Reduction in ACE2 expression in peripheral blood mononuclear cells during COVID-19 - implications for post COVID-19 conditions.

BACKGROUND: Severe COVID-19 is uncommon, restricted to 19% of the total population. In response to the first virus wave (alpha variant of SARS-CoV-2), we investigated whether a biomarker indicated severity of disease and, in particular, if variable expression of angiotensin converting enzyme 2 (ACE2) in blood might clarify this difference in risk and of post COVID -19 conditions (PCC). METHODS: The IRB-approved study compared patients hospitalized with severe COVID-19 to healthy controls. Severe infection was defined requiring oxygen or increased oxygen need from baseline at admission with positive COVID-19 PCR. A single blood sample was obtained from patients within a day of admission. ACE2 RNA expression in blood cells was measured by an RT-PCR assay. Plasma ACE1 and ACE2 enzyme activities were quantified by fluorescent peptides. Plasma TIMP-1, PIIINP and MMP-9 antigens were quantified by ELISA. Data were entered into REDCap and analyzed using STATA v 14 and GraphPad Prism v 10. RESULTS: Forty-eight patients and 72 healthy controls were recruited during the pandemic. ACE2 RNA expression in peripheral blood mononuclear cells (PBMC) was rarely detected acutely during severe COVID-19 but common in controls (OR for undetected ACE2: 12.4 [95% CI: 2.62-76.1]). ACE2 RNA expression in PBMC did not determine plasma ACE1 and ACE2 activity, suggesting alternative cell-signaling pathways. Markers of fibrosis (TIMP-1 and PIIINP) and vasculopathy (MMP-9) were additionally elevated. ACE2 RNA expression during severe COVID-19 often responded within hours to convalescent plasma. Analogous to oncogenesis, we speculate that potent, persistent, cryptic processes following COVID-19 (the renin-angiotensin system (RAS), fibrosis and vasculopathy) initiate or promote post-COVID-19 conditions (PCC) in susceptible individuals. CONCLUSIONS: This work elucidates biological and temporal plausibility for ACE2, TIMP1, PIIINP and MMP-9 in the pathogenesis of PCC. Intersection of these independent systems is uncommon and may in part explain the rarity of PCC.

Metagenomic next-generation sequencing for the etiological diagnosis of rabies virus in cerebrospinal fluid.

Background: Rabies is a highly fatal disease. Once symptoms develop, death usually occurs within days. Survivors were occasionally reported in the literatures. Ante-mortem diagnosis remains a challenge in most rabies endemic countries. A novel, accurate diagnostic assay is highly desirable. Methods: We used metagenomic next-generation sequencing (mNGS) to examine the cerebrospinal fluid (CSF) samples of a 49-year-old patient with rabies and validated the results by TaqMan PCR and RT-PCR/Sanger sequencing. Results: Metagenomic next-generation sequencing identified sequence reads uniquely aligned to the rabies virus (RABV). PCR confirmed the presence of the partial RABV N gene in the CSF. Phylogenetic analysis showed that the RABV grouped as an Asian clade, which is the most broadly distributed clade in China. Conclusion: Metagenomic next-generation sequencing may be a useful screening tool for the etiological diagnosis of rabies, especially in the absence of timely rabies laboratory testing or in patients with no exposure history.

Collagen-Specific HSP47+ Myofibroblasts and CD163+ Macrophages Identify Profibrotic Phenotypes in Deceased Hearts With SARS-CoV-2 Infections.

Background Cardiac fibrosis complicates SARS-CoV-2 infections and has been linked to arrhythmic complications in survivors. Accordingly, we sought evidence of increased HSP47 (heat shock protein 47), a stress-inducible chaperone protein that regulates biosynthesis and secretion of procollagen in heart tissue, with the goal of elucidating molecular mechanisms underlying cardiac fibrosis in subjects with this viral infection. Methods and Results Using human autopsy tissue, immunofluorescence, and immunohistochemistry, we quantified Hsp47+ cells and collagen α 1(l) in hearts from people with SARS-CoV-2 infections. Because macrophages are also linked to inflammation, we measured CD163+ cells in the same tissues. We observed irregular groups of spindle-shaped HSP47+ and CD163+ cells as well as increased collagen α 1(I) deposition, each proximate to one another in "hot spots" of ≈40% of hearts after SARS-CoV-2 infection (HSP47+ P<0.05 versus nonfibrotics and P<0.001 versus controls). Because HSP47+ cells are consistent with myofibroblasts, subjects with hot spots are termed "profibrotic." The remaining 60% of subjects dying with COVID-19 without hot spots are referred to as "nonfibrotic." No control subject exhibited hot spots. Conclusions Colocalization of myofibroblasts, M2(CD163+) macrophages, and collagen α 1(l) may be the first evidence of a COVID-19-related "profibrotic phenotype" in human hearts in situ. The potential public health and diagnostic implications of these observations require follow-up to further define mechanisms of viral-mediated cardiac fibrosis.

The Amazonian Tropical Bites Research Initiative, a hope for resolving zoonotic neglected tropical diseases in the One Health era.

BACKGROUND: Neglected tropical diseases (NTDs) disproportionately affect populations living in resource-limited settings. In the Amazon basin, substantial numbers of NTDs are zoonotic, transmitted by vertebrate (dogs, bats, snakes) and invertebrate species (sand flies and triatomine insects). However, no dedicated consortia exist to find commonalities in the risk factors for or mitigations against bite-associated NTDs such as rabies, snake envenoming, Chagas disease and leishmaniasis in the region. The rapid expansion of COVID-19 has further reduced resources for NTDs, exacerbated health inequality and reiterated the need to raise awareness of NTDs related to bites. METHODS: The nine countries that make up the Amazon basin have been considered (Bolivia, Brazil, Colombia, Ecuador, French Guiana, Guyana, Peru, Surinam and Venezuela) in the formation of a new network. RESULTS: The Amazonian Tropical Bites Research Initiative (ATBRI) has been created, with the aim of creating transdisciplinary solutions to the problem of animal bites leading to disease in Amazonian communities. The ATBRI seeks to unify the currently disjointed approach to the control of bite-related neglected zoonoses across Latin America. CONCLUSIONS: The coordination of different sectors and inclusion of all stakeholders will advance this field and generate evidence for policy-making, promoting governance and linkage across a One Health arena.

High-Dose IV Hydroxocobalamin (Vitamin B12) in Septic Shock: A Double-Blind, Allocation-Concealed, Placebo-Controlled Single-Center Pilot Randomized Controlled Trial (The Intravenous Hydroxocobalamin in Septic Shock Trial).

BACKGROUND: Elevated hydrogen sulfide (H2S) contributes to vasodilatation and hypotension in septic shock, and traditional therapies do not target this pathophysiologic mechanism. High-dose IV hydroxocobalamin scavenges and prevents H2S formation, which may restore vascular tone and may accentuate recovery. No experimental human studies have tested high-dose IV hydroxocobalamin in adults with septic shock. RESEARCH QUESTION: In adults with septic shock, is comparing high-dose IV hydroxocobalamin with placebo feasible? STUDY DESIGN AND METHODS: We conducted a phase 2 single-center, double-blind, allocation-concealed, placebo-controlled, parallel-group pilot randomized controlled trial comparing high-dose IV hydroxocobalamin with placebo in critically ill adults with septic shock. Patients meeting Sepsis 3 criteria were randomized 1:1 to receive a single 5-g dose of high-dose IV hydroxocobalamin or equivalent volume 0.9% saline solution as placebo. The primary outcome was study feasibility (enrollment rate, clinical and laboratory compliance rate, and contamination rate). Secondary outcomes included between-group differences in plasma H2S concentrations and vasopressor dose before and after infusion. RESULTS: Twenty patients were enrolled over 19 months, establishing an enrollment rate of 1.05 patients per month. Protocol adherence rates were 100% with zero contamination. In the high-dose IV hydroxocobalamin group, compared to placebo, there was a greater reduction in vasopressor dose between randomization and postinfusion (-36% vs 4%, P < .001) and randomization and 3-h postinfusion (-28% vs 10%, P = .019). In the high-dose IV hydroxocobalamin group, the plasma H2S level was reduced over 45 mins by -0.80 ± 1.73 µM, as compared with -0.21 ± 0.64 µM in the placebo group (P = .3). INTERPRETATION: This pilot trial established favorable feasibility metrics. Consistent with the proposed mechanism of benefit, high-dose IV hydroxocobalamin compared with placebo was associated with reduced vasopressor dose and H2S levels at all time points and without serious adverse events. These data provide the first proof of concept for feasibility of delivering high-dose IV hydroxocobalamin in septic shock. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT03783091; URL: www. CLINICALTRIALS: gov.

Rabies: who should care?

Rabies is the deadliest viral infection known, with no reliable treatment, and although it is entirely preventable, rabies continues to kill more than 60,000 people every year, mostly children in countries where dog rabies is endemic. America is only 1 generation away from the time when rabies killed more than 10,000 animals and 50 Americans every year, but 3 to 5 Americans continue to die annually from rabies. Distressingly, > 50,000 Americans undergo rabies prevention therapy every year after exposure to potentially rabid animals. While enormous progress has been made, more must be done to defeat this ancient but persistent, fatal zoonosis. In the US, lack of public awareness and ambivalence are the greatest dangers imposed by rabies, resulting in unnecessary exposures, anxiety, and risk. Veterinarians have a special role in informing and reassuring the public about prevention and protection from rabies. This summary of current facts and future advances about rabies will assist veterinarians in informing their clients about the disease.

Blastomycosis in 64 Wisconsin Children: Unanticipated Infection Risk and Severity in Urban Residents.

BACKGROUND: Blastomycosis, an endemic mycosis of immunocompetent individuals, is typically seen after exposure to waterways within rural wooded regions. It is not considered a disease of urban environments. Infection can be solely pneumonic or disseminate to skin, bone or central nervous system. Unknown factors influence disease acquisition and severity in children. METHODS: We analyzed acquisition risks and disease characteristics of blastomycosis in children seen at a tertiary care center from 1998 to 2018 to identify potential exposure sources, measure disease severity and assess the effect of race upon disease severity. RESULTS: Of 64 infected children, mean age was 12.9 years, with median time to diagnosis 38.5 days. About 72% were male, 38% resided in urban counties and 50% had typical environmental exposure. Isolated pulmonary infection occurred in 33 (52%). The remainder had evidence of dissemination to skin (N = 13), bone (N = 16; 7 clinically silent) and cranium (N = 7; 3 clinically silent). Infection was moderate/severe in 19 (30%). Two children (3%) died. About 79% of children with moderate/severe disease (P = 0.008) and 71% of urban children (P = 0.007) lacked typical environmental exposure. Comparing children from urban counties to other residences, 63% versus 5% were black (P < 0.001) and 71% versus 35% developed extrapulmonary dissemination (P = 0.006). Moderate/severe disease was seen in 7/17 (42%) black children but only 12/47 (26%) children of other races (P = 0.23). CONCLUSIONS: Blastomycosis, can be endemic in urban children in the absence of typical exposure history, have frequent, sometimes clinically silent, extrapulmonary dissemination and possibly produces more severe disease in black children.

High-Dose Vitamin B12 in Vasodilatory Shock: A Narrative Review.

Vasodilatory shock, as observed in postoperative states and sepsis, is hallmarked by low systemic vascular resistance and low blood pressure compensated by increased cardiac output. Gasotransmitters, such as nitric oxide and hydrogen sulfide, are implicated in the development and perpetuation of vasodilatory shock. Established therapies do not target these physiologic drivers of vasodilation. Due to their nontoxic and pleotropic effects, micronutrients are being used as rescue therapy in postoperative vasoplegia and septic shock. Here, we outline the pathophysiology of vasodilatory shock, describe the rationale for vitamin B12 (hydroxocobalamin) in vasodilatory shock, and identify literature evaluating its use in vasoplegic states.

Impact of American Academy of Pediatrics Palivizumab Guidance for Children ≥29 and <35 Weeks of Gestational Age.

OBJECTIVES: To evaluate the impact of the American Academy of Pediatrics revised recommendations (2014) for palivizumab prophylaxis on respiratory syncytial virus (RSV) admissions and severity of illness among children ≥29 weeks and <35 weeks of gestational age. STUDY DESIGN: We evaluated patients hospitalized with RSV infection from October 1, 2012, through April 30, 2017. RSV hospitalizations, community RSV activity, duration of hospitalization, disease severity, and mortality were reviewed. Data were compared before and after implementation of the guideline changes. RESULTS: A total of 91 patients were born at ≥29 weeks and <35 weeks of gestational age and hospitalized within the first year of life during the evaluation period. Gestational age, birth weight, age at diagnosis, and sex remained constant over the seasons evaluated. RSV hospitalizations and activity in the community were unchanged over 5 years. Duration of hospitalization increased. There was no difference in need for intensive care, supplemental oxygen, or mechanical ventilation or mortality. CONCLUSIONS: Implementation of the 2014 American Academy of Pediatrics guidelines regarding eligibility for palivizumab prophylaxis in older infants born preterm did not increase RSV hospitalizations or disease severity among children hospitalized for RSV at our hospital. Our data support continued adherence to the guidelines.

NMR metabolomics of cerebrospinal fluid differentiates inflammatory diseases of the central nervous system.

BACKGROUND: Myriad infectious and noninfectious causes of encephalomyelitis (EM) have similar clinical manifestations, presenting serious challenges to diagnosis and treatment. Metabolomics of cerebrospinal fluid (CSF) was explored as a method of differentiating among neurological diseases causing EM using a single CSF sample. METHODOLOGY/PRINCIPAL FINDINGS: 1H NMR metabolomics was applied to CSF samples from 27 patients with a laboratory-confirmed disease, including Lyme disease or West Nile Virus meningoencephalitis, multiple sclerosis, rabies, or Histoplasma meningitis, and 25 controls. Cluster analyses distinguished samples by infection status and moderately by pathogen, with shared and differentiating metabolite patterns observed among diseases. CART analysis predicted infection status with 100% sensitivity and 93% specificity. CONCLUSIONS/SIGNIFICANCE: These preliminary results suggest the potential utility of CSF metabolomics as a rapid screening test to enhance diagnostic accuracies and improve patient outcomes.

Successful Application of Whole Genome Sequencing in a Medical Genetics Clinic.

A pilot program was initiated using whole genome sequencing (WGS) to diagnose suspected genetic disorders in the Genetics Clinic at Children's Hospital of Wisconsin. Twenty-two patients underwent WGS between 2010 and 2013. Initially, we obtained a 14% (3/22) diagnosis rate over 2 years; with subsequent reanalysis, this increased to 36% (8/22). Disease causing variants were identified in SKIV2L, CECR1, DGKE, PYCR2, RYR1, PDGFRB, EFTUD2, and BCS1L. In 75% (6/8) of diagnosed cases, the diagnosis affected treatment and/or medical surveillance. Additionally, one case demonstrated a homozygous A18V variant in VLDLR that appears to be associated with a previously undescribed phenotype.

Implementation of American Academy of Pediatrics guidelines for palivizumab prophylaxis in a pediatric hospital.

PURPOSE: One hospital's implementation of revised American Academy of Pediatrics (AAP) guidelines for palivizumab prophylaxis of respiratory syncytial virus (RSV) infection is described. METHODS: Revised AAP guidelines for RSV prophylaxis in infants and young children at increased risk for RSV infection recommend that up to five doses of palivizumab be administered during the RSV season. The guidelines also recommend that inpatients not receive monthly palivizumab prophylaxis and that infants and young children eligible for prophylaxis during the RSV season receive a dose of palivizumab two or three days before discharge or promptly after discharge. To ensure compliance with the revised AAP guidelines, a 296-bed hospital implemented a quality-improvement project including (1) efforts by the antimicrobial stewardship pharmacist and the chief medical officer to notify and educate healthcare providers regarding institutional adoption of the guidelines, (2) reinforcement of guideline adherence by clinical pharmacists during daily bedside rounds and via prospective review of all palivizumab orders, and (3) a medication-use evaluation (MUE) to assess adherence to the guidelines. The MUE results showed that during the 2014-15 RSV season (after implementation of the practice changes), the number of palivizumab doses administered at the hospital declined by 56% from the previous RSV season, with 97% of doses administered for appropriate indications. CONCLUSION: Standardized, comprehensive guidelines with defined criteria for palivizumab prophylaxis of RSV infection resulted in $303,227 of cost savings without a discernible change in nosocomial transmission, or morbidity, or mortality. Hospital infection-control practices controlled nosocomial RSV transmission.

Rabies: Rare Human Infection - Common Questions.

Rabies is an acute, rapidly progressive encephalitis that is almost always fatal. Prophylaxis is highly effective but economics limits disease control. The mechanism of death from rabies is unclear. It is poorly cytopathic and poorly inflammatory. Rabies behaves like an acquired metabolic disorder. There may be a continuum of disease severity. History of animal bite is rare. The diagnosis is often missed. Intermittent encephalopathy, dysphagia, hydrophobia and aerophobia, and focal paresthesias or myoclonic jerks suggest rabies. Laboratory diagnosis is cumbersome but sensitive. Treatment is controversial but survivors are increasingly reported, with good outcomes in 4 of 8 survivors.

A Case of Necrotizing Epiglottitis Due to Nontoxigenic Corynebacterium diphtheriae.

Diphtheria is a rare cause of infection in highly vaccinated populations and may not be recognized by modern clinicians. Infections by nontoxigenic Corynebacterium diphtheriae are emerging. We report the first case of necrotizing epiglottitis secondary to nontoxigenic C diphtheriae. A fully vaccinated child developed fever, poor oral intake, and sore throat and was found to have necrotizing epiglottitis. Necrotizing epiglottitis predominantly occurs in the immunocompromised host. Laboratory evaluation revealed pancytopenia, and bone marrow biopsy was diagnostic for acute lymphoblastic leukemia. Clinicians should be aware of aggressive infections that identify immunocompromised patients. This case highlights the features of a reemerging pathogen, C diphtheriae.

Clinical guidance for smallpox vaccine use in a postevent vaccination program.

This report outlines recommendations for the clinical use of the three smallpox vaccines stored in the U.S. Strategic National Stockpile for persons who are exposed to smallpox virus or at high risk for smallpox infection during a postevent vaccination program following an intentional or accidental release of the virus. No absolute contraindications exist for smallpox vaccination in a postevent setting. However, several relative contraindications exist among persons with certain medical conditions. CDC recommendations for smallpox vaccine use were developed in consideration of the risk for smallpox infection, risk for an adverse event following vaccination, and benefit from vaccination. Smallpox vaccines are made from live vaccinia viruses that protect against smallpox disease. They do not contain variola virus, the causative agent of smallpox. The three smallpox vaccines stockpiled are ACAM2000, Aventis Pasteur Smallpox Vaccine (APSV), and Imvamune. Surveillance and containment activities including vaccination with replication-competent smallpox vaccine (i.e., vaccine viruses capable of replicating in mammalian cells such as ACAM2000 and APSV) will be the primary response strategy for achieving epidemic control. Persons exposed to smallpox virus are at high risk for developing and transmitting smallpox and should be vaccinated with a replication-competent smallpox vaccine unless severely immunodeficient. Because of a high likelihood of a poor immune response and an increased risk for adverse events, smallpox vaccination should be avoided in persons with severe immunodeficiency who are not expected to benefit from vaccine, including bone marrow transplant recipients within 4 months of transplantation, persons infected with HIV with CD4 cell counts <50 cells/mm3, and persons with severe combined immunodeficiency, complete DiGeorge syndrome, and other severely immunocompromised states requiring isolation. If antivirals are not immediately available, it is reasonable to consider the use of Imvamune in the setting of a smallpox virus exposure in persons with severe immunodeficiency. Persons without a known smallpox virus exposure might still be at high risk for developing smallpox infection depending on the magnitude of the outbreak and the effectiveness of the public health response. Such persons will be defined by public health authorities and should be screened for relative contraindications to smallpox vaccination. Relative contraindications include atopic dermatitis (eczema), HIV infection (CD4 cell counts of 50-199 cells/mm3), other immunocompromised states, and vaccine or vaccine-component allergies. Persons with relative contraindications should be vaccinated with Imvamune when available and authorized for use by the Food and Drug Administration. These recommendations will be updated as new data on smallpox vaccines become available and further clinical guidance for other medical countermeasures including antivirals is developed.

Virology, immunology and pathology of human rabies during treatment.

BACKGROUND: Rabies is an acute fatal encephalitis caused by all members of the Lyssavirus genus. The first human rabies survivor without benefit of prior vaccination was reported from Milwaukee in 2005. We report a second unvaccinated patient who showed early recovery from rabies and then died accidentally during convalescence, providing an unparalleled opportunity to examine the histopathology as well as immune and virological correlates of early recovery from human rabies. METHODS: Case report, rapid fluorescent focus inhibition test, enzyme-linked immunosorbent assay, indirect and direct fluorescent antibody assays, reverse-transcriptase polymerase chain reaction, phylogenetic reconstruction, isolation in tissue culture, pathology and immunohistochemistry. RESULTS: The 9 year old died 76 days after presenting with rabies of vampire bat phylogeny transmitted by cat bite. Antibody response in serum and cerebrospinal fluid was robust and associated with severe cerebral edema. No rabies virus was cultured at autopsy. Rabies virus antigen was atypical in size and distribution. Rabies virus genome was present in neocortex but absent in brainstem. CONCLUSIONS: Clinical recovery was associated with detection of neutralizing antibody and clearance of infectious rabies virus in the central nervous system by 76 days but not clearance of detectable viral subcomponents such as nucleoprotein antigen or RNA in brain.