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.

Establishment and Preliminary Application of a Rapid Fluorescent Focus Inhibition Test (RFFIT) for Rabies Virus

The World Health Organization (WHO) standard assay for determining levels of the rabies virus neutralization antibody (RVNA) is the rapid fluorescent focus inhibition test (RFFIT), which is used to evaluate the immunity effect after vaccination against rabies. For RFFIT, CVS-11 was used as the challenge virus, BSR cells as the adapted cells, and WHO rabies immunoglobulin (WHO STD) as the reference serum in this study. With reference to WHO and Pasteur RFFIT procedures, a micro-RFFIT procedure adapted to our laboratory was produced, and its specificity and reproducibility were tested. We tested levels of RVNA in human serum samples after immunization with different human rabies vaccines (domestic purified Vero cell rabies vaccine (PVRV) and imported purified chick embryo cell vaccine (PCECV)) using different regimens (Zagreb regimen and Essen regimen). We analyzed the levels of RVNA, and compared the immune efficacy of domestic PVRV and imported PCECV using different immunization regimens. The results showed that the immune efficacy of domestic PVRV using the Zagreb regimen was as good as that of the imported PCECV, but virus antibodies were generated more rapidly with the Zagreb regimen than with the Essen regimen. The RFFIT procedure established in our laboratory will enhance the comprehensive detection ability of institutions involved in rabies surveillance in China.

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].

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.

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.

Norovirus Infection and Histo-blood Group Antigens in Children Hospitalized with Diarrhea in Lulong and Chenzhou in China

Norovirus （NOV） is a pathogen that commonly causes viral diarrhea in children. Studies indicate that NoV recognizes human histo-blood group antigens （HBGAs） as cell attachment factors. In order to explore the correlation between of NoV infection and HBGAs, a cross-sectional study was conducted in children less than five years old who were hospitalized with diarrhea in two areas of China between November 2014 and February 2015. Of the paired stool and saliva samples taken from 424 children,

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%。

Gene Knockdown in Human Rhinovirus 1B Using 2′-OMe-modified si RNAs Results in the Reactivation of the Interferon Response

The aim of this study was to investigate the knockdown efficiency of 2＇-O-methylated （2＇-OMe）-modified small interfering RNAs （siRNAs） on human rhinovirus 1B （HRV1B） replication and the interferon response. Thus, 24 2＇-OMe-modified siRNAs were designed to target HRV1B. The RNA levels of HRV1B, Toll-like receptor 3, melanoma differentiation-associated gene 5, retinoic acid inducible gene-I, and interferons were determined in HRV1B-infected HeLa and BEAS-2B epithelial cells transfected with 2＇-OMe-modified siRNAs. The results revealed that all 2＇-OMe-modified siRNAs interfered with the replication of HRVIB in a cell-specific and transfection efficiency-dependent manner. Viral activation of Toll-like receptor 3, melanoma differentiation-associated gene 5, retinoic acid inducible gene-1, and the interferon response was detected. In conclusion, the 2＇-OMe-modified siRNAs used in this study could interfere with HRV1B replication, possibly leading to the reactivation of the interferon response.

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.

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.

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.

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.

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.

Serological Investigation of Laboratory-Confirmed and Suspected Ebola Virus Disease Patients During the Late Phase of the Ebola Outbreak in Sierra Leone

This study aimed to investigate the serological characteristics of Ebola virus(EBOV) infection during the late phase of the Ebola outbreak in Sierra Leone. In total, 877 blood samples from 694 suspected Ebola virus disease(EVD) cases assessed from March to December 2015, were analyzed via real-time reverse transcription polymerase chain reaction(RT-PCR) for viral RNA and enzyme-linked immunosorbent assay(ELISA) and Luminex to detect antibodies against EBOV. Viral load and EBOV-specific IgM/IgG titers displayed a declining trend during March to December 2015. Viral RNA load decreased rapidly at earlier stages after disease onset, while EBOV-specific IgM and IgG still persisted in 58.1%(18/31) and 93.5%(29/31) of the confirmed EVD patients and in 3.8%(25/663) and 17.8%(118/663) of the RNA-negative suspected patients in the later phase, respectively. Dynamic analysis of longitudinally collected samples from eight EVD patients revealed typically reversed trends of declining viral load and increasing IgM and/or IgG titers in response to the EBOV infection.The present results indicate that certain populations of Sierra Leone developed immunity to an EBOV infection in the late phase of the outbreak, providing novel insights into the risk assessment of EBOV infections among human populations.

Epidemiological Characteristics of Dengue Fever-China,2005–2023

Introduction:The global incidence of dengue fever has increased significantly over the past two decades,and China faces a significant upward trend in dengue control challenges.Methods:Data were obtained from China’s NNDRS from 2005 to 2023.Joinpoint regression software was used to analyze temporal trends,while SaTScan software was used to analyze spatial,seasonal,and spatiotemporal distributions.ArcGIS software was used to visualize clusters.Results:A total of 117,892 dengue cases were reported from 2005 to 2023,with significant fluctuation in annual reported cases.Dengue was not endemic in China.Autochthonous outbreaks most likely occurred in the southwestern,southeastern coastal,and inland areas of China.These outbreaks have occurred between June and November,generally peaking in September or October,around epidemiological week(EW)40.Conclusions:Dengue challenges in China are increasing.Timely case monitoring,proactive control interventions,and staff mobilization should be implemented before June to ensure a timely response to autochthonous outbreaks.

Genomic Surveillance for SARS-CoV-2 Variants: Dominance of XBB Replacement — China, January–June 2023

Introduction:In the first half of 2023,a global shift was observed towards the predominance of XBB variants.China faced a significant epidemic between late 2022 and early 2023 due to Omicron subvariants BA.5.2 and BF.7.This study aims to depict the evolving variant distribution among provincial-level administrative divisions(PLADs)in China and explore the factors driving the predominance of XBB replacement.Methods:Sequences from local and imported coronavirus disease 2019(COVID-19)cases recorded between January 1 and June 30,2023,were included.The study analyzed the changing distribution of viral variants and assessed how the prior dominance of specific variants,XBB subvariants,and imported cases influenced the prevalence of the XBB replacement variant.Results:A total of 56,486 sequences were obtained from local cases,and 8,669 sequences were from imported cases.Starting in April,there was a shift in the prevalence of XBB from imported to local cases,with varying dominance among PLADs.In PLADs previously high in BF.7,the rise of XBB was delayed.A positive correlation was found between XBB proportions in imported cases from January to March and local cases in April.The distribution pattern of XBB subvariants differed between local and imported cases within the same PLAD.No significant differences were noted in the replacement rates of XBB subvariants.Conclusions:The timing of XBB dominance differed among various PLADs in China in the first half of 2023,correlating closely with the prevalence of XBB variants among imported cases.

Molecular Epidemiology of Echovirus 18 Circulating in China's Mainland from 2015 to 2016

Echovirus 18(E18), a serotype of Enterovirus B(EV-B) species, is an important pathogen in aseptic meningitis. E18 had rarely been detected in China's Mainland, but became the predominant pathogen associated with viral encephalitis(VE) and meningitis in Hebei province for the first time in 2015. To investigate the molecular epidemiology and genetic characteristics of E18 in China's Mainland, sixteen E18 strains from patient throat swabs with hand, foot, and mouth disease(HFMD) in six provinces in China collected between 2015 and 2016, and four E18 strains isolated from 18 patient cerebrospinal fluid specimens with VE in Hebei Province in 2015 were obtained and sequenced. Combined with the sequences from the GenBank database, we performed an extensive genetic analysis. Phylogenetic analysis of VP1 gene sequences revealed that all E18 strains from China's Mainland after 2015 belonged to subgenotype C2. There were no obvious specific differences in phylogenetic and variation analyses of E18 genome sequences between HFMD and VE/meningitis strains. Potential multiple recombination may have occurred in the 50-untranslated region and in the P2 and P3 nonstructural protein-encoding regions of E18 strains from China. The current E18 strains were potential multiplerecombinant viruses. Overall, these findings supported that E18 caused HFMD, VE, and meningitis, although there were no significant associations between clinical features and viral genomic characteristics.

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.