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.

Developing a one-step triplet-repeat primed PCR assay for diagnosingmyotonic dystrophy

Myotonic dystrophy type 1 (DM1),or Steiner’s disease,is an autosomal dominant disorder caused by the expansion of unstable trinucleotide repeats (CTG) in the 3’ untranslated region of the myotonic dystrophy protein kinase gene (DMPK) (Brook et al.,1992;Mahadevan et al.,1992).The number of CTG repeats observed in normal individuals is in a range of 5-34,while the individuals with 35-49 CTG repeats are usually asymptomatic but at risk of

Combination of Loop-Mediated Isothermal Amplification Assay and Nested PCR for Detection of Borrelia burgdorferi sensu lato in Human Serum Samples

A set of universal loop-mediated isothermal amplification （LAMP） primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato （B. burgdorferi s.I.） in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.I. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.I. in human serum samples.

Feasibility of the Routine Clinical Use of a Multiplex Virus Polymerase Chain Reaction Assay Based on Blood Virus Detection in Hematopoietic Stem Cell-Transplanted Patients

Background: Multiplex virus assays are useful in immunocompromised hosts but still challenging in routine clinical settings in terms of their sensitivity, specificity, reproducibility, and time and cost performances. In recent years, we developed a qualitative multiplex virus PCR assay capable of the simultaneous detection of 13 virus species within 3 h. However, because of the multiple and concomitant nature of this virus assay, it should be validated for qualitative reliability. Materials and Methods: As a preclinical examination, this multiplex PCR was able to detect 1.25 × 103 copies/mL of 13 synthesized virus genomes and preserved same virus DNAs by the serial dilution method. Blood samples from 40 patients who underwent hematopoietic stem cell transplantation were then examined by multiplex PCR for 13 virus species, followed by quantitative real-time PCR for all 13 virus species as reference PCR when these patients developed symptoms suggestive of viral infection. Results: In 421 cumulative qualitative-quantitative tests, the multiplex PCR certainly detected 1.0 × 103 copies/mL of 5 viruses (CMV, JCV, BKV, HHV-6, ADV) that were frequently detected and thus reasonably analyzed. The positive and negative predictive values of multiplex PCR were 84.2% - 93.3% and 90.7% - 99.0%, respectively, and sensitivity and specificity were 59.0% - 83.3% and 97.2% - 99.2%, respectively, for these 5 viruses. Conclusion: From these performances, the multiplex PCR assay may be acceptable in a routine clinical laboratory setting.

Identified Bacteria and Virus in the Cerebrospinal Fluid of under Five Years Hospitalized Children for Clinical Meningitis at Panzi Hospital in the Eastern Part of DRC

Background: Meningitis remains a leading cause of death among children below 5 years of age in the Democratic Republic of the Congo (DR Congo). Distinguishing children with bacterial meningitis from those with viral meningitis in the emergency department is sometimes difficult. Here we identified bacteria and virus in the cerebral spinal fluid (CSF) of children with meningitis. Material and Methods: This is a prospective, analytical study carried out in the Pediatrics department of Panzi Hospital in the South-Kivu province of DR Congo. Between April 2021 and March 2022, 150 of 251 collected CSF from children aged from 1 to 59 months hospitalised due to clinical meningitis at Panzi referral university hospital, Bukavu, Eastern DR Congo were sent to the Lancet laboratory for bacteria identification by a multiplex real-time PCR assay for detection of the most different viruses and bacterial species causing meningitis. Result: The used multiplex real-time PCR assay allowed us to identify germs in 24.7% of cases (37/150). We isolated bacteria in 25/37 (67.5%) cases, and viruses in 9/37 (24.3%) while virus and bacteria co-infection was detected in 3/37 (8.1%). The most frequently identified bacteria were Streptococcus pneumoniae 14/37 (37.8%) followed by Haemophilus influenzae 6/37 (16.2%). The main virus was cytomegalovirus 5/37 (3.5%). Despite the age, the most found bacterial are common in children from rural areas and unvaccinated children. Bacterial and virus co-infection were identified in 66.7% of children aged between 25 - 60 months, mainly among male children, and in all children from rural areas (100%). The overall case fatality rate was 30% and was very high among cases with co-infection CMV-Pneumococcal (66.7%), followed by Streptococcus pneumoniae (50%). Conclusion: Meningitis remains frequent among children aged from one to 59 months among Bukavu Infants. We noticed that, Children with co-infection with bacteria and viruses might need higher attention when having meningitis symptoms, as this could lead to fatal outcomes. The introduction of molecular techniques, such as multiplex real-time PCR, has the potential to improve diagnosis and patient outcomes.

A multiplex microsatellite PCR method for evaluating genetic diversity in grass carp(Ctenopharyngodon idellus)

Grass carp is one of the most important cultured fishes all over the world.The genetic diversity of grass carp plays an important role whatever in selective breeding progress or ecological conservation purposes.However,some genetic diversity researches were not accuracy and cannot be compared with each other due to different molecular markers,sample size and detection methods.In this study,we constructed five multiplex PCR assays contained 20 microsatellites with highly polymorphism and heterozygosity for evaluating genetic information of grass carp.We used nine cultured populations consisting of 507 individuals to detect stability of the five multiplex PCR assays.The results showed that the number of alleles(Na),effective number of alleles(Ne),observed heterozygosity(Ho),expected heterozygosity(He)and polymorphism information content(PIC)of the 20 microsatellite loci were relative high compared with the genetic diversity parameters of microsatellite loci developed by other researchers.Six loci were significantly deviated from Hardy-Weinberg equilibrium(P<0.01).And with exception of the Shaoguan,Indian and Nepal cultured population,all other cultured populations showed very high genetic diversity.Through the test of grass carp populations,we developed an effective and accurate multiplex SSR-PCR assays that can be as statistical powerful tool for detecting genetic information of grass carp.

A Multicenter Study of Viral Aetiology of Community-Acquired Pneumonia in Hospitalized Children in Chinese Mainland

Community-acquired pneumonia(CAP) is one of the leading causes of morbidity and mortality in children worldwide.In this study,we aimed to describe the aetiology of viral infection of pediatric CAP in Chinese mainland.During November2014 to June 2016,the prospective study was conducted in 13 hospitals.The hospitalized children under 18 years old who met the criteria for CAP were enrolled.The throat swabs or nasopharyngeal aspirates(NPAs) were collected which were then screened 18 respiratory viruses using multiplex PCR assay.Viral pathogens were present in 56.6%(1539/2721) of the enrolled cases,with the detection rate of single virus in 39.8% of the cases and multiple viruses in 16.8% of the cases.The most frequently detected virus was respiratory syncytial virus(RSV)(15.2%,414/2721).The highest detection rate of virus was in <6-month-age group(70.7%,292/413).RSV,human metapneumovirus(HMPV),human parainfluenza viruses(HPIVs) and influenza B virus(Flu B) showed the similar prevalence patterns both in north and south China,but HPIVs,Flu A,human bocavirus(HBoV),human adenovirus(HAdV) and human coronaviruses(HCoVs) showed the distinct circulating patterns in north and south China.Human enterovirus/human rhinovirus(HEV/HRV)(27.6%,27/98),HBoV(18.4%,18/98),RSV(16.3%,16/98) and HMPV(14.3%,14/98) were the most commonly detected viruses in severe pneumonia cases with single virus infection.In conclusion,viral pathogens are frequently detected in pediatric CAP cases and may therefore play a vital role in the aetiology of CAP.RSV was the most important virus in hospitalized children with CAP in Chinese mainland.

Estimating the prevalence of Echinococcus in domestic dogs in highly endemic for echinococcosis

Background:Cystic echinococcosis(CE)and alveolar echinococcosis(AE)are highly endemic in Xiji County of Ningxia Hui Autonomous Region(NHAR)in China where the control campaign based on dog de-worming with praziquantel has been undertaken over preceding decades.This study is to determine the current prevalence of Echinococcus granulosus and E.multilocularis in domestic dogs and monitor the echinococcosis transmission dynamics.Methods:Study villages were selected using landscape patterns(Geographic Information System,GIS)for Echinococcus transmission“hot spots”,combined with hospital records identifying risk areas for AE and CE.A survey of 750 domestic dogs,including copro-sampling and owner questionnaires,from 25 selected villages,was undertaken in 2012.A copro-multiplex PCR assay was used for the specific diagnosis of E.granulosus and E.multilocularis in the dogs.Data analysis,using IBM SPSS Statistics,was undertaken,to compare the prevalence of the two Echinococcus spp.in dogs between four geographical areas of Xiji by theχ^(2)test.Univariate analysis of the combinations of outcomes from the questionnaire and copro-PCR assay data was carried out to determine the significant risk factors for dog infection.Results:The highest de-worming rate of 84.0%was found in the northwest area of Xiji County,and significant differences(P<0.05)in the de-worming rates among dogs from the four geographical areas of Xiji were detected.The highest prevalence(19.7%,59/300)of E.multilocularis occurred in northwest Xiji,though the highest prevalence(18.1%,38/210)of E.granulosus occurred in southwest Xiji.There was no significant difference(P>0.05)in the prevalence of E.granulosus in dogs from the northwest,southwest,northeast,and southeast of Xiji,but there were significant differences(P<0.05)between dogs infected with E.multilocularis from the four areas.None of the other independent variables was statistically significant.Conclusions:The results from this study indicate a high prevalence of both E.granulosus and E.muiltilocularis in dogs in Xiji County,NHAR.Transmission of E.multilocularis was more impacted by geographical risk-factors in Xiji County than that of E.granulosus.Dogs have the potential to maintain the transmission of both species of Echinococcus within local Xiji communities,and the current praziquantel dosing of dogs appears to be ineffective or poorly implemented in this area.

Effect of human rhinovirus infection in pediatric patients with influenza-like illness on the 2009 pandemic influenza A（H1N1） virus

Background Some research groups have hypothesized that human rhinoviruses （HRVs） delayed the circulation of the 2009 pandemic influenza A（H1N1） virus （A（H1N1）pdm09） at the beginning of Autumn 2009 in France.This study aimed to evaluate the relationship between HRV and A（H1N1）pdm09 in pediatric patients with influenza-like illness in Beijing,China.Methods A systematic analysis to detect A（H1N1）pdm09 and seasonal influenza A virus （FLU A） was performed on 4 349 clinical samples from pediatric patients with influenza-like illness during the period June 1,2009 to February 28,2010,while a one-step real-time RT-PCR （rRT-PCR） assay was used to detect HRV in 1 146 clinical specimens selected from those 4 349 specimens.Results During the survey period,only one wave of A（H1N1）pdm09 was observed.The percentage of positive cases for A（H1N1）pdm09 increased sharply in September with a peak in November 2009 and then declined in February 2010.Data on the monthly distribution of HRVs indicated that more HRV-positive samples were detected in September （2.2％） and October （3.3％）,revealing that the peak of HRV infection in 2009 was similar to that of other years.Among the 1 146 specimens examined for HRVs,21 （1.8％） were HRV-positive,which was significantly lower than that reported previously in Beijing （15.4％ to 19.2％） （P ＜0.01）.Overall,6 samples were positive for both A（H1N1）pdm09 and HRV,which represented a positive relative frequency of 1.60％ and 2.08％ HRV,considering the A（H1N1）pdm09-positive and-negative specimens,respectively.The odds ratio was 0.87 （95％ CI 0.32; 2.44,P=0.80）.Conclusions HRVs and A （H1N1）pdm09 co-circulated in this Chinese population during September and October 2009,and the HRV epidemic in 2009 did not affect A（H1N1）pdm09 infection rates in Beijing,China as suggested by other studies.However,the presence of A（H1N1）pdm09 might explain the unexpected reduction in the percentage of HRV positive cases during the period studied.

BRACO-19和TMPyP4在HBV-1.3mer质粒感染模型对HBV转录和复制的抑制作用

目的探讨G-四链体小分子配体BRACO-19和TMPyP4对乙型肝炎病毒转录和复制的影响。方法利用含有HBV全基因组1.3倍复制子(HBV 1.3-mer)质粒转染HepG2细胞感染模型,BRACO-19和TMPyP4分别作用48 h,酶联免疫吸附试验检测上清表面抗原(HBsAg)和e抗原(HBeAg)表达水平,实时荧光定量PCR检测细胞内HBV Total RNA、3.5kbRNA以及DNA表达水平,将HBV DNA用plasmid SafeTMATP-Dependent DNase 37℃作用3 h后,定量PCR检测病毒cccDNA的表达;QGRS Mapper软件分析HBV推定的四链体形成序列(putative quadruplex-forming sequences,PQS),并在HBV 1.3-mer质粒DNA上设计PQS区域上下游引物及非PQS区域对照引物,两种小分子配体分别作用HBV 1.3-mer质粒DNA,qPCR stop试验分别定量PQS和非PQS区域DNA扩增水平,1%琼脂糖凝胶电泳可视化验证。结果20μmol/L BRACO-19和TMPyP4作用HepG2转染模型显著抑制HBsAg和HBeAg的表达水平,HBV的Total RNA、3.5 kb RNA、DNA和cccDNA水平相较于对照组均显著下调。通过不同浓度BRACO-19和TMPyP4作用HBV质粒DNA,qPCR stop试验发现随着BRACO-19和TMPyP4药物浓度递增可以显著抑制DNA聚合酶在基因组中三个HBV PQS区域的扩增。结论小分子配体BRACO-19和TMPyP4均能抑制HBV转染模型中的转录和复制,HBV基因存在富含鸟嘌呤的四链体形成序列,BRACO-19和TMPyP4特异性阻滞DNA聚合酶在HBV的PQS区域复制延伸。