Simultaneous identification of DNA and RNA pathogens using metagenomic sequencing in cases of severe acute respiratory infection.

Metagenomic next-generation sequencing (mNGS) is a valuable technique for identifying pathogens. However, conventional mNGS requires the separate processing of DNA and RNA genomes, which can be resource- and time-intensive. To mitigate these impediments, we propose a novel method called DNA/RNA cosequencing that aims to enhance the efficiency of pathogen detection. DNA/RNA cosequencing uses reverse transcription of total nucleic acids extracted from samples by using random primers, without removing DNA, and then employs mNGS. We applied this method to 85 cases of severe acute respiratory infections (SARI). Influenza virus was identified in 13 cases (H1N1: seven cases, H3N2: three cases, unclassified influenza type: three cases) and was not detected in the remaining 72 samples. Bacteria were present in all samples. Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii were detected in four influenza-positive samples, suggesting coinfections. The sensitivity and specificity for detecting influenza A virus were 73.33% and 95.92%, respectively. A κ value of 0.726 indicated a high level of concordance between the results of DNA/RNA cosequencing and SARI influenza virus monitoring. DNA/RNA cosequencing enhanced the efficiency of pathogen detection, providing a novel capability to strengthen surveillance and thereby prevent and control infectious disease outbreaks.

Characterisation of human astrovirus in a diarrhoea outbreak using nanopore and Sanger sequencing protocols.

Human astroviruses (HAstV) are etiologic agents of acute gastroenteritis that most often afflict young children and elderly adults. Most studies of HAstV have focused on epidemiology. In this study, we collected 10 stool samples from a diarrhea outbreak from a diarrhea sentinel surveillance hospital in Beijing. Samples were evaluated immediately using parallel multiplex RT-qPCR and nanopore sequencing, and were then amplified by designed primers and Sanger sequencing to obtain whole genome sequences. Six isolates were categorized as HAstV-5 and subjected to whole genome analysis to characterize their genetic variation and evolution. Full genome analysis revealed low genetic variation (99.38-100% identity) among isolates. Phylogenetic analysis showed that all isolates were closely related to domestic strains Yu/1-CHN and 2013/Fuzhou/85. The recombination breakpoint of the six isolates was located at 2741 bp in the overlap region of ORF1a and ORF1b, similar to those of Yu/1-CHN and 2013/Fuzhou/85. Overall, our study highlights the combined use of RT-qPCR and sequencing as an important tool in rapid diagnosis and acquisition of whole genome sequences of HAstV.

Altered Respiratory Microbiomes, Plasma Metabolites, and Immune Responses in Influenza A Virus and Methicillin-Resistant Staphylococcus aureus Coinfection.

Influenza A virus (IAV)-methicillin-resistant Staphylococcus aureus (MRSA) coinfection causes severe respiratory infections. The host microbiome plays an important role in respiratory tract infections. However, the relationships among the immune responses, metabolic characteristics, and respiratory microbial characteristics of IAV-MRSA coinfection have not been fully studied. We used specific-pathogen-free (SPF) C57BL/6N mice infected with IAV and MRSA to build a nonlethal model of IAV-MRSA coinfection and characterized the upper respiratory tract (URT) and lower respiratory tract (LRT) microbiomes at 4 and 13 days postinfection by full-length 16S rRNA gene sequencing. Immune response and plasma metabolism profile analyses were performed at 4 days postinfection by flow cytometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The relationships among the LRT microbiota, the immune response, and the plasma metabolism profile were analyzed by Spearman's correlation analysis. IAV-MRSA coinfection showed significant weight loss and lung injury and significantly increased loads of IAV and MRSA in bronchoalveolar lavage fluid (BALF). Microbiome data showed that coinfection significantly increased the relative abundances of Enterococcus faecalis, Enterobacter hormaechei, Citrobacter freundii, and Klebsiella pneumoniae and decreased the relative abundances of Lactobacillus reuteri and Lactobacillus murinus. The percentages of CD4+/CD8+ T cells and B cells in the spleen; the levels of interleukin-9 (IL-9), interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), IL-6, and IL-8 in the lung; and the level of mevalonolactone in plasma were increased in IAV-MRSA-coinfected mice. L. murinus was positively correlated with lung macrophages and natural killer (NK) cells, negatively correlated with spleen B cells and CD4+/CD8+ T cells, and correlated with multiple plasma metabolites. Future research is needed to clarify whether L. murinus mediates or alters the severity of IAV-MRSA coinfection. IMPORTANCE The respiratory microbiome plays an important role in respiratory tract infections. In this study, we characterized the URT and LRT microbiota, the host immune response, and plasma metabolic profiles during IAV-MRSA coinfection and evaluated their correlations. We observed that IAV-MRSA coinfection induced severe lung injury and dysregulated host immunity and plasma metabolic profiles, as evidenced by the aggravation of lung pathological damage, the reduction of innate immune cells, the strong adaptation of the immune response, and the upregulation of mevalonolactone in plasma. L. murinus was strongly correlated with immune cells and plasma metabolites. Our findings contribute to a better understanding of the role of the host microbiome in respiratory tract infections and identified a key bacterial species, L. murinus, that may provide important references for the development of probiotic therapies.

Topography of respiratory tract and gut microbiota in mice with influenza A virus infection.

Introduction: Influenza A virus (IAV)-induced dysbiosis may predispose to severe bacterial superinfections. Most studies have focused on the microbiota of single mucosal surfaces; consequently, the relationships between microbiota at different anatomic sites in IAV-infected mice have not been fully studied. Methods: We characterized respiratory and gut microbiota using full-length 16S rRNA gene sequencing by Nanopore sequencers and compared the nasopharyngeal, oropharyngeal, lung and gut microbiomes in healthy and IAV-infected mice. Results: The oropharyngeal, lung and gut microbiota of healthy mice were dominated by Lactobacillus spp., while nasopharyngeal microbiota were comprised primarily of Streptococcus spp. However, the oropharyngeal, nasopharyngeal, lung, and gut microbiota of IAV-infected mice were dominated by Pseudomonas, Escherichia, Streptococcus, and Muribaculum spp., respectively. Lactobacillus murinus was identified as a biomarker and was reduced at all sites in IAV-infected mice. The microbiota composition of lung was more similar to that of the nasopharynx than the oropharynx in healthy mice. Discussion: These findings suggest that the main source of lung microbiota in mice differs from that of adults. Moreover, the similarity between the nasopharyngeal and lung microbiota was increased in IAV-infected mice. We found that IAV infection reduced the similarity between the gut and oropharyngeal microbiota. L. murinus was identified as a biomarker of IAV infection and may be an important target for intervention in post-influenza bacterial superinfections.

Application of nanopore adaptive sequencing in pathogen detection of a patient with Chlamydia psittaci infection.

Introduction: Nanopore sequencing has been widely used in clinical metagenomic sequencing for pathogen detection with high portability and real-time sequencing. Oxford Nanopore Technologies has recently launched an adaptive sequencing function, which can enrich on-target reads through real-time alignment and eject uninteresting reads by reversing the voltage across the nanopore. Here we evaluated the utility of adaptive sequencing in clinical pathogen detection. Methods: Nanopore adaptive sequencing and standard sequencing was performed on a same flow cell with a bronchoalveolar lavage fluid sample from a patient with Chlamydia psittacosis infection, and was compared with the previous mNGS results. Results: Nanopore adaptive sequencing identified 648 on-target stop receiving reads with the longest median read length(688bp), which account for 72.4% of all Chlamydia psittaci reads and 0.03% of total reads in enriched group. The read proportion matched to C. psittaci in the stop receiving group was 99.85%, which was much higher than that of the unblock (<0.01%) and fail to adapt (0.02%) groups. Nanopore adaptive sequencing generated similar data yield of C. psittaci compared with standard nanopore sequencing. The proportion of C. psittaci reads in adaptive sequencing is close to that of standard nanopore sequencing and mNGS, but generated lower genome coverage than mNGS. Discussion: Nanopore adaptive sequencing can effectively identify target C. psittaci reads in real-time, but how to increase the targeted data of pathogens still needs to be further evaluated.

Murine models of neonatal susceptibility to a clinical strain of enterovirus A71.

Enterovirus A71 (EV-A71) is neurotropic and one of the primary enteric pathogens responsible for severe central nervous system infection in infants and young children. Neonatal mice are ideal models for studying the pathogenesis of infection caused by EV-A71. In this study, we assessed the susceptibility of neonatal BALB/c, C57BL/6, ICR, Kunming, and NIH mice to a clinically isolated EV-A71 strain. One-day-old mice were challenged with a clinical isolate of EV-A71 via intraperitoneal injection, then observed for 13 days for mortality, body-weight changes, and limb paralysis. RT-qPCR was performed to quantify viral RNA in the brain, spinal cord, skeletal muscle, and lungs of BALB/c and C57BL/6 mice. The expression of murine scavenger receptor class B member 2 (mSCARB2) was measured by western blotting. Finally, lesions were assessed by histological examination. We found that neonatal BALB/c and C57BL/6 mice were both susceptible to EV-A71, leading to decreased survival rate, greater body weight loss, and prominent hind-limb paralysis. Tissue viral loads of C57BL/6J mice were markedly higher than those of BALB/c mice, indicating that EV-A71 replicated more efficiently in C57BL/6 mice. Increased expression of mSCARB2 was observed 5 days after infection in C57BL/6 mice, which coincided with the peak in EV-A71 replication. Histological examination indicated that infection caused obvious pathogenic lesions. In conclusion, C57BL/6 are most susceptible to infection caused by EV-A71 and can be used as a model for studying its pathogenesis and test therapeutic options.

Using a grey relational analysis in an improved Grunow-Finke assessment tool to detect unnatural epidemics.

The Grunow-Finke epidemiological assessment tool (GFT) has several limitations in its ability to differentiate between natural and man-made epidemics. Our study aimed to improve the GFT and analyze historical epidemics to validate the model. Using a gray relational analysis (GRA), we improved the GFT by revising the existing standards and adding five new standards. We then removed the artificial weights and final decision threshold. Finally, by using typically unnatural epidemic events as references, we used the GRA to calculate the unnatural probability and obtain assessment results. Using the advanced tool, we conducted retrospective and case analyses to test its performance. In the validation set of 13 historical epidemics, unnatural and natural epidemics were divided into two categories near the unnatural probability of 45%, showing evident differences (p < 0.01) and an assessment accuracy close to 100%. The unnatural probabilities of the Ebola virus disease of 2013 and Middle East Respiratory Syndrome of 2012 were 30.6% and 36.1%, respectively. Our advanced epidemic assessment tool improved the accuracy of the original GFT from approximately 55% to approximately 100% and reduced the impact of human factors on these outcomes effectively.

Comparison of the respiratory tract microbiome in hospitalized COVID-19 patients with different disease severity.

Little is known about the characteristics of respiratory tract microbiome in Coronavirus disease 2019 (COVID-19) inpatients with different severity. We conducted a study that expected to clarify these characteristics as much as possible. A cross-sectional study was conducted to characterize respiratory tract microbial communities of 69 COVID-19 inpatients from 64 nasopharyngeal swabs and 5 sputum specimens using 16S ribosomal RNA gene V3-V4 region sequencing. The bacterial profiles were analyzed to find potential biomarkers by the two-step method, the combination of random forest model and the linear discriminant analysis effect size, and explore the connections with clinical characteristics by Spearman's rank test. Compared with mild COVID-19 patients, severe patients had significantly decreased bacterial diversity (p-values were less than 0.05 in the alpha and beta diversity) and relative lower abundance of opportunistic pathogens, including Actinomyces, Prevotella, Rothia, Streptococcus, Veillonella. Eight potential biomarkers including Treponema, Leptotrichia, Lachnoanaerobaculum, Parvimonas, Alloprevotella, Porphyromonas, Gemella, and Streptococcus were found to distinguish the mild COVID-19 patients from the severe COVID-19 patients. The genera of Actinomyces and Prevotella were negatively correlated with age in two groups. Intensive care unit admission, neutrophil count, and lymphocyte count were significantly correlated with different genera in the two groups. In addition, there was a positive correlation between Klebsiella and white blood cell count in two groups. The respiratory tract microbiome had significant differences in COVID-19 patients with different severity. The value of the respiratory tract microbiome as predictive biomarkers for COVID-19 severity deserves further exploration.

Rapid PCR-Based Nanopore Adaptive Sequencing Improves Sensitivity and Timeliness of Viral Clinical Detection and Genome Surveillance.

Nanopore sequencing has been widely used for the real-time detection and surveillance of pathogens with portable MinION. Nanopore adaptive sequencing can enrich on-target sequences without additional pretreatment. In this study, the performance of adaptive sequencing was evaluated for viral genome enrichment of clinical respiratory samples. Ligation-based nanopore adaptive sequencing (LNAS) and rapid PCR-based nanopore adaptive sequencing (RPNAS) workflows were performed to assess the effects of enrichment on nasopharyngeal swab samples from human adenovirus (HAdV) outbreaks. RPNAS was further applied for the enrichment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from nasopharyngeal swab samples to evaluate sensitivity and timeliness. The RPNAS increased both the relative abundance (7.87-12.86-fold) and data yield (1.27-2.15-fold) of HAdV samples, whereas the LNAS increased only the relative abundance but had no obvious enrichment on the data yield. Compared with standard nanopore sequencing, RPNAS detected the SARS-CoV-2 reads from two low-abundance samples, increased the coverage of SARS-CoV-2 by 36.68-98.92%, and reduced the time to achieve the same coverage. Our study highlights the utility of RPNAS for virus enrichment directly from clinical samples, with more on-target data and a shorter sequencing time to recover viral genomes. These findings promise to improve the sensitivity and timeliness of rapid identification and genomic surveillance of infectious diseases.

Comparison of reverse-transcription qPCR and droplet digital PCR for the detection of SARS-CoV-2 in clinical specimens of hospitalized patients.

Accurate detection of severe acute respiratory syndrome coronavirus 2 is not only necessary for viral load monitoring to optimize treatment in hospitalized coronavirus disease 2019 patients, but also critical for deciding whether the patient could be discharged without any risk of viral shedding. Digital droplet PCR (ddPCR) is more sensitive than reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and is usually considered the superior choice. In the current study, we compared the clinical performance of RT-qPCR and ddPCR using oropharyngeal swab samples from patients hospitalized in the temporary Huoshenshan Hospital, Wuhan, Hubei, China. Results demonstrated that ddPCR was indeed more sensitive than RT-qPCR. Negative results might be caused by poor sampling technique or recovered patients, as the range of viral load in these patients varied significantly. In addition, both methods were highly correlated in terms of their ability to detect all three target genes as well as the ratio of copies of viral genes to that of the IC gene. Furthermore, our results evidenced that both methods detected the N gene more easily than the ORF gene. Taken together, these findings imply that the use of ddPCR, as an alternative to RT-qPCR, is necessary for the accurate diagnosis of hospitalized coronavirus disease 2019 patients.

Rapid Identification and Source Tracing of a Salmonella Typhimurium Outbreak in China by Metagenomic and Whole-Genome Sequencing.

Salmonella spp. are among the most prevalent foodborne pathogens. Rapid identification of etiologic agents during foodborne outbreaks is of great importance. In this study, we report a traceback investigation of a Salmonella outbreak in China. Metagenomic sequencing of suspected food samples was performed on MinION and MiSeq platforms. Real-time nanopore sequencing analysis identified reads belonging to the Enterobacteriaceae family. MiSeq sequencing identified 63 reads specifically mapped to Salmonella. Conventional methods including quantitative-PCR and culture-based isolation confirmed as Salmonella enterica serovar Typhimurium. The foodborne outbreak of Salmonella Typhimurium was further recognized by whole-genome sequencing and pulsed-field gel electrophoresis analysis. Our study demonstrates the ability of metagenomic sequencing to rapidly identify enteric pathogens directly from food samples. These results highlight the capacity of metagenomic sequencing to deliver actionable information rapidly and to expedite the tracing and identification of etiologic agents during foodborne outbreaks.

A CRISPR-based nucleic acid detection method for severe fever with thrombocytopenia syndrome virus.

OBJECTIVE: Fever with thrombocytopenia syndrome virus (SFTS) is a tick-borne infection now known to spread among humans as an aerosol, which has resulted in several outbreaks across Asia over the past decade. As mortality is substantial, it is vital to establish a rapid, on-site nucleic acid detection method for diagnosis. Here we describe such a method for SFTSV (Dabie bandavirus) based on CRISPR-Cas13a. METHODS: Specific recombinase-aided amplification (RAA) primers and CRISPR (cr)RNA nucleic acid detection targets were designed and synthesized for the conserved sequence of the SFTSV genome, and fluorescent CRISPR detection was used to screen for high-sensitivity crRNAs. Colloidal immunochromatography test paper was used to read CRISPR detection results. Sensitivity and specificity were evaluated by running tests on gradient dilutions of SFTSV nucleic acid and the nucleic acids of other pathogens with similar transmission routes or clinical manifestations. RESULTS: One crRNA with high detection sensitivity was screened out of 5 crRNAs with conserved sequences from the SFTSV genome. This CRISPR nucleic acid-based detection method was able to detect a single crRNA copy per microliter but not the nucleic acids of similar pathogens. CONCLUSION: This CRISPR test strip detection method permits rapid, sensitive, and specific diagnosis of SFTS without the need for advanced nucleic acid detection equipment, thus allowing for on-site application.

Environmental contamination with SARS-CoV-2 in COVID-19 hospitals in Wuhan, China, 2020.

Coronavirus disease 2019 (COVID-19) pandemic has caused high number of infections and deaths of healthcare workers globally. Distribution and possible transmission route of SARS-CoV-2 in hospital environment should be clarified. We herein collected 431 environmental (391 surface and 40 air) samples in the intensive care unit (ICU) and general wards (GWs) of three hospitals in Wuhan, China from February 21 to March 4, 2020, and detected SARS-CoV-2 RNA by real-time quantitative PCR. The viral positive rate in the contaminated areas was 17.8% (28/157), whereas there was no virus detected in the clean areas. Higher positive rate (22/59, 37.3%) was found in ICU than that in GWs (3/63, 4.8%). The surfaces of computer keyboards and mouse in the ICU were the most contaminated (8/10, 80.0%), followed by the ground (6/9, 66.7%) and outer glove (2/5, 40.0%). From 17 air samples in the contaminated areas, only one sample collected at a distance of around 30 cm from the patient was positive. Enhanced surface disinfection and hand hygiene effectively decontaminated the virus from the environment. This finding might help understand the transmission route and contamination risk of SARS-CoV-2 and evaluate the effectiveness of infection prevention and control measures in healthcare facilities.

Metagenomic diagnosis of severe psittacosis using multiple sequencing platforms.

BACKGROUND: Chlamydia psittaci is an avian pathogen that can cause lethal human infections. Diagnosis of C. psittaci pneumonia is often delayed due to nonspecific clinical presentations and limited laboratory diagnostic techniques. RESULTS: The MinION platform established the diagnosis in the shortest time, while BGISEQ-500 generated additional in-depth sequence data that included the rapid characterization of antibiotic susceptibility. Cytopathy appeared only in cell cultures of BALF. BALF yielded a higher bacterial load than sputum or blood, and may be the most suitable clinical specimen for the genomic diagnosis of severe pneumonia. CONCLUSIONS: This study indicated that the benefits of metagenomic sequencing include rapid etiologic diagnosis of unknown infections and the provision of additional relevant information regarding antibiotic susceptibility. The continued optimization and standardization of sampling and metagenomic analysis promise to enhance the clinical utility of genomic diagnosis.

Genomic Elucidation of a COVID-19 Resurgence and Local Transmission of SARS-CoV-2 in Guangzhou, China.

While China experienced a peak and decline in coronavirus disease 2019 (COVID-19) cases at the start of 2020, regional outbreaks continuously emerged in subsequent months. Resurgences of COVID-19 have also been observed in many other countries. In Guangzhou, China, a small outbreak, involving less than 100 residents, emerged in March and April 2020, and comprehensive and near-real-time genomic surveillance of SARS-CoV-2 was conducted. When the numbers of confirmed cases among overseas travelers increased, public health measures were enhanced by shifting from self-quarantine to central quarantine and SARS-CoV-2 testing for all overseas travelers. In an analysis of 109 imported cases, we found diverse viral variants distributed in the global viral phylogeny, which were frequently shared within households but not among passengers on the same flight. In contrast to the viral diversity of imported cases, local transmission was predominately attributed to two specific variants imported from Africa, including local cases that reported no direct or indirect contact with imported cases. The introduction events of the virus were identified or deduced before the enhanced measures were taken. These results show the interventions were effective in containing the spread of SARS-CoV-2, and they rule out the possibility of cryptic transmission of viral variants from the first wave in January and February 2020. Our study provides evidence and emphasizes the importance of controls for overseas travelers in the context of the pandemic and exemplifies how viral genomic data can facilitate COVID-19 surveillance and inform public health mitigation strategies.

Rapid identification and metagenomics analysis of the adenovirus type 55 outbreak in Hubei using real-time and high-throughput sequencing platforms.

The rise in human adenovirus (HAdV) infections poses a serious challenge to public health in China. Real-time (RT) sequencing provides solutions for achieving rapid pathogen identification during outbreaks, whereas high-throughput sequencing yields higher sequence accuracy. In the present study, we report the outcomes of applying nanopore and BGI platforms in the identification and genomic analysis of an HAdV outbreak in Hubei province, China in May of 2019. A mixed sample of nine nasopharyngeal swabs and one single sample were submitted to direct nanopore sequencing (MinION device), generating their first HAdV-55 reads within 13 and 20 min, respectively. The sequences were confirmed by RT-polymerase chain reaction (PCR). Ten HAdV-positive samples were further sequenced using next-generation high-throughput sequencing (BGISEQ-500 device). Phylogenetic analysis revealed that the outbreak strain had a close genetic relation to strains isolated in Sichuan province. Metagenomic analysis showed that HAdV-55 was not a dominant species in samples from which the whole HAdV-55 genome could not be assembled. The present results highlight the value of combining sequencing platforms and using mixed samples for nucleic acid enrichment in pathogen detection of infectious disease outbreaks.

Nosocomial Outbreak of Carbapenemase-Producing Proteus mirabilis With Two Novel Salmonella Genomic Island 1 Variants Carrying Different bla NDM-1 Gene Copies in China.

NDM-1-producing multidrug-resistant Proteus mirabilis brings formidable clinical challenges. We report a nosocomial outbreak of carbapenem-resistant P. mirabilis in China. Six P. mirabilis strains collected in the same ward showed close phylogenetic relatedness, indicating clonal expansion. Illumina and MinION sequencing revealed that three isolates harbored a novel Salmonella genomic island 1 carrying a bla NDM-1 gene (SGI1-1NDM), while three other isolates showed elevated carbapenem resistance and carried a similar SGI1 but with two bla NDM-1 gene copies (SGI1-2NDM). Four new single nucleotide mutations were present in the genomes of the two-bla NDM-1-harboring isolates, indicating later emergence of the SGI1-2NDM structure. Passage experiments indicated that both SGI variants were stably persistent in this clone without bla NDM-1 copy number changes. This study characterizes two novel bla NDM-1-harboring SGI1 variants in P. mirabilis and provides a new insight into resistance gene copy number variation in bacteria.

Clinical features and antibody response of patients from a COVID-19 treatment hospital in Wuhan, China.

Coronavirus disease 2019 (COVID-19) has rapidly evolved into a global pandemic. A total of 1578 patients admitted into a newly built hospital specialized for COVID-19 treatment in Wuhan, China, were enrolled. Clinical features and the levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin (Ig)M and IgG were analyzed. In total, 1532 patients (97.2%) were identified as laboratory-confirmed cases. Seventy-seven patients were identified as asymptomatic carriers (n = 64) or SARS-CoV-2 RNA positive before symptom onset (n = 13). The positive rates of SARS-CoV-2 IgM and IgG were 80.4% and 96.8%, respectively. The median of IgM and IgG titers were 37.0A U/ml (interquartile range [IQR]: 13.4-81.1 AU/ml) and 156.9 AU/ml (IQR: 102.8-183.3 AU/ml), respectively. The IgM and IgG levels of asymptomatic patients (median titers, 8.3 AU/ml and 100.3 AU/ml) were much lower than those in symptomatic patients (median titers, 38.0 AU/ml and 158.2 AU/ml). A much lower IgG level was observed in critically ill patients 42-60 days after symptom onset. There were 153 patients with viral RNA shedding after IgG detection. These patients had a higher proportion of critical illness during hospitalization (p < .001) and a longer hospital stay (p < .001) compared to patients with viral clearance after IgG detection. Coronary heart disease (odds ratio [OR], 1.89 [95% confidence interval [CI], 1.11-3.24]; p = .020), and intensive care unit admission (OR, 2.47 [95% CI, 1.31-4.66]; p = .005) were independent risk factors associated with viral RNA shedding after IgG detection. Symptomatic patients produced more antibodies than asymptomatic patients. The patients who had SARS-CoV-2 RNA shedding after developing IgG were more likely to be sicker patients.

A Novel sRNA in Shigella flexneri That Regulates Tolerance and Virulence Under Hyperosmotic Pressure.

Regulation of the environmental stress response and virulence of Shigella flexneri may involve multiple signaling pathways; however, these mechanisms are not well-defined. In bacteria, small regulatory RNAs (sRNAs) regulate bacterial growth, metabolism, virulence, and environmental stress response. Therefore, identifying novel functional sRNAs in S. flexneri could help elucidate pathogenic adaptations to host micro-environmental stresses and associated virulence. The aim of this study was to confirm the presence of an sRNA, Ssr54, in S. flexneri and to determine its functions and possible mechanism of action. Ssr54 was found to regulate tolerance and virulence under hyperosmotic pressure. Its expression was verified by qRT-PCR and Northern blotting, and its genomic position was confirmed by 5'-rapid amplification of cDNA ends. Ssr54 expression was significantly decreased (~ 80%) under hyperosmotic conditions (680 mM NaCl), and the survival rate of the Ssr54 deletion strain increased by 20% under these conditions. This suggested that Ssr54 has been selected to promote host survival under hyperosmotic conditions. Additionally, virulence assessment, including guinea pig Sereny test and competitive invasion assays in mouse lungs, revealed that Ssr54 deletion significantly decreased S. flexneri virulence. Two-dimensional gel analyses suggest that Ssr54 may modulate the expression of tolC, ompA, and treF genes, which may affect the virulence and survival of S. flexneri under osmotic pressures. Furthermore, treF expression has been shown to improve the survival of S. flexneri under osmotic pressures. These results suggest that Ssr54 has a broad range of action in S. flexneri response to hyperosmotic environmental stresses and in controlling its virulence to adapt to environmental stresses encountered during host infection.