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.

Epidemiological Characteristics of Notifiable Infectious Diseases among Foreign Cases in China,2004–2017

Objective We aimed to assess the features of notifiable infectious diseases found commonly in foreign nationals in China between 2004 and 2017 to improve public health policy and responses for infectious diseases.Methods We performed a descriptive study of notifiable infectious diseases among foreigners reported from 2004 to 2017 in China using data from the Chinese National Notifiable Infectious Disease Reporting System(NNIDRIS). Demographic, temporal-spatial distribution were described and analyzed.Results A total of 67,939 cases of 33 different infectious diseases were reported among foreigners.These diseases were seen in 31 provinces of China and originated from 146 countries of the world. The infectious diseases with the highest incidence number were human immunodeficiency virus(HIV) of18,713 cases, hepatitis B(6,461 cases), hand, foot, and mouth disease(6,327 cases). Yunnan province had the highest number of notifiable infectious diseases in foreigners. There were different trends of the major infectious diseases among foreign cases seen in China and varied among provinces.Conclusions This is the first description of the epidemiological characteristic of notifiable infectious diseases among foreigners in China from 2004 to 2017. These data can be used to better inform policymakers about national health priorities for future research and control strategies.

A Core Genome Multilocus Sequence Typing Scheme for Proteus mirabilis

Objective A core genome multilocus sequence typing(cgMLST)scheme to genotype and identify potential risk clonal groups(CGs)in Proteus mirabilis.Methods In this work,we propose a publicly available cgMLST scheme for P.mirabilis using chew BBACA.In total 72 complete P.mirabilis genomes,representing the diversity of this species,were used to set up a cgMLST scheme targeting 1,842 genes,635 unfinished(contig,chromosome,and scaffold)genomes were used for its validation.Results We identified a total of 205 CGs from 695 P.mirabilis strains with regional distribution characteristics.Of these,159 unique CGs were distributed in 16 countries.CG20 and CG3 carried large numbers of shared and unique antibiotic resistance genes.Nine virulence genes(papC,papD,papE,papF,papG,papH,papI,papJ,and papK)related to the P fimbrial operon that cause severe urinary tract infections were only found in CG20.These CGs require attention due to potential risks.Conclusion This research innovatively performs high-resolution molecular typing of P.mirabilis using whole-genome sequencing technology combined with a bioinformatics pipeline(chewBBACA).We found that the CGs of P.mirabilis showed regional distribution differences.We expect that our research will contribute to the establishment of cgMLST for P.mirabilis.

Protein Containing the GGDEF Domain Affects Motility and Biofilm Formation in Vibrio cholerae and is Negatively Regulated by Fur and HapR

Objective This study aimed to investigate whether the VCA0560 gene acts as an active diguanylate cyclase(DGC)in Vibrio cholerae and how its transcription is regulated by Fur and Hap R.Methods The roles of VCA0560 was investigated by utilizing various phenotypic assays,including colony morphological characterization,crystal violet staining,Cyclic di-GMP(c-di-GMP)quantification,and swimming motility assay.The regulation of the VCA0560 gene by Fur and Hap R was analyzed by luminescence assay,electrophoretic mobility shift assay,and DNase I footprinting.Results VCA0560 gene mutation did not affect biofilm formation,motility,and c-di-GMP synthesis in V.cholerae,and its overexpression remarkably enhanced biofilm formation and intracellular c-di-GMP level but reduced motility capacity.The transcription of the VCA0560 gene was directly repressed by Fur and the master quorum sensing regulator Hap R.Conclusion Overexpressed VCA0560 functions as an active DGC in V.cholerae,and its transcription is repressed by Fur and Hap R.

Species-level Microbiota of Biting Midges and Ticks from Poyang Lake

Objective The purpose of this study was to investigate the bacterial communities of biting midges and ticks collected from three sites in the Poyang Lake area,namely,Qunlu Practice Base,Peach Blossom Garden,and Huangtong Animal Husbandry,and whether vectors carry any bacterial pathogens that may cause diseases to humans,to provide scientific basis for prospective pathogen discovery and disease prevention and control.Methods Using a metataxonomics approach in concert with full-length 16S rRNA gene sequencing and operational phylogenetic unit(OPU)analysis,we characterized the species-level microbial community structure of two important vector species,biting midges and ticks,including 33 arthropod samples comprising 3,885 individuals,collected around Poyang Lake.Results A total of 662 OPUs were classified in biting midges,including 195 known species and 373 potentially new species,and 618 OPUs were classified in ticks,including 217 known species and 326 potentially new species.Surprisingly,OPUs with potentially pathogenicity were detected in both arthropod vectors,with 66 known species of biting midges reported to carry potential pathogens,including Asaia lannensis and Rickettsia bellii,compared to 50 in ticks,such as Acinetobacter lwoffii and Staphylococcus sciuri.We found that Proteobacteria was the most dominant group in both midges and ticks.Furthermore,the outcomes demonstrated that the microbiota of midges and ticks tend to be governed by a few highly abundant bacteria.Pantoea sp7 was predominant in biting midges,while Coxiella sp1 was enriched in ticks.Meanwhile,Coxiella spp.,which may be essential for the survival of Haemaphysalis longicornis Neumann,were detected in all tick samples.The identification of dominant species and pathogens of biting midges and ticks in this study serves to broaden our knowledge associated to microbes of arthropod vectors.Conclusion Biting midges and ticks carry large numbers of known and potentially novel bacteria,and carry a wide range of potentially pathogenic bacteria,which may pose a risk of infection to humans and animals.The microbial communities of midges and ticks tend to be dominated by a few highly abundant bacteria.

Advances in the Application of Molecular Diagnostic Techniques to Brucellosis

Brucellosis is a zoonotic infectious and allergic disease caused by Brucella bacteria.Brucellosis occurs worldwide and has had a huge economic impact on the livestock industry in many countries and regions.It has become a major public health problem.Brucella is an endoparasitic,non-motile Gram-negative bacterium capable of surviving within a diverse range of domestic animal hosts.

Molecular Investigation of Theileria in Ixodid Ticks from Changzhi,Shanxi Province,China

Theileria is a tick-borne intracellular protozoan that typically parasitizes erythrocytes,lymphocytes,and macrophages and causes acute clinical symptoms in host animals,including high fever,anemia,jaundice,and swelling of superficial lymph nodes[1].Theileriosis can lead to high mortality rates,and its rising global prevalence has resulted in significant economic losses to livestock husbandry and the national economy[2].

Seven-day triple therapy is a better choice for Helicobacter pylori eradication in regions with low antibiotic resistance

AIM: To investigate whether 7-d triple therapies are still valid in populations with low levels of resistance.METHODS: A total of 1106 Helicobacter pylori(H. pylori)-positive patients were divided into three groups,each of which received one type of 7-d triple therapy. Therapeutic outcomes of the patients were assessed by the 13C-urea breath test at 8 wk after treatment. The susceptibility of H. pylori to antibiotics was determined by an agar-dilution method. Data analysis was performed by χ2 tests.RESULTS: The eradication rates in groups A,B and C were 90.71%(332/366),90.46%(313/346) and 90.87%(189/208),respectively(P = 0.986). The resistance rates were 8.91% for clarithromycin,14.78% for levofloxacin and 0% for amoxicillin. The eradication rate was significantly different between clarithromycin-and levofloxacin-resistant patients(P < 0.05) in group A. Patients whose treatment failed in group A also had a higher clarithromycin resistance rate than did successive patients(P = 0.034). However,levofloxacin resistance had no obvious influence on the eradication rate. Furthermore,three main antibiotics(clarithromycin,levofloxacin and amoxicillin) had lower DID(defined daily dose per 1000 inhabitants per day) in this city.CONCLUSION: Clarithromycin resistance is the main reason for the failure of 7-d triple therapy. In populations with low levels of resistance,a 7-d triple therapy is a viable choice. The choice of therapy should not be influenced by conditions in high antibiotic resistance regions.

Seroepidemiological Investigation of Lyme Disease and Human Granulocytic Anaplasmosis among People Living in Forest Areas of Eight Provinces in China

Objective Lyme disease and Human granulocytic anaplasmosis are tick-borne diseases caused by Borrelia burgdorferi and Anoplasrna phagocytophilum respectively. We have investigated infection and co-infection of the two diseases in the population of forest areas of eight provinces in China by measuring seroprevalence of antibodies against B. burgdorferi and A. phagocytophilum. Methods Forest areas in 8 provinces were chosen for investigation using whole sampling and questionnaire survey methods. 3 669 serum samples from people in the forest areas were tested for the presence of antibodies by indirect immunofluorescent assay （IFA）. Results Seroprevalence against B. burgdorferi was 3% to 15% and against A. phagocytophilum was 2% to 18% in the study sites in the 8 provinces in China. We also found co-infection of B. burgdorferi and A. phagocytophilum in 7 of the 8 provinces （the exception being the Miyun area in Beijing）. The seroprevalence for both B. burgdorferi and A. phagocytophilum was significantly higher among people exposed to ticks than among people who were not exposed to ticks. Conclusion We conclude that both pathogens are endemic in the forest areas in the eight provinces, but the prevalence of B. burgdorferi and A. phagocytophilum differs between the provinces.

A Study of the Technique of Western Blot for Diagnosis of Lyme Disease caused by Borrelia afzelii in China

Objective To study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure. Methods FP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and irnmunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples （159 patients with Lyme disease and 292 controls）, all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index. Results Criteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively.

Development and application of a real-time polymerase chain reaction method for Campylobacter jejuni detection

AIM:To develop a real-time polymerase chain reaction(PCR) method to detect and quantify Campylobacter jejuni(C.jejuni) from stool specimens.METHODS:Primers and a probe for real-time PCR were designed based on the specific DNA sequence of the hipO gene in C.jejuni.The specificity of the primers and probe were tested against a set of Campylobacter spp.and other enteric pathogens.The optimal PCR conditions were determined by testing a series of conditions with standard a C.jejuni template.The detection limits were obtained using purified DNA from bacterial culture and extracted DNA from the stool specimen.Two hundred and forty-two specimens were analyzed for the presence of C.jejuni by direct bacterial culture and real-time PCR.RESULTS:The optimal PCR system was determined using reference DNA templates,1 × uracil-DNA glycosylase,3.5 mmol/L MgCl 2,1.25 U platinum Taq polymerase,0.4 mmol/L PCR nucleotide mix,0.48 μmol/L of each primer,0.2 μmol/L of probe and 2 μL of DNA template in a final volume of 25 μL.The PCR reaction was carried as follows:95 ℃ for 4 min,followed by 45 cycles of 10 s at 95 ℃ and 30 s at 59 ℃.The detection limit was 4.3 CFU/mL using purified DNA from bacterial culture and 10 3 CFU/g using DNA from stool specimens.Twenty(8.3%,20/242) C.jejuni strains were isolated from bacterial culture,while 41(16.9%,41/242) samples were found to be positive by realtime PCR.DNA sequencing of the PCR product indicated the presence of C.jejuni in the specimen.One mixed infection of C.jejuni and Salmonella was detected in one specimen and the PCR test for this specimen was positive.CONCLUSION:The sensitivity of detection of C.jejuni from stool specimens was much higher using this PCR assay than using the direct culture method.

Evaluation of a New Real-time PCR Assay for Detection of Mycoplasma Pneumoniae in Clinical Specimens

Objective To establish and evaluate a real-time PCR assay to detect Mycoplasma pneumoniae （M.pneumoniae） in clinical specimens. Methods By analysing the whole pl gene sequence of 60 M.pneurnoniae clinical isolates in Beijing of China, an optimized real-time PCR assay （MpP1） using pl gene conserved region was designed. The specificity and sensitivity of this assay were evaluated and compared with other two reported assays （RepMpl and Mp181） using 40 positive and 100 negative clinical specimens. Results The detection limit of the new assay was 8.1 fg （about 1-3CFU） M.pneumoniae DNA. The sensitivity of MpP1, RepMpl, and Mp181 assays appeared to be 100%, 100%, and 85%, respectively. Conclusion MpP1 assay is suitable for the detection of M.pneumoniae in Chinese clinical specimens.

MicroRNA-135a in ABCA1-labeled Exosome is a Serum Biomarker Candidate for Alzheimer’s Disease

Objective In the present study,the ABCA1 was used as a label to capture specific exosomes,the level of ABCA1-labeled exosomal microRNA-135 a(miR-135 a)was evaluated for the diagnosis of Alzheimer’s disease(AD),especially in patients with early stages of AD.Methods This is a preliminary research focused on the levels of ABCA1 in WBCs,RBCs,HT-22 cells,and neuron cells.The diagnostic value of ABCA1-labeled exosomal miR-135 a was examined using the CSF and serum of APP/PS1 double transgenic mice,and 152 patients with SCD,131 patients with MCI,198 patients with DAT,and 30 control subjects.Results The level of ABCA1 exosomes harvested from HT-22 cells and neuron culture medium was significantly higher compared to that of RBCs and WBCs(P<0.05).The levels of ABCA1-labeled exosomal miR-135 a increased in the CSF of MCI and DAT group compared to those of control group(P<0.05),slightly increased(P>0.05)in the serum of SCD patient group,and significantly increased in MCI and DAT patient groups compared to those of the control group(P<0.05).Conclusion This study outlines a method to capture specific exosomes and detect them using immunological methods,which is more efficient for early diagnosis of AD.

Evaluation of Multidrug Resistant Loop-mediated Isothermal Amplification Assay for Detecting the Drug Resistance of Mycobacterium tuberculosis

Objective To evaluate multidrug resistant loop-mediated isothermal amplification(MDR-LAMP)assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the rpoB,katG,and inhA genes at the Chinese Center for Disease Control and Prevention.Methods MDR-LAMP assay was evaluated using 100 Mycobacterium tuberculosis(Mtb)isolates obtained from the National Reference Laboratory for Tuberculosis in China.Phenotypic resistance to isoniazid and rifampicin and whole-genome sequencing served as reference standards.Results The sensitivity,specificity,positive predictive value(PPV),and negative predictive value(NPV)of MDR-LAMP were 85.5%,93.6%,96.7%,and 74.4%for the detection of resistance to isoniazid and rifampicin,respectively,and 80.5%,92.3%,98.6%,and 41.4%for the detection of Mtb cultured from smear-positive sputum samples,respectively.When DNA sequencing was used as the reference standard,the sensitivity,specificity,PPV,and NPV of MDR-LAMP were 93.1%,92.3%,97.2%,and 82.8%for the detection of katG and inhA gene mutations,respectively,and 89.1%,88.9%,93.4%,and 81.1%for the detection of rpoB gene mutation,respectively.Conclusion MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of Mtb isolates.

Development of An Enzyme-Linked Immunosorbent Assay for Determination of the Furaltadone Etabolite,3-Amino-5-Morpholinomethyl-2-Oxazolidinone(AMOZ) in Animal Tissues

Objective To determine 3-amino-5-morpholinomethyl-2-oxazolidinone （AMOZ） residues released from protein bound AMOZ in animal tissues. Methods Polyclonal and monoclonal antibodies were produced in this study. A rapid, sensitive, and specific competitive direct enzyme-linked immunosorbent assay （cdELISA） was developed. Results Rabbit polyclonal antibodies were used in the optimized cdELISA method, and exhibited negligible cross-reactivity with other compounds structurally related to AMOZ. The IC50 of the polycional antibody was 0.16 ng/mL The method limit of detection in four different types of animal and fish tissues was less than 0.06 μg/kg. Recoveries ranged from 80% to 220% for fortified samples with the coefficient of variation values less than 15%. The results of the cdELISA method were in good agreement with the results from an established liquid chromatography-tandem mass spectrometry confirmatory method used for AMOZ residues. Conclusion The cdELISA method developed in the present study is a convenient practical tool for screening large numbers of animal and fish tissue samples for the the detection of released protein bound AMOZ residues.

Rapid and Sensitive Chemiluminescent Enzyme Immunoassay for the Determination of Neomycin Residues in Milk

Immunoassays greatly contribute to veterinary drug residue analysis. However, there are few reports on detecting neomycin residues by immunoassay. Here, a rapid and sensitive chemiluminescent enzyme immunoassay （CLIEA） was successfully developed for neomycin residue analysis. CLIEA demonstrated good cross-reactivity for neomycin, and the IC50 value was 2.4 ng/mL in buffer.

Screening Pathogenic Microorganism Standard Strains for Disinfection Efficacy Evaluation

Infectious diseases would always threaten human health,especially emerging infectious diseases(EIDs),such as the coronavirus disease-2019(COVID-19).In 2019,COVID-19 spread rapidly worldwide.On January 30,2020,World Health Organization(WHO)declared its outbreak a public health emergency of international concern.

A New High-throughput Real-time PCR Assay for the Screening of Multiple Antimicrobial Resistance Genes in Broiler Fecal Samples from China

Objective Antimicrobial resistance(AMR)has become a global concern and is especially severe in China.To effectively and reliably provide AMR data,we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology,and screened multiple AMR genes in broiler fecal samples.Methods A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection.A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.Results The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction.The sensitivity rate,specificity rate,positive predictive value,negative predictive value and correct indices were 99.30%,98.08%,95.31%,99.79%,and 0.9755,respectively.Utilizing this assay,we demonstrate that AMR genes are widely spread,with positive detection rates ranging from 0 to 97.07%in 273 broiler fecal samples.bla CTX-M,bla TEM,mcr-1,fex A,cfr,optr A,and int I1 showed over 80%prevalence.The dissemination of AMR genes was distinct between the two farms.Conclusions We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples.The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.

Optimization of Pulse-Field Gel Electrophoresis for Borrelia burgdorferi Subtyping

Objective To optimize the performance of Pulsed-Field Gel Electrophoresis （PFGE） for the comparison of inter-laboratory results and information exchange of Borrelia burgdorferi subtypingo Methods A panel of 34 strains of B. burgdorferi were used to optimize PFGE for subtyping. In order to optimize the electrophoretic parameters （EPs）, all 34 strains of B. burgdorferi were analyzed using four EPs, yielding different Simpson diversity index （D） values and the epidemiological concordance was also evaluated. Results The EP of a switch time of l s to 25 s for13 h and l s to10 s for 6 h produced the highest D value and was declared to be optimal for Mlul and 5mal PFGE of B. burgdorferi. Mlul and Smal were selected as the first and second restriction enzymes for PFGE subtyping of B. burgdorferi according to discrimination and consistency with epidemiological data. Conclusion PFGE can be used as a valuable test for routine genospecies identification of B burgdorferi.

Establishment of Multiple Locus Variable-number Tandem Repeat Analysis Assay for Genotyping of Borrelia burgdorferi sensu lato Detected in China

Objective Human Lyme Borreliosis （LB）, which is caused by Borrefia burgdorferi sensu lato （B. burgdorferi）, has been identified as a major arthropod-borne infectious disease in China. We aimed to develop a multiple locus variable-number tandem repeat （VNTR） analysis （MLVA） assay for the genotyping of Borrelia burgdorJ：eri strains detected in China. Methods B. garinii PBi complete 904.246 kb chromosome and two plasmids （cp26 and Ip54） were screened by using Tandem Repeats Finder program for getting potential VNTR loci, the potential VNTR loci were analyzed and identified with PCR and the VNTR loci data were analyzed and MLVA clustering tree were constrcted by using the categorical coefficient and the unweighted pair-group method with arithmetic means （UPGMA）. Results We identified 5 new VNTR loci through analyzing 47 potential VNTR loci. We used the MLVA protocol to analyse 101 B. burgdorferi strains detected in China and finally identified 51 unique genotypes in 4 major clusters including B. burgdorferi sensu stricto （B.b.s.s）, B. garinii, B. a[zelii, and B. valaisiana, consistent with the current MLSA phylogeny studies. The allele numbers of VNTR-1, VNTR-2, VNTR-3, VNTR-4, and VNTR-5 were 7, 3, 9, 7, and 6. The Hunter-Gaston index （HGI） of five VNTR loci were 0.79, 0.22, 0.77, 0.71, and 0.67, respectively. The combined HGI of five VNTR loci was 0.96. Clustering of the strains of Xinjiang, Inner Mongolia and Heilongjiang was confirmed, and this situation was consistent with the close geographical distribution of those provinces. Conclusion The MLVA protocol esytablished in this study is easy and can show strains＇ phylogenetic relationships to distinguish the strains of Borrelia species. It is useful for further phylogenetic and epidemiological analyses of Borrelia strains.