Trends of Antimicrobial Susceptibility of Neisseria gonorrhoeae Isolates Between 2012 and 2023: Results From an Open Italian Cohort.

BACKGROUND: Neisseria gonorrhoeae (Ng) is a public health priority because of the rapid evolution of antimicrobial resistance, the emergence of antibiotic resistance, and the absence of a vaccine against Ng. The aim of this study was to investigate trends in the minimum inhibitory concentration and resistance (R) or reduced susceptibility (DS) of Ng cases to ceftriaxone (CRO), azithromycin (AZM), tetracycline (TET), benzylpenicillin (PenG), and ciprofloxacin (CIP) during a 10-year period. METHODS: We conducted a retrospective analysis on an open cohort of Ng cases diagnosed on rectal, urethral, and pharyngeal samples at San Raffaele Scientific Institute, between September 2012 and February 2023. Minimum inhibitory concentrations of antibiotics were determined by gradient-test strips. Bivariate linear regression models were applied on logarithmic minimum inhibitory concentrations values; Cochran-Armitage test was used to determine a linear trend in the proportions of resistant strains. RESULTS: A total of 436 Ng isolates from 352 individuals were analyzed. Minimum inhibitory concentrations of CRO and PenG reduced over time ( P < 0.001, P = 0.030), AZM increased ( P = 0.001), and CIP and TET did not change ( P = 0.473, P = 0.272). The percentages of resistant strains were as follows: PenG, 89.9%; TET, 90.8%; CIP, 48.2%; AZM, and 4.4%. CRO-DS strains were 8.7%, and only 1 case of CRO-R was identified. The proportion of resistant strains increased over time for AZM ( P = 0.007), TET ( P = 0.001), and CIP ( P < 0.001), whereas it decreased for PenG ( P < 0.001) and CRO-DS/R strains ( P < 0.001). CONCLUSIONS: Ng strains showed high susceptibility to CRO, although we identified cases of DS/R and observed high levels of susceptibility to AZM. Overall, the recommended primary regimen for Ng treatment was confirmed to be effective.

SARS-CoV-2 before and after Omicron: two different viruses and two different diseases?

For the first time in the history of medicine, it has been possible to describe-after a spillover-the evolution of a new human virus spreading in a non-immune population. This allowed not only to observe the subsequent emersion of variants endowed with features providing the virus with an evolutionary advantage, but also the shift of the pathways of virus replication and the acquisition of immunoevasive features. These characteristics had a remarkable influence on the diffusion of the SARS-CoV-2 and on the clinical presentation and prognosis of COVID-19, aspects that are described and commented in this review.

Weak correlation between antibody titers and neutralizing activity in sera from SARS-CoV-2 infected subjects.

Plenty of serologic tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed so far, thus documenting the importance of evaluating the relevant features of the immune response to this viral agent. The performance of these assays is currently under investigation. Amongst them, LIAISON® SARS-CoV-2 S1/S2 IgG by DiaSorin and Elecsys Anti-SARS-CoV-2 cobas® by Roche are currently used by laboratory medicine hospital departments in Italy and many other countries. In the present study, we firstly compared two serologic tests on serum samples collected at two different time points from 46 laboratory-confirmed coronavirus disease-2019 (COVID-19) subjects. Secondly, 85 negative serum samples collected before the SARS-CoV-2 pandemic were analyzed. Thirdly, possible correlations between antibody levels and the resulting neutralizing activity against a clinical isolate of SARS-CoV-2 were evaluated. Results revealed that both tests are endowed with low sensitivity on the day of hospital admission, which increased to 97.8% and 100% for samples collected after 15 days for DiaSorin and Roche tests, respectively. The specificity evaluated for the two tests ranges from 96.5% to 100%, respectively. Importantly, a poor direct correlation between antibody titers and neutralizing activity levels was evidenced in the present study. These data further shed light on both potentials and possible limitations related to SARS-CoV-2 serology. In this context, great efforts are still necessary for investigating antibody kinetics to develop novel diagnostic algorithms. Moreover, further investigations on the role of neutralizing antibodies and their correlate of protection will be of paramount importance for the development of effective vaccines.

Interferon-ß-1a Inhibition of Severe Acute Respiratory Syndrome-Coronavirus 2 In Vitro When Administered After Virus Infection.

The ongoing coronavirus disease 2019 pandemic has forced the clinical and scientific community to try drug repurposing of existing antiviral agents as a quick option against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). Under this scenario, interferon (IFN) ß-1a, whose antiviral potential is already known, and which is a drug currently used in the clinical management of multiple sclerosis, may represent as a potential candidate. In this report, we demonstrate that IFN-ß-1a was highly effective in inhibiting in vitro SARS-CoV-2 replication at clinically achievable concentration when administered after virus infection.

Cell-to-Cell Spread Blocking Activity Is Extremely Limited in the Sera of Herpes Simplex Virus 1 (HSV-1)- and HSV-2-Infected Subjects.

Herpes simplex virus 1 (HSV-1) and HSV-2 can evade serum antibody-mediated neutralization through cell-to-cell transmission mechanisms, which represent one of the central steps in disease reactivation. To address the role of humoral immunity in controlling HSV-1 and HSV-2 replication, we analyzed serum samples from 44 HSV-1 and HSV-2 seropositive subjects by evaluating (i) their efficiency in binding both the purified viral particles and recombinant gD and gB viral glycoproteins, (ii) their neutralizing activity, and (iii) their capacity to inhibit the cell-to-cell virus passage in vitro All of the sera were capable of binding gD, gB, and whole virions, and all sera significantly neutralized cell-free virus. However, neither whole sera nor purified serum IgG fraction was able to inhibit significantly cell-to-cell virus spreading in in vitro post-virus-entry infectious assays. Conversely, when spiked with an already described anti-gD human monoclonal neutralizing antibody capable of inhibiting HSV-1 and -2 cell-to-cell transmission, each serum boosted both its neutralizing and post-virus-entry inhibitory activity, with no interference exerted by serum antibody subpopulations.IMPORTANCE Despite its importance in the physiopathology of HSV-1 and -2 infections, the cell-to-cell spreading mechanism is still poorly understood. The data shown here suggest that infection-elicited neutralizing antibodies capable of inhibiting cell-to-cell virus spread can be underrepresented in most infected subjects. These observations can be of great help in better understanding the role of humoral immunity in controlling virus reactivation and in the perspective of developing novel therapeutic strategies, studying novel correlates of protection, and designing effective vaccines.

Detection of low-level HCV variants in DAA treated patients: comparison amongst three different NGS data analysis protocols.

BACKGROUND: Notwithstanding the efforts of direct-acting antivirals (DAAs) for the treatment of chronically infected hepatitis C virus (HCV) patients, concerns exist regarding the emergence of resistance-associated substitutions (RAS) related to therapy failure. Sanger sequencing is still the reference technique used for the detection of RAS and it detects viral variants present up to 15%, meaning that minority variants are undetectable, using this technique. To date, many studies are focused on the analysis of the impact of HCV low variants using next-generation sequencing (NGS) techniques, but the importance of these minority variants is still debated, and importantly, a common data analysis method is still not defined. METHODS: Serum samples from four patients failing DAAs therapy were collected at baseline and failure, and amplification of NS3, NS5A and NS5B genes was performed on each sample. The genes amplified were sequenced using Sanger and NGS Illumina sequencing and the data generated were analyzed with different approaches. Three different NGS data analysis methods, two homemade in silico pipeline and one commercially available certified user-friendly software, were used to detect low-level variants. RESULTS: The NGS approach allowed to infer also very-low level virus variants. Moreover, data processing allowed to generate high accuracy data which results in reduction in the error rates for each single sequence polymorphism. The results improved the detection of low-level viral variants in the HCV quasispecies of the analyzed patients, and in one patient a low-level RAS related to treatment failure was identified. Importantly, the results obtained from only two out of the three data analysis strategies were in complete agreement in terms of both detection and frequency of RAS. CONCLUSIONS: These results highlight the need to find a robust NGS data analysis method to standardize NGS results for a better comprehension of the clinical role of low-level HCV variants. Based on the extreme importance of data analysis approaches for wet-data interpretation, a detailed description of the used pipelines and further standardization of the in silico analysis could allow increasing diagnostic laboratory networking to unleash true potentials of NGS.

Virus-induced preferential antibody gene-usage and its importance in humoral autoimmunity.

It is known that even the adaptive components of the immune system are based on genetic traits common to all individuals, and that diversity is shaped by the lifelong contacts with different non-self antigens, including those found on infectious pathogens. Besides the individual differences, some of these common traits may be more prone to react against a given antigen, and this may be exploited by the infectious pathogens. Indeed, viral infections can deregulate immune response by subverting antibody (Ab) gene usage, leading to the overexpression of specific Ab subfamilies. This overexpression often results in a protective antiviral response but, in some cases, also correlates with a higher likelihood of developing humoral autoimmune disorders. These aspects of virus-induced autoimmunity have never been thoroughly reviewed, and this is the main purpose of this review. An accurate examination of virus specific Ab subfamilies elicited during infections may help further characterize the complex interplay between viruses and the humoral immune response, and be useful in the design of future monoclonal antibody (mAb)-based anti-infective prophylactic and therapeutic strategies.

Chimeric antigen receptor (CAR)-engineered T cells redirected against hepatitis C virus (HCV) E2 glycoprotein.

OBJECTIVE: The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2). DESIGN: Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3ζ domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc). RESULTS: In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon γ, interleukin 2 and tumour necrosis factor α. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes. CONCLUSIONS: Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool.

Evaluation of resistance against bacterial microleakage of a new conical implant-abutment connection versus conventional connections: an in vitro study.

The aim of the present in vitro study was to evaluate bacterial microleakage from inside to outside the implant-abutment assembly in a new design of internal conical connection compared to eight different internal connections. The design of this connection should prevent or limit microbiologic leakage into the surrounding implant tissue, that could contribute to infections without bone loss (mucositis) or with bone loss (peri-implantits). In order to investigate bacterial microleakage, the inner part of each system was inoculated with an Escherichia coli suspension. Eight different groups were considered; each group was composed of 10 dental implants, for a total of 80 implants. Groups 1-7 were considered controls, while group 8 was the test connection (an internal connection characterized by a double taper principle). Results showed that in control implants (Group 1 to 7), little microleakage was observed after the first 6 hours (500 CFU/ µl) and, after 24 hours of incubation, they showed a significant bacterial contamination in all samples (>100.000 CFU/ µl). In group 8 (test connection) no contamination was found in the first 6 hours, with 7 out of 10 implants showing no contamination even after 96 hours. Statistically significant differences were found between Group 8 and the other groups (p<0.05), whereas no significant differences were found among implants of the control groups (from group 1 to 7). Within the limits of the present study, the new connection studied presented significantly less microleakage at 96 h in comparison with the other control internal connections.

Heterosubtypic protection conferred by the human monoclonal antibody PN-SIA28 against influenza A virus lethal infections in mice.

PN-SIA28 is a human monoclonal antibody (Hu-MAb) targeting highly conserved epitopes within the stem portion of the influenza virus hemagglutinin (HA) (N. Clementi, et al, PLoS One 6:e28001, 2011, http://dx.doi.org/10.1371/journal.pone.0028001). Previous in vitro studies demonstrated PN-SIA28 neutralizing activities against phylogenetically divergent influenza A subtypes. In this study, the protective activity of PN-SIA28 was evaluated in mice inoculated with lethal influenza A/WSN/33 (H1N1), A/Quebec/144147/09 (H1N1)pdm09, and A/Victoria/3/75 (H3N2) viruses. At 24 h postinoculation (p.i.), animals received PN-SIA28 intraperitoneally (1 or 10 mg/kg of body weight) or 10 mg/kg of unrelated Hu-MAb (mock). Body weight loss and mortality rate (MR) were recorded for 14 days postinfection (p.i.). Lung viral titers (LVT) were determined at day 5 p.i. In A/WSN/33 (H1N1)-infected groups, all untreated and mock-receiving mice died, whereas MRs of 87.5% and 25% were observed in mice that received PN-SIA28 1 and 10 mg/kg, respectively. In influenza A(H1N1) pdm09-infected groups, an MR of 75% was recorded for untreated and mock-treated groups, whereas the PN-SIA28 1-mg/kg and 10-mg/kg groups had rates of 62.5% and 0%, respectively. In A/Victoria/3/75 (H3N2)-infected animals, untreated and mock-treated animals had MRs of 37.5% and 25%, respectively, and no mortalities were recorded after PN-SIA28 treatments. Accordingly, PN-SIA28 treatments significantly reduced weight losses and resulted in a ≥ 1-log reduction in LVT compared to the control in all infection groups. This study confirms that antibodies targeting highly conserved epitopes in the influenza HA stem region, like PN-SIA28, not only neutralize influenza A viruses of clinically relevant subtypes in vitro but also, more importantly, protect from a lethal influenza virus challenge in vivo.

Potential impact of a microarray-based nucleic acid assay for rapid detection of Gram-negative bacteria and resistance markers in positive blood cultures.

We evaluated the Verigene Gram-negative blood culture (BC-GN) test, a microarray that detects Gram-negative bacteria and several resistance genes. A total of 102 positive blood cultures were tested, and the BC-GN test correctly identified 97.9% of the isolates within its panel. Resistance genes (CTX-M, KPC, VIM, and OXA genes) were detected in 29.8% of the isolates, with positive predictive values of 95.8% (95% confidence interval [CI], 87.7% to 98.9%) in Enterobacteriaceae and 100% (95% CI, 75.9% to 100%) in Pseudomonas aeruginosa and negative predictive values of 100% (95% CI, 93.9% to 100%) and 78.6% (95% CI, 51.0% to 93.6%), respectively.

Could Dental Material Reuse Play a Significant Role in Preservation of Raw Materials, Water, Energy, and Costs? Microbiological Analysis of New versus Reused Healing Abutments: An In Vitro Study.

AIM: The objective of this in vitro study was to compare reused and sterilized versus new healing abutments to assess whether a decontamination and sterilization process performed on resued healing abutments was sufficient to remove residual proteins. The two groups were comparable with respect to patient safety. MATERIALS AND METHODS: During the period from September 2022 to October 2023, healing abutment screws were selected and divided into two groups according to whether they were new or previously used in patients. The samples were subjected to a decontamination and sterilization protocol, and results from sample sterility evaluation and assessment of surface protein levels were recorded. RESULTS: The obtained results revealed a significant difference in the OD562 nm values between new and reused healing abutment samples. The assay demonstrates how treated healing abutments were still contaminated by residual proteins. CONCLUSION: Within the limitations of the present study, although from an infectious point of view sterilization results in the total eradication of pathogens, surface proteins remain on reused healing abutments.

Comparative evaluation of the Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry systems for identification of yeasts of medical importance.

We report the first comparative evaluation between the Bruker Biotyper MS (BMS) and the Vitek MS (VMS) for the identification of yeasts. The rate of correct identifications at the species level was comparable using the commercial databases (89.8% versus 84.3%; P = 0.712), but higher for BMS using an in-house-extended database (100% versus 84.3%; P = 0.245). Importantly, the rate of misidentification was significantly higher for VMS (1% versus 12.1%; P < 0.0001), including the rate of major errors (0% versus 4.5%; P = 0.0036).

In vitro phenotypes to elvitegravir and dolutegravir in primary macrophages and lymphocytes of clonal recombinant viral variants selected in patients failing raltegravir.

OBJECTIVES: The cross-resistance profiles of elvitegravir and dolutegravir on raltegravir-resistant variants is still controversial or not available in macrophages and lack extensive evaluations on wide panels of clonal variants. Thus, a complete evaluation in parallel with all currently available integrase inhibitors (INIs) was performed. METHODS: The integrase coding region was RT-PCR-amplified from patient-derived plasma samples and cloned into an HIV-1 molecular clone lacking the integrase region. Twenty recombinant viruses bearing mutations to all primary pathways of resistance to raltegravir were phenotypically evaluated with each integrase inhibitor in freshly purified CD4+ T cells or monocyte-derived macrophages. RESULTS: Y143R single mutants conferred a higher level of raltegravir resistance in macrophages [fold change (FC) 47.7-60.24] compared with CD4+ T cells (FC 9.55-11.56). All other combinations had similar effects on viral susceptibility to raltegravir in both cell types. Elvitegravir displayed a similar behaviour both in lymphocytes and macrophages with all the tested patterns. When compared with raltegravir, none to modest increases in resistance were observed for the Y143R/C pathways. Dolutegravir maintained its activity and cross-resistance profile in macrophages. Only Q148H/R variants had a reduced level of susceptibility (FC 5.48-18.64). No variations were observed for the Y143R/C (+/-T97A) or N155H variants. CONCLUSIONS: All INIs showed comparable antiretroviral activity in both cell types even if single mutations were associated with a different level of susceptibility in vitro to raltegravir and elvitegravir in macrophages. In particular, dolutegravir was capable of inhibiting with similar potency infection of raltegravir-resistant variants with Y143 or N155 pathways in both HIV-1 major cell reservoirs.

HCV proteins and immunoglobulin variable gene (IgV) subfamilies in HCV-induced type II mixed cryoglobulinemia: a concurrent pathogenetic role.

The association between hepatitis C virus (HCV) infection and type II mixed cryoglobulinemia (MCII) is well established, but the role played by distinct HCV proteins and by specific components of the anti-HCV humoral immune response remains to be clearly defined. It is widely accepted that HCV drives the expansion of few B-cell clones expressing a restricted pool of selected immunoglobulin variable (IgV) gene subfamilies frequently endowed with rheumatoid factor (RF) activity. Moreover, the same IgV subfamilies are frequently observed in HCV-transformed malignant B-cell clones occasionally complicating MCII. In this paper, we analyze both the humoral and viral counterparts at the basis of cryoglobulins production in HCV-induced MCII, with particular attention reserved to the single IgV subfamilies most frequently involved.