Divergent androgenic modulation of SARS-CoV-2 infection cooperates with dysregulated immune response to dictate worse COVID-19 outcomes in men.

BACKGROUND: Sex-determined differences are rarely addressed in the management of diseases, despite well-known contrasting outcomes between female and male patients. In COVID-19 there is a remarkable disparity, with higher rates of mortality and more severe acute disease in men compared to women, who are mostly affected by long COVID-19. Furthermore, whether androgens play a protective or detrimental role in COVID-19 is still a matter of debate. Hence, the adequate management of the disease, especially regarding men presenting acute disease aggravation, still needs important data to elucidate the interplay between sex hormones and host immune responses that drive the worse evolution in male patients. METHODS: A cohort of 92 controls and 198 non-severe and severe COVID-19 patients, from both sexes, was assessed for clinical outcomes, plasma steroids, gonadotropins, sex hormone binding globulin (SHBG) and immune mediators, before vaccination. These data were correlated with the global gene expression of blood leukocytes. The androgen receptor (AR) signaling pathway was investigated by transcriptomics and tracheal aspirate was obtained from severe patients for SARS-COV-2 quantification in the respiratory tract. The interplay among clinical, endocrine and immunological data deciphered the sex differences in COVID-19. Importantly, statistical analyses, using 95% confidence interval, considered confounding factors such as age and comorbidities, to definitely parse the role of androgens in the disease outcome. RESULTS: There were notable contrasting levels of testosterone and dihydrotestosterone (DHT) throughout the disease course in male but not female patients. Inflammatory mediators presented significant negative correlations with testosterone, which was partially dependent on age and diabetes in men. Male subjects with severe COVID-19 had a significant up regulation of the AR signaling pathway, including modulation of TMPRSS2 and SRD5A1 genes, which are related to the viral infection and DHT production. Indeed, men had a higher viral load in the tracheal aspirate and levels of DHT were associated with increased relative risk of death. In contrast, the testosterone hormone, which was notably reduced in severe disease, was significantly related with susceptibility to COVID-19 worsening in male patients. Secondary hypogonadism was ruled out in the male severe COVID-19 subjects, as FSH, LH, and SHBG levels were not significantly altered. Instead, these subjects tended to have increased gonadotropin levels. Most interestingly, in this study we identified, for the first time, combined sets of clinical and immunoendocrine parameters that together predicted progression from non-severe to severe COVID-19 in men. One of the limitations of our study was the low or undetectable levels of DHT in many patients. Then, the evaluation of enzymes related to biosynthesis and signaling by androgens was mandatory and reiterated our findings. CONCLUSIONS: These original results unraveled the disease immunoendocrine regulation, despite vaccination or comorbidities and pointed to the fundamental divergent role of the androgens testosterone and DHT in the determination of COVID-19 outcomes in men. Therefore, sex-specific management of the dysregulated responses, treatments or public health measures should be considered for the control of COVID-19 pandemic.

Plasma Sphingomyelin Disturbances: Unveiling Its Dual Role as a Crucial Immunopathological Factor and a Severity Prognostic Biomarker in COVID-19.

SARS-CoV-2 infection triggers distinct patterns of disease development characterized by significant alterations in host regulatory responses. Severe cases exhibit profound lung inflammation and systemic repercussions. Remarkably, critically ill patients display a "lipid storm", influencing the inflammatory process and tissue damage. Sphingolipids (SLs) play pivotal roles in various cellular and tissue processes, including inflammation, metabolic disorders, and cancer. In this study, we employed high-resolution mass spectrometry to investigate SL metabolism in plasma samples obtained from control subjects (n = 55), COVID-19 patients (n = 204), and convalescent individuals (n = 77). These data were correlated with inflammatory parameters associated with the clinical severity of COVID-19. Additionally, we utilized RNAseq analysis to examine the gene expression of enzymes involved in the SL pathway. Our analysis revealed the presence of thirty-eight SL species from seven families in the plasma of study participants. The most profound alterations in the SL species profile were observed in patients with severe disease. Notably, a predominant sphingomyelin (SM d18:1) species emerged as a potential biomarker for COVID-19 severity, showing decreased levels in the plasma of convalescent individuals. Elevated SM levels were positively correlated with age, hospitalization duration, clinical score, and neutrophil count, as well as the production of IL-6 and IL-8. Intriguingly, we identified a putative protective effect against disease severity mediated by SM (d18:1/24:0), while ceramide (Cer) species (d18:1/24:1) and (d18:1/24:0)were associated with increased risk. Moreover, we observed the enhanced expression of key enzymes involved in the SL pathway in blood cells from severe COVID-19 patients, suggesting a primary flow towards Cer generation in tandem with SM synthesis. These findings underscore the potential of SM as a prognostic biomarker for COVID-19 and highlight promising pharmacological targets. By targeting sphingolipid pathways, novel therapeutic strategies may emerge to mitigate the severity of COVID-19 and improve patient outcomes.

Efficacy and safety of Ixekizumab vs. low-dose IL-2 vs. Colchicine vs. standard of care in the treatment of patients hospitalized with moderate-to-critical COVID-19: A pilot randomized clinical trial (STRUCK: Survival Trial Using Cytokine Inhibitors).

BACKGROUND: Cases of coronavirus disease 2019 (COVID-19) requiring hospitalization continue to appear in vulnerable populations, highlighting the importance of novel treatments. The hyperinflammatory response underlies the severity of the disease, and targeting this pathway may be useful. Herein, we tested whether immunomodulation focusing on interleukin (IL)-6, IL-17, and IL-2, could improve the clinical outcomes of patients admitted with COVID-19. METHODS: This multicenter, open-label, prospective, randomized controlled trial was conducted in Brazil. Sixty hospitalized patients with moderate-to-critical COVID-19 received in addition to standard of care (SOC): IL-17 inhibitor (ixekizumab 80 mg SC/week) 1 dose every 4 weeks; low-dose IL-2 (1.5 million IU per day) for 7 days or until discharge; or indirect IL-6 inhibitor (colchicine) orally (0.5 mg) every 8 hours for 3 days, followed by 4 weeks at 0.5 mg 2x/day; or SOC alone. The primary outcome was accessed in the "per protocol" population as the proportion of patients with clinical improvement, defined as a decrease greater or equal to two points on the World Health Organization's (WHO) seven-category ordinal scale by day 28. RESULTS: All treatments were safe, and the efficacy outcomes did not differ significantly from those of SOC. Interestingly, in the colchicine group, all participants had an improvement of greater or equal to two points on the WHO seven-category ordinal scale and no deaths or patient deterioration were observed. CONCLUSIONS: Ixekizumab, colchicine, and IL-2 were demonstrated to be safe but ineffective for COVID-19 treatment. These results must be interpreted cautiously because of the limited sample size.

The turning point of COVID-19 severity is associated with a unique circulating neutrophil gene signature.

COVID-19 has a broad spectrum of clinical manifestations associated with the host immune response heterogeneity. Despite the advances in COVID-19 research, it is still crucial to seek a panel of molecular markers that enable accurate stratification of COVID-19 patients. Here, we performed a study that combined analysis of blood transcriptome, demographic data, clinical aspects and laboratory findings from 66 participants classified into different degrees of COVID-19 severity and healthy subjects. We identified a perturbation in blood-leukocyte transcriptional profile associated with COVID-19 aggravation, which was mainly related to processes that disfavoured lymphocyte activation and favoured neutrophil activation. This transcriptional profile stratified patients according to COVID-19 severity. Hence, it enabled identification of a turning point in transcriptional dynamics that distinguished disease outcomes and non-hospitalized from hospitalized moderate patients. Central genes of this unique neutrophil signature were S100A9, ANXA3, CEACAM6, VNN1, OLFM4, IL1R2, TCN1 and CD177. Our study indicates the molecular changes that are linked with the differing clinical aspects presented by humans when suffering from COVID-19, which involve neutrophil activation.

The Interplay among Glucocorticoid Therapy, Platelet-Activating Factor and Endocannabinoid Release Influences the Inflammatory Response to COVID-19.

COVID-19 is associated with a dysregulated immune response. Currently, several medicines are licensed for the treatment of this disease. Due to their significant role in inhibiting pro-inflammatory cytokines and lipid mediators, glucocorticoids (GCs) have attracted a great deal of attention. Similarly, the endocannabinoid (eCB) system regulates various physiological processes including the immunological response. Additionally, during inflammatory and thrombotic processes, phospholipids from cell membranes are cleaved to produce platelet-activating factor (PAF), another lipid mediator. Nonetheless, the effect of GCs on this lipid pathway during COVID-19 therapy is still unknown. This is a cross-sectional study involving COVID-19 patients (n = 200) and healthy controls (n = 35). Target tandem mass spectrometry of plasma lipid mediators demonstrated that COVID-19 severity affected eCBs and PAF synthesis. This increased synthesis of eCB was adversely linked with systemic inflammatory markers IL-6 and sTREM-1 levels and neutrophil counts. The use of GCs altered these lipid pathways by reducing PAF and increasing 2-AG production. Corroborating this, transcriptome analysis of GC-treated patients blood leukocytes showed differential modulation of monoacylglycerol lipase and phospholipase A2 gene expression. Altogether, these findings offer a breakthrough in our understanding of COVID-19 pathophysiology, indicating that GCs may promote additional protective pharmacological effects by influencing the eCB and PAF pathways involved in the disease course.

Efficacy and safety of Ixekizumab vs. low-dose IL-2 vs. Colchicine vs. standard of care in the treatment of patients hospitalized with moderate-to-critical COVID-19: A pilot randomized clinical trial (STRUCK: Survival Trial Using Cytokine Inhibitors)

ABSTRACT Background: Cases of coronavirus disease 2019 (COVID-19) requiring hospitalization continue to appear in vulnerable populations, highlighting the importance of novel treatments. The hyperinflammatory response underlies the severity of the disease, and targeting this pathway may be useful. Herein, we tested whether immunomodulation focusing on interleukin (IL)-6, IL-17, and IL-2, could improve the clinical outcomes of patients admitted with COVID-19. Methods: This multicenter, open-label, prospective, randomized controlled trial was conducted in Brazil. Sixty hospitalized patients with moderate-to-critical COVID-19 received in addition to standard of care (SOC): IL-17 inhibitor (ixekizumab 80 mg SC/week) 1 dose every 4 weeks; low-dose IL-2 (1.5 million IU per day) for 7 days or until discharge; or indirect IL-6 inhibitor (colchicine) orally (0.5 mg) every 8 hours for 3 days, followed by 4 weeks at 0.5 mg 2x/day; or SOC alone. The primary outcome was accessed in the "per protocol" population as the proportion of patients with clinical improvement, defined as a decrease greater or equal to two points on the World Health Organization's (WHO) seven-category ordinal scale by day 28. Results: All treatments were safe, and the efficacy outcomes did not differ significantly from those of SOC. Interestingly, in the colchicine group, all participants had an improvement of greater or equal to two points on the WHO seven-category ordinal scale and no deaths or patient deterioration were observed. Conclusions: Ixekizumab, colchicine, and IL-2 were demonstrated to be safe but ineffective for COVID-19 treatment. These results must be interpreted cautiously because of the limited sample size.

Efferocytosis of SARS-CoV-2-infected dying cells impairs macrophage anti-inflammatory functions and clearance of apoptotic cells.

COVID-19 is a disease of dysfunctional immune responses, but the mechanisms triggering immunopathogenesis are not established. The functional plasticity of macrophages allows this cell type to promote pathogen elimination and inflammation or suppress inflammation and promote tissue remodeling and injury repair. During an infection, the clearance of dead and dying cells, a process named efferocytosis, can modulate the interplay between these contrasting functions. Here, we show that engulfment of SARS-CoV-2-infected apoptotic cells exacerbates inflammatory cytokine production, inhibits the expression of efferocytic receptors, and impairs continual efferocytosis by macrophages. We also provide evidence supporting that lung monocytes and macrophages from severe COVID-19 patients have compromised efferocytic capacity. Our findings reveal that dysfunctional efferocytosis of SARS-CoV-2-infected cell corpses suppresses macrophage anti-inflammation and efficient tissue repair programs and provides mechanistic insights for the excessive production of pro-inflammatory cytokines and accumulation of tissue damage associated with COVID-19 immunopathogenesis.

Acetylcholine, Fatty Acids, and Lipid Mediators Are Linked to COVID-19 Severity.

Lipid and cholinergic mediators are inflammatory regulators, but their role in the immunopathology of COVID-19 is still unclear. Here, we used human blood and tracheal aspirate (TA) to investigate whether acetylcholine (Ach), fatty acids (FAs), and their derived lipid mediators (LMs) are associated with COVID-19 severity. First, we analyzed the perturbation profile induced by SARS-CoV-2 infection in the transcriptional profile of genes related to the ACh and FA/LM pathways. Blood and TA were used for metabolomic and lipidomic analyses and for quantification of leukocytes, cytokines, and ACh. Differential expression and coexpression gene network data revealed a unique transcriptional profile associated with ACh and FA/LM production, release, and cellular signaling. Transcriptomic data were corroborated by laboratory findings: SARS-CoV-2 infection increased plasma and TA levels of arachidonic acid, 5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid, 11-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid, and ACh. TA samples also exhibited high levels of PGE2, thromboxane B2, 12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acid, and 6-trans-leukotriene B4 Bioinformatics and experimental approaches demonstrated robust correlation between transcriptional profile in Ach and FA/LM pathways and parameters of severe COVID-19. As expected, the increased neutrophil-to-lymphocyte ratio, neutrophil counts, and cytokine levels (IL-6, IL-10, IL-1ß, and IL-8) correlated with worse clinical scores. Glucocorticoids protected severe and critical patients and correlated with reduced Ach levels in plasma and TA samples. We demonstrated that pulmonary and systemic hyperinflammation in severe COVID-19 are associated with high levels of Ach and FA/LM. Glucocorticoids favored the survival of patients with severe/critical disease, and this effect was associated with a reduction in ACh levels.

Matrix Metalloproteinases on Severe COVID-19 Lung Disease Pathogenesis: Cooperative Actions of MMP-8/MMP-2 Axis on Immune Response through HLA-G Shedding and Oxidative Stress.

Patients with COVID-19 predominantly have a respiratory tract infection and acute lung failure is the most severe complication. While the molecular basis of SARS-CoV-2 immunopathology is still unknown, it is well established that lung infection is associated with hyper-inflammation and tissue damage. Matrix metalloproteinases (MMPs) contribute to tissue destruction in many pathological situations, and the activity of MMPs in the lung leads to the release of bioactive mediators with inflammatory properties. We sought to characterize a scenario in which MMPs could influence the lung pathogenesis of COVID-19. Although we observed high diversity of MMPs in lung tissue from COVID-19 patients by proteomics, we specified the expression and enzyme activity of MMP-2 in tracheal-aspirate fluid (TAF) samples from intubated COVID-19 and non-COVID-19 patients. Moreover, the expression of MMP-8 was positively correlated with MMP-2 levels and possible shedding of the immunosuppression mediator sHLA-G and sTREM-1. Together, overexpression of the MMP-2/MMP-8 axis, in addition to neutrophil infiltration and products, such as reactive oxygen species (ROS), increased lipid peroxidation that could promote intensive destruction of lung tissue in severe COVID-19. Thus, the inhibition of MMPs can be a novel target and promising treatment strategy in severe COVID-19.

SARS-CoV-2 productively infects primary human immune system cells in vitro and in COVID-19 patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a hyperinflammatory state and lymphocytopenia, a hallmark that appears as both signature and prognosis of disease severity outcome. Although cytokine storm and a sustained inflammatory state are commonly associated with immune cell depletion, it is still unclear whether direct SARS-CoV-2 infection of immune cells could also play a role in this scenario by harboring viral replication. We found that monocytes, as well as both B and T lymphocytes, were susceptible to SARS-CoV-2 infection in vitro, accumulating double-stranded RNA consistent with viral RNA replication and ultimately leading to expressive T cell apoptosis. In addition, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from coronavirus disease 2019 (COVID-19) patients. The rates of SARS-CoV-2-infected monocytes in peripheral blood mononuclear cells from COVID-19 patients increased over time from symptom onset, with SARS-CoV-2-positive monocytes, B cells, and CD4+ T lymphocytes also detected in postmortem lung tissue. These results indicated that SARS-CoV-2 infection of blood-circulating leukocytes in COVID-19 patients might have important implications for disease pathogenesis and progression, immune dysfunction, and virus spread within the host.

Sensitive LC-MS/MS Methods for Amphotericin B Analysis in Cerebrospinal Fluid, Plasma, Plasma Ultrafiltrate, and Urine: Application to Clinical Pharmacokinetics.

Neurocryptococcosis, a meningoencephalitis caused by Cryptococcus spp, is treated with amphotericin B (AmB) combined with fluconazole. The integrity of the brain-blood barrier and the composition of the cerebrospinal fluid (CSF) may change due to infectious and/or inflammatory diseases such as neurocryptococcosis allowing for the penetration of AmB into the central nervous system. The present study aimed to develop LC-MS/MS methods capable of quantifying AmB in CSF at any given time of the treatment in addition to plasma, plasma ultrafiltrate, with sensitivity compatible with the low concentrations of AmB reported in the CSF. The methods were successfully validated in the four matrices (25 µl, 5-1,000 ng ml-1 for plasma or urine; 100 µl, 0.625-250 ng ml-1 for plasma ultrafiltrate; 100 µl, 0.1-250 ng ml-1 for CSF) using protein precipitation. The methods were applied to investigate the pharmacokinetics of AmB following infusions of 100 mg every 24 h for 16 days administered as a lipid complex throughout the treatment of a neurocryptococcosis male patient. The methods allowed for a detailed description of the pharmacokinetic parameters in the assessed patient in the beginning (4th day) and end of the treatment with AmB (16th day), with total clearances of 7.21 and 4.25 L h-1, hepatic clearances of 7.15 and 4.22 L h-1, volumes of distribution of 302.94 and 206.89 L, and unbound fractions in plasma ranging from 2.26 to 3.25%. AmB was quantified in two CSF samples collected throughout the treatment with concentrations of 12.26 and 18.45 ng ml-1 on the 8th and 15th days of the treatment, respectively. The total concentration of AmB in plasma was 31 and 20 times higher than in CSF. The unbound concentration in plasma accounted for 77 and 44% of the respective concentrations in CSF. In conclusion, the present study described the most complete and sensitive method for AmB analysis in plasma, plasma ultrafiltrate, urine, and CSF applied to a clinical pharmacokinetic study following the administration of the drug as a lipid complex in one patient with neurocryptococcosis. The method can be applied to investigate the pharmacokinetics of AmB in CSF at any given time of the treatment.

sTREM-1 Predicts Disease Severity and Mortality in COVID-19 Patients: Involvement of Peripheral Blood Leukocytes and MMP-8 Activity.

Uncontrolled inflammatory responses play a critical role in coronavirus disease (COVID-19). In this context, because the triggering-receptor expressed on myeloid cells-1 (TREM-1) is considered an intrinsic amplifier of inflammatory signals, this study investigated the role of soluble TREM-1 (sTREM-1) as a biomarker of the severity and mortality of COVID-19. Based on their clinical scores, we enrolled COVID-19 positive patients (n = 237) classified into mild, moderate, severe, and critical groups. Clinical data and patient characteristics were obtained from medical records, and their plasma inflammatory mediator profiles were evaluated with immunoassays. Plasma levels of sTREM-1 were significantly higher among patients with severe disease compared to all other groups. Additionally, levels of sTREM-1 showed a significant positive correlation with other inflammatory parameters, such as IL-6, IL-10, IL-8, and neutrophil counts, and a significant negative correlation was observed with lymphocyte counts. Most interestingly, sTREM-1 was found to be a strong predictive biomarker of the severity of COVID-19 and was related to the worst outcome and death. Systemic levels of sTREM-1 were significantly correlated with the expression of matrix metalloproteinases (MMP)-8, which can release TREM-1 from the surface of peripheral blood cells. Our findings indicated that quantification of sTREM-1 could be used as a predictive tool for disease outcome, thus improving the timing of clinical and pharmacological interventions in patients with COVID-19.

Cryptococcosis by Cryptococcus neoformans/Cryptococcus gattii Species Complexes in non-HIV-Infected Patients in Southeastern Brazil.

INTRODUCTION: The clinical manifestations of cryptococcosis are usually associated with the infecting agents Cryptococcus neoformans (CN) and C. gattii (CG) species complexes and the host. In this study, non-HIV-infected patients, at a university hospital in southeastern Brazil, had epidemiological and clinical data associated with cryptococcal disease and isolated Cryptococcus species: CN - 24 patients and CG - 12 patients. METHODS: The comparison was comprised of demographic data, predisposing factors, clinical and laboratory manifestations, and outcomes of cryptococcosis patients treated between 2000 and 2016. Immunocompetent and immunosuppressed patients were also compared, irrespective of the infecting species. Cryptococcus spp. were genotyped by PCR-RFLP analysis of the URA5 gene. RESULTS: Infections by the CN species complex (100% VNI genotype) were associated with drug immunosuppression and fungemia, and patients infected with the CG species complex (83% VG II and 17% VGI genotypes) had more evident environmental exposure and higher humoral response. CN and CG affected patients with or without comorbidities. CONCLUSIONS: Diabetes mellitus, other chronic non-infectious diseases, and alcoholism were likely predisposing factors for infection by both CN and CG species. Immunocompetent patients, independent of the infecting Cryptococcus species complexes, showed a higher occurrence of meningitis and a trend toward less fungal dissemination and longer survival than immunosuppressed hosts.

Beneficial effects of colchicine for moderate to severe COVID-19: a randomised, double-blinded, placebo-controlled clinical trial.

OBJECTIVE: To evaluate whether the addition of colchicine to standard treatment for COVID-19 results in better outcomes. DESIGN: We present the results of a randomised, double-blinded, placebo-controlled clinical trial of colchicine for the treatment of moderate to severe COVID-19, with 75 patients allocated 1:1 from 11 April to 30 August 2020. Colchicine regimen was 0.5 mg thrice daily for 5 days, then 0.5 mg twice daily for 5 days. The primary endpoints were the need for supplemental oxygen, time of hospitalisation, need for admission and length of stay in intensive care unit and death rate. RESULTS: Seventy-two patients (36 for placebo and 36 for colchicine) completed the study. Median (and IQR) time of need for supplemental oxygen was 4.0 (2.0-6.0) days for the colchicine group and 6.5 (4.0-9.0) days for the placebo group (p<0.001). Median (IQR) time of hospitalisation was 7.0 (5.0-9.0) days for the colchicine group and 9.0 (7.0-12.0) days for the placebo group (p=0.003). At day 2, 67% versus 86% of patients maintained the need for supplemental oxygen, while at day 7, the values were 9% versus 42%, in the colchicine and the placebo groups, respectively (log rank; p=0.001). Two patients died, both in placebo group. Diarrhoea was more frequent in the colchicine group (p=0.26). CONCLUSION: Colchicine reduced the length of both, supplemental oxygen therapy and hospitalisation. The drug was safe and well tolerated. Once death was an uncommon event, it is not possible to ensure that colchicine reduced mortality of COVID-19. TRIAL REGISTRATION NUMBER: RBR-8jyhxh.

Inflammasomes are activated in response to SARS-CoV-2 infection and are associated with COVID-19 severity in patients.

Severe cases of COVID-19 are characterized by a strong inflammatory process that may ultimately lead to organ failure and patient death. The NLRP3 inflammasome is a molecular platform that promotes inflammation via cleavage and activation of key inflammatory molecules including active caspase-1 (Casp1p20), IL-1ß, and IL-18. Although participation of the inflammasome in COVID-19 has been highly speculated, the inflammasome activation and participation in the outcome of the disease are unknown. Here we demonstrate that the NLRP3 inflammasome is activated in response to SARS-CoV-2 infection and is active in COVID-19 patients. Studying moderate and severe COVID-19 patients, we found active NLRP3 inflammasome in PBMCs and tissues of postmortem patients upon autopsy. Inflammasome-derived products such as Casp1p20 and IL-18 in the sera correlated with the markers of COVID-19 severity, including IL-6 and LDH. Moreover, higher levels of IL-18 and Casp1p20 are associated with disease severity and poor clinical outcome. Our results suggest that inflammasomes participate in the pathophysiology of the disease, indicating that these platforms might be a marker of disease severity and a potential therapeutic target for COVID-19.

Cryptococcosis by Cryptococcus neoformans/Cryptococcus gattii Species Complexes in non-HIV-Infected Patients in Southeastern Brazil

Abstract INTRODUCTION: The clinical manifestations of cryptococcosis are usually associated with the infecting agents Cryptococcus neoformans (CN) and C. gattii (CG) species complexes and the host. In this study, non-HIV-infected patients, at a university hospital in southeastern Brazil, had epidemiological and clinical data associated with cryptococcal disease and isolated Cryptococcus species: CN - 24 patients and CG - 12 patients. METHODS: The comparison was comprised of demographic data, predisposing factors, clinical and laboratory manifestations, and outcomes of cryptococcosis patients treated between 2000 and 2016. Immunocompetent and immunosuppressed patients were also compared, irrespective of the infecting species. Cryptococcus spp. were genotyped by PCR-RFLP analysis of the URA5 gene. RESULTS: Infections by the CN species complex (100% VNI genotype) were associated with drug immunosuppression and fungemia, and patients infected with the CG species complex (83% VG II and 17% VGI genotypes) had more evident environmental exposure and higher humoral response. CN and CG affected patients with or without comorbidities. CONCLUSIONS: Diabetes mellitus, other chronic non-infectious diseases, and alcoholism were likely predisposing factors for infection by both CN and CG species. Immunocompetent patients, independent of the infecting Cryptococcus species complexes, showed a higher occurrence of meningitis and a trend toward less fungal dissemination and longer survival than immunosuppressed hosts.

SARS-CoV-2-triggered neutrophil extracellular traps mediate COVID-19 pathology.

Severe COVID-19 patients develop acute respiratory distress syndrome that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that neutrophil extracellular traps (NETs) have been described as important mediators of tissue damage in inflammatory diseases, we investigated whether NETs would be involved in COVID-19 pathophysiology. A cohort of 32 hospitalized patients with a confirmed diagnosis of COVID-19 and healthy controls were enrolled. The concentration of NETs was augmented in plasma, tracheal aspirate, and lung autopsies tissues from COVID-19 patients, and their neutrophils released higher levels of NETs. Notably, we found that viable SARS-CoV-2 can directly induce the release of NETs by healthy neutrophils. Mechanistically, NETs triggered by SARS-CoV-2 depend on angiotensin-converting enzyme 2, serine protease, virus replication, and PAD-4. Finally, NETs released by SARS-CoV-2-activated neutrophils promote lung epithelial cell death in vitro. These results unravel a possible detrimental role of NETs in the pathophysiology of COVID-19. Therefore, the inhibition of NETs represents a potential therapeutic target for COVID-19.

Minimum concentration of Amphotericin B in serum according to the formulation, dose, and daily or prolonged intermittent therapeutic regimen.

INTRODUCTION: The therapeutic efficacy of daily amphotericin B infusion is related to its maximum concentration in blood; however, trough levels may be useful in intermittent regimens of this antifungal drug. METHODS: : High performance liquid chromatography (HPLC) was used to determine the minimum concentration (Cmin) of amphotericin B in the serum of patients receiving deoxycholate (D-Amph) or liposomal amphotericin B (L-AmB) for the treatment of cryptococcal meningitis (n=28), histoplasmosis (n=8), paracoccidioidomycosis (n=1), and leishmaniasis (n=1). RESULTS: Daily use of D-Amph 30 to 50 mg or L-AmB 50 mg resulted in a similar Cmin, but a significant increase ocurred with L-AmB 100 mg/day. The geometric mean Cmin tended to decrease with a reduction in the dose and frequency of intermittent L-AmB infusions: 357 ng/mL (100 mg 4 to 5 times/week) > 263 ng/mL (50 mg 4 to 5 times/week) > 227 ng/mL (50 mg 1 to 3 times/week). The impact on Cmin was variable in patients whose dose or therapeutic scheme was changed, especially when administered the intermittent infusion of amphotericin B. The mean Cmin for each L-AmB schedule of intermittent therapy was equal or higher than the minimum inhibitory concentration of amphotericin B against Cryptococcus isolates from 10/12 patients. The Cmin of amphotericin B in patients with cryptococcal meningitis was comparable between those that survived or died. CONCLUSIONS: By evaluating the Cmin of amphotericin B, we demonstrated the therapeutic potential of its intermittent use including in the consolidation phase of neurocryptococcosis treatment, despite the great variability in serum levels among patients.

Infection of human lymphomononuclear cells by SARS-CoV-2.

Although SARS-CoV-2 severe infection is associated with a hyperinflammatory state, lymphopenia is an immunological hallmark, and correlates with poor prognosis in COVID-19. However, it remains unknown if circulating human lymphocytes and monocytes are susceptible to SARS-CoV-2 infection. In this study, SARS-CoV-2 infection of human peripheral blood mononuclear cells (PBMCs) was investigated both in vitro and in vivo . We found that in vitro infection of whole PBMCs from healthy donors was productive of virus progeny. Results revealed that monocytes, as well as B and T lymphocytes, are susceptible to SARS-CoV-2 active infection and viral replication was indicated by detection of double-stranded RNA. Moreover, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from COVID-19 patients, and less frequently in CD4 + T lymphocytes. The rates of SARS-CoV-2-infected monocytes in PBMCs from COVID-19 patients increased over time from symptom onset. Additionally, SARS-CoV-2-positive monocytes and B and CD4+T lymphocytes were detected by immunohistochemistry in post mortem lung tissue. SARS-CoV-2 infection of blood circulating leukocytes in COVID-19 patients may have important implications for disease pathogenesis, immune dysfunction, and virus spread within the host.

Minimum concentration of Amphotericin B in serum according to the formulation, dose, and daily or prolonged intermittent therapeutic regimen

Abstract INTRODUCTION: The therapeutic efficacy of daily amphotericin B infusion is related to its maximum concentration in blood; however, trough levels may be useful in intermittent regimens of this antifungal drug. METHODS : High performance liquid chromatography (HPLC) was used to determine the minimum concentration (Cmin) of amphotericin B in the serum of patients receiving deoxycholate (D-Amph) or liposomal amphotericin B (L-AmB) for the treatment of cryptococcal meningitis (n=28), histoplasmosis (n=8), paracoccidioidomycosis (n=1), and leishmaniasis (n=1). RESULTS: Daily use of D-Amph 30 to 50 mg or L-AmB 50 mg resulted in a similar Cmin, but a significant increase ocurred with L-AmB 100 mg/day. The geometric mean Cmin tended to decrease with a reduction in the dose and frequency of intermittent L-AmB infusions: 357 ng/mL (100 mg 4 to 5 times/week) > 263 ng/mL (50 mg 4 to 5 times/week) > 227 ng/mL (50 mg 1 to 3 times/week). The impact on Cmin was variable in patients whose dose or therapeutic scheme was changed, especially when administered the intermittent infusion of amphotericin B. The mean Cmin for each L-AmB schedule of intermittent therapy was equal or higher than the minimum inhibitory concentration of amphotericin B against Cryptococcus isolates from 10/12 patients. The Cmin of amphotericin B in patients with cryptococcal meningitis was comparable between those that survived or died. CONCLUSIONS: By evaluating the Cmin of amphotericin B, we demonstrated the therapeutic potential of its intermittent use including in the consolidation phase of neurocryptococcosis treatment, despite the great variability in serum levels among patients.