Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology

Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

Analyses of the Duration and Dynamics of the COVID-19 Epidemic in 11 Severely Affected Countries

The COVID-19 pandemic has affected 220 countries,areas,and territories worldwide,with more than 65 million confirmed cases and 1.5 million deaths[1].By the beginning of May,2020,only a few countries,such as China,South Korea,Australia,and New Zealand,seemed to be close to the epidemic’s late stage with a few daily reported new cases[1].

A Novel Early Warning Model for Hand, Foot and Mouth Disease Prediction Based on a Graph Convolutional Network

Objectives Hand,foot and mouth disease(HFMD)is a widespread infectious disease that causes a significant disease burden on society.To achieve early intervention and to prevent outbreaks of disease,we propose a novel warning model that can accurately predict the incidence of HFMD.Methods We propose a spatial-temporal graph convolutional network(STGCN)that combines spatial factors for surrounding cities with historical incidence over a certain time period to predict the future occurrence of HFMD in Guangdong and Shandong between 2011 and 2019.The 2011-2018 data served as the training and verification set,while data from 2019 served as the prediction set.Six important parameters were selected and verified in this model and the deviation was displayed by the root mean square error and the mean absolute error.Results As the first application using a STGCN for disease forecasting,we succeeded in accurately predicting the incidence of HFMD over a 12-week period at the prefecture level,especially for cities of significant concern.Conclusions This model provides a novel approach for infectious disease prediction and may help health administrative departments implement effective control measures up to 3 months in advance,which may significantly reduce the morbidity associated with HFMD in the future.

Global Profiles of Acetylated Proteins in Brains of Scrapie Agents 139A- and ME7-Infected Mice Collected at Mid-Early, Mid-Late, and Terminal Stages

Objective To describe the global profiles of acetylated proteins in the brains of scrapie agents 139Aand ME7-infected mice collected at mid-early,mid-late,and terminal stages.Methods The acetylated proteins from the cortex regions of scrapie agent(139A-and ME7)-infected mice collected at mid-early(80 days postinfection,dpi),mid-late(120 dpi),and terminal(180 dpi) stages were extracted,and the global profiles of brain acetylated proteins were assayed with proteomic mass spectrometry.The proteins in the infected mice showing 1.5-fold higher or lower levels than that of agematched normal controls were considered as differentially expressed acetylated peptides(DEAPs).Results A total of 118,42,and 51 DEAPs were found in the brains of 139A-80,139A-120,and 139A-180dpi mice,respectively.Meanwhile,390,227,and 75 DEAPs were detected in the brains of ME7-80,ME7-120,and ME7-180 dpi mice,respectively.The overwhelming majority of DEAPs in the mid-early stage were down-regulated,and more portions of DEAPs in the mid-late and late stages were up-regulated.Approximately 22.1%(328/1,485) of acetylated peptides mapped to 74 different proteins were mitochondrial associated.Kyoto Encyclopedia of Genes and Genomes pathway analysis identified 39(80dpi),13(120 dpi),and 10(180 dpi) significantly changed pathways in 139A-infected mice.Meanwhile,55,25,and 18 significantly changed pathways were observed in the 80,120,and 180 dpi samples of139A-and ME7-infected mice(P < 0.05),respectively.Six pathways were commonly involved in all tested samples.Moreover,many steps in the citrate cycle(tricarboxylic acid cycle) were affected,represented by down-regulated acetylation for relevant enzymes in the mid-early stage and upregulated acetylation in the mid-late and late stages.Conclusion Our data here illustrated the changes in the global profiles for brain acetylated proteins during prion infection,showing remarkably inhibited acetylation in the early stage and relatively enhanced acetylation in the late stage.

Seasonal and Genetic Characteristics of Human Metapneumovirus Circulating—Henan Province,China,2017-2023

Introduction:This study examines the seasonal and genetic characteristics of human metapneumovirus(HMPV)in Henan from 2017 to 2023.Methods:Samples from patients with acute respiratory infection(ARI)testing positive for HMPV were subjected to real-time reverse transcription polymerase chain reaction The G gene was amplified and sequenced from these samples for epidemiological and phylogenetic analysis.Results:We enrolled 2,707 ARI patients from October 2017 to March 2023,finding an HMPV positivity rate of 6.17%(167/2,707).Children under five exhibited the highest infection rate at 7.78%(138/1,774).The 2018 and 2019 HMPV outbreaks predominantly occurred in spring(March to May),with peak positivity rates of 31.11%in May 2018 and 19.57%in May 2019.A notable increase occurred in November 2020,when positivity reached a historic high of 42.11%,continuing until January 2021.From February 2021 through March 2023,no significant seasonal peaks were observed,with rates ranging from 0%to 8.70%.Out of 81 G gene sequences analyzed,46.91%(38/81)were identified as subtype A(A2c:45.67%,37/81;A2b:1.23%,1/81)and 53.09%(43/81)as subtype B(B1:9.88%,8/81;B2:43.21%,35/81).Notably,an AAABBA switch pattern was observed in HMPV subtypes.The dominant strains were A2c111nt-dup in subtype A and B2 in subtype B.Conclusions:Six years of surveillance in Henan Province has detailed the seasonal and genetic dynamics of HMPV,contributing valuable insights for the control and prevention of HMPV infections in China.These findings support the development of targeted HMPV vaccines and immunization strategies.

Epidemiology and genetic characteristics of coxsackievirus A16 associated with hand-foot-and-mouth disease in Yantai city,China in 2018-2021

In 2008,China launched a national surveillance system for hand‐foot‐and‐mouth disease(HFMD).Several million cases of HFMD are reported every year,coxsackievirus A16(CVA16)was the leading cause of HFMD epidemic in Yantai city,China in recent years,but the information of epidemiology and molecular characterization of CVA16 in Yantai is limited.The aim of this study is to investigate the epidemiological characteristics and pathogenic spectrum of HFMD,and most importantly,the molecular characterization of CVA16 in Yantai from 2018 to 2021.A total of 2,000 clinical samples were collected in Yantai city from 2018 to 2021 and the enterovirus typing was performed using real‐time reverse transcriptase–polymerase chain reaction(qRT‐PCR).VP1 coding regions of 41 CVA16 isolates were amplified and Sanger sequenced,and phylogenetic analysis was performed.During the study period,HFMD became prevalent from May to August each year.It peaked in June and declined in September.The incidence was highest in children aged 1 to 5 years,while more common in males than females.1,617 out of 2,000 clinical collection of samples were tested positive for enterovirus.Among them,614 were identified as CVA16,45 were enterovirus A71(EV A17),and 958 were other enterovirus serotypes.All 41 CVA16 strains belonged to the Bla and B1b genotypes.Homology analysis showed that 41 CVA16 isolates shared 83.2%–100%nucleotide and 93.7%–100%amino acid similarity among themselves.The results of this study update molecular epidemiology of CVA16 and provide a reference for HFMD prevention and control.

Safety and Effectiveness of SA58 Nasal Spray Against COVID-19 Infection in Medical Personnel:An Open-Label,Blank-Controlled Study—Hohhot City,Inner Mongolia Autonomous Region,China,2022

Summary What is already known about this topic?The active ingredient of the SA58 Nasal Spray is a broad-spectrum neutralizing antibody with a high neutralizing capacity against different Omicron subvariants in vitro studies.What is added by this report?This study demonstrated the safety and effectiveness of SA58 Nasal Spray against coronavirus disease 2019(COVID-19)infection in medical personnel for the first time.What are the implications for public health practice?This study provides an effective approach for the public to reduce their risk of COVID-19 infection.The findings of this research have the potential to significantly reduce the risk of infection and limit human-to-human transmission in the event of a COVID-19 outbreak.

A Reverse-transcription Recombinase-aided Amplification Assay for the Rapid Detection of the Far-Eastern Subtype of Tick-borne Encephalitis Virus

Objective Tick-borne encephalitis virus(TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis(TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease. Methods A reverse-transcription recombinase-aided amplification(RT-RAA) assay was developed. This assay can be completed in one closed tube at 39℃ within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type(WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay. Results The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units(pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay. Conclusion A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.

Rapid Internal Control Reference Recombinase-Aided Amplification Assays for EBV and CMV Detection

Epstein-Barr virus(EBV)and cytomegalovirus(CMV),two of the most prevalent human herpesviruses,cause a wide spectrum of diseases and symptoms and are associated with serious health problem.In this study,we developed an internal control reference recombinase-aided amplification(ICR-RAA)assay for the rapid detection of EBV and CMV within 30 min.The assay had a sensitivity of 5 and 1 copies/test for EBV and CMV,respectively,with no cross reaction with other pathogens.In comparison with those of the commercial quantitative polymerase chain reaction(qPCR),the sensitivity of the EBV and CMV ICR-RAAs using extracted DNA was 93.33%and 84.84%,respectively;the specificity was 98.75%and 100.00%,respectively;and the Kappa values were 0.930 and 0.892(P<0.05),respectively.In comparison with those of qPCR,the sensitivity of the EBV and CMV ICR-RAAs using the DNA by thermal lysis was 72.22%and 80.00%,respectively;the specificity was 100.00%。

Monkeypox virus:past and present

Background The objective of this paper is to analyze the current status of monkeypox worldwide.In the face of this public health threat,our purpose is to elucidate the clinical characteristics and epidemiology of monkeypox,the developmental progress of monkeypox-related drugs and the vaccines available.Data sources The literature review was performed in databases including PubMed,Science Direct and Google Scholar up to July 2022.Results Since May 2022,the World Health Organization has reported more than 45,000 confirmed cases from 92 nonendemic countries,including nine deaths.Although some women and children have been infected so far,most cases have occurred among men who have sex with other men,especially those with multiple sexual partners or anonymous sex.Conclusions Pediatric monkeypox infection has been associated with a higher likelihood of severe illness and mortality than in adults.Severe monkeypox illness in pediatrics often requires adjunctive antiviral therapy.It is crucial for all countries to establish sound monitoring and testing systems and be prepared with emergency preparedness.

A one-step reverse-transcription recombinase aided PCR assay for the rapid and sensitive detection of human enteroviruses

Human enteroviruses(HEVs)include many different types that cause a wide range of diseases,and an effective method of genus-level identification has therefore significant clinical implications.However,quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR),the gold-standard method,still has shortfalls in diagnostic sensitivity and timeliness.Here we established a one-step real-time reverse-transcription recombinase-aided PCR assay(RT-RAP)to detect HEV fragment within an hour.The RT-RAP assay showed a detection limit of 5 copies/μL using recombinant plasmids and was extensively verified using 15 HEV strains.Among 15 types of HEV(species A-C),the sensitivity of RT-RAP was approximately 2-8 folds lower than that of the qRT-PCR in 9 types,and no-cross reaction with other viruses was observed.RT-RAP was further applied to analyze CSF and fecal specimens;the clinical performance demonstrated that the RT-RAP and the commercial qRT-PCR kit provided consistent results.These results indicated that RT-RAP assay may be a promising approach for rapid and sensitive detection of HEV.

Surveillance, epidemiology, and pathogen spectrum of hand, foot, and mouth disease in mainland of China from 2008 to 2017

Hand,foot and mouth disease(HFMD)was reported in May 2,2008 to be the 38th legally notifiable disease in China's National Notifiable Disease Reporting and Surveillance System.In order to solve the infection,an extensive three-level HFMD surveillance laboratory network was established.In this study,the framework of that network is assessed and the incidence of HFMD in China from 2008 to 2017 is reported using a descriptive epidemiologic method.During these 10 years,a series of techniques have been widely applied in all the network laboratories.Using information and material obtained from the network,a virus bank and database containing 18,238 viruses were established.Nationally,18,184,834 HFMD cases,including 152,436 severe cases and 3633 fatal cases,were reported in mainland of China.The average annual incidence in the population was 133.99/100,000 people,with a maximum incidence of 205.06/100,000 people in 2014.The incidence and mortality rates of HFMD were the highest in children aged 1–2 years.The numbers of reported cases fluctuated,with a high incidence observed every 2 years.An overall increase in the number of reported cases was also observed throughout the study period.Despite this,the incidence of severe cases and the mortality rate have been decreasing.High-risk regions are located in southern,eastern,and central China.Two peaks of HFMD infection cases were observed annually except for Northeast China.Different proportions of enterovirus serotypes were observed during the studied years.The predominant enterovirus varies from year to year,but the disease severity is always closely related to the specific serotype.EV-A71 is the dominant serotype associated with severe and fatal cases,with constituent ratios of 70.03%and 92.23%,respectively.The studied highly sensitive and efficient surveillance network provides information that is critical for prevention and control of the disease.It is extremely necessary and important to continuously conduct extensive virological surveillance for HFMD.

Multicenter evaluation of a simple and sensitive nucleic acid self-testing for SARS-CoV-2

A rapid and accurate COVID-19 diagnosis is a prerequisite for blocking the source of infection as soon as possible and taking the appropriate medical action.Herein,we developed GeneClick,a device for nucleic acid self-testing of SARS-CoV-2,consisting of three modules:a sampling kit,a microfluidic chip-based disposable cartridge,and an amplification reader.In addition,we evaluated the clinical performance of GeneClick using 2162 nasal swabs collected at three medical institutions,using three commercial RT-qPCR kits and an antigen self-test as references.Compared to RT-qPCR,the sensitivity and specificity of the GeneClick assay were 97.93%and 99.72%,respectively,with a kappa value of 0.979(P<0.01).Of the 2162 samples,2076 were also tested for SARS-CoV-2 antigens.Among the 314 positive samples identified by GeneClick assay,63 samples were undetected by antigen tests.Overall,the GeneClick nucleic acid self-test demonstrated higher accuracy than the antigen-based detection.Based on the additional features,including simple operation,affordable price,portable device,and reliability of smartphone APP-driven sampling and result reporting,GeneClick offers a powerful tool for field-based SARS-CoV-2 detection in primary healthcare institutions or at-home use.

Establishment of a Special Platform for the Research of Prion and the Diagnosis of Human Prion Disease —China’s Studies

The studies of prions and prion disease usually need many special platforms and techniques that differ from those for classical microbes.Search of new biomarkers and establishment of new methods for the diagnosis of human prion diseases are priorities in the field of prion study.

Activation of Innate Immunity and Autophagy in Brain Tissues with Prion Disease and Degradation of Abnormal PrPs in Cells—China’s Studies

Unlike infectious diseases caused by conventional microbes,there are no detectable specific humoral or cellular immunoresponses to prion infection.However,extensive and active gliosis is observable in affected brain regions along with significant deposits of scrapielike prion protein(PrP^(Sc)).Here,we summarize our studies of vibrant activation of host non-specific immunocomponents and autophagy in the microenvironment of prion infected brains.Activation of the brain’s innate immunity and autophagy upon prion infection reflect non-specific host defense systems attempt to dispose of accumulated prions.Vibrant elevation of neuroinflammation leads to neuron injury.

IP10, KC and M-CSF Are Remarkably Increased in the Brains from the Various Strains of Experimental Mice Infected with Different Scrapie Agents

Activation of inflammatory cells and upregulations of a number of cytokines in the central nervous system(CNS)of patients with prion diseases are frequently observed.To evaluate the potential changes of some brain cytokines that were rarely addressed during prion infection,the levels of 17 different cytokines in the brain homogenates of mice infected with different scrapie mouse-adapted agents were firstly screened with Luminex assay.Significant upregulations of interferon gamma-induced protein 10(IP10),keratinocyte chemoattractant(KC)and macrophage colony stimulating factor(M-CSF)were frequently detected in the brain lysates of many strains of scrapie infected mice.The upregulations of those three cytokines in the brains of scrapie infected mice were further validated by the individual specific ELISA and immunohistochemical assay.Increased specific mRNAs of IP10,M-CSF and KC in the brains of scrapie infected mice were also detected by the individual specific qRT-PCRs and IP10-specific digital PCR.Dynamic analyses of the brain samples collected at different time points post infection revealed the time-dependent increases of those three cytokines,particularly IP10 during the incubation period of scrapie infection.In addition,we also found that the levels of IP10 in cerebral spinalfluid(CSF)of 45 sporadic Creutzfeldt–Jakob disease(sCJD)patients were slightly but significantly higher than those of the cases who were excluded the diagnosis of prion diseases.These data give us a better understanding of inflammatory reaction during prion infection and progression of prion disease.

Therapeutic implications of prion diseases

Prions are unconventional infectious agents that cause lethal transmissible neurodegenerative diseases in human and animals.Prions can be distinguished fromother knownpathogens by their lack of nucleic acids.The most essential process for prion propagation is conversion fromnormal cellular prion protein on the cell membrane to insoluble,limited protease digestion-resistant,pathogenic scrapie prion protein.For dozens of years,many pharmacological tools and interventions targeting different stages of disease progression have been developed and evaluated,and a few have been entered clinical trials.However,no approved prophylactic or therapeutic drugs for prion diseases are available.In this review,we summarize the current concepts in prion research and discuss advances in the research and development of drugs for the prevention and treatment of prion disease.

Enhancement of a recombinase-aided amplification assay using betaine and pullulan

Background:Nucleic acid amplification enhancers suitable for use in a recombinase-aided amplification(RAA)assay were studied for the first time,and amplification of a long-fragment(509 bp)was initially explored.Methods:Using recombinant plasmids and clinical samples,RAA fluorescence and basic methods were used to evaluate the efficacy.The fluorescence method was evaluated by threshold time and fluorescence value,and the basic method was characterized by 2%agarose gel electrophoresis.Results:Taking a previously established RAA assay for HPV18 as an example,we demonstrated that the addition of 0.2 M,0.4 M,and 0.6 M betaine and 10%pullulan could enhance the RAA.The new RAA assays with betaine and pullulan were named B-RAA and P-RAA,respectively.Using the B-RAA and P-RAA fluorescence methods,the threshold time values could be shortened by 1.72-2.32 minutes and 2.60 minutes,respectively,and the fluorescence values could be enhanced by 8847.25-9094.37 mv and 5250 mv,respectively.Using the basic method,the sensitivity could be increased 10-fold.We successfully amplified a long-fragment of 509 bp using a P-RAA assay with a sensitivity of 102 copies/μL(compared with 103 copies/μL in the RAA assay).Conclusions:Thus,we concluded that betaine and pullulan are effective additives to enhance the sensitivity of RAA assays.

Possible processes and origin-tracing methods of“human-to-item”contamination and“item-to-human”infection with SARS-CoV-2

1.Introduction The first wave of coronavirus disease 2019(COVID-19)outbreak in China's Mainland was declared controlled after April 2020.Since then,the origins of all local outbreaks were imported cases of COVID-19 infection or imported articles contaminated with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)[1],[2],[3].The volume of import and export trade in China is enormous,and the entry of SARS-CoV-2-contaminated articles into China is inevitable.Additionally,SARS-CoV-2 can survive on the surface of noncold-chain products for prolonged periods in cold environments during fall and winter[4],[5],[6].

Revealing the Mysterious Veil of Prion Diseases Under the Framework of China CDC

Prion disease(PrD)or transmissible spongiform encephalopathy(TSE)is a group of transmissible and fatal neurodegenerative diseases affecting humans and many animal species.Clinically,PrD,either in humans or in animals,has been documented for more than hundreds of years,e.g.,scrapie in the early 18th century and human Creutzfeldt-Jakob disease(CJD)in the early 20th century(1–2).However,the hypothesis and conception of“prions”have been gradually accepted only since the 1980s.As an unconventional infectious agent without nucleic acids,the principle of the“prion”is unsolved conformational changes from host normal membrane protein PrPc to abnormal pathogenic scrapie-like prion protein(PrP^(Sc))((3).The hypothesis of the prion concept mostly explains the pathogenesis of PrD;however,there are still many gaps that need to be filled.More importantly,“prion theory”opens a completely new window in biology,possibly highlighting a new type of life.