Hemoglobin scavenger receptor CD163 as a potential biomarker of hemolysis induced hepatobiliary injury in sickle cell disease.

Sickle cell disease (SCD) associated chronic hemolysis promotes oxidative stress, inflammation and thrombosis leading to organ damage, including liver damage. Hemoglobin scavenger receptor CD163 plays a protective role in SCD by scavenging both hemoglobin-haptoglobin complexes and cell free hemoglobin. A limited number of studies in the past have shown a positive correlation of CD163 expression with poor disease outcomes in patients with SCD. However, the role and regulation of CD163 in SCD related hepatobiliary injury has not been fully elucidated yet. Here, we show that chronic liver injury in SCD patients is associated with elevated levels of hepatic membrane bound CD163. Hemolysis and increase in hepatic heme, hemoglobin and iron levels elevate CD163 expression in the SCD mouse liver. Mechanistically we show that HO-1 positively regulates membrane bound CD163 expression independent of NRF2 signaling in SCD liver. We further demonstrate that of the interaction between CD163 and HO-1 is not dependent on CD163-hemoglobin binding. These findings indicate that CD163 is a potential biomarker of SCD associated hepatobiliary injury. Understanding the role of HO-1 in membrane bound CD163 regulation may help identify novel therapeutic targets for hemolysis induced chronic liver injury.

Liver-to-lung microembolic NETs promote gasdermin D-dependent inflammatory lung injury in sickle cell disease.

Acute lung injury, referred to as the acute chest syndrome, is a major cause of morbidity and mortality in patients with sickle cell disease (SCD), which often occurs in the setting of a vaso-occlusive painful crisis. P-selectin antibody therapy reduces hospitalization of patients with SCD by ∼50%, suggesting that an unknown P-selectin-independent mechanism promotes remaining vaso-occlusive events. In patients with SCD, intraerythrocytic polymerization of mutant hemoglobin promotes ischemia-reperfusion injury and hemolysis, which leads to the development of sterile inflammation. Using intravital microscopy in transgenic, humanized mice with SCD and in vitro studies with blood from patients with SCD, we reveal for the first time that the sterile inflammatory milieu in SCD promotes caspase-4/11-dependent activation of neutrophil-gasdermin D (GSDMD), which triggers P-selectin-independent shedding of neutrophil extracellular traps (NETs) in the liver. Remarkably, these NETs travel intravascularly from liver to lung, where they promote neutrophil-platelet aggregation and the development of acute lung injury. This study introduces a novel paradigm that liver-to-lung embolic translocation of NETs promotes pulmonary vascular vaso-occlusion and identifies a new GSDMD-mediated, P-selectin-independent mechanism of lung injury in SCD.

Impaired hemoglobin clearance by sinusoidal endothelium promotes vaso-occlusion and liver injury in sickle cell disease.

Sickle cell disease (SCD) is a monogenic disorder that affects 100,000 African Americans and millions of people worldwide. Intra-erythrocytic polymerization of sickle hemoglobin (HbS) promotes erythrocyte sickling, impaired rheology, ischemia and hemolysis, leading to the development of progressive liver injury in SCD. Liver resident macrophages and monocytes are known to enable the clearance of HbS, however, the role of liver sinusoidal endothelial cells (LSECs) in HbS clearance and liver injury in SCD remains unknown. Using real-time intravital (in vivo) imaging in the mice liver as well as flow cytometric analysis and confocal imaging of primary human LSECs, we show for the first time that liver injury in SCD is associated with accumulation of HbS and iron in the LSECs, leading to LSEC senescence. Hb uptake by LSECs was mediated by micropinocytosis. Hepatic monocytes were observed to attenuate LSECsenescence by accelerating HbS clearance in the liver of SCD mice, however, this protection was impaired in P-selectin-deficient SCD mice secondary to reduced monocyte recruitment in the liver. These findings are the first to suggest that LSECs contribute to HbS clearance and HbS induced LSEC-senescence promotes progressive liver injury in SCD mice. Our results provide a novel insight into the pathogenesis of hemolysis induced chronic liver injury in SCD caused by LSEC senescence. Identifying the regulators of LSEC mediated HbS clearance may lead to new therapies to prevent the progression of liver injury in SCD.

Phenotypic & genotypic profile of antimicrobial resistance in Pseudomonas species in hospitalized patients.

Background & objectives: Nosocomial infections caused by multidrug-resistant, Pseudomonas species have become a major clinical and public health concern. The aim of this study was to characterize phenotypic and genotypic profile of antimicrobial resistance (AMR) in Pseudomonas spp. isolated from hospitalized patients. Methods: A total of 126 consecutive, non-duplicate isolates of Pseudomonas spp. isolated from various clinical samples were included in the study over a period of two years. Identification and antimicrobial sensitivity was performed using automated culture system according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. Phenotypic detection of extended-spectrum ß-lactamases (ESBLs), Amp-C ß-lactamase (AmpC) and metallo-ß-lactamases (MBLs) were done by various combinations of disc-diffusion and E-test methods, followed by polymerase chain reaction-based detection of ß-lactamase-encoding genes. Results: Among 126 clinical isolates, 121 (96.1%) isolates were identified as Pseudomonas aeruginosa. Most of the isolates were recovered from pus sample, 35 (27.8%) followed by urine, 25 (19.84%); endotracheal aspirate, 24 (19.04%); blood, 14 (11.11%) and sputum, four (3.17%). The highest rate of resistance was against ticarcillin-clavulanic acid, 113 (89.7%) followed by meropenem, 92 (72.5%) and ceftazidime, 91 (72.3%). Overall, ESBLs, AmpC and carbapenemase production was detected in 109 (96.4%), 64 (50.8%) and 105 (94.6%) isolates by phenotypic methods. The most prevalent ESBL gene was blaTEMin 72 (57.1%) and the least prevalent was blaSHVin 19 (15.1%) isolates. AmpC gene was seen less compared to ESBL gene. The most prevalent carbapenemases gene was blaNDM-141 (46.06%) followed by blaVIM and blaOXA-1. Interpretation & conclusions: Our findings suggested that a high rate of ESBLs and carbapenemases production was observed in Pseudomonas spp. Therefore, phenotypic and genotypic detection of AMR needs to be combined for better characterization of resistance patterns in Pseudomonas spp.

Lung microvascular occlusion by platelet-rich neutrophil-platelet aggregates promotes cigarette smoke-induced severe flu.

Cigarette smoking is associated with a higher risk of ICU admissions among patients with flu. However, the etiological mechanism by which cigarette smoke (CS) exacerbates flu remains poorly understood. Here, we show that a mild dose of influenza A virus promotes a severe lung injury in mice preexposed to CS but not room air for 4 weeks. Real-time intravital (in vivo) lung imaging revealed that the development of acute severe respiratory dysfunction in CS- and flu-exposed mice was associated with the accumulation of platelet-rich neutrophil-platelet aggregates (NPAs) in the lung microcirculation within 2 days following flu infection. These platelet-rich NPAs formed in situ and grew larger over time to occlude the lung microvasculature, leading to the development of pulmonary ischemia followed by the infiltration of NPAs and vascular leakage into the alveolar air space. These findings suggest, for the first time to our knowledge, that an acute onset of platelet-driven thrombo-inflammatory response in the lung contributes to the development of CS-induced severe flu.

Sulforaphane mitigates genotoxicity induced by radiation and anticancer drugs in human lymphocytes.

Sulforaphane, present in cruciferous vegetables such as broccoli, is a dietary anticancer agent. Sulforaphane, added 2 or 20 h following phytohemaglutinin stimulation to cultured peripheral blood lymphocytes of individuals accidentally exposed to mixed γ and ß-radiation, reduced the micronucleus frequency by up to 70%. Studies with whole blood cultures obtained from healthy volunteers confirmed the ability of sulforaphane to ameliorate γ-radiation-induced genotoxicity and to reduce micronucleus induction by other DNA-damaging anticancer agents, such as bleomycin and doxorubicin. This reduction in genotoxicity in lymphocytes treated at the G(0) or G(1) stage suggests a role for sulforaphane in modulating DNA repair. Sulforaphane also countered the radiation-induced increase in lymphocyte HDAC activity, to control levels, when cells were treated 2 h after exposure, and enhanced histone H4 acetylation status. Sulforaphane post-irradiation treatment enhanced the CD 34(+)Lin(-) cell population in culture. Sulforaphane has therapeutic potential for management of the late effects of radiation.

Long-Term L-Glutamine Treatment Reduces Hemolysis without Ameliorating Hepatic Vaso-Occlusion and Liver Fibrosis in a Mouse Model of Sickle Cell Disease.

Sickle cell disease (SCD) is an autosomal recessive monogenic disorder caused by a homozygous mutation in the ß-globin gene, which leads to erythrocyte sickling, hemolysis, vaso-occlusion, and sterile inflammation. The administration of oral L-glutamine has been shown to reduce the frequency of pain in SCD patients; however, the long-term effect of L-glutamine in SCD remains to be determined. To understand the long-term effect of L-glutamine administration in the liver we used quantitative liver intravital microscopy and biochemical analysis in humanized SCD mice. We here show that chronic L-glutamine administration reduces hepatic hemoglobin-heme-iron levels but fails to ameliorate ischemic liver injury. Remarkably, we found that this failure in the resolution of hepatobiliary injury and persistent liver fibrosis is associated with the reduced expression of hepatic Kupffer cells post-L-glutamine treatment. These findings establish the importance of investigating the long-term effects of L-glutamine therapy on liver pathophysiology in SCD patients.

An outbreak of Burkholderia cepacia bloodstream infections in a tertiary-care facility in northern India detected by a healthcare-associated infection surveillance network.

OBJECTIVE: The burden of healthcare-associated infections (HAIs) is higher in low- and middle-income countries, but HAIs are often missed because surveillance is not conducted. Here, we describe the identification of and response to a cluster of Burkholderia cepacia complex (BCC) bloodstream infections (BSIs) associated with high mortality in a surgical ICU (SICU) that joined an HAI surveillance network. SETTING: A 780-bed, tertiary-level, public teaching hospital in northern India. METHODS: After detecting a cluster of BCC in the SICU, cases were identified by reviewing laboratory registers and automated identification and susceptibility testing outputs. Sociodemographic details, clinical records, and potential exposure histories were collected, and a self-appraisal of infection prevention and control (IPC) practices using assessment tools from the World Health Organization and the US Centers for Disease Control and Prevention was conducted. Training and feedback were provided to hospital staff. Environmental samples were collected from high-touch surfaces, intravenous medications, saline, and mouthwash. RESULTS: Between October 2017 and October 2018, 183 BCC BSI cases were identified. Case records were available for 121 case patients. Of these 121 cases, 91 (75%) were male, the median age was 35 years, and 57 (47%) died. IPC scores were low in the areas of technical guidelines, human resources, and monitoring and evaluation. Of the 30 environmental samples, 4 grew BCC. A single source of the outbreak was not identified. CONCLUSIONS: Implementing standardized HAI surveillance in a low-resource setting detected an ongoing Burkholderia cepacia outbreak. The outbreak investigation and use of a multimodal approach reduced incident cases and informed changes in IPC practices.

A self-reported survey on the implementation of infection prevention and control elements in Indian hospitals, part of a HAI surveillance network: Results from 23 hospitals conducting a standardized IPC assessment.

BACKGROUND: Healthcare-associated infections (HAIs) are one of the most common adverse events in patient care that account for substantial morbidity and mortality. We evaluate the existing Infection Prevention and Control (IPC) practices in hospitals participating in the nationally representative HAI Surveillance network. METHODS: This cross-sectional survey was conducted in 23 hospitals across 22 states of India from October-2015 to September-2018 in the HAI surveillance network. The World Health Organization (WHO) IPC core components assessment tool for health-care facility level (IPCAT-H) was adapted from IPC assessment tool developed by US Centers for Disease Control and Prevention (US CDC) under the Epidemiology and Laboratory Capacity (ELC) Infection Control Assessment and Response (ICAR) Program. Mann-Whitney U test was used to calculate the significant difference between scores (P < .05). RESULTS: Amongst the participating hospitals, 7 were private sectors and 16 were public health care facilities. Infection IPCAT-H average score per multimodal strategy was less than 50% for programmed IPC activities (45.7); implementation of health care workers (HCWs) immunization programme (43.5%); monitoring and evaluation component (38.30%). CONCLUSIONS: There is potential for improvement in Human Resources, Surveillance of HAIs as well as Monitoring and Evaluation components.

High Prevalence of Fungal and NDM-OXA Producing Gram-Negative Bacterial Superinfections in the Second Wave of Coronavirus Disease 2019 in India: Experience from a Dedicated Coronavirus Disease 2019 Hospital in North India.

Introduction: During the second wave of coronavirus disease 2019 (COVID-19), superinfection caused by fungus and multidrug-resistant bacteria worsened the severity of illness in COVID-19 patients. Limited studies from India reported the antimicrobial resistance pattern of secondary infections. In this study, we aim to study the epidemiology of pathogens causing superinfections and genotyping of Gram-negative isolates in COVID-19 patients. Methods: This retrospective study was conducted at a dedicated COVID-19 center, India. The identification of bacteria/fungi was done by Vitek2® and matrix-assisted laser desorption/ionization-time of flight mass spectrometry system. Identification of beta-lactamase genes was done using thermal cycler. The diagnosis of mucormycosis was based on 10% potassium hydroxide direct microscopy. Statistical analyses were performed using STATA version 15.1 (StataCorp., College Station, TX, USA). For continuous variables, mean and standard deviation were computed. For comparing proportions of secondary infections across admission location and outcomes, the Chi-squared test of independence was used. To compare the mean and median between intensive care units and outcomes, an independent t-test and a Mann-Whitney test were used. Results: Of all the clinical samples, 45.4% of samples were cultured positive for secondary infections. Acinetobacter baumannii (35%) was the most common Gram-negative pathogen, while among Gram positive, it was Enterococcus faecium (40%). Among fungus, Candida spp. (61%) predominates followed by molds. Colistin and tigecycline proved effective against these pathogens. blaNDM was the most prevalent gene followed by the blaOX among the carbapenemase genes. Conclusions: The mortality rate among COVID-19 patients with secondary infection was significantly higher compared to the overall mortality rate in COVID-19 patients.

Defenestrated endothelium delays liver-directed gene transfer in hemophilia A mice.

Hemophilia A is an inherited bleeding disorder caused by defective or deficient coagulation factor VIII (FVIII) activity. Until recently, the only treatment for prevention of bleeding involved IV administration of FVIII. Gene therapy with adeno-associated vectors (AAVs) has shown some efficacy in patients with hemophilia A. However, limitations persist due to AAV-induced cellular stress, immunogenicity, and reduced durability of gene expression. Herein, we examined the efficacy of liver-directed gene transfer in FVIII knock-out mice by AAV8-GFP. Surprisingly, compared with control mice, FVIII knockout (F8TKO) mice showed significant delay in AAV8-GFP transfer in the liver. We found that the delay in liver-directed gene transfer in F8TKO mice was associated with absence of liver sinusoidal endothelial cell (LSEC) fenestration, which led to aberrant expression of several sinusoidal endothelial proteins, causing increased capillarization and decreased permeability of LSECs. This is the first study to link impaired liver-directed gene transfer to liver-endothelium maladaptive structural changes associated with FVIII deficiency in mice.

Self-reported survey on infection prevention and control structures in healthcare facilities part of a national level healthcare associated infection surveillance network in India, 2019.

BACKGROUND: Healthcare associated infections (HAIs) are prevalent and difficult to treat worldwide. Most HAIs can be prevented by effective implementation of Infection Prevention and Control (IPC) measures. A survey was conducted to assess the existing IPC practices across a network of Indian Hospitals using the World Health Organization designed self-assessment IPC Assessment Framework (IPCAF) tool. METHODS: This was a cross sectional observation study. Thirty-two tertiary care public and private facilities, part of the existing Indian HAI surveillance network was included. Data collected was analyzed by a central team at All India Institute of Medical Sciences, New Delhi, a tertiary care hospital of India. The WHO questionnaire tool was used to understand the capacity and efforts to implement IPC practices across the network. RESULTS: The overall median score of IPCAF across the network was 620. Based on the final IPCAF score of the facilities; 13% hospitals had basic IPC practices, 28% hospitals had intermediate and 59% hospitals had advanced IPC practices. The component multimodal strategies had the broadest range of score while the component IPC guidelines had the narrowest one. CONCLUSIONS: Quality improvement training for IPC nurses and healthcare professionals are needed to be provided to health facilities.

Antimicrobial Resistance in Enterobacteriaceae Bacteria Causing Infection in Trauma Patients: A 5-Year Experience from a Tertiary Trauma Center.

Introduction Multiple drug resistance emergences among bacteria at an alarming rate worldwide are posing a serious threat to the treatment benefits that have been achieved with antibiotics. This crisis is due to the inappropriate and overuse of existing antibiotics. We evaluated the antimicrobial resistance pattern of Enterobacteriaceae pathogens isolated from intensive care units (ICUs), wards, and outpatient department (OPD) patients. Objectives The aim of the study is to determine the antimicrobial resistance pattern in bacteria of Enterobacteriaceae family. Material and Methods This is a retrospective study conducted at a tertiary care level-1 trauma center in the capital city of India. We collected all the retrospective data of 5 years from the laboratory information system software of the microbiology laboratory. The retrospective data included patients' details, samples detail, organism's identification, and their antimicrobial susceptibility testing, done by Vitek2 compact system and disk diffusion test according to each year's Clinical and Laboratory Standards Institute (CLSI) guidelines. This study included the interpretation of zone diameters and minimum inhibitory concentrations of all isolates according to CLSI guidelines, 2018. Results Among all the Enterobacteriaceae , Klebsiella spp. was the most commonly isolated pathogen, followed by Escherichia coli and Enterobacter spp. in ICUs and wards, while in OPD patients E. coli was the most commonly isolated pathogen, followed by Klebsiella spp. and Enterobacter spp. Enterobacteriaceae isolates remained resistant to all classes of cephalosporins in all settings. In addition, ß lactam and ß-lactamase inhibitor remained less effective. Carbapenems showed less resistance than quinolones and aminoglycosides. Among the different antimicrobial agents, tigecycline proved most effective in all settings; however, it showed more resistance than other studies. Conclusion Tigecycline proved effective among different multidrug resistance bacteria. Multidrug resistance in bacteria leads to prolonged hospital stays as well as makes the treatment less cost effective. Proper and judicious use of antimicrobials is the need of the hour.

Multiple importations and transmission of colistin-resistant Klebsiella pneumoniae in a hospital in northern India.

OBJECTIVE: Resistance to colistin, a last resort antibiotic, has emerged in India. We investigated colistin-resistant Klebsiella pneumoniae(ColR-KP) in a hospital in India to describe infections, characterize resistance of isolates, compare concordance of detection methods, and identify transmission events. DESIGN: Retrospective observational study. METHODS: Case-patients were defined as individuals from whom ColR-KP was isolated from a clinical specimen between January 2016 and October 2017. Isolates resistant to colistin by Vitek 2 were confirmed by broth microdilution (BMD). Isolates underwent colistin susceptibility testing by disk diffusion and whole-genome sequencing. Medical records were reviewed. RESULTS: Of 846 K. pneumoniae isolates, 34 (4%) were colistin resistant. In total, 22 case-patients were identified. Most (90%) were male; their median age was 33 years. Half were transferred from another hospital; 45% died. Case-patients were admitted for a median of 14 days before detection of ColR-KP. Also, 7 case-patients (32%) received colistin before detection of ColR-KP. All isolates were resistant to carbapenems and susceptible to tigecycline. Isolates resistant to colistin by Vitek 2 were also resistant by BMD; 2 ColR-KP isolates were resistant by disk diffusion. Moreover, 8 multilocus sequence types were identified. Isolates were negative for mobile colistin resistance (mcr) genes. Based on sequencing analysis, in-hospital transmission may have occurred with 8 case-patients (38%). CONCLUSIONS: Multiple infections caused by highly resistant, mcr-negative ColR-KP with substantial mortality were identified. Disk diffusion correlated poorly with Vitek 2 and BMD for detection of ColR-KP. Sequencing indicated multiple importation and in-hospital transmission events. Enhanced detection for ColR-KP may be warranted in India.

Mitigation of hematopoietic radiation injury by diallyl sulphide.

The development of mitigating agents to counter injuries induced by radiation is as important as the development of radioprotective agents. This study reports the ability of diallyl sulphide (DAS), a naturally occurring organosulfur compound, to mitigate radiation-induced mortality and injuries to the hematopoietic system in whole-body irradiated mice. Intraperitoneal administration of a single dose of DAS (160 mg/kg body weight) 2 hours after 9 Gy whole-body irradiation resulted in 37% animal survival as opposed to 100% mortality in the irradiation-only group, improved general conditions with no visual signs of sickness, and body weight loss. Restoration of spleen body weight index by DAS in animals exposed to sublethal dosages (2 and 5 Gy) of whole-body irradiation increased endogenous spleen colony-forming units and increased bone marrow cellularity indicated enhanced hematopoietic recovery, which was supported further by recoveries in lymphocyte count. Further studies to elucidate the mechanism of action of DAS are warranted to design effective protocols for mitigation of radiation injuries using compounds like DAS.

Radioprotective property of an aqueous extract from valeriana wallichii.

OBJECTIVES: Preparations of herbal drugs have drawn considerable interest in scientific community in recent years for the treatment of several stress related health problems including radiation-injury. MATERIALS AND METHODS: An aqueous extract from Valeriana wallichii containing hesperidin as one of its major constituent was evaluated for its ability to protect against radiation-injury in model systems like plasmid deoxyribonucleic acid (DNA) and cultured human fibroblast cells. RESULTS: The extract was found to significantly counter radiation-induced free radicals at 4 h after 5 Gy irradiation, reduced prolonged oxidative stress led increase in mitochondrial mass, enhanced reproductive viability of cultured cells and protected against radiation-induced DNA damage in solution. DISCUSSION: Further studies are required to validate the radioprotective ability of the extract and to develop a safer radioprotective agent.