Plasma SARS-CoV-2 RNA Levels as a Biomarker of Lower Respiratory Tract SARS-CoV-2 Infection in Critically Ill Patients With COVID-19.

Plasma SARS-CoV-2 viral RNA (vRNA) levels are predictive of COVID-19 outcomes in hospitalized patients, but whether plasma vRNA reflects lower respiratory tract (LRT) vRNA levels is unclear. We compared plasma and LRT vRNA levels in serially collected samples from mechanically ventilated patients with COVID-19. LRT and plasma vRNA levels were strongly correlated at first sampling (n = 33, r = 0.83, P < 10-9) and then declined in parallel in available serial samples except in nonsurvivors who exhibited delayed vRNA clearance in LRT samples. Plasma vRNA measurement may offer a practical surrogate of LRT vRNA burden in critically ill patients, especially early after ICU admission.

Contribution of Coronavirus-Specific Immunoglobulin G Responses to Complement Overactivation in Patients with Severe Coronavirus Disease 2019.

BACKGROUND: Excessive complement activation has been implicated in the pathogenesis of coronavirus disease 2019 (COVID-19), but the mechanisms leading to this response remain unclear. METHODS: We measured plasma levels of key complement markers, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and antibodies against SARS-CoV-2 and seasonal human common cold coronaviruses (CCCs) in hospitalized patients with COVID-19 of moderate (n = 18) and critical severity (n = 37) and in healthy controls (n = 10). RESULTS: We confirmed that complement activation is systemically increased in patients with COVID-19 and is associated with a worse disease outcome. We showed that plasma levels of C1q and circulating immune complexes were markedly increased in patients with severe COVID-19 and correlated with higher immunoglobulin (Ig) G titers, greater complement activation, and higher disease severity score. Additional analyses showed that the classical pathway was the main arm responsible for augmented complement activation in severe patients. In addition, we demonstrated that a rapid IgG response to SARS-CoV-2 and an anamnestic IgG response to the nucleoprotein of the CCCs were strongly correlated with circulating immune complex levels, complement activation, and disease severity. CONCLUSIONS: These findings indicate that early, nonneutralizing IgG responses may play a key role in complement overactivation in severe COVID-19. Our work underscores the urgent need to develop therapeutic strategies to modify complement overactivation in patients with COVID-19.

Severe Acute Respiratory Syndrome Coronavirus 2 Viremia Is Associated With Coronavirus Disease 2019 Severity and Predicts Clinical Outcomes.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA (vRNA) is detected in the bloodstream of some patients with coronavirus disease 2019 (COVID-19), but it is not clear whether this RNAemia reflects viremia (ie, virus particles) and how it relates to host immune responses and outcomes. METHODS: SARS-CoV-2 vRNA was quantified in plasma samples from observational cohorts of 51 COVID-19 patients including 9 outpatients, 19 hospitalized (non-intensive care unit [ICU]), and 23 ICU patients. vRNA levels were compared with cross-sectional indices of COVID-19 severity and prospective clinical outcomes. We used multiple imaging methods to visualize virions in plasma. RESULTS: SARS-CoV-2 vRNA was detected in plasma of 100%, 52.6%, and 11.1% of ICU, non-ICU, and outpatients, respectively. Virions were detected in plasma pellets using electron tomography and immunostaining. Plasma vRNA levels were significantly higher in ICU > non-ICU > outpatients (P < .0001); for inpatients, plasma vRNA levels were strongly associated with higher World Health Organization (WHO) score at admission (P = .01), maximum WHO score (P = .002), and discharge disposition (P = .004). A plasma vRNA level >6000 copies/mL was strongly associated with mortality (hazard ratio, 10.7). Levels of vRNA were significantly associated with several inflammatory biomarkers (P < .01) but not with plasma neutralizing antibody titers (P = .8). CONCLUSIONS: Visualization of virus particles in plasma indicates that SARS-CoV-2 RNAemia is due, at least in part, to viremia. The levels of SARS-CoV-2 RNAemia correlate strongly with disease severity, patient outcome, and specific inflammatory biomarkers but not with neutralizing antibody titers.

An unexpected connection: A narrative review of the associations between Gut Microbiome and Musculoskeletal Pain.

PURPOSE: Multiple diverse factors contribute to musculoskeletal pain, a major cause of physical dysfunction and health-related costs worldwide. Rapidly growing evidence demonstrates that the gut microbiome has overarching influences on human health and the body's homeostasis and resilience to internal and external perturbations. This broad role of the gut microbiome is potentially relevant and connected to musculoskeletal pain, though the literature on the topic is limited. Thus, the literature on the topic of musculoskeletal pain and gut microbiome was explored. METHODS: This narrative review explores the vast array of reported metabolites associated with inflammation and immune-metabolic response, which are known contributors to musculoskeletal pain. Moreover, it covers known modifiable (e.g., diet, lifestyle choices, exposure to prescription drugs, pollutants, and chemicals) and non-modifiable factors (e.g., gut architecture, genetics, age, birth history, and early feeding patterns) that are known to contribute to changes to the gut microbiome. Particular attention is devoted to modifiable factors, as the ultimate goal of researching this topic is to implement gut microbiome health interventions into clinical practice. RESULTS: Overall, numerous associations exist in the literature that could converge on the gut microbiome's pivotal role in musculoskeletal health. Particularly, a variety of metabolites that are either directly produced or indirectly modulated by the gut microbiome have been highlighted. CONCLUSION: The review highlights noticeable connections between the gut and musculoskeletal health, thus warranting future research to focus on the gut microbiome's role in musculoskeletal conditions.

Intractable Coronavirus Disease 2019 (COVID-19) and Prolonged Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Replication in a Chimeric Antigen Receptor-Modified T-Cell Therapy Recipient: A Case Study.

A chimeric antigen receptor-modified T-cell therapy recipient developed severe coronavirus disease 2019, intractable RNAemia, and viral replication lasting >2 months. Premortem endotracheal aspirate contained >2 × 1010 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA copies/mL and infectious virus. Deep sequencing revealed multiple sequence variants consistent with intrahost virus evolution. SARS-CoV-2 humoral and cell-mediated immunity were minimal. Prolonged transmission from immunosuppressed patients is possible.

Topographic heterogeneity of lung microbiota in end-stage idiopathic pulmonary fibrosis: the Microbiome in Lung Explants-2 (MiLEs-2) study.

BACKGROUND: Lung microbiota profiles in patients with early idiopathic pulmonary fibrosis (IPF) have been associated with disease progression; however, the topographic heterogeneity of lung microbiota and their roles in advanced IPF are unknown. METHODS: We performed a retrospective, case-control study of explanted lung tissue obtained at the time of lung transplantation or rapid autopsy from patients with IPF and other chronic lung diseases (connective tissue disease-associated interstitial lung disease (CTD-ILD), cystic fibrosis (CF), COPD and donor lungs unsuitable for transplant from Center for Organ Recovery and Education (CORE)). We sampled subpleural tissue and airway-based specimens (bronchial washings and airway tissue) and quantified bacterial load and profiled communities by amplification and sequencing of the 16S rRNA gene. FINDINGS: Explants from 62 patients with IPF, 15 patients with CTD-ILD, 20 patients with CF, 20 patients with COPD and 20 CORE patients were included. Airway-based samples had higher bacterial load compared with distal parenchymal tissue. IPF basilar tissue had much lower bacterial load compared with CF and CORE lungs (p<0.001). No microbial community differences were found between parenchymal tissue samples from different IPF lobes. Dirichlet multinomial models revealed an IPF cluster (29%) with distinct composition, high bacterial load and low alpha diversity, exhibiting higher odds for acute exacerbation or death. INTERPRETATION: IPF explants had low biomass in the distal parenchyma of all three lobes with higher bacterial load in the airways. The discovery of a distinct subgroup of patients with IPF with higher bacterial load and worse clinical outcomes supports investigation of personalised medicine approaches for microbiome-targeted interventions.

Alterations in Oral Microbiota in HIV Are Related to Decreased Pulmonary Function.

Rationale: Mechanisms of HIV-associated chronic obstructive pulmonary disease (COPD) are poorly understood. The oral microbiome shapes the lung microbiome, and gut dysbiosis can affect lung diseases; however, relationships of the oral and gut microbiome to COPD in HIV have not been explored.Objectives: To examine alterations in the oral and gut microbiome associated with pulmonary disease in persons with HIV (PWH).Methods: Seventy-five PWH and 93 HIV-uninfected men from the MACS (Multicenter AIDS Cohort Study) performed pulmonary function testing. Sequencing of bacterial 16S ribosomal RNA in saliva and stool was performed. We used nonmetric multidimensional scaling, permutational multivariate ANOVA, and linear discriminant analysis to analyze communities by HIV and lung function.Measurements and Main Results: Oral microbiome composition differed by HIV and smoking status. Alterations of oral microbial communities were observed in PWH with abnormal lung function with increases in relative abundance of Veillonella, Streptococcus, and Lactobacillus. There were no significant associations between the oral microbiome and lung function in HIV-uninfected individuals. No associations with HIV status or lung function were seen with the gut microbiome.Conclusions: Alterations of oral microbiota in PWH were related to impaired pulmonary function and to systemic inflammation. These results suggest that the oral microbiome may serve as a biomarker of lung function in HIV and that its disruption may contribute to COPD pathogenesis.

Respiratory Tract Dysbiosis Is Associated with Worse Outcomes in Mechanically Ventilated Patients.

Rationale: Host inflammatory responses have been strongly associated with adverse outcomes in critically ill patients, but the biologic underpinnings of such heterogeneous responses have not been defined.Objectives: We examined whether respiratory tract microbiome profiles are associated with host inflammation and clinical outcomes of acute respiratory failure.Methods: We collected oral swabs, endotracheal aspirates (ETAs), and plasma samples from mechanically ventilated patients. We performed 16S ribosomal RNA gene sequencing to characterize upper and lower respiratory tract microbiota and classified patients into host-response subphenotypes on the basis of clinical variables and plasma biomarkers of innate immunity and inflammation. We derived diversity metrics and composition clusters with Dirichlet multinomial models and examined our data for associations with subphenotypes and clinical outcomes.Measurements and Main Results: Oral and ETA microbial communities from 301 mechanically ventilated subjects had substantial heterogeneity in α and ß diversity. Dirichlet multinomial models revealed a cluster with low α diversity and enrichment for pathogens (e.g., high Staphylococcus or Pseudomonadaceae relative abundance) in 35% of ETA samples, associated with a hyperinflammatory subphenotype, worse 30-day survival, and longer time to liberation from mechanical ventilation (adjusted P < 0.05), compared with patients with higher α diversity and relative abundance of typical oral microbiota. Patients with evidence of dysbiosis (low α diversity and low relative abundance of "protective" oral-origin commensal bacteria) in both oral and ETA samples (17%, combined dysbiosis) had significantly worse 30-day survival and longer time to liberation from mechanical ventilation than patients without dysbiosis (55%; adjusted P < 0.05).Conclusions: Respiratory tract dysbiosis may represent an important, modifiable contributor to patient-level heterogeneity in systemic inflammatory responses and clinical outcomes.

Distinctive gene and protein characteristics of extremely piezophilic Colwellia.

BACKGROUND: The deep ocean is characterized by low temperatures, high hydrostatic pressures, and low concentrations of organic matter. While these conditions likely select for distinct genomic characteristics within prokaryotes, the attributes facilitating adaptation to the deep ocean are relatively unexplored. In this study, we compared the genomes of seven strains within the genus Colwellia, including some of the most piezophilic microbes known, to identify genomic features that enable life in the deep sea. RESULTS: Significant differences were found to exist between piezophilic and non-piezophilic strains of Colwellia. Piezophilic Colwellia have a more basic and hydrophobic proteome. The piezophilic abyssal and hadal isolates have more genes involved in replication/recombination/repair, cell wall/membrane biogenesis, and cell motility. The characteristics of respiration, pilus generation, and membrane fluidity adjustment vary between the strains, with operons for a nuo dehydrogenase and a tad pilus only present in the piezophiles. In contrast, the piezosensitive members are unique in having the capacity for dissimilatory nitrite and TMAO reduction. A number of genes exist only within deep-sea adapted species, such as those encoding d-alanine-d-alanine ligase for peptidoglycan formation, alanine dehydrogenase for NADH/NAD+ homeostasis, and a SAM methyltransferase for tRNA modification. Many of these piezophile-specific genes are in variable regions of the genome near genomic islands, transposases, and toxin-antitoxin systems. CONCLUSIONS: We identified a number of adaptations that may facilitate deep-sea radiation in members of the genus Colwellia, as well as in other piezophilic bacteria. An enrichment in more basic and hydrophobic amino acids could help piezophiles stabilize and limit water intrusion into proteins as a result of high pressure. Variations in genes associated with the membrane, including those involved in unsaturated fatty acid production and respiration, indicate that membrane-based adaptations are critical for coping with high pressure. The presence of many piezophile-specific genes near genomic islands highlights that adaptation to the deep ocean may be facilitated by horizontal gene transfer through transposases or other mobile elements. Some of these genes are amenable to further study in genetically tractable piezophilic and piezotolerant deep-sea microorganisms.

Intestinal injury and gut permeability in sickle cell disease.

BACKGROUND: Due to recurrent hypoxia-reperfusion injury induced by vaso-occlusive crises (VOC), patients with sickle cell disease (SCD) may have intestinal injury and increased permeability. These may explain the qualitative and quantitative neutrophil abnormalities observed in these patients. METHODS: Serum intestinal fatty-acid binding protein (iFABP), lipopolysaccharides (LPS), and CD62L were measured by ELISA. Multicolor flow cytometry was used to measure circulating aged neutrophils. RESULTS: Compared to controls, SCD individuals had higher iFABP (median: 1.38 ng/ml vs 0.81 ng/ml; p = 0.04) and LPS (median: 2.15 µg/ml vs 0.69 µg/ml; p = 0.03), indicating intestinal injury and increased intestinal bacterial translocation into the systemic circulation. They also had higher soluble CD62L (median: 1.38 µg/ml vs 1.11 µg/ml; p = 0.04). Among SCD individuals, soluble CD62L correlated positively with circulating aged neutrophils (R = 0.7, p = 0.03) and LPS (R = 0.66, p = 0.027). Surprisingly, serum iFABP in SCD correlated negatively with both LPS (R = - 0.7, p = 0.02) and soluble CD62L (R = - 0.56, p = 0.08). CONCLUSIONS: Since LPS translocation across the intestinal barrier may be due to increases in the intestinal bacterial density, gut permeability, or both, the negative correlations between iFABP and LPS, and CD62L raise the possibility that any damage-associated molecular patterns induced by intestinal injury may modulate the degree of bacterial translocation. Our results provide the first evidence of the presence of intestinal injury and increased gut permeability in SCD.

Metagenomic identification of severe pneumonia pathogens in mechanically-ventilated patients: a feasibility and clinical validity study.

BACKGROUND: Metagenomic sequencing of respiratory microbial communities for pathogen identification in pneumonia may help overcome the limitations of culture-based methods. We examined the feasibility and clinical validity of rapid-turnaround metagenomics with Nanopore™ sequencing of clinical respiratory specimens. METHODS: We conducted a case-control study of mechanically-ventilated patients with pneumonia (nine culture-positive and five culture-negative) and without pneumonia (eight controls). We collected endotracheal aspirates and applied a microbial DNA enrichment method prior to metagenomic sequencing with the Oxford Nanopore MinION device. For reference, we compared Nanopore results against clinical microbiologic cultures and bacterial 16S rRNA gene sequencing. RESULTS: Human DNA depletion enabled in depth sequencing of microbial communities. In culture-positive cases, Nanopore revealed communities with high abundance of the bacterial or fungal species isolated by cultures. In four cases with resistant clinical isolates, Nanopore detected antibiotic resistance genes corresponding to the phenotypic resistance in antibiograms. In culture-negative pneumonia, Nanopore revealed probable bacterial pathogens in 1/5 cases and Candida colonization in 3/5 cases. In controls, Nanopore showed high abundance of oral bacteria in 5/8 subjects, and identified colonizing respiratory pathogens in other subjects. Nanopore and 16S sequencing showed excellent concordance for the most abundant bacterial taxa. CONCLUSIONS: We demonstrated technical feasibility and proof-of-concept clinical validity of Nanopore metagenomics for severe pneumonia diagnosis, with striking concordance with positive microbiologic cultures, and clinically actionable information obtained from sequencing in culture-negative samples. Prospective studies with real-time metagenomics are warranted to examine the impact on antimicrobial decision-making and clinical outcomes.

Microbiome in lung explants of idiopathic pulmonary fibrosis: a case-control study in patients with end-stage fibrosis.

The microbiome has been proposed to play a role in the progression of idiopathic pulmonary fibrosis (IPF) based on bronchoalveolar lavage analyses, but the microbiome of lung tissue in IPF has not been explored. In a case-control study of lung explants analysed by 16S rRNA gene sequencing, we could not reliably detect bacterial DNA in basilar tissue samples from patients with either chronic or acute exacerbations of IPF, in contrast to control candidate-donor lungs or cystic fibrosis explants. Thus, our data do not indicate microbiome alterations in regions of IPF lung with advanced fibrosis.

Invasive non-typhoidal Salmonella in sickle cell disease in Africa: is increased gut permeability the missing link?

Non-typhoidal Salmonella usually induces self-limiting gastroenteritis. However, in many parts of Africa, especially in individuals who are malnourished, infected with malaria, or have sickle cell disease, the organism causes serious and potentially fatal systemic infections. Since the portal of entry of non-typhoidal Salmonella into the systemic circulation is by way of the intestine, we argue that an increased gut permeability plays a vital role in the initiation of invasive non-typhoidal Salmonella in these patients. Here, we will appraise the evidence supporting a breach in the intestinal barrier and propose the mechanisms for the increased risks for invasive non-typhoidal Salmonella infections in these individuals.

Antibiotics in early life alter the murine colonic microbiome and adiposity.

Antibiotics administered in low doses have been widely used as growth promoters in the agricultural industry since the 1950s, yet the mechanisms for this effect are unclear. Because antimicrobial agents of different classes and varying activity are effective across several vertebrate species, we proposed that such subtherapeutic administration alters the population structure of the gut microbiome as well as its metabolic capabilities. We generated a model of adiposity by giving subtherapeutic antibiotic therapy to young mice and evaluated changes in the composition and capabilities of the gut microbiome. Administration of subtherapeutic antibiotic therapy increased adiposity in young mice and increased hormone levels related to metabolism. We observed substantial taxonomic changes in the microbiome, changes in copies of key genes involved in the metabolism of carbohydrates to short-chain fatty acids, increases in colonic short-chain fatty acid levels, and alterations in the regulation of hepatic metabolism of lipids and cholesterol. In this model, we demonstrate the alteration of early-life murine metabolic homeostasis through antibiotic manipulation.

NeatFreq: reference-free data reduction and coverage normalization for De Novo sequence assembly.

BACKGROUND: Deep shotgun sequencing on next generation sequencing (NGS) platforms has contributed significant amounts of data to enrich our understanding of genomes, transcriptomes, amplified single-cell genomes, and metagenomes. However, deep coverage variations in short-read data sets and high sequencing error rates of modern sequencers present new computational challenges in data interpretation, including mapping and de novo assembly. New lab techniques such as multiple displacement amplification (MDA) of single cells and sequence independent single primer amplification (SISPA) allow for sequencing of organisms that cannot be cultured, but generate highly variable coverage due to amplification biases. RESULTS: Here we introduce NeatFreq, a software tool that reduces a data set to more uniform coverage by clustering and selecting from reads binned by their median kmer frequency (RMKF) and uniqueness. Previous algorithms normalize read coverage based on RMKF, but do not include methods for the preferred selection of (1) extremely low coverage regions produced by extremely variable sequencing of random-primed products and (2) 2-sided paired-end sequences. The algorithm increases the incorporation of the most unique, lowest coverage, segments of a genome using an error-corrected data set. NeatFreq was applied to bacterial, viral plaque, and single-cell sequencing data. The algorithm showed an increase in the rate at which the most unique reads in a genome were included in the assembled consensus while also reducing the count of duplicative and erroneous contigs (strings of high confidence overlaps) in the deliverable consensus. The results obtained from conventional Overlap-Layout-Consensus (OLC) were compared to simulated multi-de Bruijn graph assembly alternatives trained for variable coverage input using sequence before and after normalization of coverage. Coverage reduction was shown to increase processing speed and reduce memory requirements when using conventional bacterial assembly algorithms. CONCLUSIONS: The normalization of deep coverage spikes, which would otherwise inhibit consensus resolution, enables High Throughput Sequencing (HTS) assembly projects to consistently run to completion with existing assembly software. The NeatFreq software package is free, open source and available at https://github.com/bioh4x/NeatFreq .

Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons.

Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys.

The nonfermentable dietary fiber hydroxypropyl methylcellulose modulates intestinal microbiota.

Diet influences host metabolism and intestinal microbiota; however, detailed understanding of this tripartite interaction is limited. To determine whether the nonfermentable fiber hydroxypropyl methylcellulose (HPMC) could alter the intestinal microbiota and whether such changes correlated with metabolic improvements, C57B/L6 mice were normalized to a high-fat diet (HFD), then either maintained on HFD (control), or switched to HFD supplemented with 10% HPMC, or a low-fat diet (LFD). Compared to control treatment, both LFD and HPMC reduced weight gain (11.8 and 5.7 g, respectively), plasma cholesterol (23.1 and 19.6%), and liver triglycerides (73.1 and 44.6%), and, as revealed by 454-pyrosequencing of the microbial 16S rRNA gene, decreased microbial α-diversity and differentially altered intestinal microbiota. Both LFD and HPMC increased intestinal Erysipelotrichaceae (7.3- and 12.4-fold) and decreased Lachnospiraceae (2.0- and 2.7-fold), while only HPMC increased Peptostreptococcaceae (3.4-fold) and decreased Ruminococcaceae (2.7-fold). Specific microorganisms were directly linked with weight change and metabolic parameters in HPMC and HFD mice, but not in LFD mice, indicating that the intestinal microbiota may play differing roles during the two dietary modulations. This work indicates that HPMC is a potential prebiotic fiber that influences intestinal microbiota and improves host metabolism.