Circulating tumor HPV DNA in the management of HPV+ oropharyngeal cancer and its correlation with MRI.

BACKGROUND: First aim was to compare ddPCR assays of ctHPVDNA with p16 IHC and qualitative HPV PCR. Second aim was to carry out longitudinal blood sampling to test for association of ctHPVDNA with histological confirmed recurrence. Third aim was to perform a multidimensional assessment which included: (1) clinical features; (2) ctHPVDNA; (3) MRI-based tumor size measurements of primary tumor (PT) and cervical lymph node metastases (CLNM). METHODS: Plasma samples were collected before treatment and during follow-up, and ddPCR assay comprising E6 of HPV16 and HPV 33 and HPV 35 was used. RESULTS: Present study was conducted at diagnosis in 117 patients and revealed a ctHPVDNA sensitivity of 100% (95% CI 95.5-100) and a specificity of 94.4 (95% CI 81.3-99.3), positive predictive value (PPV) of 94.4 (95% CI 81.3-99.3), and negative predictive value (NPP) of 100% (95% CI 89.7-100). During follow-up ctHPVDNA had a sensitivity of 100% (95% CI 72.1-100)% and specificity of 98.4% (95% CI 91.7-100)%, PPV% of 90.9% (95% CI 62.3-98.4) and NPV% of 100% (95% CI 94.3-100) for ability to detect recurrence. Correlation between both the CLNM volume and the sum of PT and CLNM volume was observed. CONCLUSIONS: ctHPVDNA was superior to p16 in identification of HPV-OPSCC at diagnosis. Introduction of ctHPVDNA, beyond diagnostic setting, represents a great opportunity to improve follow-up protocol of OPSCC patients.

Efficacy of sodium hypochlorite in overcoming antimicrobial resistance and eradicating biofilms in clinical pathogens from pressure ulcers.

Sodium hypochlorite (NaOCl) is widely recognized for its broad-spectrum antimicrobial efficacy in skin wound care. This study investigates the effectiveness of NaOCl against a range of bacterial and fungal isolates from pressure ulcer (PU) patients. We analyzed 20 bacterial isolates from PU patients, comprising carbapenem-resistant Klebsiella pneumoniae (CRKP), multidrug-resistant Acinetobacter baumannii (MDRAB), methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), along with 5 Candida albicans isolates. Antibiotic resistance profiles were determined using standard susceptibility testing. Whole-genome sequencing (WGS) was employed to identify antimicrobial resistance genes (ARGs) and disinfectant resistance genes (DRGs). Genetic determinants of biofilm formation were also assessed. The antimicrobial activity of NaOCl was evaluated by determining the minimum inhibitory concentration (MIC) and the minimal biofilm eradication concentration (MBEC) for both planktonic and biofilm-associated cells. CRKP and MDRAB showed resistance to fluoroquinolones and carbapenems, while MRSA exhibited resistance to ß-lactams and levofloxacin. MSSA displayed a comparatively lower resistance profile. WGS identified significant numbers of ARGs in CRKP and MDRAB, with fewer DRGs compared to MRSA and MSSA. All isolates possessed genes associated with fimbriae production and adhesion, correlating with pronounced biofilm biomass production. NaOCl demonstrated substantial antimicrobial activity against both planktonic cells and biofilms. The MIC90 for planktonic bacterial cells was 0.125 mg/mL, and the MBEC90 ranged from 0.225 to 0.5 mg/mL. For planktonic C. albicans, the MIC90 was 0.150 mg/mL, and the MBEC90 was 0.250 mg/mL. These results highlight the challenge in treating biofilm-associated infections and underscore the potential of NaOCl as a robust antimicrobial agent against difficult-to-treat biofilm infections at concentrations lower than those typically found in commercial disinfectants.

Bacterial Biofilm in Chronic Wounds and Possible Therapeutic Approaches.

Wound repair and skin regeneration is a very complex orchestrated process that is generally composed of four phases: hemostasis, inflammation, proliferation, and remodeling. Each phase involves the activation of different cells and the production of various cytokines, chemokines, and other inflammatory mediators affecting the immune response. The microbial skin composition plays an important role in wound healing. Indeed, skin commensals are essential in the maintenance of the epidermal barrier function, regulation of the host immune response, and protection from invading pathogenic microorganisms. Chronic wounds are common and are considered a major public health problem due to their difficult-to-treat features and their frequent association with challenging chronic infections. These infections can be very tough to manage due to the ability of some bacteria to produce multicellular structures encapsulated into a matrix called biofilms. The bacterial species contained in the biofilm are often different, as is their capability to influence the healing of chronic wounds. Biofilms are, in fact, often tolerant and resistant to antibiotics and antiseptics, leading to the failure of treatment. For these reasons, biofilms impede appropriate treatment and, consequently, prolong the wound healing period. Hence, there is an urgent necessity to deepen the knowledge of the pathophysiology of delayed wound healing and to develop more effective therapeutic approaches able to restore tissue damage. This work covers the wound-healing process and the pathogenesis of chronic wounds infected by biofilm-forming pathogens. An overview of the strategies to counteract biofilm formation or to destroy existing biofilms is also provided.

Modulating the skin mycobiome-bacteriome and treating seborrheic dermatitis with a probiotic-enriched oily suspension.

Seborrheic dermatitis (SD) affects 2-5% of the global population, with imbalances in the skin microbiome implicated in its development. This study assessed the impact of an oily suspension containing Lactobacillus crispatus P17631 and Lacticaseibacillus paracasei I1688 (termed EUTOPLAC) on SD symptoms and the skin mycobiome-bacteriome modulation. 25 SD patients were treated with EUTOPLAC for a week. Symptom severity and skin mycobiome-bacteriome changes were measured at the start of the treatment (T0), after seven days (T8), and three weeks post-treatment (T28). Results indicated symptom improvement post-EUTOPLAC, with notable reductions in the Malassezia genus. Concurrently, bacterial shifts were observed, including a decrease in Staphylococcus and an increase in Lactobacillus and Lacticaseibacillus. Network analysis highlighted post-EUTOPLAC instability in fungal and bacterial interactions, with increased negative correlations between Malassezia and Lactobacillus and Lacticaseibacillus genera. The study suggests EUTOPLAC's potential as a targeted SD treatment, reducing symptoms and modulating the mycobiome-bacteriome composition.

It is time to improve the diagnostic workup of oropharyngeal cancer with circulating tumor HPV DNA: Systematic review and meta-analysis.

The possibility of detecting circulating tumor HPV DNA (ctHPVDNA) in plasma in patients with oropharyngeal cancer has been demonstrated in several reports. However, these data are from small cohorts and available tests for detection of ctHPVDNA are not fully validated. The aim is to evaluate sensitivity, specificity, and accuracy of ctHPVDNA by ddPCR to define its efficacy in the clinical setting for the diagnosis of HPV + OPSCC. A comprehensive search of three different databases: MEDLINE, Embase, and Cochrane Library databases. A total of 998 patients were evaluated from the 13 studies. OPSSC p16+ were 729, while controls p16- were 269. The meta-analytic study estimated the diagnostic performance of ctHPVDNA as follows: pooled sensitivity and specificity of 0.90 (95% CI: 0.82-0.94) and 0.94 (95% CI: 0.85-0.98), respectively; positive and negative likelihood ratios of 12.6 (95% CI: 4.9-32.1) and 0.05 (95% CI: 0.02-0.13), respectively. ddPCR for ctHPVDNA has good accuracy, sensitivity, and specificity for diagnosis of HPV + OPSCC. ctHPVDNA kinetic represents a great reliable opportunity to improve diagnostic and therapeutic management of cancer patients and could open new perspectives for understanding tumor biology.

Acinetobacter baumannii in the critically ill: complex infections get complicated.

Acinetobacter baumannii is increasingly associated with various epidemics, representing a serious concern due to the broad level of antimicrobial resistance and clinical manifestations. During the last decades, A. baumannii has emerged as a major pathogen in vulnerable and critically ill patients. Bacteremia, pneumonia, urinary tract, and skin and soft tissue infections are the most common presentations of A. baumannii, with attributable mortality rates approaching 35%. Carbapenems have been considered the first choice to treat A. baumannii infections. However, due to the widespread prevalence of carbapenem-resistant A. baumannii (CRAB), colistin represents the main therapeutic option, while the role of the new siderophore cephalosporin cefiderocol still needs to be ascertained. Furthermore, high clinical failure rates have been reported for colistin monotherapy when used to treat CRAB infections. Thus, the most effective antibiotic combination remains disputed. In addition to its ability to develop antibiotic resistance, A. baumannii is also known to form biofilm on medical devices, including central venous catheters or endotracheal tubes. Thus, the worrisome spread of biofilm-producing strains in multidrug-resistant populations of A. baumannii poses a significant treatment challenge. This review provides an updated account of antimicrobial resistance patterns and biofilm-mediated tolerance in A. baumannii infections with a special focus on fragile and critically ill patients.

Dynamics of humoral and cellular response to three doses of anti-SARS-CoV-2 BNT162b2 vaccine in patients with hematological malignancies and older subjects.

Background: Few data are available about the durability of the response, the induction of neutralizing antibodies, and the cellular response upon the third dose of the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in hemato-oncological patients. Objective: To investigate the antibody and cellular response to the BNT162b2 vaccine in patients with hematological malignancy. Methods: We measured SARS-CoV-2 anti-spike antibodies, anti-Omicron neutralizing antibodies, and T-cell responses 1 month after the third dose of vaccine in 93 fragile patients with hematological malignancy (FHM), 51 fragile not oncological subjects (FNO) aged 80-92, and 47 employees of the hospital (healthcare workers, (HW), aged 23-66 years. Blood samples were collected at day 0 (T0), 21 (T1), 35 (T2), 84 (T3), 168 (T4), 351 (T pre-3D), and 381 (T post-3D) after the first dose of vaccine. Serum IgG antibodies against S1/S2 antigens of SARS-CoV-2 spike protein were measured at every time point. Neutralizing antibodies were measured at T2, T3 (anti-Alpha), T4 (anti-Delta), and T post-3D (anti-Omicron). T cell response was assessed at T post-3D. Results: An increase in anti-S1/S2 antigen antibodies compared to T0 was observed in the three groups at T post-3D. After the third vaccine dose, the median antibody level of FHM subjects was higher than after the second dose and above the putative protection threshold, although lower than in the other groups. The neutralizing activity of antibodies against the Omicron variant of the virus was tested at T2 and T post-3D. 42.3% of FHM, 80,0% of FNO, and 90,0% of HW had anti-Omicron neutralizing antibodies at T post-3D. To get more insight into the breadth of antibody responses, we analyzed neutralizing capacity against BA.4/BA.5, BF.7, BQ.1, XBB.1.5 since also for the Omicron variants, different mutations have been reported especially for the spike protein. The memory T-cell response was lower in FHM than in FNO and HW cohorts. Data on breakthrough infections and deaths suggested that the positivity threshold of the test is protective after the third dose of the vaccine in all cohorts. Conclusion: FHM have a relevant response to the BNT162b2 vaccine, with increasing antibody levels after the third dose coupled with, although low, a T-cell response. FHM need repeated vaccine doses to attain a protective immunological response.

Skin dysbiosis and Cutibacterium acnes biofilm in inflammatory acne lesions of adolescents.

Acne vulgaris is a common inflammatory disorder affecting more than 80% of young adolescents. Cutibacterium acnes plays a role in the pathogenesis of acne lesions, although the mechanisms are poorly understood. The study aimed to explore the microbiome at different skin sites in adolescent acne and the role of biofilm production in promoting the growth and persistence of C. acnes isolates. Microbiota analysis showed a significantly lower alpha diversity in inflammatory lesions (LA) than in non-inflammatory (NI) lesions of acne patients and healthy subjects (HS). Differences at the species level were driven by the overabundance of C. acnes on LA than NI and HS. The phylotype IA1 was more represented in the skin of acne patients than in HS. Genes involved in lipids transport and metabolism, as well as potential virulence factors associated with host-tissue colonization, were detected in all IA1 strains independently from the site of isolation. Additionally, the IA1 isolates were more efficient in early adhesion and biomass production than other phylotypes showing a significant increase in antibiotic tolerance. Overall, our data indicate that the site-specific dysbiosis in LA and colonization by virulent and highly tolerant C. acnes phylotypes may contribute to acne development in a part of the population, despite the universal carriage of the microorganism. Moreover, new antimicrobial agents, specifically targeting biofilm-forming C. acnes, may represent potential treatments to modulate the skin microbiota in acne.

Immunogenicity of three doses of anti-SARS-CoV-2 BNT162b2 vaccine in psoriasis patients treated with biologics.

Introduction: Psoriasis has not been directly linked to a poor prognosis for COVID-19, yet immunomodulatory agents used for its management may lead to increased vulnerability to the dangerous complications of SARS-CoV-2 infection, as well as impair the effectiveness of the recently introduced vaccines. The three-dose antibody response trend and the safety of BNT162b2 mRNA vaccine in psoriasis patients treated with biologic drugs have remained under-researched. Materials and methods: Forty-five psoriatic patients on biologic treatment were enrolled to evaluate their humoral response to three doses of BNT162b2. IgG titers anti-SARS-CoV-2 spike protein were evaluated at baseline (day 0, first dose), after 3 weeks (second dose), four weeks post-second dose, at the time of the third dose administration and 4 weeks post-third dose. Seropositivity was defined as IgG ≥15 antibody-binding units (BAU)/mL. Data on vaccine safety were also collected by interview at each visit. Results: A statistically significant increase in antibody titers was observed after each dose of vaccine compared with baseline, with no significant differences between patients and controls. Methotrexate used in combination with biologics has been shown to negatively influence the antibody response to the vaccine. On the contrary, increasing body mass index (BMI) positively influenced the antibody response. No adverse effects were reported, and no relapses of psoriasis were observed in the weeks following vaccine administration in our study population. Conclusions: Our data are largely consistent with the recent literature on this topic confirming the substantial efficacy and safety of BNT162b2 mRNA vaccine on psoriatic patients treated with biologics of different types and support the recommendation to perform additional doses in this specific subgroup of patients.

Homocysteine and Inflammatory Cytokines in the Clinical Assessment of Infection in Venous Leg Ulcers.

Inflammation and biofilm-associated infection are common in chronic venous leg ulcers (VU), causing deep pain and delayed healing. Albeit important, clinical markers and laboratory parameters for identifying and monitoring persistent VU infections are limited. This study analyzed 101 patients with infected (IVU) and noninfected VUs (NVU). Clinical data were collected in both groups. The serum homocysteine (Hcys) and inflammatory cytokines from the wound fluid were measured. In addition, microbial identification, antibiotic susceptibility, and biofilm production were examined. IVU were 56 (55.4%) while NVU were 45 (44.5%). IVUs showed a significant increase in the wound's size and depth compared to NVUs. In addition, significantly higher levels of interleukin (IL)-6, IL-10, IL17A, and tumor necrosis factor-alpha (TNF-α) were found in patients with IVUs compared to those with NVUs. Notably, hyperhomocysteinemia (HHcy) was significantly more common in patients with IVUs than NVUs. A total of 89 different pathogens were identified from 56 IVUs. Gram-negative bacteria were 51.7%, while the Gram-positives were 48.3%. At the species level, Staphylococcus aureus was the most common isolate (43.8%), followed by Pseudomonas aeruginosa (18.0%). Multidrug-resistant organisms (MDROs) accounted for 25.8% of the total isolates. Strong biofilm producers (SBPs) (70.8%) were significantly more abundant than weak biofilm producers (WBP) (29.2%) in IVUs. SBPs were present in 97.7% of the IVUs as single or multispecies infections. Specifically, SBPs were 94.9% for S. aureus, 87.5% for P. aeruginosa, and 28.6% for Escherichia coli. In IVU, the tissue microenvironment and biofilm production can support chronic microbial persistence and a most severe clinical outcome even in the presence of an intense immune response, as shown by the high levels of inflammatory molecules. The measurement of local cytokines in combination with systemic homocysteine may offer a novel set of biomarkers for the clinical assessment of IVUs caused by biofilm-producing bacteria.

Immunogenicity and Safety of BNT162b2 Homologous Booster Vaccination in People Living with HIV under Effective cART.

Data on COVID-19 boosting vaccination in people living with HIV (PLWH) are scant. We investigated the immunogenicity and safety of the BNT162b2 homologous boosting vaccination. Anti-SARS-CoV-2 spike antibodies (LIAISON® SARS-CoV-2 S1/S2 IgG test, DiaSorin®), CD4+, CD8+ and viraemia were monitored at T0 (pre-vaccination), T1 (4 weeks after the second dose), T2 (pre-booster) and T3 (4 weeks after the booster dose). Humoral responses were evaluated according to sex, age, BMI, nadir and baseline CD4+ counts, as well as type of cART regimen. Forty-two subjects were included: the median age was 53 years (IQR: 48−61); the median time since HIV was 12.4 years (IQR: 6.5−18.3); the median nadir and baseline CD4+ counts were 165 (IQR: 104−291) and 687 cells/mm3 (IQR: 488−929), respectively. The booster dose was administered at a median of 5.5 months after the second dose. Median anti-SARS-CoV-2 IgG concentration had significantly decreased at T2 compared to T1 (107 vs. 377, p < 0.0001). Antibody levels elicited by the booster dose (median: 1580 AU/mL) were significantly higher compared with those of all the other time points (p < 0.0001). None of the investigated variables significantly affected antibody response induced by the booster dose. Local and systemic side-effects were referred by 23.8% and 14.3% of the subjects, respectively. One patient developed sensorineural hearing loss (SNHL) 24 h after boosting. He recovered auditory function upon endothympanic administration of corticosteroids. The BNT162b2 boosting vaccination in PLWH is safe and greatly increased the immune response with respect to the primary vaccination.

Unequal Access to Testing and Vaccination Services for the Homeless and Undocumented Population During COVID-19 Pandemic.

Objectives: To furnish a model to ensure access and use of healthcare services to the undocumented and homeless population. Methods: Between March 2020 and October 2021, public and third sector actors in Rome implemented an accessible COVID-19 screening service and vaccination program targeting the homeless and undocumented population. Results: 95.6% of the patients tested negative to both rapid and molecular tests. 0.9% tested positive to both. 0.7% were false negatives, while 2.8% were false positives. None of the participants refused the diagnostic treatment. From July to October 2021, 1384 people received a complete cycle of the COVID-19 vaccine through the program. 632 (45.6%) also agreed to perform the antibodies testing before inoculation. 318 (50.31%) of these were positive at the time of vaccination. Conclusion: We present a cost-effective model for reducing structural barriers to access diagnostic and preventive services for the homeless and undocumented population that can be applied to different public health settings.

Development of Approaches and Metrics to Measure the Impact and Improve the Clinical Outcomes of Patients With Frailty in the Era of COVID-19. The COMETA Italian Protocol.

The outbreak of the coronavirus 2 disease 2019 (COVID-19) puts an enormous burden on healthcare systems worldwide. This may worsen outcomes in patients with severe chronic diseases such as cancer, autoimmune diseases, and immune deficiencies. In this critical situation, only a few available data exist, which do not allow us to provide practical guides for the treatment of oncological or immunocompromised patients. Therefore, a further step forward is needed, addressing the specific needs and demands of frail patients in the pandemic era. Here we aim to present a protocol of a study approved by an ethical committee named "CO.M.E.TA". CO.M.E.TA protocol is a network project involving six Italian institutions and its goals are: i) to measure and compare the impact of the pandemic on the access of cancer and immunocompromised patients to therapies in three Italian regions; ii) to assess how reorganizational measures put in place in these different institutions have impacted specific metrics of performance; iii) to establish a COVID-19 Biobank of biological samples from SARS-CoV-2 infected patients to be used to study immunological alterations in patients with immune frailty.

Optimizing the Illumina COVIDSeq laboratorial and bioinformatics pipeline on thousands of samples for SARS-CoV-2 Variants of Concern tracking.

The SARS-CoV-2 Variants of Concern tracking via Whole Genome Sequencing represents a pillar of public health measures for the containment of the pandemic. The ability to track down the lineage distribution on a local and global scale leads to a better understanding of immune escape and to adopting interventions to contain novel outbreaks. This scenario poses a challenge for NGS laboratories worldwide that are pressed to have both a faster turnaround time and a high-throughput processing of swabs for sequencing and analysis. In this study, we present an optimization of the Illumina COVID-seq protocol carried out on thousands of SARS-CoV-2 samples at the wet and dry level. We discuss the unique challenges related to processing hundreds of swabs per week such as the tradeoff between ultra-high sensitivity and negative contamination levels, cost efficiency and bioinformatics quality metrics.

Role of Extracellular DNA in Dalbavancin Activity against Methicillin-Resistant Staphylococcus aureus (MRSA) Biofilms in Patients with Skin and Soft Tissue Infections.

Methicillin-resistant Staphylococcus aureus (MRSA) has become the leading cause of skin and soft tissue infections (SSTIs). Biofilm production further complicates patient treatment, contributing to increased bacterial persistence and antibiotic tolerance. The study aimed to explore the efficacy of different antibiotics on biofilm-producing MRSA isolated from patients with SSTI. A total of 32 MRSA strains were collected from patients with SSTI. The MIC and minimal biofilm eradication concentration (MBEC) were measured in planktonic and biofilm growth. The study showed that dalbavancin, linezolid, and vancomycin all inhibited MRSA growth at their EUCAST susceptible breakpoint. Of the MRSA strains, 87.5% (n = 28) were strong biofilm producers (SBPs), while only 12.5% (n = 4) were weak biofilm producers (WBPs). The MBEC90 values for dalbavancin were significantly lower than those of linezolid and vancomycin in all tested strains. We also found that extracellular DNA (eDNA) contributes to the initial microbial attachment and biofilm formation. The amount of eDNA differed among MRSA strains and was significantly higher in those isolates with high dalbavancin and vancomycin tolerance. Exogenously added DNA increased the MBEC90 and protection of biofilm cells from dalbavancin activity. Of note, the relative abundance of eDNA was higher in MRSA biofilms exposed to MBEC90 dalbavancin than in untreated MRSA biofilms and those exposed to sub-MIC90. Overall, dalbavancin was the most active antibiotic against MRSA biofilms at concentrations achievable in the human serum. Moreover, the evidence of a drug-related increase of eDNA and its contribution to antimicrobial drug tolerance reveals novel potential targets for antibiofilm strategies against MRSA. IMPORTANCE Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs) worldwide. In addition, methicillin-resistant S. aureus (MRSA) is increasingly frequent in postoperative infections and responsible for a large number of hospital readmissions and deaths. Biofilm formation by S. aureus is a primary risk factor in SSTIs, due to a higher antibiotic tolerance. Our study showed that the biofilm-forming capacity varied among MRSA strains, although strong biofilm producers were significantly more abundant than weak biofilm producer strains. Notably, dalbavancin demonstrated a potent antibiofilm activity at concentrations achievable in human serum. Nevertheless, dalbavancin activity was affected by an increased concentration of extracellular DNA in the biofilm matrix. This study provides novel insight for designing more targeted therapeutic strategies against MRSA and to prevent or eradicate harmful biofilms.

Rapid decline of humoral response to two doses of BNT162b2 vaccine in patients with solid cancer after six months: The urgent need of the additional dose!

INTRODUCTION: We previously reported on the immunogenicity and safety of BNT162b2 in a large cohort of patients with cancer after the first and second doses (Di Noia et al., 2021) [1]. Herein, we present result after six months of follow-up. METHODS: This prospective study included patients affected by solid tumors and afferent to our institution who received two doses of BNT162b2 vaccine. A cohort of vaccinated healthcare workers (HCW) was used as control-group. Both cohorts were evaluated for the titer of anti-Spike (S) IgG at prefixed time-points (TPs). Time-point 4 was scheduled at 24-26 weeks after the second dose. RESULTS: In the current analysis, 400 patients and 232 healthcare workers were evaluated. Responders (IgG > 15 AU/mL) in patients group were 86.5% compared with 94.4% among healthcare workers. Also the IgG titer at TP4 was significantly inferior in patients than in healthcare workers (70.81 vs 134.64 AU/ml, p < 0.001). There was a more rapid decline of the antibody level from TP3 to TP4 in patients than in healthcare workers (1.78 vs 1.3 fold). The estimated IgG half-life was significantly shorter for patients (73 days) than in healthcare workers (118 days) as well as the time to reach negative serological status (340 vs 532 days). CONCLUSION: The decline of humoral response to the vaccine observed in patients with solid cancer after six months from the first dose support the urgent need of an early additional dose.