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.

Identification of Mixtures of Two Types of Body Fluids Using the Multiplex Methylation System and Random Forest Models

Objective Body fluid mixtures are complex biological samples that frequently occur in crime scenes,and can provide important clues for criminal case analysis.DNA methylation assay has been applied in the identification of human body fluids,and has exhibited excellent performance in predicting single-source body fluids.The present study aims to develop a methylation SNaPshot multiplex system for body fluid identification,and accurately predict the mixture samples.In addition,the value of DNA methylation in the prediction of body fluid mixtures was further explored.Methods In the present study,420 samples of body fluid mixtures and 250 samples of single body fluids were tested using an optimized multiplex methylation system.Each kind of body fluid sample presented the specific methylation profiles of the 10 markers.Results Significant differences in methylation levels were observed between the mixtures and single body fluids.For all kinds of mixtures,the Spearman’s correlation analysis revealed a significantly strong correlation between the methylation levels and component proportions(1:20,1:10,1:5,1:1,5:1,10:1 and 20:1).Two random forest classification models were trained for the prediction of mixture types and the prediction of the mixture proportion of 2 components,based on the methylation levels of 10 markers.For the mixture prediction,Model-1 presented outstanding prediction accuracy,which reached up to 99.3%in 427 training samples,and had a remarkable accuracy of 100%in 243 independent test samples.For the mixture proportion prediction,Model-2 demonstrated an excellent accuracy of 98.8%in 252 training samples,and 98.2%in 168 independent test samples.The total prediction accuracy reached 99.3%for body fluid mixtures and 98.6%for the mixture proportions.Conclusion These results indicate the excellent capability and powerful value of the multiplex methylation system in the identification of forensic body fluid mixtures.

New serological biomarkers of inflammatory bowel disease

Serological biomarkers in inflammatory bowel disease (IBD) are a rapidly expanding list of non-invasive tests for objective assessments of disease activity, early diagnosis, prognosis evaluation and surveillance. This review summarizes both old and new biomarkers in IBD, but focuses on the development and character-ization of new serological biomarkers (identifi ed since 2007). These include fi ve new anti-glycan antibodies, anti-chitobioside IgA (ACCA), anti-laminaribioside IgG (ALCA), anti-manobioside IgG (AMCA), and antibod-ies against chemically synthesized (∑) two major oligomannose epitopes, Man α-1,3 Man α-1,2 Man (∑Man3) and Man α-1,3 Man α-1,2 Man α-1,2 Man (∑Man4). These new biomarkers serve as valuable complementary tools to existing biomarkers not only in differentiating Crohn's disease (CD), ulcerative colitis (UC), normal and other non-IBD gut diseases, but also in predicting disease involvement (ileum vs colon), IBD risk (as subclinical biomarkers), and disease course (risk of complication and surgery). Interestingly, the prevalence of the antiglycan antibodies, including anti-Saccharomyces cerevisiae antibodies (ASCA), ALCA and AMCA, was found to be associated with single nucleotide polymorphisms (SNPs) of IBD susceptible genes such as NOD2/CARD15, NOD1/CARD4, toll-likereceptors (TLR) 2 and 4, and β-defensin-1. Further-more, a gene dosage effect was observed: anti-glycan positivity became more frequent as the number of NOD2/CARD15 SNPS increased. Other new serum/ plasma IBD biomarkers reviewed include ubiquitination factor E4A (UBE4A), CXCL16 (a chemokine), resistin, and apolipoprotein A-IV. This review also discusses the most recent studies in IBD biomarker discovery by the application of new technologies such as proteomics, fourier transform near-infrared spectroscopy, and mul-tiplex enzyme-linked immunosorbent assay (ELISA)'s (with an emphasis on cytokine/chemokine profiling). Finally, the prospects of developing more clinically use-ful novel diagnostic algorithms by incorporating new technologies in serological biomarker profiling and integrating multiple biomarkers with bioinformatics analysis/modeling are also discussed.

Multiplexed micro RNA TG-FRET assay with isothermal and amplification-free single-step

Human genome encodes more than 2000 distinct micro RNAs(Micro RNA database:http://www.mirbase.org/),a class of single-strand noncoding RNAs with a short length of 19 to 25 nucleotides[1].They take part as regulatory factors in almost all biological processes from cell proliferation,differentiation to death.Since the alternation in the micro RNA expression level can be associated with a num-

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.

Point-of-Care Immunoassays with Tunable Detection Range for Detecting Infection in Intensive Care Unit

One of the difficulties of a multiplexed analytical assay is matching the concentration range of different markers.Here,the authors develop an automatic,multiplexed point-of-care(POC)immunoassay that allows detecting C-reactive protein(CRP),procalcitonin(PCT),and interleukin 6(IL-6)in 50μL serum samples with limits of detection 1.87μg/mL,0.17 ng/mL,and 49.75 pg/mL,respectively.The authors use electrospun fibers and surface chemistry of gold nanoparticles(AuNPs)to adjust the detection range of different biomarkers.The authors have proposed some new diagnostic indicators(mScore and mScorePlus)that combine the results of CRP,PCT,IL-6,and complete blood counts(CBCs).The authors also tracked changes in CRP,PCT,and IL-6 of patients in an intensive care unit(ICU).The authors find that mScore and mScorePlus have advantages in improving the diagnostic accuracy and providing more analytical information.mScore and mScorePlus are effective tools to detect infections,differentiate bacterial and viral infections,and monitor disease.Integrating multiple markers into one straightforward parameter is an effective method for analytical applications.The authors believe that this method provides a general way for chemists to develop increasingly accurate detection methods and indicators.

Twenty-seven continental ancestry-informative SNP analysis of bone remains to resolve a forensic case

We employed our previously developed 27-plex ancestry-informative single nucleotide polymorphism(SNP)panel to infer the ancestral components of bone remains of a possible foreign pilot found in south-western China.For ancestry assignment of this unknown individual,we first obtained the 27-SNP genotype of the individual.Then,based on a reference database of 3081 individuals from 33 populations,we calculated the match probability and likelihood ratio using the self-developed software program Forensic Intelligence.Inferred ancestral components of this individual were calculated by structure at K=3.A complete profile was obtained for the individual using our multiplexed SNP assay.The European population was within one order of magnitude of the highest likelihood.The major ancestral component of this individual was 97.6%European.

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.