Objective:To explore the feasibility of rapid detection of AAC(6′)-Ⅰb-cr enzyme-producing Enterobacteriales using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods:A total of 55 Enterobacteriales strains isolated from the Department of Clinical Laboratory of Wenzhou TCM Hospital from January to July 2022 were collected. Ciprofloxacin and norfloxacin solutions were used as reaction substrates, incubated with the test strains for 1 hour, followed by centrifugation, and the supernatant was detected by MALDI-TOF MS. In addition, 128 clinical strains isolated from the clinical laboratory of our hospital from March to May 2023 were prospectively collected for the validation study of the method. Results:After ciprofloxacin and norfloxacin were acetylated by the AAC(6′)-Ⅰb-cr enzyme, a mass shift of relative molecular mass 42 occurred, resulting in the appearance of three mass spectral peaks representing the acetylated drugs. No such peak was observed in enzyme-negative strains. Besides, compared with gene sequencing results, the MALDI-TOF MS results of 128 strains used in the validation test showed 100% both in specificity and sensitivity.Conclusion:MALDI-TOF MS can rapidly and accurately detect AAC(6′)-Ⅰb-cr enzyme-producing Enterobacteriaceae bacteria.

Objective:To explore the feasibility of rapid detection of AAC(6′)-Ⅰb-cr enzyme-producing Enterobacteriales using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods:A total of 55 Enterobacteriales strains isolated from the Department of Clinical Laboratory of Wenzhou TCM Hospital from January to July 2022 were collected. Ciprofloxacin and norfloxacin solutions were used as reaction substrates, incubated with the test strains for 1 hour, followed by centrifugation, and the supernatant was detected by MALDI-TOF MS. In addition, 128 clinical strains isolated from the clinical laboratory of our hospital from March to May 2023 were prospectively collected for the validation study of the method. Results:After ciprofloxacin and norfloxacin were acetylated by the AAC(6′)-Ⅰb-cr enzyme, a mass shift of relative molecular mass 42 occurred, resulting in the appearance of three mass spectral peaks representing the acetylated drugs. No such peak was observed in enzyme-negative strains. Besides, compared with gene sequencing results, the MALDI-TOF MS results of 128 strains used in the validation test showed 100% both in specificity and sensitivity.Conclusion:MALDI-TOF MS can rapidly and accurately detect AAC(6′)-Ⅰb-cr enzyme-producing Enterobacteriaceae bacteria.

Objective:To explore the molecular pathogenesis of a family with hereditary factor Ⅴ (FⅤ) deficiency.Methods:All the exons, flanking sequences, 5′ and 3′ untranslated regions of the F5 of the proband, and the corresponding mutation sites of the family members were analyzed via direct DNA sequencing. The CAT measurement was used to detect the amount of thrombin produced. The ClustalX software was used to analyze the conservation of mutation sites. The online bioinformatics software, Mutation Taster, PolyPhen-2, PROVEAN, LRT, and SIFT were applied to predict the effects of mutation sites on protein function. The Swiss-PdbViewer software was used to analyze the changes in the protein model and intermolecular force before and after amino acid variation.Results:The proband had a heterozygous missense mutation c.1258G>T (p.Gly392Cys) in exon 8 of the F5, and a heterozygous deletion mutation c.4797delG (p.Glu1572Lys fsX19) in exon 14, which results in a frameshift and produces a truncated protein. Her grandfather and father had p.Gly392Cys heterozygous variation, whereas her maternal grandmother, mother, little aunt, and cousin all had p.Glu1572LysfsX19 heterozygous variation. The ratio of proband's thrombin generation delay to peak time was significantly increased. Conservation analysis results showed that p.Gly392 was located in a conserved region among the 10 homologous species. Five online bioinformatics software predicted that p.Gly392Cys was pathogenic, and Mutation Taster also predicted p.Glu1572Lys fsX19 as a pathogenic variant. Protein model analysis showed that the replacement of Gly392 by Cys392 can lead to the extension of the original hydrogen bond and the formation of a new steric hindrance, which affected the stability of the protein structure.Conclusion:The c.1258G>T heterozygous missense mutation in exon 8 and the c.4797delG heterozygous deletion mutation in exon 14 of the F5 may be responsible for the decrease of FⅤ levels in this family.

Objective To elaborate the changes of the soluble B cell-activating factor of the tumor necrosis factor family (BAFF) in the peripheral blood of chronic human immunodeficiency virus (HIV)-infected patients ,and to study the correlation between the soluble BAFF in HIV-infected patients and the progressions of acquired immune deficiency syndrome (AIDS).Methods Fifty untreated HIV outpatients and 30 healthy controls were recruited .According to the counts of CD4+T lymphocytes ,HIV-infected patients were divided into three groups ,＜ 200 cells／μL group , (200 － 350 ) cells／μL group and ＞350 cells／μL group .B cell counts and the BAFF levels were compared among the three groups and the healthy controls .The correlation analysis was conducted for the levels of BAFF ,the counts of CD4+T lymphocytes and B cells ,and viral load in HIV-infected patients .The value of BAFF in staging of HIV disease was identified by receiver operating characteristic (ROC) curve.Results The B cell counts were (90.3 ± 43.1)cells／μL in ＜200 cells／μL group ,(114 .4 ± 28 .8) cells／μL in (200 －350) cells／μL group ,and (162 .1 ± 29 .5) cells／μL in ＞350 cells／μL group and (307.1 ± 97 .0) cells／μL in healthy controls ,which was significantly different among the four groups (F＝47.92 ,P＜0.05).The concentrations of BAFF in the four groups were (1 737.5 ± 719.7) ,(962.8 ± 341.1) ,(859.8 ± 270.4) ,and (456.9 ± 163.7) ng／L ,with significant difference among the groups (F＝36.72 ,P＜0.05).The level of BAFF was negatively correlated with both B cell counts and CD4+T lymphocyte counts (r＝ －0.722 and －0.568 ,respectively ;both P＜0.05) ,and positively correlated with viral load (r＝0.607 ,P＜0 .05).The area under the ROC curve was 0 .881.If the level of BAFF was 1 281.5 ng／L ,the sensitivity and specificity to predict the period of AIDS were 74 .1% and 87.0%,respectively .Conclusion The levels of soluble BAFF in HIV-infected patients are significantly increased and related with the reduction of B cell counts and disease progression.

Objective To study the correlation between CD169 expression of monocytes and disease progression in human immunodeficiency virus (HIV )-infected patients .Methods Sixty HIV-infected patients and 30 healthy controls were recruited .According to the CD4 + T lymphocyte counts ,HIV-infected patients were divided into three groups including < 200 cells/μL ,200 — 350 cells/μL and > 350 cells/μL groups . The differences in monocytes counts ,the proportions of CD16 + and CD169 + monocytes were analyzed among the three groups and healthy controls .The correlations between proportion of CD169 + monocytes and CD4 + T lymphocyte counts ,viral load ,and proportion of CD16 + monocytes were analyzed .Results The monocyte counts in CD4 + T lymphocytes < 200 cells/μL group , (200 — 350 ) cells/μL group , >350 cells/μL group and healthy control group were (342 ± 99) ,(396 ± 145) ,(365 ± 80) ,and (404 ± 106)/μL ,respectively ,which were not significantly different (F= 2 .55 , P > 0 .05) .The proportions of CD16 + monocytes in the four groups were (19 .8 ± 8 .8)％ ,(14 .3 ± 2 .8)％ ,(9 .7 ± 2 .0)％ and (4 .0 ± 0 .8)％ ,respectively ,which were significantly different ( F = 30 .90 , P < 0 .05 ) . The proportions of CD169 + monocytes in the four groups were (72 .6 ± 11 .4)％ ,(59 .4 ± 14 .7)％ ,(33 .3 ± 14 .5)％ and (2 .6 ± 0 .8)％ ,respectively ,which were significantly different (F = 152 .40 , P< 0 .05) .The proportion of CD169 + monocytes was negatively correlated with CD4 + T lymphocyte counts (r = 0 .792 , P< 0 .05) , while positively correlated with both viral load (r= 0 .485 ,P< 0 .05) and proportion of CD16 + monocytes (r= 0 .395 , P< 0 .05) .Conclusions The CD169 expressions of monocytes in HIV-infected patients are significantly increased and correlated with both monocyte activation and disease progression .