Multimodal and spatially resolved profiling identifies distinct patterns of T cell infiltration in nodal B cell lymphoma entities.

The redirection of T cells has emerged as an attractive therapeutic principle in B cell non-Hodgkin lymphoma (B-NHL). However, a detailed characterization of lymphoma-infiltrating T cells across B-NHL entities is missing. Here we present an in-depth T cell reference map of nodal B-NHL, based on cellular indexing of transcriptomes and epitopes, T cell receptor sequencing, flow cytometry and multiplexed immunofluorescence applied to 101 lymph nodes from patients with diffuse large B cell, mantle cell, follicular or marginal zone lymphoma, and from healthy controls. This multimodal resource revealed quantitative and spatial aberrations of the T cell microenvironment across and within B-NHL entities. Quantitative differences in PD1+ TCF7- cytotoxic T cells, T follicular helper cells or IKZF3+ regulatory T cells were linked to their clonal expansion. The abundance of PD1+ TCF7- cytotoxic T cells was associated with poor survival. Our study portrays lymphoma-infiltrating T cells with unprecedented comprehensiveness and provides a unique resource for the investigation of lymphoma biology and prognosis.

The remission status of AML patients after allo-HCT is associated with a distinct single-cell bone marrow T-cell signature.

ABSTRACT: Acute myeloid leukemia (AML) is a hematologic malignancy for which allogeneic hematopoietic cell transplantation (allo-HCT) often remains the only curative therapeutic approach. However, incapability of T cells to recognize and eliminate residual leukemia stem cells might lead to an insufficient graft-versus-leukemia (GVL) effect and relapse. Here, we performed single-cell RNA-sequencing (scRNA-seq) on bone marrow (BM) T lymphocytes and CD34+ cells of 6 patients with AML 100 days after allo-HCT to identify T-cell signatures associated with either imminent relapse (REL) or durable complete remission (CR). We observed a higher frequency of cytotoxic CD8+ effector and gamma delta (γδ) T cells in CR vs REL samples. Pseudotime and gene regulatory network analyses revealed that CR CD8+ T cells were more advanced in maturation and had a stronger cytotoxicity signature, whereas REL samples were characterized by inflammatory tumor necrosis factor/NF-κB signaling and an immunosuppressive milieu. We identified ADGRG1/GPR56 as a surface marker enriched in CR CD8+ T cells and confirmed in a CD33-directed chimeric antigen receptor T cell/AML coculture model that GPR56 becomes upregulated on T cells upon antigen encounter and elimination of AML cells. We show that GPR56 continuously increases at the protein level on CD8+ T cells after allo-HCT and confirm faster interferon gamma (IFN-γ) secretion upon re-exposure to matched, but not unmatched, recipient AML cells in the GPR56+ vs GPR56- CD8+ T-cell fraction. Together, our data provide a single-cell reference map of BM-derived T cells after allo-HCT and propose GPR56 expression dynamics as a surrogate for antigen encounter after allo-HCT.

Real-World Data Regarding Dalbavancin Use before and during the COVID-19 Pandemic-A Single-Center Retrospective Study.

Dalbavancin is a semisynthetic lipoglycopeptide, which possesses great potential for bactericidal activity similar to antimicrobials with the same mechanism of action, such as vancomycin and teicoplanin. Due to its very prolonged half-life, it can be used in a single or two-dose regimen to treat infections by Gram-positive microorganisms, even resistant ones, such as methicillin-resistant Staphylococcus aureus (MRSA). Currently, it is approved only for the treatment of acute bacterial skin and skin structure infections (ABSSSIs). The aim of this study was to investigate the clinical and microbiological characteristics of patients to whom dalbavancin was administered at the University Hospital of Heraklion and evaluate its use in regard to the COVID-19 pandemic. In total, 146 patients were included in this retrospective cohort study evaluating the use of dalbavancin from the first time it was used in 2017 until the end of 2022. The median age was 68 years (range: 21-96 years), and 86 (59%) patients were male. The most common indications for dalbavancin use were osteoarticular infections in 43%, followed by ABSSSIs in 37%, and cardiovascular infections in 10%. Dalbavancin was used empirically in one out of three patients, most commonly with the indication of ABSSSIs, and most commonly in the post-COVID-19 era. The most frequently isolated pathogens were coagulase-negative staphylococci in 70%, S. aureus in 27%, Enterococcus spp. in 22%, and Streptococcus spp. in 8%, while one out of three infections were polymicrobial. In 12% of patients, the infection was not cured, but no patients died. For patients with ABSSSIs, endocarditis and vascular infections, and bacteremia, the cure rates were more than 90%, and in osteoarticular infections, the cure rate was 76%. Thus, dalbavancin has great potential for use in complicated and invasive infections that may require prolonged intravenous antimicrobial treatment. However, further studies are required to formally investigate its role in such infections.

A carbapenem-focused antimicrobial stewardship programme implemented during the COVID-19 pandemic in a setting of high endemicity for multidrug-resistant Gram-negative bacteria.

BACKGROUND: Greece is among the countries characterized by high rates of antimicrobial resistance and high consumption of antibiotics, including carbapenems. OBJECTIVES: To measure the impact of a carbapenem-focused antimicrobial stewardship programme (ASP) on the antibiotic consumption and patient outcomes in a Greek tertiary hospital during the COVID-19 pandemic. METHODS: A quasi-experimental, before-after study, comparing a 12 month pre-intervention period with a 12 month intervention period in which a carbapenem-focused ASP was implemented. RESULTS: A total of 1268 patients were enrolled. The proportion of admitted patients who received carbapenems decreased from 4.1% (842 of 20 629) to 2.3% (426 of 18 245) (-1.8%; P < 0.001). A decrease of -4.9 DDD/100 patient-days (PD) (95% CI -7.3 to -2.6; P = 0.007) in carbapenem use and an increase in the use of piperacillin/tazobactam [+2.1 DDD/100 PD (95% CI 1.0-3.3; P = 0.010)] were observed. Thirty-day mortality following initiation of carbapenem treatment and all-cause in-hospital mortality remained unaltered after ASP implementation. In contrast, length of hospital stay increased (median 17.0 versus 19.0 days; P < 0.001), while the risk of infection-related readmission within 30 days of hospital discharge decreased (24.6% versus 16.8%; P = 0.007). In the post-implementation period, acceptance of the ASP intervention was associated with lower daily hazard of in-hospital death [cause-specific HR (csHR) 0.49; 95% CI 0.30-0.80], lower odds of 30 day mortality (OR 0.36; 95% CI 0.18-0.70) and higher rate of treatment success (csHR 2.45; 95% CI 1.59-3.77). CONCLUSIONS: Implementing and maintaining a carbapenem-focused ASP is feasible, effective and safe in settings with high rates of antimicrobial resistance, even during the COVID-19 pandemic.

Asymptomatic Bacteriuria in Patients with Type 2 Diabetes Mellitus.

Objectives: Asymptomatic bacteriuria (ASB) is a common finding in patients with diabetes. Moreover, patients with diabetes and ASB have a greater risk for symptomatic urinary tract infections and associated severe complications. The aim of this study was to estimate the prevalence of ASB, as well as to identify independent risk factors and related pathogens associated with ASB in female and male patients with type 2 diabetes mellitus (T2D). Methods: This prospective case-control study was performed at the University hospital, and the Venezeleion General Hospital, Heraklion, Greece between 2012 and 2019. All patients with T2D attending the diabetes and hypertension outpatient clinics at both hospitals were enrolled, and data regarding their medical history and clinical and laboratory profiles were recorded. Asymptomatic patients with positive urine cultures were assigned as cases while those with negative urine cultures were designated as controls. Results: A total of 437 adult patients of which 61% were female and 39% were male patients with a mean age of 70.5 ± 9.6 years, were enrolled. The prevalence of ASB was 20.1%, in total. ASB was noted in 27% of female participants and 9.4% of male participants. Higher glycated hemoglobin (OR = 3.921, 95%CI: 1.521−10.109, p < 0.001) and urinary tract infection within the previous year (OR = 13.254, 95%CI: 2.245−78.241, p < 0.001) were independently positively associated with ASB, while higher levels of vitamin B12 were independently negatively associated with ASB (OR = 0.994 per ng/mL, 95%CI: 0.989−0.999, p < 0.001). Conclusions: Development of ASB was associated with specific factors, some of which may be modifiable. Interestingly, high B12 was found to be negatively associated with ASB.

Antimicrobial Prescribing before and after the Implementation of a Carbapenem-Focused Antimicrobial Stewardship Program in a Greek Tertiary Hospital during the COVID-19 Pandemic.

BACKGROUND: Irrational use of antimicrobials poses a significant risk for public health by aggravating antimicrobial resistance. The aim of this repeated point prevalence survey (PPS) was to evaluate the impact of a carbapenem-focused antimicrobial stewardship program (ASP) on overall antimicrobial use and quality of antimicrobial prescribing during the COVID-19 pandemic. METHODS: All adult inpatients in the University Hospital of Heraklion in Greece were audited twice, before and after the implementation of the ASP, in October 2019 and October 2020, respectively. Patient characteristics, indications and diagnoses for antimicrobial administration, antimicrobials prescribed, and compliance with treatment guidelines were recorded. RESULTS: Of 743 adult inpatients on the days of the two surveys, 398 (53.6%) were on antimicrobials for 437 diagnoses. Following implementation of the ASP, there was substantial decrease in the utilization of carbapenems (4.9% of all antibacterials prescribed in the second PPS compared to 10.3% in the first PPS). A significant improvement was observed for all indicators of the quality of antimicrobial prescribing. CONCLUSIONS: Our study demonstrated a positive impact of an ASP implementation during the first stages of the COVID-19 pandemic on reducing the use of last-line antimicrobials and improving overall quality of antimicrobial prescribing.

Knowledge and Perceptions about COVID-19 among Health Care Workers: Evidence from COVID-19 Hospitals during the Second Pandemic Wave.

Health care workers (HCWs) face a higher risk of infection, since they work at the front line of COVID-19 patients' management. Misinterpretations of current scientific evidence among HCWs may impact the delivery of appropriate care to COVID-19 patients and increase the risk of SARS-CoV-2 transmission in the hospital setting. Moreover, knowledge may affect HCWs perceptions depending on their broad beliefs and past experiences. The aim of this study was to explore the knowledge and perceptions of HCWs regarding COVID-19 issues during the second wave of the pandemic. A cross-sectional survey, involving a printed questionnaire, was conducted from 21 October 2020 to 31 January 2021 in four tertiary care hospitals located at four distant geographical regions in Greece. In total, 294 HCWs participated in this study. The majority of HCWs provided precise responses regarding general knowledge, perceptions, and practices concerning the COVID-19 pandemic. However, responses on hand hygiene and antimicrobial use in HCWs with COVID-19 were mistaken. This study reveals a certain degree of misconceptions and knowledge gaps in HCWs everyday practice, especially regarding hand hygiene and antimicrobial use in COVID-19 patients.

Plasmodium falciparum malaria drives epigenetic reprogramming of human monocytes toward a regulatory phenotype.

In malaria-naïve children and adults, Plasmodium falciparum-infected red blood cells (Pf-iRBCs) trigger fever and other symptoms of systemic inflammation. However, in endemic areas where individuals experience repeated Pf infections over many years, the risk of Pf-iRBC-triggered inflammatory symptoms decreases with cumulative Pf exposure. The molecular mechanisms underlying these clinical observations remain unclear. Age-stratified analyses of uninfected, asymptomatic Malian individuals before the malaria season revealed that monocytes of adults produced lower levels of inflammatory cytokines (IL-1ß, IL-6 and TNF) in response to Pf-iRBC stimulation compared to monocytes of Malian children and malaria-naïve U.S. adults. Moreover, monocytes of Malian children produced lower levels of IL-1ß and IL-6 following Pf-iRBC stimulation compared to 4-6-month-old infants. Accordingly, monocytes of Malian adults produced more IL-10 and expressed higher levels of the regulatory molecules CD163, CD206, Arginase-1 and TGM2. These observations were recapitulated in an in vitro system of monocyte to macrophage differentiation wherein macrophages re-exposed to Pf-iRBCs exhibited attenuated inflammatory cytokine responses and a corresponding decrease in the epigenetic marker of active gene transcription, H3K4me3, at inflammatory cytokine gene loci. Together these data indicate that Pf induces epigenetic reprogramming of monocytes/macrophages toward a regulatory phenotype that attenuates inflammatory responses during subsequent Pf exposure. Trial Registration: ClinicalTrials.gov NCT01322581.

Doctors' Perceptions, Attitudes and Practices towards the Management of Multidrug-Resistant Organism Infections after the Implementation of an Antimicrobial Stewardship Programme during the COVID-19 Pandemic.

BACKGROUND: Greece is among the European countries with the highest consumption of antibiotics, both in community and hospital settings, including last-line antibiotics, such as carbapenems. We sought to explore doctors' perceptions, attitudes and practices towards the management of patients with multidrug-resistant organism (MDRO) infections after the implementation of an antimicrobial stewardship programme (ASP) in a tertiary academic hospital during the COVID-19 pandemic. METHODS: A self-administered, internet-based questionnaire survey was completed by doctors of the University Hospital of Heraklion in Crete, Greece. RESULTS: In total, 202 (59.1%) hospital doctors fully completed the questionnaire. Most of them agreed that the prospective audit and feedback ASP strategy is more effective and educational than the preauthorization ASP strategy. ASP implementation prompted most respondents to monitor the continuously evolving microbiological data of their patients more closely and affected them towards a multidisciplinary and personalised care of patients with infections caused by MDROs and towards a more rigorous implementation of infection prevention and control measures. The vast majority of participants (98.5%) stated that ASP must be continued and further developed during the COVID-19 pandemic. CONCLUSION: The ASP implementation in our hospital had a beneficial impact on doctors' perceptions, attitudes and practices with regard to the management of infections due to MDROs.

Candida parapsilosis endocarditis. Report of cases and review of the literature.

INTRODUCTION: Infective endocarditis (IE) due to Candida species is a rare disease representing about 1-2% of all IE cases and carries a high mortality rate. Given the rarity of the disease, there are no clear guidelines on the type and duration of antifungal therapy. Thus, long-term or even life-long antifungal treatment is commonly used. CASE REPORT: We report two patients with prosthetic valve C. parapsilosis IE and persistent candidemia that failed conservative treatment and ultimately developed heart failure. They underwent prosthetic valve replacement and prolonged antifungal treatment with favorable outcome. DISCUSSION: Candida IE commonly occurs in the setting of underlying malignancy, chronic liver disease, previous endocarditis, previous antimicrobial exposure, previous abdominal surgery, intravenous drug use, presence of a central venous catheter, and previous cardiac surgery. Both present patients had undergone a cardiac surgery and had a prosthetic heart valve, while one patient had an underlying autoimmune disease that could be associated with higher risk of IE. In both patients transthoracic ultrasound failed to diagnose IE. In our patients, conservative treatment alone was not enough to control the infection, thus, both patients underwent valve replacement and were subsequently treated with antifungals for 6 weeks. Furthermore, both patients were put on long-term antifungal suppression treatment. CONCLUSIONS: Given the absence of controlled randomized trials, the treatment of Candida endocarditis mostly relies on experts' opinion, and, thus, future studies focusing on the type and duration of antifungal treatment are required.

Plasmodium falciparum malaria drives epigenetic reprogramming of human monocytes toward a regulatory phenotype.

In malaria-naïve children and adults, Plasmodium falciparum -infected red blood cells ( Pf -iRBCs) trigger fever and other symptoms of systemic inflammation. However, in endemic areas where individuals experience repeated Pf infections over many years, the risk of Pf -iRBC-triggered inflammatory symptoms decreases with cumulative Pf exposure. The molecular mechanisms underlying these clinical observations remain unclear. Age-stratified analyses of monocytes collected from uninfected, asymptomatic Malian individuals before the malaria season revealed an inverse relationship between age and Pf -iRBC-inducible inflammatory cytokine (IL-1ß, IL-6 and TNF) production, whereas Malian infants and malaria-naïve U.S. adults produced similarly high levels of inflammatory cytokines. Accordingly, monocytes of Malian adults produced more IL-10 and expressed higher levels of the regulatory molecules CD163, CD206, Arginase-1 and TGM2. These observations were recapitulated in an in vitro system of monocyte to macrophage differentiation wherein macrophages re-exposed to Pf -iRBCs exhibited attenuated inflammatory cytokine responses and a corresponding decrease in the epigenetic marker of active gene transcription, H3K4me3, at inflammatory cytokine gene loci. Together these data indicate that Pf induces epigenetic reprogramming of monocytes/macrophages toward a regulatory phenotype that attenuates inflammatory responses during subsequent Pf exposure. These findings also suggest that past malaria exposure could mitigate monocyte-associated immunopathology induced by other pathogens such as SARS-CoV-2. AUTHOR SUMMARY: The malaria parasite is mosquito-transmitted and causes fever and other inflammatory symptoms while circulating in the bloodstream. However, in regions of high malaria transmission the parasite is less likely to cause fever as children age and enter adulthood, even though adults commonly have malaria parasites in their blood. Monocytes are cells of the innate immune system that secrete molecules that cause fever and inflammation when encountering microorganisms like malaria. Although inflammation is critical to initiating normal immune responses, too much inflammation can harm infected individuals. In Mali, we conducted a study of a malaria-exposed population from infants to adults and found that participants' monocytes produced less inflammation as age increases, whereas monocytes of Malian infants and U.S. adults, who had never been exposed to malaria, both produced high levels of inflammatory molecules. Accordingly, monocytes exposed to malaria in the laboratory became less inflammatory when re-exposed to malaria again later, and these monocytes 'turned down' their inflammatory genes. This study helps us understand how people become immune to inflammatory symptoms of malaria and may also help explain why people in malaria-endemic areas appear to be less susceptible to the harmful effects of inflammation caused by other pathogens such as SARS-CoV-2.

Treatment pattern, prognostic factors, and outcome in patients with infection due to pan-drug-resistant gram-negative bacteria.

The present study investigated the clinical course, treatment pattern, prognostic factors, and outcome of patients with pun-drug resistant (PDR) infections. This was a retrospective single-center cohort study including consecutive eligible patients with a PDR infection hospitalized at the University Hospital of Heraklion, Crete, Greece, between January 2010 and June 2018. In total, 65 patients with infections due to PDR gram-negative pathogens were identified. The median age was 64 years (interquartile range, IQR: 45.5-74.5) and the median Charlson comorbidity index 3.0 (IQR: 1.0-5.75). Of the 65 PDR isolates, 31 (48%) were Klebsiella pneumoniae, 28 (43%) Acinetobacter baumannii, and 6 (9%) Pseudomonas aeruginosa. The most common empirical therapy was colistin-based combination (n = 32; 49%), followed by non-colistin, non-tigecycline combination (n = 25; 39%), and carbapenemes + tigecycline (n = 8; 12%). The empirical therapy was effective in 50%, 37.5%, and 8% of patients receiving colistin combination, carbapenemes - tigecycline, and non-colistin, non-tigecycline combination, respectively (p value = 0.003). The infection-related in-hospital mortality was 32% (95% confidence interval, CI: 21-45%). Three factors were significantly associated with infection-related in-hospital mortality in multivariate analysis: Charlson comorbidity index (odds ratio, OR: 1.5, 95% CI: 1.0-2.3, p value = 0.030), prior steroid use (OR: 4.1, 95% CI: 1.0-17.0, p value = 0.049), and empirical treatment with non-colistin, non-tigecycline combination (OR: 7.5; 95% CI: 1.7-32.8, p value = 0.008). Infections due to PDR pathogens are associated with considerable mortality. Our results support the use of colistin and/or tigecycline-based combinations as empirical therapy when infection due to PDR pathogens is suspected.