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Host-microbiome interaction is known to play a pivotal role in the cancer ecosystem, yet the associations have not been systematically investigated at the pan-cancer and the multi-omics level. Here, we evaluated nearly 10,000 samples across 32 cancer types collected from The Cancer Genome Atlas (TCGA), to investigate the association between the tumor microbiome (taxa, n=1,630) and tumor microenvironment composition (cell types, n=20), epigenome (CpG island methylation, n=30,716), transcriptome (gene expression, n=10,216) and proteome (protein expression, n=193). We identified 836,738 candidate associations between the tumor microbiome and host molecular aberrations across multiple cancers. Besides cancer-specific associations, we also revealed recurrent pan-cancer associations between microbes (Lachnoclostridium,Flammeovirga,TerrabacterandCampylobacter) and immune cells, as well as between microbes (CollimonasandSutterella) and fibroblasts, which were further validated by cell type estimations derived from pathological images and methylation data. We also identified several potential microbe and gene/protein expression associations mediated by DNA methylation using the sequential mediation analysis. Furthermore, our survival analysis demonstrated that tumor microbes may affect the patient’s overall survival and progression-free survival. Finally, a user-friendly web portal, Multi-Omics and Microbiome Associations in Cancer (MOMAC) was constructed for users to explore potential host-microbe interactions in cancer.Graphical abstract

Pseudomonas aeruginosa is an opportunistic pathogen with multiple strategies to interact with other microbes and host cells, gaining fitness in complicated infection sites. The contact-dependent type VI secretion system (T6SS) is one critical secretion apparatus involved in both interbacterial competition and pathogenesis. To date, only limited numbers of T6SS-effectors have been clearly characterized in P. aeruginosa laboratory strains, and the importance of T6SS diversity in the evolution of clinical P. aeruginosa remains unclear. Recently, we characterized a P. aeruginosa clinical strain LYSZa7 from a COVID-19 patient, which adopted complex genetic adaptations toward chronic infections. Bioinformatic analysis has revealed a putative type VI secretion system (T6SS) dependent lipase effector in LYSZa7, which is a homologue of TseL in Vibrio cholerae and is widely distributed in pathogens. We experimentally validated that this TseL homologue belongs to the Tle2, a subfamily of T6SS-lipase effectors; thereby, we name this effector TseL (TseL

The 2022 monkeypox outbreak outside Africa is ongoing. Cases have been reported in Hong Kong and Chongqing, China. In order to better prevent and control the potential spread of monkeypox virus in China, the development of sensitive and reliable detection commercial kits is imminent. This correspondence reviews the existing laboratory assays and related technologies for nucleic acid (PCR) and serological assays for the diagnosis of monkeypox virus to provide reference for the management and decision-making departments. Due to the serological cross-reactivity of orthopoxviruses, PCR is the laboratory test of choice to confirm monkeypox virus infection. We recommend a dual-target PCR approach in which one assay targets a conserved sequence of the Orthopoxvirus genus and the other targets a monkeypox virus specific sequence.

Bedaquiline has been widely used as a part of combination dosage regimens for the treatment of multidrug-resistant tuberculosis (MDR-TB) patients with limited options. Although the effectiveness and safety of bedaquiline have been demonstrated in clinical trials, limited studies have investigated the significant pharmacokinetics and the impact of genotype on bedaquiline disposition. Here, we developed a population pharmacokinetic model of bedaquiline to describe the concentration-time data from Chinese adult patients diagnosed with MDR-TB. A total of 246 observations were collected from 99 subjects receiving the standard recommended dosage. Bedaquiline disposition was well described by a one-compartment model with first-order absorption. Covariate modeling identified that gamma-glutamyl transferase (GGT) and the single-nucleotide polymorphism (SNP) rs319952 in the

The clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection can be combined with recombinase-aided amplification (RAA) to enable rapid, accurate, and early detection of SARS-CoV-2. Current CRISPR-based approaches to detecting viral nucleic acid typically require immense manual operations to transfer RPA amplicons for CRISPR detection or suffer from compromised sensitivity by mixing the competing RPA amplification and CRISPR detection. Here, we develop dual-CRISPR/Cas12a-assisted RT-RAA assay and a ″sample-to-answer″ centrifugal microfluidic platform that can automatically detect 1 copy/μL of the SARS-CoV-2 within 30 min. This chip separates the amplification (RAA) from detection (CRISPR), such that sensitivity is maximized and the time consumption is decreased by a factor of 3. For the 26 positive and 8 negative clinical SARS-CoV-2 samples, this automated centrifugal microfluidics achieved 100% accuracy compared to the gold-standard RT-PCR technique. This point-of-care test, with the advantages of being one-step, automated, rapid, and sensitive, will have a significant potential for clinical diagnosis and disease prevention.

Background Co‐infections, secondary bacterial or fungal infections, are important risk factors for poor outcomes in viral infections. The prevalence of co‐infection and secondary infection in patients infected with SARS‐CoV‐2 is not well understood. Aims To investigate the role of co‐infections and secondary infections in disease severity of hospitalized individuals with COVID‐19. Materials and Methods A retrospective study was carried out between 11 January 2020 and 1 March 2020 among 408 laboratory confirmed COVID‐19 patients in China. These patients were divided into three groups based on disease severity: mild or moderate, severe, or critically ill. Microbiological pathogens in blood, urine, and respiratory tract specimens were detected by the combination of culture, serology, polymerase chain reaction, and metagenomic next‐generation sequencing (mNGS). Results The median age of participants was 48 years (IQR 34–60 years). Fifty‐two patients (12.7%) had at least one additional pathogen, 8.1% were co‐infected, and 5.1% had a secondary infection. There were 13 Mycoplasma pneumoniae cases, 8 Haemophilus influenzae cases, 8 respiratory viruses, and 3 Streptococcus pneumoniae cases, primarily detected in mild and moderate COVID‐19 patients. Hospital‐acquired infection pathogens were more common in critically ill patients. Compared to those without additional pathogens, patients with co‐infections and/or secondary infections were more likely to receive antibiotics (p < 0.001) and have elevated levels of d‐dimer (p = 0.0012), interleukin‐6 (p = 0.0027), and procalcitonin (p = 0.0002). The performance of conventional culture was comparable with that of mNGS in diagnosis of secondary infections. Conclusion Co‐infections and secondary infections existed in hospitalized COVID‐19 patients and were relevant to the disease severity. Screening of common respiratory pathogens and hospital infection control should be strengthened.

The members of the glutathione S-transferase (GST) superfamily often exhibit functional overlap and can compensate for each other. Their concentrations in serum are considered as disease biomarkers. A global and quantitative evaluation of serum GSTs is therefore urgent, but there is a lack of efficient approaches due to technological limitations. GSH magnetic beads were examined for their affinity to enrich GSTs in serum, and the enriched GSTs were quantitatively targeted using a Q Exactive HF-X mass spectrometer in parallel reaction monitoring (PRM) mode. To optimize the quantification of GST peptides, sample types, trypsin digestion, and serum loading were carefully assessed; a biosynthetic method was employed to generate isotope-labeled GST peptides, and instrumental parameters were systematically optimized. A total of 134 clinical sera were collected for GST quantification from healthy donors and patients with four liver diseases. Using the new approach, GSTs in healthy sera were profiled: 14 GST peptides were quantified, and the abundance of five GST families was ranked GSTMGSTPGSTAMGST1GSTT1, ranging from 0.1 to 4 pmol/L. Furthermore, combining the abundance of multiple GST peptides could effectively distinguish different types of liver diseases. Quantification of serum GSTs through targeted proteomics, therefore, has apparent clinical potential for disease diagnosis.

Background & Aims Recent data on the coronavirus disease 2019 (COVID-19) outbreak caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has begun to shine light on the impact of the disease on the liver. But no studies to date have systematically described liver test abnormalities in patients with COVID-19. We evaluated the clinical characteristics of COVID-19 in patients with abnormal liver test results. Methods Clinical records and laboratory results were obtained from 417 patients with laboratory-confirmed COVID-19 who were admitted to the only referral hospital in Shenzhen, China from January 11 to February 21, 2020 and followed up to March 7, 2020. Information on clinical features of patients with abnormal liver tests were collected for analysis. Results Of 417 patients with COVID-19, 318 (76.3%) had abnormal liver test results and 90 (21.5%) had liver injury during hospitalization. The presence of abnormal liver tests became more pronounced during hospitalization within 2 weeks, with 49 (23.4%), 31 (14.8%), 24 (11.5%) and 51 (24.4%) patients having alanine aminotransferase, aspartate aminotransferase, total bilirubin and gamma-glutamyl transferase levels elevated to more than 3× the upper limit of normal, respectively. Patients with abnormal liver tests of hepatocellular type or mixed type at admission had higher odds of progressing to severe disease (odds ratios [ORs] 2.73; 95% CI 1.19–6.3, and 4.44, 95% CI 1.93–10.23, respectively). The use of lopinavir/ritonavir was also found to lead to increased odds of liver injury (OR from 4.44 to 5.03, both p, Graphical abstract, Highlights • Of 417 patients with COVID-19, 76.3% had abnormal liver tests and 21.5% had liver injury during hospitalization. • Patients with abnormal liver tests had significantly higher odds of developing severe pneumonia. • The use of lopinavir/ritonavir increased the odds of liver injury by 4-fold.

Bacterial persistence is one of the major causes of antibiotic treatment failure and the step stone for antibiotic resistance. However, the mechanism by which persisters arise has not been well understood. Maintaining a dormant state to prevent antibiotics from taking effect is believed to be the fundamental mechanistic basis, and persisters normally maintain an intact cellular structure. Here we examined the morphologies of persisters in Acinetobacter baumannii survived from the treatment by three major classes of antibiotics (i.e. β-lactam, aminoglycoside, and fluoroquinolone) with microcopy and found that a fraction of enlarged spherical bacteria constitutes a major sub-population of bacterial survivors from β-lactam antibiotic treatment, whereas survivors from the treatment of aminoglycoside and fluoroquinolone were less changed morphologically. Further studies showed that these spherical bacteria had completely lost their cell wall structures but could survive without any osmoprotective reagent. The spherical bacteria were not the viable-but-non-culturable cells and they could revive upon the removal of β-lactam antibiotics. Importantly, these non-walled spherical bacteria also persisted during antibiotic therapy in vivo using Galleria mellonella as the infection model. Additionally, the combinational treatment on A. baumannii by β-lactam and membrane-targeting antibiotic significantly enhanced the killing efficacy. Our results indicate that in addition to the dormant, structure intact persisters, the non-wall spherical bacterium is another important type of persister in A. baumannii. The finding suggests that targeting the bacterial cell membrane during β-lactam chemotherapy could enhance therapeutic efficacy on A. baumannii infection, which might also help to reduce the resistance development of A. baumannii.

Introduction. Human adenovirus (HAdV) is an important pathogen in acute respiratory tract infections (ARTIs) and HAdV genotypes are associated with disease severity. Hypothesis. Comparative analyses of clinical features could reveal the severity of different HAdV genotypes in ARTIs. Aim. This study aimed to investigate the molecular epidemiology of HAdV infections and explore the correlations between clinical features and HAdV genotypes. Methodology. A retrospective study was conducted on ARTIs at Beijing Chao-Yang Hospital during the period 2011–2016. A standardized data form was used to record the clinical information. HAdV was detected by FQ-PCR from respiratory specimens, and genotypes were determined by entire hexon gene sequencing. Results. A total of 8044 samples were collected, of which 296 (3.7 %) were HAdV-positive. Patients ≤44 years old were more likely to be positive for HAdV. There were three peak periods of adenoviral infections, with detection rates of 13.03, 9.39 and 10.38 %, respectively. Six HAdV genotypes (HAdV-55, -7, -3, -14, -50, -2) were identified, with HAdV-55 and HAdV-7 being the most prevalent (50.6 and 21.5 %). Compared with HAdV-7 and other types, patients infected with HAdV-55 had a longer duration of fever (P=0.0428). Infections with HAdV-55 and HAdV-7 were more severe compared to those caused by other types, with higher rates of oxygen therapy and mechanical ventilation (P=0.0172 and P=0.0144). All five deaths were caused by HAdV‐55. Conclusion. This study describes the epidemiological characteristics of HAdV infections in North China, revealing the higher severity of HAdV-55 and HAdV-7 in ARTIs. Thus, strengthened surveillance of HAdV genotypes is warranted.

BACKGROUND: Sepsis is a life-threatening condition of organ dysfunction caused by the host’s disordered immune response to infection. It has a high fatality rate and seriously endangers human health. Rapid and accurate treatment plays an important role in the reduction of septic mortality. This study aimed to investigate the clinical value of hematological parameters neutrophil (NEU)-X, NEU-Y, monocyte (MON)-X, and MON-Y in sepsis, and compare their values with that of with C-reactive protein (CRP). METHODS: We collected dipotassium ethylenediaminetetraacetic acid (EDTA-K2) anticoagulant blood samples from a total of 267 patients with positive bacterial culture and 260 healthy physical check-up patients. Participants were divided into three groups: a normal control group (n=260), bacterial infection group (n=196), and a sepsis group (n=71). RESULTS: Median values of NEU-X, NEU-Y, MON-X, MON-Y, and CRP in the sepsis group were significantly higher than those in the control group and the bacterial infection group (P

Background: Analysis of the molecular characteristics of isolates is very important for clinical and epidemiological study of community-acquired pneumonia (CAP) caused by Mycoplasma pneumoniae. Methods: Between 2010 and 2012, an epidemic period, M. pneumoniae was isolated from oropharyngeal swabs of consecutive CAP patients. Minimum inhibitory concentrations of macrolides, 23S rRNA gene sequencing, P1 gene and multilocus variable-number tandem-repeat analysis (MLVA) genotyping was conducted. Results: 88.3% (181/205) of the isolates were macrolide-resistant M. pneumoniae (MRMP) and all harbored an A2063 G mutation. The strains were clustered into 7 MLVA types, and P1 type 1 and type 2 lineages were co-circulated (86.3% and 13.7%). Compared with adults, no specific MLVA type contributed to higher M. pneumoniae infection in children (p = 0.14). Similar macrolide profile and genotypes of M. pneumoniae was found between outpatients and inpatients. Significant differences in proportion of P1 types and two main MLVA types 4/5/7/2 and 3/5/6/2 were observed between MRMP and macrolide-sensitive M. pneumoniae (MSMP) (p

The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes., Control of CRISPR-Cas9 activity allows for fine-tuning of editing and gene expression. Here the authors use gRNAs modified with RNA aptamers to enable small molecule control in bacterial systems.

The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs, has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to humans1-6. However, the difficulty of establishing effective CRISPRi systems across bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish 'Mobile-CRISPRi', a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in gammaproteobacteria and Bacillales Firmicutes at the individual gene scale, by examining drug-gene synergies, and at the library scale, by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microorganism interactions.

COVID-19 pandemic has caused millions of death globally and caused huge impact on the health of infected patients. Shift in the lung microbial ecology upon such viral infection often worsens the disease and increases host susceptibility to secondary infections. Recent studies have indicated that bacterial coinfection is an unignorable factor contributing to the aggravation of COVID-19 and posing great challenge to clinical treatments. However, there is still a lack of in-depth investigation on the coinfecting bacteria in COVID-19 patients for better treatment of bacterial coinfection. With the knowledge thatPseudomonas aeruginosais one of the top coinfecting pathogens, we analyzed the adaptation and convergent evolution of nosocomialP. aeruginosaisolated from two critical COVID-19 patients in this study. We sequenced and compared the genomes and transcriptomes ofP. aeruginosaisolates longitudinally and parallelly for its evolutionary traits.P. aeruginosaoverexpressed alginate and attenuated Type VI secretion system (T6SS) during coinfection for excessive biofilm formation and suppressed virulence. Results of bacterial competition assay and macrophage cytotoxicity test indicated thatP. aeruginosareduced its virulence towards both prokaryotic competitors and eukaryotic host through inhibiting its T6SS during evolution.P. aeuginosaT6SS is thus one of the reasons for its advantage to cause coinfection in COVID-19 patients while the attenuation of T6SS could cause a shift in the microecological composition in the lung. Our study will contribute to the development of therapeutic measures and the discovery of novel drug target to eliminateP. aeruginosacoinfection in COVID-19 patient.

ObjectiveThe etiology and epidemiology of co-infection and secondary infection in COVID-19 patients remain unknown. The study aims to investigate the occurrence and characteristics of co-infection and secondary infection in COVID-19 patients, mainly focusing on Streptococcus pneumoniae co-infections.MethodsThis study was a prospective, observational cohort study of the inpatients diagnosed with COVID-19 in two designated hospitals in south China enrolled between Jan 11 and Feb 22, 2020. The urine specimen was collected on admission and applied for pneumococcal urinary antigen tests (PUATs). Demographic, clinical and microbiological data of patients were recorded simultaneously.ResultA total of 146 patients with a confirm diagnosis of COVID-19 at the median age of 50.0 years (IQR 36.0-61.0) were enrolled, in which, 16 (11.0%) were classified as severe cases and 130 (89.0%) as non-severe cases. Of the enrolled patients, only 3 (2.1%) were considered to present the co-infection, in which 1 was co-infected with S.pneumoniae, 1 with B. Ovatus infection and the other one with Influenza A virus infection. Secondary infection occurred in 16 patients, with S. maltophilia as the most commonly isolated pathogen (43.8%), followed by P. aeruginosa (25.0%), E. aerogenes (25.0%), C. parapsilosis (25.0%) and A. fumigates (18.8%).ConclusionPatients with confirmed COVID-19 were rarely co-infected with Streptococcus pneumoniae or other pathogens, indicating that the application of antibiotics against CAP on admission may not be necessary in the treatment of COVID-19 cases.

This study characterized a genetically adapted Pseudomonas aeruginosa small colony variant isolated from a COVID-19 patient who suffered persistent bacterial coinfection and eventually recovered from critical illness. Specification and modification of the isolates discovered at genomic and transcriptomic levels with aligned phenotypic observations indicated that these isolates formed excessive biofilm with elevated quorum sensing systems.

Objectives The aim of this study was to identify the parameters in routine blood tests that can be used to evaluate the severity of coronavirus disease 2019 (COVID-19) and thus assist in clinically predicting the extent of progression. Methods This study retrospectively analyzed the epidemiological, clinical symptom and laboratory examination data of 159 COVID-19 patients. The percentage of lymphocytes (Lym%) and hemoglobin (HGB) were integrated into a joint parameter, Lym%&HGB, by binary logistic regression. Results Both Lym% and HGB gradually decreased with disease progression whereas the joint parameter Lym%&HGB increased gradually with disease progression. When using Lym%, HGB, and Lym%&HGB to predict COVID-19 severity, the area under the receiver operating characteristic (ROC) curve (AUC) was 0.89, 0.79, and 0.92, respectively. The dynamic change curves showed that Lym% and HGB continued to decline while Lym%&HGB continued to increase with disease progression in patients with severe COVID. The change in Lym%&HGB was more prominent than the changes in Lym% and HBG. Conclusions The joint parameter Lym%&HGB can serve as a good tool to differentiate severe and nonsevere COVID-19, and it has a higher sensitivity and specificity than either Lym% or HGB alone.

Bacterial persistence is one of the major causes of antibiotic treatment failure and the step stone for antibiotic resistance. However, the mechanism by which persisters arise has not been well understood. Maintaining a dormant state to prevent antibiotics from taking effect is believed to be the fundamental mechanistic basis, and persisters normally maintain an intact cellular structure. Here we examined the morphologies of persisters in Acinetobacter baumannii survived from the treatment by three major classes of antibiotics (i.e. β-lactam, aminoglycoside, and fluoroquinolone) with microcopy and found that a fraction of enlarged spherical bacteria constitutes a major sub-population of bacterial survivors from β-lactam antibiotic treatment, whereas survivors from the treatment of aminoglycoside and fluoroquinolone were less changed morphologically. Further studies showed that these spherical bacteria had completely lost their cell wall structures but could survive without any osmoprotective reagent. The spherical bacteria were not the viable-but-non-culturable cells and they could revive upon the removal of β-lactam antibiotics. Importantly, these non-walled spherical bacteria also persisted during antibiotic therapy in vivo using Galleria mellonella as the infection model. Additionally, the combinational treatment on A. baumannii by β-lactam and membrane-targeting antibiotic significantly enhanced the killing efficacy. Our results indicate that in addition to the dormant, structure intact persisters, the non-wall spherical bacterium is another important type of persister in A. baumannii. The finding suggests that targeting the bacterial cell membrane during β-lactam chemotherapy could enhance therapeutic efficacy on A. baumannii infection, which might also help to reduce the resistance development of A. baumannii.

IMPORTANCEHow to appropriately care for patients who become PCR-negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still not known. Patients who have recovered from coronavirus disease 2019 (COVID-19) could profoundly impact the health care system if a subset were to be PCR-positive again with reactivated SARS-CoV-2.OBJECTIVETo characterize a single center COVID-19 cohort with and without recurrence of PCR positivity, and develop an algorithm to identify patients at high risk of retest positivity after discharge to inform health care policy and case management decision-making.DESIGN, SETTING, AND PARTICIPANTSA cohort of 414 patients with confirmed SARS-CoV-2 infection, at The Second Affiliated Hospital of Southern University of Science and Technology in Shenzhen, China from January 11 to April 23, 2020.EXPOSURESPolymerase chain reaction (PCR) and IgM-IgG antibody confirmed SARS-CoV-2 infection.MAIN OUTCOMES AND MEASURESUnivariable and multivariable statistical analysis of the clinical, laboratory, radiologic image, medical treatment, and clinical course of admission/quarantine/readmission data to develop an algorithm to predict patients at risk of recurrence of PCR positivity.RESULTS16.7% (95CI: 13.0%-20.3%) patients retest PCR positive 1 to 3 times after discharge, despite being in strict quarantine. The driving factors in the recurrence prediction model included: age, BMI; lowest levels of the blood laboratory tests during hospitalization for cholinesterase, fibrinogen, albumin, prealbumin, calcium, eGFR, creatinine; highest levels of the blood laboratory tests during hospitalization for total bilirubin, lactate dehydrogenase, alkaline phosphatase; the first test results during hospitalization for partial pressure of oxygen, white blood cell and lymphocyte counts, blood procalcitonin; and the first test episodic Ct value and the lowest Ct value of the nasopharyngeal swab RT PCR results. Area under the ROC curve is 0.786.CONCLUSIONS AND RELEVANCEThis case series provides clinical characteristics of COVID-19 patients with recurrent PCR positivity, despite strict quarantine, at a 16.7% rate. Use of a recurrence prediction algorithm may identify patients at high risk of PCR retest positivity of SARS-CoV-2 and help modify COVID-19 case management and health policy approaches.Key PointsQuestionWhat are the characteristics, clinical presentations, and outcomes of COVID-19 patients with PCR retest positivity after resolution of the initial infection and consecutive negative tests? Can we identify recovered patients, prior to discharge, at risk of the recurrence of SARS-CoV-2 PCR positivity?FindingsIn this series of 414 COVID-19 inpatients discharged to a designated quarantine center, 69 retest positive (13 with 2 readmissions, and 3 with 3 readmissions). A multivariable model was developed to predict the risk of the recurrence of SARS-CoV-2 PCR positivity.MeaningRate and timing of the recurrence of PCR positivity following strict quarantine were characterized. Our prediction algorithm may have implications for COVID-19 clinical treatment, patient management, and health policy.

We profiled the serological responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein and spike (S) glycoprotein. The majority of the patients developed robust antibody responses between 17 and 23 days after illness onset. Delayed, but stronger, antibody responses were observed in critical patients.

Background: In December 2019, a cluster of acute respiratory illness caused by SARS-CoV-2, now known as Coronavirus Disease 2019 (COVID-19), occurred in Wuhan

BACKGROUND: Recurrence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive detection in infected but recovered individuals has been reported. Patients who have recovered from coronavirus disease 2019 (COVID-19) could profoundly impact the health care system if a subset were to be polymerase chain reaction (PCR)-positive again with reactivated SARS-CoV-2. We sought to define the kinetics and relevance of PCR-positive recurrence during recovery from acute COVID-19 to better understand risks for prolonged infectivity and reinfection. METHODS: A series of COVID-19 414 patients, at The Second Affiliated Hospital of Southern University of Science and Technology in Shenzhen, China from January 11 to April 23, 2020. Univariable and multivariable statistical analysis of inpatient data were performed to develop an algorithm to predict patients at risk of recurrence of PCR positivity. FINDINGS: 16·7% recovered patients with PCR positive recurring one to three times, despite being in strict quarantine. Younger patients with mild pulmonary respiratory syndrome had higher risk of PCR positivity recurrence. The recurrence prediction model had an area under the ROC curve of 0·786. INTERPRETATION: This case series provides clinical characteristics of recovered COVID-19 patients with recurrent SARS-CoV-2 positivity, despite strict quarantine, at a 16·7% rate. Use of a recurrence prediction algorithm may identify patients at high risk of recurrent SARS-CoV-2 positivity and help understand reactivation and reinfection possibilities to establish protocols for health policy. FUNDING STATEMENT: This work was supported by grants from Sanming Project of Medicine in Shenzhen (Jia Huang, No. SZSM201812065); Bill & Melinda Gates Foundations (Lei Liu); and from National Natural Science Foundation of China (Jia Huang, No. 81501651) DECLARATION OF INTERESTS: The authors declare no competing interests. ETHICS APPROVAL STATEMENT: This study was approved by the Ethics Committee of the Second Affiliated Hospital of Southern University of Science and Technology. Written informed consent was obtained from all patients.

Background/Purpose During the routine screening of vancomycin-resistant Enterococci faecium (VREfm) we found that VanA phenotype– vanA genotype and VanB phenotype– vanA genotype isolates had an identical Tn 1546 -like element structure. This study aimed to evaluate the genetic background and vanA gene expression to identify the mechanisms of the development of the VanB phenotype– vanA genotype VREfm. Methods Twelve VREfm isolates were collected from a 1500-bed tertiary-care teaching hospital in Beijing. Genetic variations of the Tn 1546 -like element were determined by an overlapping polymerase chain reaction assay and sequencing. The genetic background was determined by pulsed-field gel electrophoresis and mutilocus sequence typing. vanA gene expression was evaluated using a TaqMan quantitative real-time polymerase chain reaction. Results For the 12 isolates, six isolates with the VanA phenotype– vanA genotype and six with the VanB phenotype– vanA genotype were identified. According to the structure analysis of the Tn 1546 -like elements, our isolates were divided into two types. In the four isolates of type A, IS 1542 and IS 1216V were inserted into the orf2-vanR and vanX-vanY regions, respectively. In the eight isolates of type D, a similar insertion as type A occurred, except for an ISE fa4 insertion into IS 1542 . A significant difference in vanA gene expression was observed between the VanA and VanB phenotype isolates in type A, but not in type D. Mutilocus sequence typing and pulsed-field gel electrophoresis analysis showed that these isolates have a different genetic background. Conclusion Our results indicated that the occurrence of the VanB phenotype– vanA genotype might not completely depend on the structure of Tn 1546 -like elements and vanA gene expression.

Introduction The long-term mortality rate in adolescent and middle-aged patients with low-medium risk community-acquired pneumonia (CAP) remains unelucidated. Objective Understanding the long-term mortality rate in adolescent and middle-aged patients with low-medium risk CAP in Beijing, China, to provide a basis for the long-term management of CAP patient. Methods A follow-up survey was conducted telephonically from December 2017 to January 2018 to assess the survival status among 808 patients from a CAP-China cohort enrolled from 10 general hospitals in Beijing between November 2010 and April 2012. The all-cause mortality rate was determined and Cox's proportional hazard model was performed to identify potential factors predicting mortality. Results Among the 808 patients, the mean age (SD) was 45.4 (19.6) year-old and the median (IQR) pneumonia severity index (PSI) score was 42.0 (35.8). Survival status for a total of 426 (52.7%) were determined during the follow-up and the non-follow-up patients were with slightly larger PSI score. The mean age (SD) for the follow-up patients were 44.6 (18.7) year-old and the median (IQR) PSI score was 42.0 (33.3). Over a median of 7 years, 32 participants died and the cumulative 1-, 3-, 5- and 7-year all-cause mortality rates were 1.6%, 4.2%, 5.9% and 7.5%, respectively. The average annual standardized mortality rate among the study participants was 9.79‰, which was significantly higher than the mortality rate of 5.20‰ among Beijing residents in 2016. Multivariable Cox proportional hazards analyses revealed that age, comorbidity and PSI were independent prognostic factors associated with long-term mortality, with hazard ratios of 4.953 (95% confidence interval [CI]3.270-7.502), 2.393 (95% CI 1.148-4.985) and 3.553 (95% CI 2.607-4.843), respectively. [Correction added on 20 August 2020, after first online publication: "9.79%" has been corrected to "9.79‰".] CONCLUSION: The long-term mortality rate is higher among patients with CAP compared with the age-adjusted general population in the same city. Age, comorbidity and initial PSI class are independently prognostic factors for the long-term mortality rate.

To understand functional role of PD-1-expressing MAIT cells during tuberculosis infection in humans, sorted PD-1+ and PD-1- MAIT cells from pleural effusions of patients with pleural tuberculosis were subjected to transcriptome sequencing. PD-1-expressing MAIT cells were analysed by flow cytometry and their phenotypic and functional features were investigated. Transcriptome sequencing identified 144 genes that were differentially expressed between PD-1+ and PD-1- MAIT cells from tuberculous pleural effusions and CXCL13 was the gene with highest fold difference. The level of PD-1-expressing MAIT cells was associated with extent of TB infection in humans. PD-1-expressing MAIT cells had increased production of CXCL13 and IL-21 as determined by flow cytometry. PD-1high CXCR5- MAIT cells were significantly expanded in pleural effusions from patients with pleural tuberculosis as compared with those from peripheral blood of both patients with tuberculosis and healthy controls. Although PD-1high CXCR5- MAIT cells from tuberculous pleural effusions had reduced IFN-γ level and increased expression of Tim-3 and GITR, they showed activated phenotype and had higher glucose uptake and lipid content. It is concluded that PD-1-expressing MAIT cells had reduced IFN-γ level but increased production of both CXCL13 and IL-21.

Pathogens express a set of proteins required for establishing and maintaining an infection, termed virulence life-style genes (VLGs). Due to their outsized importance in pathogenesis, VLG products are attractive targets for the next generation of antimicrobials. However, precise manipulation of VLG expression in the context of infection is technically challenging, limiting our ability to understand the roles of VLGs in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of gene knockdown tools that are transferred by conjugation and stably integrate into pathogen genomes that we call “Mobile-CRISPRi”. Here we show the efficacy of Mobile-CRISPRi in controlling VLG expression in a murine infection model. We optimize Mobile-CRISPRi inPseudomonas aeruginosafor use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid non-specific toxicity. As a proof of principle, we demonstrate that knockdown of a VLG encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of VLGs across many bacterial species and pathogenesis models.ImportanceAntibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically these genetic drivers have been difficult to manipulate, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study virulence life-style genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.