Objective To explore the T/NK cell-related differentially expressed genes(T/NK-DEGs)related to the prognosis of liver cancer based on the single-cell RNA-seq data,gene expression data and clinical information in the GEO and TCGA databases,and construct the prognostic model of liver cancer.Methods The single-cell RNA-seq data and gene expression matrices of liver cancer were obtained from the GEO database.The TCGA-LIHC cohorts,including mRNA expression data,clinical information,survival infor-mation,and somatic mutation data,were obtained from the TCGA database.Based on the two databases,the prognostic model of liver cancer patients was constructed by the bioinformatics method,and the performance of the model was predicted.Results Two T/NK-DEGs,PTTG1 and UBE2C,were identified to be associated with the prognosis of liver cancer and a prognostic model of liver cancer was constructed based on them.According to the risk score,the patients were divided into the high-risk score group and low-risk score group,in which the patients with high-risk score had a worse prognosis than those with low-risk score.The areas under the receiv-er operating characteristics(ROC)curve(AUCROC)of the prognostic model at 1-year,3-year and 5-year time points were 0.685,0.647 and 0.594,respectively.The higher risk score was correlated with the advanced pathological stage(Ⅰstage,Ⅱstage,andⅢstage)and T-stage(T1,T2,and T3)(P＜0.05).The prognostic model was able to predict the proportion of tumor infiltrating immune cells,and the sensitivity of immunotherapy and chemotherapy drugs in patients with liver cancer.Conclusion The constructed prog-nostic model in this study has an important role in the prediction of individualized survival and clinical treatment response of patients with liver cancer.

Objective To analyze the results of newborn screening for galactocerebrosidase(GALC)gene and enzyme activity and pro-vide a basis for the clinical diagnosis and genetic counseling of Krabbe disease.Methods The dried blood samples on filter paper from 12 744 newborns born at Nanjing Women and Children's Healthcare Hospital from March 18,2022 to December 31,2022 were collect-ed.The pathogenic variant sites of GALC gene were detected using the chip capture next-generation sequencing technology and Sanger sequencing was used for family validation.The tandem mass spectrometry was used to determine the enzyme activity of GALC in dried blood spots.The comparison of GALC enzyme activity between the carriers with GALC variants and negative group was conducted using the independent sample t-test.Results Among the 12 744 newborns,315 were identified as the carriers with GALC variants,with a carrier rate of 1/40.The most common variant was c.1901T＞C(269 cases,1/47),followed by c.1592G＞A(12 cases,1/1 062).Four newborns(P1-P4)were found to have two pathogenic variant sites and 2 cases were homozygous variants of c.1901T＞C.Family validation showed that the two variant sites in the four newborns were inherited from their parents,leading to a diagnosis of Krabbe dis-ease.Meanwhile,the family validation of the potential P2 patient's sister revealed that they had the same genotype,and the diagnosis of Krabbe disease was made.A preliminary threshold of 0.42 μmol/(L·h)for the enzyme activity of GALC detected by the tandem mass spectrometry was established based on 200 newborn samples.The GALC enzyme activities of 4 children with Krabbe disease were 0.74,0.21,0.17,and 0.29 μmol/(L·h),respectively.The GALC enzyme activity of the P2 patient's sister was 0.28 μmol/(L·h).Except for the P1 patient,the GALC enzyme activities of the P2,P3,and P4 patients and the P2 patient's sister were reduced,indica-ting a positive result.Conclusion The carrier rate of pathogenic variants in the GALC gene is relatively high,with the hotspot mutation being c.1901T＞C.The estimated prevalence of Krabbe disease is 1/3 186.The screening strategy using genetic testing as the primary screening and enzyme activity testing as the second screening can effectively improve the diagnostic efficiency of Krabbe disease,provi-ding a reference for the clinical diagnosis and genetic counseling of Krabbe disease.

Objective To investigate the clinical application value of blood routine parameters such as white blood cells(WBC),neu-trophils(NEU),lymphocytes(LYM),monocytes(MON),platelets(PLT),neutrophil to lymphocyte ratio(NLR),and platelet to lymphocyte ratio(PLR)and C-reactive protein(CRP)in children with influenza A(Flu A)virus infection.Methods 317 children with upper respiratory tract symptoms visited Department of Pediatrics,Affiliated Jiangning Hospital of Nanjing Medical University from September 2023 to May 2024 were retrospectively analyzed.According to the six kinds of nucleic acid test results of upper respiratory tract,the children were divided into the Flu A group(n=150)and disease control group(n=167).In addition,97 healthy children who underwent physical examination in our hospital during the same period were selected as the healthy control group.The finger blood of the children was collected for the detection of blood routine parameters and CRP.The Kruskal-Wallis H non-parametric test was used to compare the differences of these parameters among the three groups.The receiver operating characteristic(ROC)curve was drawn,and the area under the ROC curve(AUCROC)was used to evaluate the diagnostic value of each indicator for influenza A virus infection.Results The NLR and PLR in the Flu A group were significantly higher than those in the disease control group and healthy control group,while the levels of WBC and LYM were significantly lower than those in the disease control group and healthy control group(P＜0.05).The level of CRP in the Flu A group was higher than that in the healthy control group but lower than that in the disease control group(P＜0.05).The indicator with the best diagnostic efficacy for influenza A virus infection was LYM,whose AUCROC was 0.79.When the optimal diagnostic threshold was 1.55×109/L,its sensitivity and specificity were 0.620 and 0.837,respectively.Next were PLR and NLR,with AUCROC of 0.735 and 0.723,specificity of 0.754 and 0.826,and sensitivity of 0.693 and 0.553,respectively.The diagnostic efficacy of MON,PLT,and CRP was relatively low.The AUCROC of the combined detection of LYM,NLR,PLR,MON,PLT,and CRP for the diagnosis of influenza A virus infection could increase to 0.838.Conclusion The blood routine parameters of children with influenza A virus infection show a decrease in the level of LYM and an increase in NLR and PLR.The combined detection of LYM,NLR,PLR,MON,PLT,and CRP can improve the accuracy of early diagnosis of influenza A virus infection in children.

Objective To analyze the genomic characteristics of Klebsiella pneumoniae carrying blaNDM-1 and Atlanta subterranean carry-ing blaIMP-4 isolated from the stool sample of the same patient and elucidate their resistance mechanisms.Methods The minimum in-hibitory concentrations(MIC)of the isolated strains were detected by the micro-broth dilution method.The NG-Test CARBA-5 was used to detect the phenotype of carbapenemases.The genomic characteristics of the strains were analyzed by the whole genome sequen-cing(WGS)and bioinformatics analysis.Results The isolated Klebsiella pneumoniae was resistant to most antibiotics.Atlanta subter-ranean was resistant to meropenem and ertapenem,but sensitive to imipenem.The two strains were susceptible to polymyxin,tigecy-cline,amikacin,ciprofloxacin,and aztreonam.The WGS analysis showed that Klebsiella pneumoniae belonged to ST1040 type and car-ried 8 resistance genes,including blaNDM-1,aac(6')-Ib-cr.v2,aadA16,aadA2,qnrS1,arr-3,sul1 and dfrA27,and 4 plasmid repli-cons such as lncR,lncU,lncFIA(Hl1)and lncFIB(K).Atlanta subterranean carried two resistance genes(blaIMP-4 and qnrS1)and 1 plasmid replicon(lncN).The analysis results of virulence factors showed that Klebsiella pneumoniae carried virulence factors such as fimbrium,capsule,and siderophore.Atlanta subterranean carried virulence factors such as fimbrium,flagella,lipopolysaccharides,and endotoxins.Conclusion The Atlanta subterranean producing blaIMP-4 enzyme and Klebsiella pneumoniae producing blaNDM-1 enzyme are isolated from the stool sample of the same patient,and their genomic characteristics are analyzed.The emergence of new carbapen-em-resistant strains poses a huge challenge to clinical treatment and infection prevention and control.The screening and detection of carbapenemase genes should be strengthened.

Objective To develop a high throughput sequencing method for pathogenic microbial metagenomics and verify its detection performance.Methods First,the library construction conditions with different input levels of nucleic acid were studied.Three differ-ent input levels of nucleic acid such as 5 ng,50 ng,and 100 ng,three fragmentation times such as 20 min,25 min,and 30 min,and three amplification cycles such as 7,5,and 3 cycles were tested.The optimal library construction conditions were determined by the li-brary concentration and fragment size.The representative bacteria covering fungi and gram-positive/negative bacteria,including Candi-da albicans,Staphylococcus aureus,Staphylococcus epidermidis,Escherichia coli and Cryptococcus Gattii,were selected.Three different concentrations of mixed bacterial suspensions were prepared at a 1∶1 ratio.Then,106/mL of T-cell suspensions were added to prepare reference samples.The performance of the self-established method(Method 1)was verified by the reference samples,and its consis-tency with Method 2(DNA sample library construction kit combined with probe anchored polymerase chain reaction sequencing method)was compared.Results The optimal library construction conditions of the sequencing method for pathogenic microbial metagenomics were determined as follows:a nucleic acid input of 50 ng,a nucleic acid fragmentation time of 20 min,and 5 amplification cycles for PCR.The positive coincidence rate(accuracy)and negative coincidence rate(specificity)of Method 1 were 100%,and its detection limit was 500 CFU/mL.The correlation coefficients(r)of the reads per million(RPM)in linear samples of various microorganisms were greater than 0.9.The CV of bacterial detection were less than 10%,while those of fungal detection were around 20%.The consis-tency between Method 1 and Method 2 was 100%.Conclusion A sequencing method for pathogenic microbial metagenomics is estab-lished successfully,which can acquire accurate,stable,and reliable results and is suitable for the pathogen detection of alveolar lavage fluid samples.

Objective To detect and identify the differentially expressed tsRNA in the serum of patients with Sj?gren's syndrome(SS),and preliminarily explore their potential application value in the diagnosis of SS.Methods The serum samples from SS patients diagnosed at the Rheumatology Department of Nanjing Drum Tower Hospital and healthy controls(HCs)matched age and gender were collected,and the differentially expressed tsRNA were screened by small RNA sequencing.Fluorescence quantitative real-time PCR(qRT-PCR)was used to valid the sequence data.The receiver operating characteristic(ROC)curve was used to evaluate the diagnos-tic efficiency of tRNA-Gly-GCC-1-1.KEGG and GO analysis were used to predict the potential mechanism of tRNA-GLy-GCC-1-1 in the occurrence and development of SS.Results Compared with HCs(13.34[10.45,18.74]fmol/L),the levels of serum tRNA-Gly-GCC-1-1 in SS patients(8.82[7.26,12.20]fmol/L)were significantly reduced(P＜0.000 1),and its area under the ROC curve(AUCROC)in distinguishing SS patients from HCs was 0.766(95%CI:0.671-0.861).When the cut-off value was 9.97 fmol/L,its sensitivity and specificity were 83.33%and 64.58%,respectively.Bioinformatics analysis showed that serum tRNA-Gly-GCC-1-1 might be involved in the regulation of signaling pathways such as PI3K-Akt.Conclusion Serum tRNA-Gly-GCC-1-1 in SS patients is significantly down-regulated,which has potential clinical application value in assisting the diagnosis of SS.

Objective To investigate the detection rate of Klebsiella pneumoniae(KP)in the intestinal tract of intensive care unit(ICU)patients and community healthy adults,and analyze its virulence genes and drug resistance.Methods Fecal swabs or fecal specimens from ICU patients admitted to the Third Affiliated Hospital of Wenzhou Medical University and community healthy adults from December 2020 to December 2021 were collected for screening KP.The hypermucoviscosity(HM)phenotype was identified by the wire drawing test.The Vitek 2 automatic microbial analysis system and K-B disk diffusion method were used for detecting drug sus-ceptibility.PCR was used to screen extended spectrum β-lactamases(ESBLs),carbapenem resistance genes,9 virulence related genes,including allS,entB,fimH,iutA,kfu,magA,mrkD,rmpA,and ybtS,and 6 highly virulent capsule serotypes such as K1,K2,K5,K20,K54,and K57.Meanwhile,their multilocus sequence typing(MLST)was analyzed.Statistical analysis was conducted using SPSS 23.0 software.Results A total of 448 fecal samples were collected,including 140 from ICU patients and 308 from commu-nity healthy adults.A total of 89 strains of KP were isolated,including 36(25.7%)from ICU patients and 53(17.2%)from commu-nity healthy adults.The detection rate of KP in ICU patients was significantly higher than that in community healthy adults(χ2=4.375,P＜0.05).There were 9 strains(25%)of HMKP in ICU patients and 19 strains(35.8%)in community healthy adults,and there was no significant difference in the detection rate between them(χ2=1.170,P＞0.05).HMKP and KP detected in healthy adults were natu-rally resistant to ampicillin and still highly sensitive to commonly used antibiotics.The KP from ICU patients showed varying degrees of resistance to commonly used drugs.The resistance of the KP from ICU patients to cephalosporins,penicillinase inhibitors,carbapenem,quinolones,amtronam,gentamicin,amicacin and cotrimoxazole except for tobramycin was significantly higher than that from healthy a-dults(all P＜0.05).In addition,18 strains(50%)of KP producing ESBLs were detected,all of which were non-HM phenotypes.A-mong them,6 strains were carbapenem resistant KP(CRKP).Five strains of CRKP carrying KPC enzyme all belonged to ST11 type and 1 carrying NDM enzyme to ST1855 type.A total of 27(30.3%)strains of KP with highly virulent capsule serotypes were detected,mainly K1,K2,and K57,but no K5.There were no significant differences in the detection rates of 9 virulence genes and 5 highly vir-ulent capsule serotypes between ICU patients and healthy adults(all P＞0.05).High-risk ST types were detected in both ICU patients and healthy adults.Conclusion High virulence and high-risk KP strains are detected from the intestinal tract of healthy adults in this area,which increases the risk of infection transferring from community to hospital.In addition,carbapenemase-resistant ST11 is detec-ted from the intestinal tract of ICU patients,so the spread of drug-resistant plasmids should be highly vigilant.

Objective To investigate the risk factors for mortality and bleeding complications in extracorporeal membrane oxygenation(ECMO)treated patients and to evaluate the impact of thrombocytopenia severity on the prognosis of ECMO therapy.Methods A total of 153 patients who received ECMO treatment at Peking University Third Hospital between January 2013 and September 2024 were en-rolled in this study.The patients were divided into death group(n=97)and recovery group(n=56)based on their final outcomes.Additionally,the patients were categorized into bleeding group(n=104)and non-bleeding group(n=49)based on the occurrence of bleeding complications during ECMO.Clinical baseline characteristics and extreme laboratory values during ECMO were compared be-tween groups.Logistic regression was used to analyze the risk factors for mortality and bleeding.The patients were further divided,based on the initial platelet(PLT)values on the day of catheter placement and the lowest platelet count during ECMO,into normal group(PLT≥ 100× 109/L),moderate reduction group[PLT=(50～99)× 109/L],and severe reduction group(PLT＜50× 109/L).Kaplan-Meier analysis was used to compare survival rates among these groups.The patients in the moderate and severe reduction groups were further divided into a platelet transfusion group and a non-transfusion group,and the outcomes and complication rates were com-pared.Results The recovery group had a higher proportion of myocarditis,higher minimum values of PLT,Hb,and Fib,and higher initial PLT values,while the maximum values of lactic dehydrogenase(LDH),total bilirubin(T-Bil),prothrombin time(PT),and procalcitonin(PCT)were lower(all P＜0.05)with significant differences.Logistic regression showed that age and maximum PCT were independent risk factors for mortality(OR=1.025 and 1.015 respectively,all P＜0.05).The bleeding group had longer ECMO dura-tions,more plasma transfusions,lower minimum Hb values,and higher maximum values of WBC,neutrophils(Neu),and APTT(all P＜0.05)with statistical differences.The minimum PLT value,maximum WBC value,and maximum APTT value were independent risk factors for bleeding complications(OR=0.986,1.062,and 1.004 respectively,all P＜0.05).Kaplan-Meier analysis showed that the patients in the severe reduction group had lower survival rates,regardless of whether the grouping was based on initial or minimum platelet counts(all P＜0.05).Platelet transfusion improved the mortality in the severe reduction group(P＜0.05)but had no effect on the moderate reduction group.Conclusion Age and peak value of PCT are the risk factors for mortality in ECMO patients,while mini-mum PLT count,peak value of WBC and APTT are the risk factors for bleeding complications.Early intervention for infection and in-flammation during ECMO may improve the outcome of patients.Severe thrombocytopenia during ECMO therapy increased the risk of mortality,and targeted platelet transfusion may improve the survival of these patients.

Objective To prepare in-house coagulation factor Ⅷ(F Ⅷ)inhibitor-positive control material and evaluate its perform-ance.Methods Frozen plasma samples from hemophilia A patients with positive factor Ⅷ inhibitors were pooled,and diluted with Owren's Veronal Buffer(OVB)to 1 BU/mL of the inhibitor concentration in the mixture,then aliquoted and freeze-stored.The homo-geneity and stability of the in-house quality control material were verified,and its suitability was further assessed through intra-laborato-ry reproducibility among different technologists and inter-laboratory comparisons.Results Twenty-one aliquots were randomly tested for homogeneity assessment,yielding an average of 1.05 BU/mL(range 0.9-1.15 BU/mL),with a standard deviation(SD)of 0.083 and coefficient of variation(CV)of 7.90％.The freshly prepared inhibitor-positive control samples contained a concentration of 1.03 BU/mL.After storage at-80℃ for 24 hours,1 week,1 month,2 months,3 months,4 months,5 months,6 months,7 months,8 months,and 9 months,thawed the samples showed relative deviations of 9％,0％,10％,9％,14％,15％,6％,0％,-10％,-5％,and 2％,respectively.The intra-laboratory CV value from different technologists at this center was 7.28％,and the inter-labora-tory CV across different centers was 18.75％.Conclusion The prepared in-house positive control material of Factor Ⅷ inhibitor ex-hibited adequate uniformity and stability.

Objective To analyze the clinical value of fungus(1,3)-β-D glucan test magnetic particle chemiluminescence immunoas-say(G-CLIA)for diagnosis of invasive fungal disease(IFD).Methods A total of 509 patients with clinically suspected IFD in Qilu Hospital of Shandong University from 1 March to 30 April,2023 were collected.According to the inclusion criteria,the 509 patients were grouped into IFD group(141 patients)and non-IFD group(368 patients).The sensitivity,specificity,accuracy,positive predic-tive value and negative predictive value of G-CLIA were analyzed,and the consistency of the results of G-CLIA with G test colorimetric method,fungal smear microscopy or culture and metagenomics next-generation sequencing(mNGS)was comparatively analyzed.Re-sults The sensitivity and specificity of G-CLIA were 88.65％and 96.47％,respectively,and the positive percentage agreement of G-CLIA with G test colorimetric assay,fungal smear microscopy or culture,and mNGS were 92.19％,75.86％,and 75.00％,respective-ly,and the consistency of G-CLIA with G test colorimetric assay was the highest(kappa value≥ 0.75).Conclusion G-CLIA has high sensitivity and specificity for detecting IFD with excellent diagnostic value.Combined with the fully automated chemiluminescence analy-zer,G-CLIA test is fast and has a high throughput,which provides a new option for the clinical diagnosis of IFD.