OBJECTIVE: To explore the genotype-phenotype correlation in a Chinese family with structural brain abnormalities due to variant of the TUBB gene. METHODS: A family undergoing prenatal diagnosis at Beijing Obstetrics and Gynecology Hospital in October 2024 was selected as the study subject. Clinical data were collected. Amniotic fluid sample was subjected to chromosomal copy number variation sequencing (CNV-seq). Trio whole-exome sequencing (Trio-WES) was carried out on the amniotic fluid and parental blood samples, and candidate variant was verified by Sanger sequencing. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: 2023-KY-076-01). RESULTS: Both prenatal ultrasound and fetal MRI showed deviation of brain midline, unilateral lateral ventriculomegaly, and bilateral gyral asymmetry. Trio-WES revealed that the fetus has harbored a maternally derived heterozygous missense variant of the TUBB gene [NM_178014.4: c.155A>G (p.N52S)]. Sanger sequencing confirmed that the woman and a previously terminated fetus both harbored the same variant. Both the proband and two fetuses exhibited similar neuroimaging abnormalities including midline deviation and asymmetrical gyri. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as likely pathogenic (PM2_Supporting+PS2_Moderate+PS3). CONCLUSION The heterozygous c.155A>G (p.N52S) variant was the TUBB gene probably underlay the pathogenesis of the structural brain abnormalities in this family. Above findings have expanded the phenotypic spectrum associated with the variant and facilitated the prenatal diagnosis for this family.
Humans ; Female ; Pregnancy ; Prenatal Diagnosis ; Tubulin/genetics* ; Adult ; Brain/diagnostic imaging* ; Male ; Pedigree ; DNA Copy Number Variations/genetics* ; Exome Sequencing ; Genetic Association Studies ; Magnetic Resonance Imaging

Objective To analyze the distribution and drug resistance patterns of pathogenic bacteria in bile and blood cultures obtained from patients with biliary stones accompanied by acute biliary tract infection,to evaluate the clinical appropriate-ness of antibiotic use based on drug sensitivity results,and to provide evidence for empirical antibiotic treatment in such patients.Methods The clinical data of 161 patients with biliary calculi complicated by acute biliary tract infection who were admitted to the First People's Hospital of Neijiang from 2017 to 2023 were retrospectively analyzed.The results of microbial culture,drug sensitivity analysis,and patient characteristics were assessed to evaluate the appropriateness of clinical antimicrobial therapy.Results Among the 161 patients with positive cultures,212 strains of pathogenic bacteria were detected.The predominant patho-gens were Escherichia coli,Klebsiella pneumoniae subspecies,and Enterococcus faecium.Age and underlying diseases significantly affected the distribution of Escherichia coli and Klebsiella pneumoniae subspecies.Within the gram-negative bacterial group,Esche-richia coli and Klebsiella pneumoniae subspecies exhibited higher drug resistance to commonly used broad-spectrum penicillin,third-generation cephalosporin and quinolones but lower resistance rates to piperacillin and tazobactam;furthermore,elderly indi-viduals aged ≥65 years showed higher resistance rates to ceftriaxone than those under age 65 while people with drug exposure history had higher ceftazidime resistance rates that were statistically significant.In contrast to Enterococcus faecalis which displayed low antimicrobial resistance rates for most drugs tested in this study,Enterococcus faecium demonstrated high levels of antibiotic resistance;however,both Enterococcus faecalis and Enterococcus faecium exhibited zero-resistance rates against vancomycin and tigecycline although this may be attributed to their small sample size in our study cohort.Finally,we found that empirical anti-in-fective drugs,as well as target anti-infective drugs,were not prescribed rationally among these patients due mainly to inappropriate combinations of antibiotics or incorrect dosages.Conclusions The predominant pathogens in patients with acute biliary tract infection are gram-negative bacteria,Gram-positive bacteria,and fungi;however,the potential involvement of anaerobic bacteria should not be overlooked.Vancomycin exhibits sensitivity against gram-positive bacteria,yet the overall rationality of antibiotic usage remains suboptimal.Enhanced clinical testing for pathogenic microorganisms is imperative in the management of biliary stones accompanied by acute biliary tract infection.In contrast,clinical pharmacists should provide comprehensive training on anti-infective drugs to clinicians to facilitate their judicious selection of antibiotics based on drug sensitivity results and prevent the e-mergence of multidrug-resistant bacteria.

Objective To analyze the value of actin-related protein 2(ACTR2)and DEAD-box RNA helicase 3X-linked(DDX3X)expression in evaluating the severity of Rotavirus gastroenteritis(RVGE)in children.Methods A total of 153 children with rotavirus gastroenteritis admitted to Handan Maternal and Child Health Hospital from September 2022 to September 2024 were selected as the research objects.According to the severity of RVGE,the children with RVGE were divided into mild group(n=60),moderate group(n=71)and severe group(n=22).In addition,60 healthy children were randomly selected as the healthy group.The clinical data of the light group,the medium group and the heavy group were compared.The expression levels of ACTR2 and DDX3X in serum of each group were detected by real-time fluorescence quantitative PCR.ROC curve was used to analyze the evaluation value of combined detection of serum ACTR2 and DDX3X in children with severe RVGE.Results The age of children with RVGE in the severe group was significantly lower than that in the mild group and the moderate group,and the differences were statistically significant(Z=8.307,5.885,all P＜0.001).There were statistically significant differences in dehydration and diarrhea among the three groups(F=9.434,126.080,all P＜0.05).The expression levels of ACTR2(1.20±0.28)and DDX3X(1.22±0.37)in RVGE group were significantly higher than those in control group(1.01±0.02,1.04±0.09),and the differences were statistically significant(t=5.071,3.584,all P＜0.05).The expression levels of ACTR2[1.11±0.23,1.23±0.21,1.42(1.25,1.57)]and DDX3X[1.14±0.22,1.25±0.24,1.32(1.23,1.62)]in the serum of the mild group,the moderate group and the severe group increased in turn,and the differences were statistically significant(Z=27.196,18.013,all P＜0.05).The ROC curve analysis results showed that the combined detection of serum ACTR2 and DDX3X predicted the AUC(95%CI)of critically ill RVGE patients,with higher sensitivity and specificity than the detection of the two alone(Z=2.573,2.101,P=0.014,0.034).Conclusion The expression levels of serum ACTR2 and DDX3X are closely related to the severity of RVGE.The combined detection of the two has a high predictive value for the diagnosis of the severity of RVGE.

Objective:To explore the clinical features and molecular characteristics of myeloproliferative neoplasms (MPNs) patients with NFE2 gene mutations.Methods:Gene targeted sequencing was used to detect NFE2 gene mutation in 723 patients diagnosed with MPNs who were admitted to Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College between April 2021 and June 2023. The association between NFE2 gene mutations and clinical features and molecular characteristics of MPNs patients were retrospectively analyzed.Results:Among 723 patients with MPNs, NFE2 gene mutations were found in 41 cases (5.7%) . NFE2 gene mutations were predominantly frameshift mutations (44.4%) , followed by nonsense mutations (33.3%) . The median number of mutations in patients with NFE2 gene mutations (4 [2,5]) was higher compared to the group without NFE2 gene mutations (2, [1,3]) ( P<0.001) . NFE2 gene mutations frequently co-occurred with mutations in MPL, ATM, PPM1D, and TET1. NFE2 gene mutations were mostly sub-clonal events, with 80.5% occurring after MPNs driver mutations (JAK2, CALR, or MPL) . NFE2 mutations were correlated with older age [median age: 60 (54, 67) years vs 54 (41, 63) years, P=0.001]. Patients with NFE2 gene mutations had a higher incidence of pre-diagnosis thrombosis (39.0% vs 22.0%, P=0.012) and pre-diagnosis arterial thrombosis (36.6% vs 20.4%, P=0.014) . Using a logistic regression analysis model adjusting for age and comorbidities (including chronic infections, malignancies, and autoimmune diseases) , NFE2 gene mutation was identified as an independent determinant of elevated tumor necrosis factor-alpha (TNF-α) ( OR=2.747, 95% CI: 1.143-6.605, P=0.024) , interferon-gamma (IFN-γ) ( OR=2.689, 95% CI: 1.191-6.076, P=0.017) , IL-10 ( OR=3.219, 95% CI: 1.343-7.717, P=0.009) , IL-12P70 ( OR=3.397, 95% CI:1.003-11.508, P=0.049) , IL-17 ( OR=2.284, 95% CI: 1.017-5.127, P=0.045) . In polycythaemia vera (PV) patients with the NFE2 gene mutation, the proportion of those classified as high-risk is notably higher in both the IWG-PV and mutation-enhanced international prognostic systems for PV (MIPSS-PV) (66.7% vs 25.3% for IWG-PV, P=0.033; 22.2% vs 2.0% for MIPSS-PV, P=0.013) . Similarly, for essential thrombocythaemia (ET) patients, the proportion in the high-risk group of the mutation-enhanced international prognostic systems for ET (MIPSS-ET) is significantly higher (15.4% vs 6.1%, P=0.021) . No statistically significant differences were observed in overall survival or cumulative incidence of thrombosis between NFE2-mutated (38 cases) and non-mutated MPNs patients (671 cases, P>0.05) . Conclusion:NFE2 gene mutations in MPNs were predominantly frameshift mutations. NFE2 gene mutations were correlated with older age, elevated levels of several inflammatory factors (including TNF-α、IFN-γ、IL-10、IL-12P70、IL-17) , and they mostly occurred in late-stage of MPNs.

OBJECTIVES: To detect prfA and hly toxin genes of Listeria monocytogenes using polymerase chain reaction (PCR) and colloidal gold technology. METHODS: L. monocytogenes DNA was extracted by boiling method. With prfA and hly of L. monocytogenes as the target genes, the 5' ends of upstream and downstream primers of prfA gene were labeled with 6-FAM and biotin, and the 5' ends of upstream and downstream primers of hly gene were labeled with digoxin and biotin, respectively, to establish the toxin gene detection method. Using cloning transformation, sequencing analysis, cloning of positive control products, the detection kid was developed and its specificity, sensitivity, reproducibility and stability were tested, followed by verification with sample testing. RESULTS: The concentration of L. monocytogenes DNA extracted by boiling method was 148.81±0.97 ng/μL, and the A₂₆₀/A₂₈₀ ratio ranged from 1.8 to 2.0. The PCR products showed a 100% homology with the gene sequences in GenBank database after cloning, transformation and sequencing. The colloidal gold strip yielded positive results only for L. monocytogenes samples without cross-reactions with Staphylococcus aureus, Escherichia coli or Bacillus cereus, and its minimum detection limit was 10^-2 ng/μL, demonstrating a 10-fold greater sensitivity of the test than agarose gel electrophoresis. The test also showed good reproducibility of the results when performed by different operators with good stability of the test strips after storage for 6 to 12 months. The test results showed that this kit could accurately and quickly detect L.monocytogenes in the test samples. CONCLUSIONS The detection kit developed in this study can simultaneously detect prfA and hly toxin genes of L. monocytogenes with good specificity, sensitivity, reproducibility and stability for use in food safety inspection.
Listeria monocytogenes/isolation & purification* ; Gold Colloid ; Bacterial Toxins/genetics* ; Polymerase Chain Reaction/methods* ; Hemolysin Proteins/genetics* ; Bacterial Proteins/genetics* ; DNA, Bacterial/genetics* ; Food Microbiology ; Heat-Shock Proteins

Objective:To establish a method of dual nueleic acid test strips for rapid detection of Bacillus cereus cytK and nhe toxin genes based on polymerase chain reaction(PCR)and colloidal gold technique,and to evaluate its specificity,sensitivity,repeatability and stability.Methods:Bacillus cereus DNA was extracted by boiling method.Specific primers were designed with Bacillus cereus cytK and nhe as the target genes.Clonal transformation was used to identify the PCR products.The optimal labeling amounts of colloidal gold-labeled streptavidin,quality control line(C line),cytK detection line(T1)and nhe detection line(T2)were determined.The nucleic acid test strips were assembled and its specificity,sensitivity,reproducibility and stability were evaluated.Results:The DNA concentration of Bacillus cereus was 248 mg·L-1,and the purities were 1.8-2.0.After cloning and plasmid sequencing,the similarities between the PCR products and the sequences of cytK and nhe registered in the GenBank database were 100％.Under the condition of pH 7.0,the optimal amount of streptavidin labeling per 200 μL of colloidal gold solution was 6.0 μL;the optimal marking amount was 2.00 g·L-1 for the quality control line(C line),0.550 g·L-1 for cytK gene detection line(T1)and 0.2 g·L-1 for nhe gene detection line(T2).In the specificity test,positive result on the test strips was seen only for Bacillus cereus,and no cross-reactivity was observed for Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa and Bacillus subtilis,which were consistent with the electrophoresis results.Sensitivity assay showed that even when DNA concentration was reduced to 10-2 mg·L-1,three bands(C line,T1 line and T2 line)could be observed,and the detection limit of the test strip was one-tenth of agarose gel electrophoresis(10-1 mg·L-1).The nucleic acid test strips were verified by different operators in different laboratories,and the results were consistent.The stability of the test strips was verified at the 6th,9th and 12th months,and the results showed good stability.Conclusion:The dual nucleic acid test strip method established in this study can simutaneously detect the cytK and nhe toxin genes of Bacillus cereus with high sensitivity and specificity,achieving short-term visual detection.

Objective:To establish a rapid detection method for pathogenic microorganisms by combining loop-mediated isothermal amplification(LAMP)and clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 12a(Cas12a)(CRISPR-Cas12a)system,and to evaluate its efficacy for detecting the thermolabile hemolysin(tlh)gene of Vibrio parahaemolyticus(Vp).Methods:Using the tlh gene of Vp as the target gene,LAMP primers and CRISPR RNA(crRNA)were designed to construct and optimize the optimal concentration ratio of each component in the LAMP-CRISPR detection system.Bacillus cereus,Staphylococcus aureus,and Escherichia coli were used as control groups,and the specificity,sensitivity,reproducibility and positive conformity rate were verified to establish a rapid LAMP-CRISPR/Cas12a method for detecting the tlh gene of Vp.Results:The method specifically detected Vp,while Bacillus cereus,Staphylococcus aureus,and Escherichia coli yielded negative results.The DNA extraction concentration of Vp was 190.67 mg·L-1 with an A(260)/(A280)ratio of 1.84.Under the reaction conditions of 37℃ with 80 cycles for 40 min using quantitative PCR(qPCR)method,when the concentrations of Cas12a protein and crRNA in the LAMP-CRISPR/Cas12a system were 50 nmol·L-1,the visual brightness and relative fluorescence intensity peaks were high.The sensitivity of LAMP CRISPR/Cas12a for detecting Vp DNA concentration could reach 10-6 mg·L-1.The reproducibility test results showed that different experimenters had consistent results in different experimental environments and times.Conclusion:The established LAMP-CRISPR/Cas12a method can rapidly detect the tlh gene of Vp with high sensitivity and specificity,and can achieve short-term visual detection in the field.

Objective:To explore the relationship between corrected serum calcium level and clinical characteristics in patients with newly-diagnosed multiple myeloma (MM), as well as the influencing factors of concomitant hypercalcemia.Methods:A retrospective case cohort study was conducted. The clinical data of newly-diagnosed MM patients who were admitted to Shanxi Province Cancer Hospital from January 2020 to December 2023 were collected. When serum albumin (ALB) was less than 40 g/L, the serum calcium level was corrected. The hypercalcemia group was defined as the corrected serum calcium level >2.60 mmol/L, the normal blood calcium group was defined as 2.20-2.60 mmol/L, and the hypocalcemia group was defined as <2.20 mmol/L; the combination of normal blood calcium group and hypocalcemia group was the non-hypercalcemia group. The age, gender, immune typing, ALB, blood urea nitrogen (BUN), serum creatinine (Scr), β 2-microglobulin (β 2-MG), and alkaline phosphatase (ALP) of patients in the hypercalcemia group and the non-hypercalcemia group were compared. Pearson correlation analysis was used for the correlation analysis. The logistic regression model was used to conduct multivariate analysis on the influencing factors of hypercalcemia. Results:Before correction, there were 31 cases (26.5%), 48 cases (40.0%) and 38 cases (32.5%) of MM patients in the hypocalcemia group, normal blood calcium group and hypercalcemia group, respectively. After correction, there were 17 cases (14.5%), 58 cases (49.6%) and 42 cases (35.9%), respectively. The corrected serum calcium level for male MM patients was (2.58± 0.41) mmol/L, while for females it was (2.53±0.38) mmol/L, and there was no statistically significant difference between the two groups ( t = 0.79, P > 0.05). The corrected serum calcium levels for patients in stage Ⅰ, stage Ⅱ and stage Ⅲ of the International Staging System (ISS) were (2.31±0.15) mmol/L, (2.52±0.37) mmol/L and (2.65±0.42) mmol/L, respectively, and the difference between the three groups was statistically significant ( F = 53.62, P < 0.01). Corrected serum calcium showed a negative correlation with patients' serum ALB ( r = -0.201, P < 0.05), and positive correlations with Scr, BUN and β 2-MG levels ( r values were 0.470, 0.247 and 0.469, respectively, all P < 0.01), but it had no correlation with age or ALP ( r values were -0.013 and 0.078, respectively, both P > 0.05). There were 15 cases (17.1%), 48 cases (54.5%) and 25 cases (28.4%) of patients with normal renal function in the hypocalcemia group, normal blood calcium group and hypercalcemia group, respectively; there were 2 cases (6.9%), 10 cases (34.5%) and 17 cases (58.6%) of patients with impaired renal function in the hypocalcemia group, normal blood calcium group and hypercalcemia group, respectively; there were statistically significant differences between the hypocalcemia group and the hypercalcemia group, as well as between the normal blood calcium group and the hypercalcemia group ( χ2 = 4.57, P < 0.05; χ2 = 6.67, P < 0.05), but there was no statistically significant difference between the hypocalcemia group and the normal calcium group ( χ2 = 0.29, P>0.05). The patients with approximately normal bone status in the hypocalcemia group, normal blood calcium group and hypercalcemia group were 3 cases (13.0%), 17 cases (74.0%) and 3 cases (13.0%), respectively, and the patients with bone damage were 14 cases (14.9%), 41 cases (43.6%), and 39 cases (41.5%), respectively, and there was a statistically significant difference between the normal blood calcium group and the hypercalcemia group ( χ2 = 7.48, P < 0.01), while there was no statistically significant difference between the hypocalcemia group and the hypercalcemia group ( χ2 = 1.46, P > 0.05). The proportions of patients with Scr > 177 μmol/L and β 2-MG > 3.5 mg/L in the hypercalcemia group were higher than those in the non-hypercalcemia group, and the differences were statistically significant (both P < 0.05). The results of logistic regression multivariate analysis showed that β 2-MG >3.5 mg/L was an independent risk factor for hypercalcemia in newly-diagnosed MM patients ( OR = 1.178, 95% CI: 1.058-1.311, P = 0.003). Conclusions:Corrected serum calcium level may be an important indicator for evaluating the severity of disease, renal function and bone status in newly-diagnosed MM patients.

Objective:To evaluate the long-term protective efficacy of the recombinant Chinese hamster ovary cell-derived hepatitis B vaccine（CHO-HepB）26 years post-vaccination in the rural China.Methods:Zhengding county，Hebei province was designated as a rural monitoring site for CHO-HepB efficacy. Study participants included individuals born between 1997 and 1999 who had completed the three-dose CHO-HepB primary series without booster doses. A cross-sectional survey was conducted in late 2024 using random sampling. Demographic and vaccination history data were collected via questionnaires，and hepatitis B virus（HBV）serological markers were detected using chemiluminescence. Historical surveillance data were integrated to infer infection statuses of HBsAg-positive individuals and evaluate longitudinal trends in anti-HBs seropositivity and antibody titers.Results:Among 178 participants（mean time since vaccination：26.2 years），the seroprevalence rates were 0.6% for HBsAg（95% CI：0.0%-1.6%），64.6% for anti-HBs（95% CI：57.6%-71.6%），and 1.1% for anti-HBc（95% CI：0.0%-2.7%）. Compared to the pre-vaccination baseline HBsAg positivity of 11.3% in children under 10 years of age，the estimated vaccine protection rate was 95%. Two notable cases were identified：one with concurrent HBsAg and anti-HBc positivity and one with anti-HBs and anti-HBc positivity，suggestive of transient HBV exposure（1999—2009）without chronicity. Natural immune boosting was inferred for the latter case based on anti-HBs titer dynamics. Longitudinal analysis of four prior cross-sectional surveys（2005，2009，2013，and 2017）revealed no significant upward trends in HBsAg and anti-HBc positivity（both P>0.05）over 26 years，while anti-HBs seropositivity declined significantly（ P<0.05）from 6 to 26 years post-vaccination. Conclusion:The CHO-HepB vaccine demonstrates sustained immunological persistence and robust long-term protection up to 26 years post-immunization. Continued emphasis on rigorous implementation of mother-to-child transmission prevention strategies is critical for future hepatitis B control.

Objective:To analyze the hepatitis C virus（HCV）infection status and genotype of patients with digestive system tumors in Shanxi Cancer Hospital from 2020 to 2024 and provide a basis for formulating and adjusting prevention and control strategies，optimizing diagnosis and treatment plans.Methods:Selecting Shanxi Cancer Hospital as the research site，a continuous sampling method was used from January 2020 to December 2024 to investigate the population of patients with digestive system tumors who met the inclusion criteria during the survey period. Blood samples were collected and HCV antibodies were detected by enzyme-linked immunosorbent assay（ELISA）. HCV RNA was detected by reverse transcription nested polymerase chain reaction（RT-PCR），and genotyping was performed. The positive rate of HCV antibodies and the characteristics of HCV RNA genotypes were analyzed.Results:A total of 2 068 outpatients of Shanxi Cancer Hospital were investigated from 2020 to 2024. The total number of HCV antibody positive cases was 59，with an HCV antibody positive rate of 2.85%；Among the 59 HCV antibody positive patients，38 were HCV-RNA positive，accounting for 64.41% of HCV antibody positive patients；the basic information of HCV infected patients showed that the proportion of males was higher than females，and the age group with the highest proportion was 40 to 59 years old，followed by those aged 60 and above，and the proportion of patients in rural areas was higher than that in urban areas，and the HCV infection rate of liver cancer patients was the highest，followed by gastric cancer and colorectal cancer；the overall HCV antibody positivity rate showed a downward trend from 2020 to 2024（ χ2=4.718， P<0.05）. Among the 59 HCV antibody positive patients，38 were HCV RNA positive，accounting for 64.41% of the HCV antibody positive patients. Among the 38 HCV RNA positive patients，24 were genotype 1b（63.16%），5 were genotype 2a（13.16%），2 were genotype 3a（5.26%），4 were genotype 3b（10.53%），and 3 were genotype 6a（7.89%）. Genotypes 4，5，and mixed genotypes were not found. The distribution of genotypes 1b，2a，3a，3b，and 6a among patients of different genders showed statistically significant differences（ χ2=14.710， P<0.05）. The distribution of genotypes 1b，2a，3a，3b，and 6a among patients of different age groups also showed statistically significant differences（ χ2=15.179， P<0.05）. The distribution of genotypes 1b，2a，3a，3b，and 6a among patients from different residential areas also showed statistically significant differences（ χ2=10.126， P<0.05）. The average HCV RNA quantification of genotype 1b patients was（8.91±0.83）IU/ml，that of genotype 2a patients was（4.23±0.65）IU/ml，and that of patients with other genotypes was（5.06±0.98）IU/ml. The HCV RNA quantification of genotype 1b patients was significantly higher than that of genotype 2a patients（ t=11.813， P<0.001）. There were no statistically significant differences in HCV RNA quantification among genotype 1b，2a，and other genotypes（ F=0.815， P=0.665）. Conclusion:From 2020 to 2024，the HCV antibody positivity rate among patients with digestive system tumors at Shanxi Cancer Hospital was relatively high. Male，middle-aged and elderly，and rural populations were high-risk factors for HCV infection，and the HCV-RNA genotype was mainly genotype 1b，with differences in genotype distribution among different genders，ages，and residential areas.