host species. In order to unravel its low but almost unknown microbial community multiplicity, the newborn mouse gut, characterized by a “very” low complexity, was herein selected as model to design a LC–MS 2 -based shotgun metaproteomic approach, potentially suitable to study onset and shaping in human newborns. A microbiological semi-automatic computational analysis was performed to infer gut phylotypes; such as proof of evidence, related OTUs were compared to axenic-culture-based MALDI-TOF MS IDs showing consistency at family and phyla levels for the bacterial cultivable fraction. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.