INTRODUCTION: Mounting evidence indicates that an individual's humoral adaptive immune response plays a critical role in the setting of SARS-CoV-2 infection, and that the efficiency of the response correlates with disease severity. The relationship between the adaptive immune dynamics in the lower airways with those in the systemic circulation, and how these relate to an individual's clinical response to SARS-CoV-2 infection, are less understood and are the focus of this study. MATERIAL AND METHODS: We investigated the adaptive immune response to SARS-CoV-2 in paired samples from the lower airways and blood from 27 critically ill patients during the first wave of the pandemic (median time from symptom onset to intubation 11 days). Measurements included clinical outcomes (mortality), bronchoalveolar lavage fluid (BALF) and blood specimen antibody levels, and BALF viral load. RESULTS: While there was heterogeneity in the levels of the SARS-CoV-2-specific antibodies, we unexpectedly found that some BALF specimens displayed higher levels than the paired concurrent plasma samples, despite the known dilutional effects common in BALF samples. We found that survivors had higher levels of anti-spike, anti-spike-N-terminal domain and anti-spike-receptor-binding domain IgG antibodies in their BALF (p<0.05), while there was no such association with antibody levels in the systemic circulation. DISCUSSION: Our data highlight the critical role of local adaptive immunity in the airways as a key defence mechanism against primary SARS-CoV-2 infection.

Mortality among patients with COVID-19 and respiratory failure is high and there are no known lower airway biomarkers that predict clinical outcome. We investigated whether bacterial respiratory infections and viral load were associated with poor clinical outcome and host immune tone. We obtained bacterial and fungal culture data from 589 critically ill subjects with COVID-19 requiring mechanical ventilation. On a subset of the subjects that underwent bronchoscopy, we also quantified SARS-CoV-2 viral load, analyzed the microbiome of the lower airways by metagenome and metatranscriptome analyses and profiled the host immune response. We found that isolation of a hospital-acquired respiratory pathogen was not associated with fatal outcome. However, poor clinical outcome was associated with enrichment of the lower airway microbiota with an oral commensal ( Mycoplasma salivarium ), while high SARS-CoV-2 viral burden, poor anti-SARS-CoV-2 antibody response, together with a unique host transcriptome profile of the lower airways were most predictive of mortality. Collectively, these data support the hypothesis that 1) the extent of viral infectivity drives mortality in severe COVID-19, and therefore 2) clinical management strategies targeting viral replication and host responses to SARS-CoV-2 should be prioritized.

Mortality among patients with COVID-19 and respiratory failure is high and there are no known lower airway biomarkers that predict clinical outcome. We investigated whether bacterial respiratory infections and viral load were associated with poor clinical outcome and host immune tone. We obtained bacterial and fungal culture data from 589 critically ill subjects with COVID-19 requiring mechanical ventilation. On a subset of the subjects that underwent bronchoscopy, we also quantified SARS-CoV-2 viral load, analyzed the microbiome of the lower airways by metagenome and metatranscriptome analyses and profiled the host immune response. We found that isolation of a hospital-acquired respiratory pathogen was not associated with fatal outcome. However, poor clinical outcome was associated with enrichment of the lower airway microbiota with an oral commensal ( Mycoplasma salivarium ), while high SARS-CoV-2 viral burden, poor anti-SARS-CoV-2 antibody response, together with a unique host transcriptome profile of the lower airways were most predictive of mortality. Collectively, these data support the hypothesis that 1) the extent of viral infectivity drives mortality in severe COVID-19, and therefore 2) clinical management strategies targeting viral replication and host responses to SARS-CoV-2 should be prioritized.

At the start of the COVID-19 pandemic, the Centers for Disease Control and Prevention (CDC) designed, manufactured, and distributed the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel for SARS-CoV-2 detection. The diagnostic panel targeted three viral nucleocapsid gene loci (N1, N2, and N3 primers and probes) to maximize sensitivity and to provide redundancy for virus detection if mutations occurred. After the first distribution of the diagnostic panel, state public health laboratories reported fluorescent signal in the absence of viral template (false-positive reactivity) for the N3 component and to a lesser extent for N1. This report describes the findings of an internal investigation conducted by the CDC to identify the cause(s) of the N1 and N3 false-positive reactivity. For N1, results demonstrate that contamination with a synthetic template, that occurred while the "bulk" manufactured materials were located in a research lab for quality assessment, was the cause of false reactivity in the first lot. Base pairing between the 3' end of the N3 probe and the 3' end of the N3 reverse primer led to amplification of duplex and larger molecules resulting in false reactivity in the N3 assay component. We conclude that flaws in both assay design and handling of the "bulk" material, caused the problems with the first lot of the 2019-nCoV Real-Time RT-PCR Diagnostic Panel. In addition, within this study, we found that the age of the examined diagnostic panel reagents increases the frequency of false positive results for N3. We discuss these findings in the context of improvements to quality control, quality assurance, and assay validation practices that have since been improved at the CDC.

Respiratory failure is associated with increased mortality in COVID-19 patients. There are no validated lower airway biomarkers to predict clinical outcome. We investigated whether bacterial respiratory infections were associated with poor clinical outcome of COVID-19 in a prospective, observational cohort of 589 critically ill adults, all of whom required mechanical ventilation. For a subset of 142 patients who underwent bronchoscopy, we quantified SARS-CoV-2 viral load, analysed the lower respiratory tract microbiome using metagenomics and metatranscriptomics and profiled the host immune response. Acquisition of a hospital-acquired respiratory pathogen was not associated with fatal outcome. Poor clinical outcome was associated with lower airway enrichment with an oral commensal (Mycoplasma salivarium). Increased SARS-CoV-2 abundance, low anti-SARS-CoV-2 antibody response and a distinct host transcriptome profile of the lower airways were most predictive of mortality. Our data provide evidence that secondary respiratory infections do not drive mortality in COVID-19 and clinical management strategies should prioritize reducing viral replication and maximizing host responses to SARS-CoV-2.

Cronobacter spp. are emerging infectious foodborne bacteria that can cause acute meningitis and necrotizing enterocolitis in neonates and immunocompromised individuals. Although, little is known about its reservoirs or transmission routes, it has been linked to powdered infant formula worldwide. Three Cronobacter spp. (C. sakazakii, C. malonaticus, and C. turicensis) have been described as more virulent, and isolated frequently from infant meningitis cases. The estimated mortality rates are as high as 80% in infants. Thus, surveillance and typing of Cronobacter spp. isolated from food and environmental samples is essential to prevent contamination and spread of this pathogen. In this study, we have characterized 83 Cronobacter isolates recovered from various environmental samples by conventional microbiologic protocols. Species identification was accomplished by VITEK 2 system and real-time PCR analysis. Subsequently, these isolates were analyzed using VITEK MS system. Single locus sequence typing (SLST) was achieved by characterizing the regions of 16S rRNA and rpoB genes. Multilocus sequence typing (MLST) was performed by sequence characterization of seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, and pps) using ABI 3500XL Genetic Analyzer. VITEK MS system identified, the majority of isolates as Cronobacter sakazakii with a high confidence value (99.9%). MLST analysis ascertained 12 distinct clonal complexes (CC1, CC4, CC8, CC13, CC17, CC21, CC31, CC40, CC52, CC64, CC73, and CC83) for the recovered C. sakazakii isolates. The results suggest that the MALDI-TOF MS is a reliable diagnostic tool for rapid species identification whereas 7-loci MLST is a powerful technique to discriminate and differentiate Cronobacter spp. isolates.

The objective of this study was to describe the epidemiology of COVID-19 in Nigeria with a view of generating evidence to enhance planning and response strategies. A national surveillance dataset between 27 February and 6 June 2020 was retrospectively analysed, with confirmatory testing for COVID-19 done by real-time polymerase chain reaction (RT-PCR). The primary outcomes were cumulative incidence (CI) and case fatality (CF). A total of 40 926 persons (67% of total 60 839) had complete records of RT-PCR test across 35 states and the Federal Capital Territory, 12 289 (30.0%) of whom were confirmed COVID-19 cases. Of those confirmed cases, 3467 (28.2%) had complete records of clinical outcome (alive or dead), 342 (9.9%) of which died. The overall CI and CF were 5.6 per 100 000 population and 2.8%, respectively. The highest proportion of COVID-19 cases and deaths were recorded in persons aged 31-40 years (25.5%) and 61-70 years (26.6%), respectively; and males accounted for a higher proportion of confirmed cases (65.8%) and deaths (79.0%). Sixty-six per cent of confirmed COVID-19 cases were asymptomatic at diagnosis. In conclusion, this paper has provided an insight into the early epidemiology of COVID-19 in Nigeria, which could be useful for contextualising public health planning.

INTRODUCTION: Workers' exposure to metalworking fluid (MWF) has been associated with respiratory disease. As part of a public health investigation of a manufacturing facility, we performed paired environmental and human sampling to evaluate cross-pollination of microbes between environment and host and possible effects on lung pathology present among workers. METHODS: Workplace environmental microbiota was evaluated in air and MWF samples. Human microbiota was evaluated in lung tissue samples from workers with respiratory symptoms found to have lymphocytic bronchiolitis and alveolar ductitis with B-cell follicles and emphysema, lung tissue controls, and in skin, nasal and oral samples from 302 workers from different areas of the facility. In vitro effects of MWF exposure on murine B-cells were assessed. RESULTS: Increased similarity of microbial composition was found between MWF samples and lung tissue samples of case workers compared to controls. Among workers in different locations within the facility, those that worked in machine shop area had skin, nasal and oral microbiota more closely related to the microbiota present in MWF samples. Lung samples from four index cases, and skin and nasal samples from workers in machine shop area were enriched with Pseudomonas, the dominant taxa in MWF. Exposure to used MWF stimulated murine B-cell proliferation in vitro, a hallmark cell subtype found in pathology of index cases. CONCLUSIONS: Evaluation of a manufacturing facility with a cluster of workers with respiratory disease supports cross-pollination of microbes from MWF to humans and suggests the potential for exposure to these microbes to be a health hazard.

Campylobacteriosis is an infectious gastrointestinal disease caused by Campylobacter spp.In most cases, it is either underdiagnosed or underreported due to poor diagnostics and limited databases. Several DNA-based molecular diagnostic techniques, including 16S ribosomal RNA (rRNA) sequence typing, have been widely used in the species identification of Campylobacter. Nevertheless, these assays are time-consuming and require a high quality of bacterial DNA. Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS is an emerging diagnostic technology that can provide the rapid identification of microorganisms by using their intact cells without extraction or purification. In this study, we analyzed 24 American Type Culture Collection reference isolates of 16 Campylobacter spp. and five unknown clinical bacterial isolates for rapid identification utilizing two commercially available MADI-TOF MS platforms, namely the bioM&#233;rieux VITEK&#174; MS and Bruker Biotyper systems. In addition, 16S rRNA sequencing was performed to confirm the species-level identification of the unknown clinical isolates. Both MALDI-TOF MS systems identified the isolates of C. jejuni, C. coli, C. lari, and C. fetus. The results of this study suggest that the MALDI-TOF MS technique can be used in the identification of Campylobacter spp. of public health importance.