Assessing Vulnerability to COVID-19 in High-Risk Populations: The Role of SARS-CoV-2 Spike-Targeted Serology.

Individuals at increased risk for severe coronavirus disease-2019 (COVID-19) outcomes, due to compromised immunity or other risk factors, would benefit from objective measures of vulnerability to infection based on vaccination or prior infection. The authors reviewed published data to identify a specific role and interpretation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike-targeted serology testing. Specific recommendations are provided for an evidence-based and clinically-useful interpretation of SARS-CoV-2 spike-targeted serology to identify vulnerability to infection and potential subsequent adverse outcomes. Decreased vaccine effectiveness among immunocompromised individuals is linked to correspondingly high rates of breakthrough infections. Negative results on SARS-CoV-2 antibody tests are associated with increased risk for subsequent infection. "Low-positive" results on semiquantitative SARS-CoV-2 spike-targeted antibody tests may help identify persons at increased risk as well. Standardized SARS-CoV-2 spike-targeted antibody tests may provide objective information on the risk of SARS-CoV-2 infection and associated adverse outcomes. This holds especially for high-risk populations that demonstrate a relatively high rate of seronegativity. The widespread availability of such tests presents an opportunity to refine risk assessment for individuals with suboptimal SARS-CoV-2 antibody levels and to promote effective interventions. Interim federal guidance would support physicians and patients while additional investigations are pursued.

Susceptibility of Fat Tissue to SARS-CoV-2 Infection in Female hACE2 Mouse Model.

The coronavirus disease (COVID-19) is a highly contagious viral illness caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 has had a catastrophic effect globally causing millions of deaths worldwide and causing long-lasting health complications in COVID-19 survivors. Recent studies including ours have highlighted that adipose tissue can act as a reservoir where SARS-CoV-2 can persist and cause long-term health problems. Here, we evaluated the effect of SARS-CoV-2 infection on adipose tissue physiology and the pathogenesis of fat loss in a murine COVID-19 model using humanized angiotensin-converting enzyme 2 (hACE2) mice. Since epidemiological studies reported a higher mortality rate of COVID-19 in males than in females, we examined hACE2 mice of both sexes and performed a comparative analysis. Our study revealed for the first time that: (a) viral loads in adipose tissue and the lungs differ between males and females in hACE2 mice; (b) an inverse relationship exists between the viral loads in the lungs and adipose tissue, and it differs between males and females; and (c) CoV-2 infection alters immune signaling and cell death signaling differently in SARS-CoV-2 infected male and female mice. Overall, our data suggest that adipose tissue and loss of fat cells could play important roles in determining susceptibility to CoV-2 infection in a sex-dependent manner.

The importance of antimicrobial resistance in medical mycology.

Prior to the SARS-CoV-2 pandemic, antibiotic resistance was listed as the major global health care priority. Some analyses, including the O'Neill report, have predicted that deaths due to drug-resistant bacterial infections may eclipse the total number of cancer deaths by 2050. Although fungal infections remain in the shadow of public awareness, total attributable annual deaths are similar to, or exceeds, global mortalities due to malaria, tuberculosis or HIV. The impact of fungal infections has been exacerbated by the steady rise of antifungal drug resistant strains and species which reflects the widespread use of antifungals for prophylaxis and therapy, and in the case of azole resistance in Aspergillus, has been linked to the widespread agricultural use of antifungals. This review, based on a workshop hosted by the Medical Research Council and the University of Exeter, illuminates the problem of antifungal resistance and suggests how this growing threat might be mitigated.

Determinants of fluconazole resistance and echinocandin tolerance in C. parapsilosis isolates causing a large clonal candidemia outbreak among COVID-19 patients in a Brazilian ICU.

Patients presenting with severe COVID-19 are predisposed to acquire secondary fungal infections such as COVID-19-associated candidemia (CAC), which are associated with poor clinical outcomes despite antifungal treatment. The extreme burden imposed on clinical facilities during the COVID-19 pandemic has provided a permissive environment for the emergence of clonal outbreaks of multiple Candida species, including C. auris and C. parapsilosis. Here we report the largest clonal CAC outbreak to date caused by fluconazole resistant (FLZR) and echinocandin tolerant (ECT) C. parapsilosis. Sixty C. parapsilosis strains were obtained from 57 patients at a tertiary care hospital in Brazil, 90% of them were FLZR and ECT. Although only 35.8% of FLZR isolates contained an ERG11 mutation, all of them contained the TAC1L518F mutation and significantly overexpressed CDR1. Introduction of TAC1L518F into a susceptible background increased the MIC of fluconazole and voriconazole 8-fold and resulted in significant basal overexpression of CDR1. Additionally, FLZR isolates exclusively harboured E1939G outside of Fks1 hotspot-2, which did not confer echinocandin resistance, but significantly increased ECT. Multilocus microsatellite typing showed that 51/60 (85%) of the FLZR isolates belonged to the same cluster, while the susceptible isolates each represented a distinct lineage. Finally, biofilm production in FLZR isolates was significantly lower than in susceptible counterparts Suggesting that it may not be an outbreak determinant. In summary, we show that TAC1L518F and FKS1E1393G confer FLZR and ECT, respectively, in CAC-associated C. parapsilosis. Our study underscores the importance of antifungal stewardship and effective infection control strategies to mitigate clonal C. parapsilosis outbreaks.

Genomic Epidemiology and Serology Associated with a SARS-CoV-2 R.1 Variant Outbreak in New Jersey.

Examining the neutralizing capacity of monoclonal antibodies (MAbs) used to treat COVID-19, as well as antibodies recovered from unvaccinated, previously vaccinated, and infected individuals, against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) remains critical to study. Here, we report on a SARS-CoV-2 nosocomial outbreak caused by the SARS-CoV-2 R.1 variant harboring the E484K mutation in a 281-bed psychiatric facility in New Jersey among unvaccinated inpatients and health care professionals (HCPs). A total of 81 inpatients and HCPs tested positive for SARS-Cov-2 by reverse transcription (RT)-PCR from 29 October 9 to 30 November 2020. The R.1 variant exhibits partial or complete resistance to two MAbs in clinical use, as well as 2 receptor binding domain MAbs and 4 N-terminal domain (NTD) MAbs. NTD MAbs against pseudovirus harboring single characteristic R.1 mutations highlight the role of S255F in loss of activity. Additionally, we note dampened neutralization capacity by plasma from individuals with previous SARS-CoV-2 infection or sera from vaccinated individuals. The relative resistance of the R.1 variant is likely lower than that of B.1.351 and closer to that of P.1 and B.1.526. The R.1 lineage has been reported in 47 states in the United States and 40 countries. Although high proportions exhibited symptoms (26% and 61% among patients and HCPs, respectively) and relative antibody resistance, we detected only 10 R.1 variants from over 2,900 samples (~0.34%) collected from January to October 2021. Among 3 vaccinated individuals previously infected with R.1, we observed robust neutralizing antibody responses against SARS-CoV-2 wild type and VOCs. IMPORTANCE The neutralizing capacities of monoclonal antibodies used to treat COVID-19 and of those recovered from previously infected and vaccinated individuals against SARS-CoV-2 variants of concern (VOCs) remain important questions. We report on a nosocomial outbreak caused by a SARS-CoV-2 R.1 variant harboring an E484K mutation among 81 unvaccinated inpatients and health care professionals. We note high attack rates with symptoms in nearly 50% of infected individuals, in sharp contrast to an unrelated institutional outbreak caused by the R.1 variant among a vaccinated population. We found little evidence of significant community spillover. This variant exhibits partial or complete resistance to two monoclonal antibodies in clinical use and dampened the neutralization capacity of convalescent-phase plasma from individuals with previous infection or sera from vaccinated individuals. Among three vaccinated individuals previously infected with R.1, we observed robust neutralizing antibody responses against SARS-CoV-2 wild type and VOCs. These findings underscore the importance of vaccination for prevention of symptomatic COVID-19 disease.

An antibody class with a common CDRH3 motif broadly neutralizes sarbecoviruses.

The devastation caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made clear the importance of pandemic preparedness. To address future zoonotic outbreaks due to related viruses in the sarbecovirus subgenus, we identified a human monoclonal antibody, 10-40, that neutralized or bound all sarbecoviruses tested in vitro and protected against SARS-CoV-2 and SARS-CoV in vivo. Comparative studies with other receptor-binding domain (RBD)-directed antibodies showed 10-40 to have the greatest breadth against sarbecoviruses, suggesting that 10-40 is a promising agent for pandemic preparedness. Moreover, structural analyses on 10-40 and similar antibodies not only defined an epitope cluster in the inner face of the RBD that is well conserved among sarbecoviruses but also uncovered a distinct antibody class with a common CDRH3 motif. Our analyses also suggested that elicitation of this class of antibodies may not be overly difficult, an observation that bodes well for the development of a pan-sarbecovirus vaccine.

Sex Differences in Cardiac Pathology of SARS-CoV2 Infected and Trypanosoma cruzi Co-infected Mice.

Coronavirus disease-2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; CoV2) is a deadly contagious infectious disease. For those who survive COVID-19, post-COVID cardiac damage greatly increases the risk of cardiomyopathy and heart failure. Currently, the number of COVID-related cases are increasing in Latin America, where a major COVID comorbidity is Chagas' heart disease, which is caused by the parasite Trypanosoma cruzi. However, the interplay between indeterminate Chagas disease and COVID-19 is unknown. We investigated the effect of CoV2 infection on heart pathology in T. cruzi infected mice (coinfected with CoV2 during the indeterminate stage of T. cruzi infection). We used transgenic human angiotensin-converting enzyme 2 (huACE2/hACE2) mice infected with CoV2, T. cruzi, or coinfected with both in this study. We found that the viral load in the hearts of coinfected mice is lower compared to the hearts of mice infected with CoV2 alone. We demonstrated that CoV2 infection significantly alters cardiac immune and energy signaling via adiponectin (C-ApN) and AMP-activated protein kinase (AMPK) signaling. Our studies also showed that increased ß-adrenergic receptor (b-AR) and peroxisome proliferator-activated receptors (PPARs) play a major role in shifting the energy balance in the hearts of coinfected female mice from glycolysis to mitochondrial ß-oxidation. Our findings suggest that cardiac metabolic signaling may differently regulate the pathogenesis of Chagas cardiomyopathy (CCM) in coinfected mice. We conclude that the C-ApN/AMPK and b-AR/PPAR downstream signaling may play major roles in determining the progression, severity, and phenotype of CCM and heart failure in the context of COVID.

Candidemia Among Coronavirus Disease 2019 Patients in Turkey Admitted to Intensive Care Units: A Retrospective Multicenter Study.

Background: We evaluated the epidemiology of candidemia among coronavirus disease 2019 (COVID-19) patients admitted to intensive care units (ICUs). Methods: We conducted a retrospective multicenter study in Turkey between April and December 2020. Results: Twenty-eight of 148 enrolled patients developed candidemia, yielding an incidence of 19% and incidence rate of 14/1000 patient-days. The probability of acquiring candidemia at 10, 20, and 30 days of ICU admission was 6%, 26%, and 50%, respectively. More than 80% of patients received antibiotics, corticosteroid, and mechanical ventilation. Receipt of a carbapenem (odds ratio [OR] = 6.0, 95% confidence interval [CI] = 1.6-22.3, P = .008), central venous catheter (OR = 4.3, 95% CI = 1.3-14.2, P = .02), and bacteremia preceding candidemia (OR = 6.6, 95% CI = 2.1-20.1, P = .001) were independent risk factors for candidemia. The mortality rate did not differ between patients with and without candidemia. Age (OR = 1.05, 95% CI = 1.01-1.09, P = .02) and mechanical ventilation (OR = 61, 95% CI = 15.8-234.9, P < .0001) were independent risk factors for death. Candida albicans was the most prevalent species overall. In Izmir, Candida parapsilosis accounted for 50% (2 of 4) of candidemia. Both C parapsilosis isolates were fluconazole nonsusceptible, harbored Erg11-Y132F mutation, and were clonal based on whole-genome sequencing. The 2 infected patients resided in ICUs with ongoing outbreaks due to fluconazole-resistant C parapsilosis. Conclusions: Physicians should be aware of the elevated risk for candidemia among patients with COVID-19 who require ICU care. Prolonged ICU exposure and ICU practices rendered to COVID-19 patients are important contributing factors to candidemia. Emphasis should be placed on (1) heightened infection control in the ICU and (2) developing antibiotic stewardship strategies to reduce irrational antimicrobial therapy.

Candidemia among COVID-19 patients in Turkey admitted to ICUs: A retrospective multicenter study

Objectives We evaluated the epidemiology of candidemia among COVID-19 patients admitted to intensive care units (ICUs). Methods We conducted a retrospective multicenter study in Turkey between April- December 2020. Results Twenty-eight of 148 enrolled patients developed candidemia, yielding an incidence of 19% and incidence rate of 14/1,000 patient-days. The probability of acquiring candidemia at 10, 20 and 30 days of ICU admission was 6%, 26% and 50%, respectively. Over 80% of patients received antibiotics, corticosteroid and mechanical ventilation. Receipt of a carbapenem (odds ratio and 95% confidence interval (OR, 95% CI) of 6.0 (1.6–22.3), P=0.008), central venous catheter (4.3 (1.3–14.2), P=0.02) and bacteremia preceding candidemia (6.6 (2.1–20.1), P=0.001) were independent risk factors for candidemia. Mortality rate did not differ between patients with and without candidemia. Age (1.05 (1.01–1.09), P=0.02) and mechanical ventilation (61 (15.8–234.9), P<0.0001) were independent risk factors for death. Candida albicans was the most prevalent species overall. In Izmir, C. parapsilosis accounted for 50% (2/4) of candidemia. Both C. parapsilosis isolates were fluconazole non-susceptible, harbored Erg11-Y132F mutation, and were clonal based on whole-genome sequencing. The two infected patients resided in ICUs with ongoing outbreaks due to fluconazole-resistant C. parapsilosis. Conclusions Physicians should be aware of the elevated risk for candidemia among COVID-19 patients requiring ICU care. Prolonged ICU exposure and ICU practices rendered to COVID-19 patients are important contributing factors to candidemia. Emphasis should be placed on heightened infection control in the ICU, and developing antibiotic stewardship strategies to reduce irrational antimicrobial therapy.

Focused multivector ultraviolet (FMUV) technology rapidly eradicates SARS-CoV-2 in-vitro: Implications for hospital disinfection of COVID-19 environments.

Focused Multivector Ultraviolet technology rapidly killed the SARS-CoV-2 coronavirus in-vitro. Plates were inoculated with a mean of greater than 106 plaque forming units of USA-WA1 Washington index patient strain of SARS-CoV-2 and exposed to ultraviolet, resulting in mean reductions of 99.99% within 30 seconds, 99.999% within 60 seconds, and 99.9999% within 90 seconds. These results support the effectiveness of Focused Multivector Ultraviolet technology for SARS-CoV-2 disinfection.

Randomized, double-blind, controlled trial of human anti-LIGHT monoclonal antibody in COVID-19 acute respiratory distress syndrome.

BACKGROUNDSevere coronavirus disease 2019 (COVID-19) is associated with a dysregulated immune response, which can result in cytokine-release syndrome and acute respiratory distress syndrome (ARDS). Patients with COVID-19-associated ARDS have elevated free serum levels of the cytokine lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells (LIGHT; also known as TNFSF14). Such patients may benefit from LIGHT-neutralization therapy.METHODSThis randomized, double-blind, multicenter, proof-of-concept trial enrolled adults hospitalized with COVID-19-associated pneumonia and mild to moderate ARDS. Patients received standard of care plus a single dose of a human LIGHT-neutralizing antibody (CERC-002) or placebo. The primary endpoint was the proportion of patients receiving CERC-002 who remained alive and free of respiratory failure through day 28. Safety was assessed via adverse event monitoring.RESULTSFor most of the 83 enrolled patients, standard of care included systemic corticosteroids (88.0%) or remdesivir (57.8%). A higher proportion of patients remained alive and free of respiratory failure through day 28 after receiving CERC-002 (83.9%) versus placebo (64.5%; P = 0.044), including in patients 60 years of age or older (76.5% vs. 47.1%, respectively; P = 0.042). Mortality rates were 7.7% (CERC-002) and 14.3% (placebo) on day 28 and 10.8% and 22.5%, respectively, on day 60. Treatment-emergent adverse events were less frequent with CERC-002 than placebo.CONCLUSIONFor patients with COVID-19-associated ARDS, adding CERC-002 to standard-of-care treatment reduces LIGHT levels and might reduce the risk of respiratory failure and death.TRIAL REGISTRATIONClinicalTrials.gov NCT04412057.FUNDINGAvalo Therapeutics.

Postvaccination SARS-COV-2 among Health Care Workers in New Jersey: A Genomic Epidemiological Study.

Emergence of SARS-CoV-2 with high transmission and immune evasion potential, the so-called variants of concern (VOC), is a major concern. We describe the early genomic epidemiology of SARS-CoV-2 recovered from vaccinated health care professionals (HCP). Our postvaccination COVID-19 symptoms-based surveillance program among HCPs in a 17-hospital network identified all vaccinated HCPs who tested positive for COVID-19 after routine screening or after self-reporting. From 1 January 2021 to 30 April 2021, 23,687 HCPs received either mRNA-1273 or BNT162b2 mRNA vaccine. All available postvaccination SARS-CoV-2 samples and a random collection from nonvaccinated patients during the similar time frame were subjected to VOC screening and whole-genome sequencing (WGS). Sixty-two percent (23,697/37,500) of HCPs received at least one vaccine dose, with 60% (22,458) fully vaccinated. We detected 138 (0.58%, 138/23,697) COVID-19 cases, 105 among partially vaccinated and 33 (0.15%, 33/22,458) among fully vaccinated. Five partially vaccinated required hospitalization, four with supplemental oxygen. VOC screening from 16 fully vaccinated HCPs identified 6 (38%) harboring N501Y and 1 (6%) with E484K polymorphisms; percentage of concurrent nonvaccinated samples was 37% (523/1,404) and 20% (284/1,394), respectively. There was an upward trend from January to April for E484K/Q (3% to 26%) and N501Y (1% to 49%). WGS analysis from vaccinated and nonvaccinated individuals indicated highly congruent phylogenies. We did not detect an increased frequency of any receptor-binding domain (RBD)/N-terminal domain (NTD) polymorphism between groups (P > 0.05). Our results support robust protection by vaccination, particularly among recipients of both doses. Despite VOCs accounting for over 40% of SARS-CoV-2 from fully vaccinated individuals, the genomic diversity appears to proportionally represent VOCs among nonvaccinated populations. IMPORTANCE A number of highly effective vaccines have been developed and deployed to combat the COVID-19 pandemic. The emergence and epidemiological dominance of SARS-CoV-2 mutants with high transmission potential and immune evasion properties, the so-called variants of concern (VOC), continue to be a major concern. Whether these VOCs alter the efficacy of the administered vaccines is of great concern and a critical question to study. We describe the initial genomic epidemiology of SARS-CoV-2 recovered from partial/fully vaccinated health care professionals and probe specifically for VOC enrichment. Our findings support the high level of protection provided by full vaccination despite a steep increase in the prevalence of polymorphisms associated with increased transmission potential (N501Y) and immune evasion (E484K) in the nonvaccinated population. Thus, we do not find evidence of VOC enrichment among vaccinated groups. Overall, the genomic diversity of SARS-CoV-2 recovered postvaccination appears to proportionally represent the observed viral diversity within the community.

Elevated cytokines and chemokines in peripheral blood of patients with SARS-CoV-2 pneumonia treated with high-titer convalescent plasma.

The global SARS-CoV-2 coronavirus pandemic continues to be devastating in many areas. Treatment options have been limited and convalescent donor plasma has been used by many centers to transfer passive neutralizing antibodies to patients with respiratory involvement. The results often vary by institution and are complicated by the nature and quality of the donor plasma itself, the timing of administration and the clinical aspects of the recipients. SARS-CoV-2 infection is known to be associated with an increase in the blood concentrations of several inflammatory cytokines/chemokines, as part of the overall immune response to the virus and consequential to mediated lung pathology. Some of these correlates contribute to the cytokine storm syndrome and acute respiratory distress syndrome, often resulting in fatality. A Phase IIa clinical trial at our institution using high neutralizing titer convalescent plasma transfer gave us the unique opportunity to study the elevations of correlates in the first 10 days after infusion. Plasma recipients were divided into hospitalized COVID-19 pneumonia patients who did not (Track 2) or did (Track 3) require mechanical ventilation. Several cytokines were elevated in the patients of each Track and some continued to rise through Day 10, while others initially increased and then subsided. Furthermore, elevations in MIP-1α, MIP-1ß and CRP correlated with disease progression of Track 2 recipients. Overall, our observations serve as a foundation for further study of these correlates and the identification of potential biomarkers to improve upon convalescent plasma therapy and to drive more successful patient outcomes.

Emergence of Multiple SARS-CoV-2 Antibody Escape Variants in an Immunocompromised Host Undergoing Convalescent Plasma Treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), harboring spike protein N-terminal domain (NTD) or receptor-binding domain (RBD) mutations, exhibit reduced in vitro susceptibility to convalescent-phase serum, commercial antibody cocktails, and vaccine neutralization and have been associated with reinfections. The accumulation of these mutations could be the consequence of intrahost viral evolution due to prolonged infection in immunocompromised hosts. In this study, we document the microevolution of SARS-CoV-2 recovered from sequential tracheal aspirates from an immunosuppressed patient on steroids and convalescent plasma therapy and identify the emergence of multiple NTD and RBD mutations. SARS-CoV-2 genomes from the first swab (day 0) and from three tracheal aspirates (days 7, 21, and 27) were compared at the sequence level. We identified a mixed viral population with five different S protein mutations (141 to 144 deletion, 243 to 244 deletion, E484K, Q493K, and Q493R) at the NTD or RBD region from the second tracheal aspirate sample (day 21) and a predominance of the S protein 141 to 144 LGVY deletion and E484K mutant on day 27. The neutralizing antibodies against various S protein lentiviral pseudovirus mutants, as well as the anti-SARS-CoV-2 total Ig and IgG, showed "U" shape dynamics, in support of the endogenous development of neutralizing antibodies. The patient's compromised immune status, the antirejection regiment, convalescent plasma treatment, and the development of neutralizing antibodies may have resulted in unique selective pressures on the intrahost genomic evolution, and this observation supports the hypotheses that VOCs can independently arise and that immunocompromised patients on convalescent plasma therapy are potential breeding grounds for immune escape mutants. IMPORTANCE Over a year of the COVID-19 pandemic, distinct severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages have arisen in multiple geographic areas around the world. SARS-CoV-2 variants of concern (VOCs), i.e., B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), and B.1.617.2 (delta), harboring mutations and/or deletions in spike protein N-terminal domain (NTD) or receptor-binding domain (RBD) regions showed evidence of increased transmissibility and disease severity and possible reduced vaccine efficacy. In this study, we report the emergence of five different NTD and RBD mutations in an uncommon SARS-CoV-2 B.1.369 lineage from an immunosuppressed patient undergoing steroid and convalescent plasma therapy. The observation highlighted that VOCs can independently arise in immunocompromised populations undergoing anti-SARS-CoV-2 therapy, and enhanced measures will be required to reduce the transmission.

Antifungal Drug Susceptibility and Genetic Characterization of Fungi Recovered from COVID-19 Patients.

Fungal infections are common complications of respiratory viral infections and are associated with the increased need for intensive care and elevated mortality. Data regarding microbiological and molecular characteristics of such infections in COVID-19 patients are scarce. Here, we performed a comprehensive analysis, including species identification, antifungal susceptibility testing, molecular resistance determinants analysis, typing, and retrospective clinical data review, of fungal isolates recovered from 19 COVID-19 patients, who were hospitalized at the Hackensack University Medical Center (HUMC) in Hackensack, New Jersey, USA, in the initial phase of the pandemic from April-May 2020. In total, 17 Candida albicans, two C. parapsilosis, and two Aspergillus fumigatus were analyzed. All Candida spp. isolates were susceptible to micafungin and azole drugs (fluconazole, voriconazole, posaconazole, itraconazole, isavuconazole). A. fumigatus isolates were susceptible to micafungin and all triazole drugs except fluconazole (intrinsic resistance). Multilocus sequence typing (MLST) of C. albicans isolates revealed 15 different sequence types (STs), which clustered below the clade-defining limit of p-distance < 0.04. Pulsed-field gel electrophoresis (PFGE) karyotyping revealed no chromosomal rearrangements in these isolates. A. fumigatus isolates were of different, non-related genotypes. We speculate that virus- and drug-induced immunosuppression (94.7% of the patients received corticosteroids), together with prolonged hospital stay (median duration of 29 days) and mechanical ventilation (median duration of 24 days) likely increased the susceptibility to secondary respiratory and bloodstream infections in the studied patient population. The presence of fungi in blood or respiratory tract fluid was a prognosticator for poor clinical outcome, which presented as an 89.5% 30-day mortality in our patient cohort.

Pervasive but Neglected: A Perspective on COVID-19-Associated Pulmonary Mold Infections Among Mechanically Ventilated COVID-19 Patients.

Background: Recent studies from multiple countries have shown a high prevalence of coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) among severely ill patients. Despite providing valuable insight into the clinical management of CAPA, large-scale prospective studies are limited. Here, we report on one of the largest multicenter epidemiological studies to explore the clinical features and prevalence of COVID-19-associated pulmonary mold infections (CAPMIs) among mechanically ventilated patients. Methods: Bronchoalveolar lavage (BAL) and serum samples were collected for culture, galactomannan (GM), and ß-D-glucan (BDG) testing. Patients were classified as probable CAPMI based on the presence of host factors, radiological findings, and mycological criteria. Results: During the study period, 302 COVID-19 patients were admitted to intensive care units (ICUs), among whom 105 were mechanically ventilated for ≥4 days. Probable CAPMI was observed among 38% of patients (40/105), among whom BAL culture of 29 patients turned positive for molds, while galactomannan testing on BAL (GM index ≥1) and serum (GM index >0.5) samples were positive for 60% (24/40) and 37.5% (15/39) of patients, respectively. Aspergillus (22/29; 75.8%) and Fusarium (6/29; 20.6%) constituted 96.5% of the molds isolated. Diaporthe foeniculina was isolated from a COVID-19 patient. None of the patients who presented with CAPMI were treated with antifungal drugs. Conclusion: Despite being prevalent, the absence of appropriate antifungal treatment highlights that CAPMI is a neglected complication among mechanically ventilated COVID-19 patients admitted to ICUs. CAPMI can be caused by species other than Aspergillus.

Extracellular Vesicle Capture by AnTibody of CHoice and Enzymatic Release (EV-CATCHER): A customizable purification assay designed for small-RNA biomarker identification and evaluation of circulating small-EVs.

Circulating nucleic acids, encapsulated within small extracellular vesicles (EVs), provide a remote cellular snapshot of biomarkers derived from diseased tissues, however selective isolation is critical. Current laboratory-based purification techniques rely on the physical properties of small-EVs rather than their inherited cellular fingerprints. We established a highly-selective purification assay, termed EV-CATCHER, initially designed for high-throughput analysis of low-abundance small-RNA cargos by next-generation sequencing. We demonstrated its selectivity by specifically isolating and sequencing small-RNAs from mouse small-EVs spiked into human plasma. Western blotting, nanoparticle tracking, and transmission electron microscopy were used to validate and quantify the capture and release of intact small-EVs. As proof-of-principle for sensitive detection of circulating miRNAs, we compared small-RNA sequencing data from a subset of small-EVs serum-purified with EV-CATCHER to data from whole serum, using samples from a small cohort of recently hospitalized Covid-19 patients. We identified and validated, only in small-EVs, hsa-miR-146a and hsa-miR-126-3p to be significantly downregulated with disease severity. Separately, using convalescent sera from recovered Covid-19 patients with high anti-spike IgG titers, we confirmed the neutralizing properties, against SARS-CoV-2 in vitro, of a subset of small-EVs serum-purified by EV-CATCHER, as initially observed with ultracentrifuged small-EVs. Altogether our data highlight the sensitivity and versatility of EV-CATCHER.

A novel diagnostic test to screen SARS-CoV-2 variants containing E484K and N501Y mutations.

Spike protein mutations E484K and N501Y carried by SARS-CoV-2 variants have been associated with concerning changes of the virus, including resistance to neutralizing antibodies and increased transmissibility. While the concerning variants are fast spreading in various geographical areas, identification and monitoring of these variants are lagging far behind, due in large part to the slow speed and insufficient capacity of viral sequencing. In response to the unmet need for a fast and efficient screening tool, we developed a single-tube duplex molecular assay for rapid and simultaneous identification of E484K and N501Y mutations from nasopharyngeal swab (NS) samples within 2.5 h from sample preparation to report. Using this tool, we screened a total of 1135 clinical NS samples collected from COVID patients at 8 hospitals within the Hackensack Meridian Health network in New Jersey between late December 2020 and March 2021. Our data revealed dramatic increases in the frequencies of both E484K and N501Y over time, underscoring the need for continuous epidemiological monitoring.