Objective:To predict the pathogens of bloodstream infection (BSI) in hematopoietic stem cell transplantation (HSCT) patients by plasma microbial cell-free DNA (mcfDNA) sequencing with and without additional amplification.Methods:A total of 978 HSCT patients were enrolled in Peking University People′s Hospital from March to July 2021, and the 7 428 blood samples were prospectively collected from pretransplant conditioning period to 4 months after transplantation. The plasma samples were separated and then cryopreserved. According to blood culture results and whether there were plasma samples before BSI onset, twenty-eight HSCT patients with positive blood culture (39 plasma samples within 1-8 days before BSI onset) and 9 HSCT patients with negative blood culture (9 plasma samples) were filtered. The 39 samples were performed with mcfDNA additional and non-additional amplification sequencing, and the 9 samples were only performed with additional amplification sequencing. With the blood culture results as the gold standard, the consistency between the sequencing and the blood culture results was observed. Student t test and Wilcoxon test were used for statistical analysis. Results:Without additional amplification sequencing, only 7 samples sequencing results were consistent with the blood culture results, and the total pathogen detection rate was 17.95% (7/39). The rates within 3 days and 4-8 days were 23.81% (5/21) and 2/18, respectively. The main pathogenic type detected was gram-negative bacteria (5/7). With additional amplification sequencing, the total pathogen detection rate was 59.26% (16/27) and the rate within 3 days was 8/13. The number of gram-positive bacteria detected was elevated (13/16) and the number of additional microorganisms in additional amplification sequencing was increased significantly ( P=0.001 0), compared with non-additional amplification sequencing. Moreover, additional sequencing analysis of 9 samples from patients with negative culture result showed that no pathogen was detected in six samples, and the common Torque teno virus in HSCT patients was detected in only three samples. Conclusion:The pathogen detection rate of plasma mcfDNA additional amplification sequencing was better than that of non-additional amplification sequencing in HSCT patients before BSI onset, especially in the first three days, which has the potential to predict BSI pathogens.

Objective To evaluate the effect of bone marrow mesenchymal stem cell (BMSC) on the expression of interleukin (IL)-10 and tumor necrosis factor (TNF)-α in mice with ischemia-reperfusion acute kidney injury (IR-AKI). Methods All mice were randomly divided into the sham operation group (control group), ischemia-reperfusion injury group (IRI group) and BMSC treatment group (BMSC group), with 6 mice in each group, respectively. The renal function and pathological changes of mice were detected. The cell apoptosis of renal tissues of mice was determined. The expression levels of serum IL-10 and TNF-α of mice were quantitatively measured. The mouse BMSC was randomly divided into the control and hypoxia-reoxygenation groups (IRI group), and the expression levels of IL-10 and TNF-α in cell supernatant were determined. Results The renal structure of mice was normal in the control group, severe damage was observed in the IRI group, and mild damage occurred in the BMSC group. Compared with the control group, the renal tissue injury scores were significantly higher in the IRI and BMSC groups (both P < 0.05). Compared with the IRI group, the renal tissue injury score was significantly lower in the BMSC group (P < 0.05). Compared with the control group, the levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were remarkably up-regulated in the IRI group, and the level of BUN was significantly up-regulated in the BMSC group (all P < 0.05). Compared with the IRI group, the levels of Scr and BUN were significantly down-regulated in the BMSC group (both P < 0.05). In the IRI group, the quantity of apoptotic cells in the renal tissues was considerably higher than those in the BMSC and control groups, and the quantity of apoptotic cells in the BMSC group was significantly higher than that in the control group (all P < 0.05). Compared with the control group, the levels of serum IL-10 and TNF-α were significantly up-regulated in the IRI group, whereas the level of serum TNF-α was significantly down-regulated and the level of serum IL-10 was significantly up-regulated in the BMSC group (all P < 0.05). Compared with the IRI group, the levels of serum IL-10 and TNF-α were significantly down-regulated in the BMSC group (both P < 0.05). The levels of IL-10 and TNF-α in the cell supernatant did not significantly differ between the IRI and control groups (P=0.080、0.627). Conclusions BMSC infusion may reduce the incidence of renal IRI and inflammation, probably via the mechanism of down-regulating TNF-α expression rather than up-regulating IL-10 expression.

Objective To investigate the application value of Multi-Latex polygranular technique joint detection of kidney injury-related urinary microproteins in noninvasive diagnosis after renal transplantation. Methods Clinical data of 72 recipients undergoing renal transplantation were retrospectively analyzed. According to the level of serum creatinine (Scr), the recipients were divided into normal renal function group (group A, n=14), mild kidney injury (group B, n=37), and severe kidney injury group (group C, n=21). 20 healthy volunteers were selected as the healthy control group (HC group). The contents of urinary retinol binding protein (RBP), microalbumin (mAlb), IgG, transferrin (TRF), α₁-microglobulin (MG), and β₂-MG of subjects in each group were detected using the Multi-Latex polygranular technique. The correlation between urinary microproteins and Scr, blood urea nitrogen (BUN) was analyzed. The differences of urinary microproteins in each group were compared. And the diagnostic value of single and joint detection of urinary microproteins was evaluated. Results Six kinds of urinary microproteins in HC group and group A were significantly lower than those in group B and group C, and six kinds of urinary microproteins in group B were significantly lower than those in group C (all P < 0.01). Six kinds of urinary microproteins in renal transplant recipients were positively correlated with BUN. RBP, mAlb, α₁-MG, and β₂-MG were positively correlated with Scr. The correlations were statistically significant (P < 0.001-0.05). The diagnostic value of joint detection of urinary microproteins is better than the detection of single index, among which TRF+mAlb+RBP+α₁-MG quadruple detection had the highest diagnostic value. Conclusions Six kinds of urinary microproteins can be used as specific indicators to reflect graft renal function. The polygranular technique can simultaneously detect its contents and achieve noninvasive diagnosis. The diagnosis based on TRF+mAlb+RBP+α₁-MG quadruple detection is expected to further improve the noninvasive diagnosis system after renal transplantation.

Objective:To evaluate the safety and efficacy of eltrombopag combined with immunosuppressive therapy in patients with aplastic anemia (AA) in China.Methods:We investigated and analyzed the clinical data of AA patients from 14 hematological treatment centers who were treated with oral eltrombopag for at least 3 mon.Results:We enrolled 56 AA patients, including 19 treatment-na?ve patients and 37 IST-refractory patients. The median administration period for eltrombopag was 7 (3-31) months, and the median maximum stable dosage was 75 mg/d (50-150 mg/d) . The 3-month hematological response (HR) rate was 60%, and the complete response (CR) rate was 30% in 10 SAA patients who were treated with first-line eltrombopag and standard IST (ATG+CsA) . Eight of 9 eltrombopag and CsA ± androgen first-line treated SAA patients responded (8/9, 89%) and 4 (44%) gave CR. The overall HR and CR rates were 79% and 52.6%, respectively, among these 19 patients by the end of the follow-up period. Of the 19 AA patients who were refractory to CsA ± androgen, 11 achieved HR (57.9%) at 3 mon, and the best HR rate was 44% in standard IST (ATG+CsA) refractory 18 patients after eltrombopag treatment. Fifty-one percent of the patients experienced mild or moderate adverse events, and gastrointestinal discomfort was the most common adverse effect reported by the study subjects.Conclusion:Adding Eltrombopag in first-line IST can accelerate the acquisition and improve the quality of hematological responses in AA patients. AA with relatively more residual hematopoietic cells may be well treated with eltrombopag and non-ATG IST. Eltrombopag can be used as salvage therapy for CsA±androgen refractory patients. Eltrombopag was generally safe and well tolerated by AA patients in China.

Objective To establish TaqMan probe real‐time Fluorescent Quantitative PCR in detecting Cryptococcus neoformans genomic DNA ,and to provide important method for detection of cryptococcal meningitis .Methods According to the Cryptococcus neoformans ITS‐rDNA sequences obtained from NCBI ,specific primers and probe were designed based on the conserved sequences , a specific 114 bp fragment was amplified by primers and probe ,then recombinant plasmid was constructed .Eight different concen‐trations from 1 .42 × 10 copy/μL to 1 .42 × 10 copy/μL were used as templates by 2 μL .In the optimum reaction condition ,the sen‐sitivity ,specificity and repeatability were evaluated and standard curve was established .15 clinical cryptococcal meningitis strains i‐solated from clinical diagnosis patients were detected .Results The real‐time PCR showed high sensitivity and specificity and was a‐ble to detect 2 .84 × 102 copies of plasmid DNA .The detection sensitivity was 2 .84 × 102 copies plasmid DNA by real‐time PCR ,no amplification curve was detected with human genomic DNA ,other fungus ,bacterias and viruses .The CV of inter‐assay were 2 .86% ,1 .48% and 1 .36% respectively with excellent reproducibility .Fifteen clinical isolated strains could be detected accurately . Conclusion A method of detection of Cryptococcus neoformans DNA by real‐time PCR is established successfully with high sensi‐tivity and specificity ,and the results are stable ,could diagnose cryptococcal meningitis rapidly and early .

BACKGROUND:Cytotoxic T lymphocyte-associated antigen 4 is a newly discovered costimulatory molecule. It has been studied more in tumor and autoimmune diseases, less in the field of kidney transplantation.
OBJECTIVE:To explore the role of cytotoxic T lymphocyte-associated antigen 4 in acute rejection after renal transplantation.
METHODS:Fifty patients undergoing renal transplantation were divided into acute rejection group (20 cases) and stable graft function group (30 cases). Another 30 healthy persons served as control group. Blood samples were extracted from the peripheral blood. Cytotoxic T lymphocyte-associated antigen 4 was detected by enzyme linked immunosorbent assay and flow cytometry.
RESULTS AND CONCLUSION:The expression of cytotoxic T lymphocyte-associated antigen 4 in the serum showed significant differences in the acute rejection group, stable graft function group and healthy control group (F=70.008 1, P=0.000 0), but showed no difference in peripheral blood lymphocytes of three groups (F=1.865 6, P=0.161 7). Compared with the healthy control group, the expression levels of cytotoxic T lymphocyte-associated antigen 4 in peripheral blood lymphocytes of acute rejection group and stable graft function group were significantly decreased (P=0.000 0). In addition, the acute rejection group had a lower cytotoxic T lymphocyte-associated antigen 4 expression than the stable graft function group (P=0.000 0). In renal transplant rejection, the expression of cytotoxic T lymphocyte-associated antigen 4 in serum was reduced, showing some correlation with acute rejection after renal transplnatation. Cytotoxic T lymphocyte-associated antigen 4 might be involved in the rejection.

BACKGROUND:The role of galactose lectin family proteins in transplantation immunity has been proposed, but there is currently no galectin-7 detection for auxiliary diagnosis of renal dysfunction in the perioperative period after renal transplantation. For renal transplant recipients, monitoring of galectin-7 may contribute to early diagnosis of renal dysfunction after renal transplantation, and buy time for clinical treatment.
OBJECTIVE:To detect the expression of galactose-7 in acute antibody mediated rejection after renal transplantation. METHODS:Twenty-seven patients who were diagnosed as having acute antibody mediated rejection after renal transplantation by renal biopsy were enrol ed, and another 10 patients without acute antibody mediated rejection after renal transplantation were selected as controls. Immunohistochemical staining and western blot assay were used to detect expression of galectin-7 in tissue and serum, respectively.
RESULTS AND CONCLUSION:Results of immunohistochemistry staining showed that under light microscope, in the control group, galectin-7 distributed in the surface microvil i of proximal tubule epithelial cells, but not in glomeruli, distal tubule, col ecting duct and vein;in the acute rejection group, renal arteriole intima edema, tube wal fibrinoid necrosis, infiltration of renal glomerulus and tubule cells and mononuclear cells were found and galectin-7 only expressed in the surface microvil i of proximal tubule epithelial cells as wel as in the arterial smooth muscle. The number of galectin-7 positive cells in the acute rejection group was significantly higher than that in the control group (P<0.1). Western blot assay results showed that the protein expression of serum galectin-7 in the acute rejection group was higher than that in the control group (P<0.05). These findings indicate that renal puncture for renal transplantation is safe and reliable, has no adverse effect on the patients and renal transplant. Galectin-7 detection has an important guiding significance for the auxiliary diagnosis of renal dysfunction during the perioperative period after renal transplantation.

OBJECTIVETo apply pyrosequencing technique in the detection of the common pathogens in sepsis.

METHODSThe primers for amplification and sequencing in pyrosequencing were designed according to alignment of the bacterial 16S rRNA sequence. Bacterial genomic DNA was extracted for pyrosequencing, and the pathogen species were determined according to the sequencing data obtained.

RESULTSPyrosequencing effectively yielded the sequencing data of the 28 bp sequences of the pathogens and clearly distinguished the pathogen species of Streptococcus pyogenes, Streptococcus pneumonia, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Neisseria meningitides, and Salmonella, but failed to distinguish Staphylococcus epidermidis from Staphylococcus aureus.

CONCLUSIONPyrosequencing technique can effectively distinguish the common pathogens in sepsis at the species level.

Bacteria ; classification ; isolation & purification ; DNA Primers ; DNA, Bacterial ; genetics ; Microbiological Techniques ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; Sepsis ; microbiology

Objective To investigate the influence of BK virus (BKV) activation in renal transplant recipients on the renal allograft function.Method Recipients receiving renal transplantation during 2010.3-2011.4 were sdected as objectives,the urine and peripheral blood samples of them were taken and real-time PCR assays were performed to detect BKV DNA at 0.5,1,3,6,9,and 12 months post-transplantation.Results Among 88 recipients,BKV viruria occurred in 27 (30.68％) patients,and sustained viruria occurred in 17 patients.37.0％ (10/27) of patients with BKV viruria developed inot BKV viremia,and sustained viremia occurred in 5 patients.The viral load in plasma was higher in patients with sustained viremia than in those with transient viremia (P＜0.05),and serum creatinine concentrations were higher when BK viremia occurred (P＜0.05).Conclusion Graft function was impaired among patients with BK viremia,and regularly monitoring BK virus in renal transplant recipients and clinical imervention based on plasma PCR results can prevent transplant kidney damage effectively.

OBJECTIVETo establish real-time PCR and loop-mediated isothermal amplification (LAMP) systems for detecting Cryptococcus neoformans CAP10 gene.

METHODSSpecific primers were designed targeting CAP10 gene of Cryptococcus neoformans, and the plasmid was constructed. After optimization of the reaction condition, the plasmid was quantitatively detected using real-time PCR and LAMP, and the detection sensitivity and specificity were evaluated. Clinical samples were also detected using the two methods.

RESULTSThe detection sensitivity of real-time PCR and LAMP was 6.8×10(1) and 6.8×10(3) copies, respectively. Real-time PCR yielded a higher positivity rate than LAMP. Both of the two methods showed a high detection specificity and produced negative results in the detection of Neisseria meningitidis, Candida albicans, Candida tropicalis, Aspergillus flavus, Aspergillus niger and Escherichia coli.

CONCLUSIONReal-time PCR is highly sensitive and specific for detecting Cryptococcus neoformans CAP10 gene but requires sophisticated equipment. LAMP, though with a relatively lower sensitivity, is simple to operate without the need of special equipment, and the result can be conveniently observed. Both of the two methods are suitable for detecting Cryptococcus neoformans and evaluating the treatment outcomes.

Cryptococcus neoformans ; genetics ; Fungal Proteins ; genetics ; Nucleic Acid Amplification Techniques ; methods ; Plasmids ; RNA, Fungal ; genetics ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; methods ; Sensitivity and Specificity