[Metagenomic next-generation sequencing of plasma for the identification of bloodstream infectious pathogens in severe aplastic anemia].

Objective: To analyze the diagnostic value of cell-free plasma metagenomic next-generation sequencing (mNGS) pathogen identification for severe aplastic anemia (SAA) bloodstream infection. Methods: From February 2021 to February 2022, mNGS and conventional detection methods (blood culture, etc.) were used to detect 33 samples from 29 consecutive AA patients admitted to the Anemia Diagnosis and Treatment Center of the Hematology Hospital of the Chinese Academy of Medical Sciences to assess the diagnostic consistency of mNGS and conventional detection, as well as the impact on clinical treatment benefits and clinical accuracy. Results: ①Among the 33 samples evaluated by mNGS and conventional detection methods, 25 cases (75.76%) carried potential pathogenic microorganisms. A total of 72 pathogenic microorganisms were identified from all cases, of which 65 (90.28%) were detected only by mNGS. ②All 33 cases were evaluated for diagnostic consistency, of which 2 cases (6.06%) were Composite, 18 cases (54.55%) were mNGS only, 2 cases (6.06%) were Conventional method only, 1 case (3.03%) was both common compliances (mNGS/Conventional testing) , and 10 cases (30.3%) were completely non-conforming (None) . ③All 33 cases were evaluated for clinical treatment benefit. Among them, 8 cases (24.24%) received Initiation of targeted treatment, 1 case (3.03%) received Treatment de-escalation, 13 cases (39.39%) received Confirmation, and the remaining 11 cases (33.33%) received No clinical benefit. ④ The sensitivity of 80.77%, specificity of 70.00%, positive predictive value of 63.64%, negative predictive value of 84.85%, positive likelihood ratio of 2.692, and negative likelihood ratio of 0.275 distinguished mNGS from conventional detection methods (21/12 vs 5/28, P<0.001) . Conclusion: mNGS can not only contribute to accurately diagnosing bloodstream infection in patients with aplastic anemia, but can also help to guide accurate anti-infection treatment, and the clinical accuracy is high.

[Clinical characteristics and CT findings of Pneumocystis Jirovecii pneumonia in 46 cases with hematological diseases].

Objective: To summarize the original CT features of Pneumocystis Jirovecii pneumonia in patients with hematological diseases. Methods: A retrospective analysis was carried out in 46 patients with proven pneumocystis pneumonia (PJP) in the Hospital of Hematology, Chinese Academy of Medical Sciences between January 2014 and December 2021. All patients had multiple chests CT and related laboratory examinations, imaging typing were conducted based on the initial CT presentation, and the distinct imaging types were analyzed against the clinical data. Results: In the analysis, there were 46 patients with proven pathogenesis, 33 males, and 13 females, with a median age of 37.5 (2-65) years. The diagnosis was validated by bronchoalveolar lavage fluid (BALF) hexamine silver staining in 11 patients and clinically diagnosed in 35 cases. Of the 35 clinically diagnosed patients, 16 were diagnosed by alveolar lavage fluid macrogenomic sequencing (BALF-mNGS) and 19 by peripheral blood macrogenomic sequencing (PB-mNGS) . The initial chest CT presentation was categorized into 4 types, including ground glass (GGO) type in 25 cases (56.5%) , nodular type in 10 cases (21.7%) , fibrosis type in 4 cases (8.7%) , and mixed type in 5 cases (13.0%) . There was no substantial discrepancy in CT types among confirmed patients, BALF-mNGS diagnosed patients and PB-mNGS diagnosed patients (χ(2)=11.039, P=0.087) . The CT manifestations of confirmed patients and PB-mNGS diagnosed patients were primarily GGO type (67.6%, 73.7%) , while that of BALF-mNGS diagnosed patients were nodular type (37.5%) . Of the 46 patients, 63.0% (29/46) had lymphocytopenia in the peripheral blood, 25.6% (10/39) with positive serum G test, and 77.1% (27/35) with elevated serum lactate dehydrogenase (LDH) . There were no great discrepancies in the rates of lymphopenia in peripheral blood, positive G-test, and increased LDH among different CT types (all P>0.05) . Conclusion: The initial chest CT findings of PJP in patients with hematological diseases were relatively prevalent with multiple GGO in both lungs. Nodular and fibrosis types were also the initial imaging findings for PJP.

Study of the K_{L}→π

The rare decay K_{L}→π^{0}νν[over ¯] was studied with the dataset taken at the J-PARC KOTO experiment in 2016, 2017, and 2018. With a single event sensitivity of (7.20±0.05_{stat}±0.66_{syst})×10^{-10}, three candidate events were observed in the signal region. After unveiling them, contaminations from K^{±} and scattered K_{L} decays were studied, and the total number of background events was estimated to be 1.22±0.26. We conclude that the number of observed events is statistically consistent with the background expectation. For this dataset, we set an upper limit of 4.9×10^{-9} on the branching fraction of K_{L}→π^{0}νν[over ¯] at the 90% confidence level.

[A single-center study on the distribution and antibiotic resistance of pathogens causing bloodstream infection in adult patients with hematological disease during the period 2014-2018].

Objective: To investigate the distribution of pathogens and the antibiotic resistance profile of bloodstream infections in adult patients with hematological diseases in the period 2014-2018 to provide evidence for the rational use of antibiotics. Methods: We retrospectively analyzed the bloodstream infections in patients with hematological diseases from January 2014 to December 2018 at the institute of Hematology & Blood Diseases Hospital; this included an assessment of the clinical characteristics, distribution of pathogens, and antibiotic resistance data. Results: There were 1935 episodes of BSIs in the 1478 patients who were studied; among these, 1700 episodes occurred in the neutropenic phase. The 7-day and 30-day all-cause mortality rates were 5.5% and 8.2%, respectively. Bloodstream infection was usually accompanied by respiratory tract, perianal zone mucositis, and digestive tract symptoms; the respective proportions were 12.4%, 12.3%, and 9.1%, respectively. Total 2025 strains were isolated; 1551 (76.6%) of the pathogens were gram-negative bacteria, mainly Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa; 423 (20.9%) were gram-positive bacteria, mainly Staphylococcus spp. and Streptococcus spp. Viridans; 51 (2.5%) were fungi, mainly Candida tropicalis. The resistance rates of Enterobateriaceae to piperacillin/tazobactam, carbapenems, amikacin were <10%. The resistance rates of K. pneumoniae to cefepime, piperacillin/tazobactam and meropenem increased annually. The resistance rates of Pseudomonas aeruginosa to piperacillin/tazobactam, quinolones, Aminoglycosides were <5% even when compared to carbapenems. Eleven stains of methicillin-resistant S. aureus and 1 stain of vancomycin-resistant Enterococcus faecium were detected. Conclusion: The pathogens of bloodstream infection in adult patients with hematological diseases are widely distributed. The resistance rates of different strains vary; the rates in some species had a tendency to increase. Antibiotics should be selected rationally as per the distribution of pathogens and resistance to antibiotics in different patient groups.

[Analysis of pathogens and clinical characteristics of bloodstream infection in neutropenic children with hematological malignancies from 2014 to 2018].

Objective: To investigate the microbiologic and clinical characteristics of bloodstream infection in neutropenic pediatric patients with hematological malignancies and provide data support for the rational use of antimicrobial agents in these patients. Methods: A retrospective analysis was performed on the clinical data, pathogen species distribution, and drug sensitivity data of bloodstream infection in neutropenic pediatric patients with hematological malignancies from the Institute of Hematology & Blood Diseases Hospital from January 2014 to December 2018. Results: Total 537 episodes of bloodstream infections occurred in 427 neutropenic children with hematological malignancies; the 30-day all-cause mortality rate was 3.7%. The clinical feature of 44.7% patients with bloodstream infection was only fever, and the pathogenic bacteria were mainly enterobacteriaceae bacteria. Bloodstream infection was usually accompanied by oral mucosa (20.7%) , respiratory tract (20.5%) , and digestive tract (14.3%) symptoms. The distribution of pathogens in patients with different symptoms of bloodstream infection varied (χ(2)=40.561, P=0.001) . Total 550 strains of pathogens were isolated, and the top 5 bacteria were Streptococcus aureus (109 strains, 19.8%) , Escherichia coli (99 strains, 18.0%) , Staphylococcus epidermidis (75 strains, 13.6%) , Klebsiella pneumoniae (67 strains, 12.2%) , and Staphylococcus aureus (32 strains, 5.8%) . The resistance rates of Enterobacteriaceae and Pseudomonas aeruginosa to piperacillin/tazobactam and carbapenems were <5%. The proportion of methicillin-resistant Staphylococcus aureus (MRSA) in Staphylococcus aureus was 9.7%. Conclusion: The proportion of pathogenic bacteria gram-positive cocci and gram-negative bacilli in the bloodstream infection of neutropenic children with hematological malignancies was approximately the same, suggesting that the use of antimicrobial agents should be broad-spectrum. Carbapenems, glycopeptides, and enzyme inhibitor complexes still have good effects.

[Clinical features and laboratory data analysis of Aeromonas bacteremia with hematological diseases].

Objective: To investigate the clinical and laboratory features of Aeromonas bacteremia in patients with hematological diseases, and provide evidence for the prevention and treatment of Aeromonas infection. Methods: A retrospective study of patients with bloodstream infection of Aeromonas in our hospital from January 2014 to December 2018 was carried out. The clinical characteristics, antimicrobial susceptibility, infection seasons, antimicrobial therapy and evolution were analyzed. Results: A total of 42 patients with hematological diseases had Aeromonas bloodstream infection within 5 years. Among them, 39 cases (92.9%) of bloodstream infection occurred in the stage of neutropenia. The median time of fever was 4 (1-27) d, 22 (52.4%) patients only had fever, 6 (14.3%) with gastrointestinal symptoms (abdominal pain, diarrhea, nausea, upper gastrointestinal bleeding) , 8 (19.0%) with pulmonary infection, 13 (31.0%) with skin and soft tissue infections. Seven patients (16.7%) died with skin and soft tissue infection. The resistance of Aeromonas to carbapenems was 68.3%-70.7%, while the resistance rate to cephalosporins, quinolones and aminoglycosides were less than 10%. Conclusions: Aeromonas bacteremia in patients with hematological diseases mainly occur in the neutropenia stage, usually with symptom like fever. The mortality is increased when accompanied by skin and soft tissue infection. Antibiotic use should be based on susceptibility results, and avoid the use of carbapenems.

[Perianal swabs surveillance cultures of Carbapenem-resistant Enterobacteriaceae(CRE) can be hints for CRE bloodstream infection in patients with hematological diseases].

Objective: To analyze the hints role of surveillance cultures of Carbapenem-resistant Enterobacteriaceae (CRE) by perianal swabs in patients with hematological diseases, and seek risk factors of CRE bloodstream infection. Methods: The resistance of CRE from 2 914 patients with hematological diseases who cultured perianal swabs, CRE bloodstream infection and risk factors were analyzed during January 2016 to December 2017. Results: In this study, perianal swabs from 2 914 patients with hematological diseases were cultured, 74 patients were CRE positive, and bloodstream infection with CRE was found in 13 of these patients. A total of 87 CRE strains were isolated (The same patient only keep the first one for the same location), including 31 Klebsiella pheuminiae, 43 Escherichia coli, 8 Enterobacter cloacae and 6 other Enterobacteriaceae. The resistance rates to piperacillin / tazobactam, imipenem, meropenam, amikacin, levofloxacin, tigecycline were 91.9%, 74.4%, 98.8%, 17.6%, 74.4% and 8.0%, respectively. Resistance to carbapenem, aminoglycoside, quinolones and tegacycline were highly consistent between two sites from 13 patients, whose both perianal swabs and blood were positive in CRE cultures. Febrile neutropenic time, digestive tract symptoms and perianal infection were independent risk factors for bloodstream infection in patients with perianal swabs positive results, the odds ratios (OR) were 1.10 (P=0.029), 1.13 (P=0.005) and 1.23 (P=0.016), respectively. Conclusion: Perianal swabs surveillance cultures of CRE can be hints for CRE bloodstream infection in patients with hematological diseases, and also can provide suggestions for antibiotics. Long time of febrile neutropenic, digestive tract symptoms and perianal infection can be the early warning for CRE bloodstream infections.

[Clinical and bacteriological analysis of lymphoid tissue neoplasms patients with bacteria bloodstream infections].

Objective: To investigate the clinical status of lymphoid tissue neoplasms patients with bacteria bloodstream infections, bacteriology and drug susceptibility results, and provide the basis for rational clinical anti-infection option. Methods: A retrospectively analysis of clinical data and bacterial susceptibility test results of patients with bacteria bloodstream infections from September 2010 to December 2014 was conducted. Results: A total of 134 cases including 107 patients with bloodstream infections were enrolled. 84 cases were male, 50 cases were female, the median age was 31 (12-71) years old. 112 cases were agranulocytosis, and 106 cases were severe agranulocytosis (ANC<0.1×10(9)/L) . 27 cases underwent hematopoietic stem cell transplantation, 100 cases received chemotherapy[33 cases with VD (I) CP±L (vincristine+daunorubicin/idarubicin + cyclophosphamide + prednison±asparaginasum) induction chemotherapy, 41 cases with intensive chemotherapy of Hyper-CVAD/MA or MA (mitoxantrone+cytarabine) , 26 cases with other chemotherapy regimens], and 7 cases were infected without chemotherapy. 10 patients discharged from hospital owing to treatment abandoning, 120 cases were cured through anti-infective therapy, 2 patients died of bacteria bloodstream infections, 1 patient died of sudden cardiac, and 1 patient died of GVHD after allogenic hematopoietic stem cell transplantation. A total of 144 strains were isolated, including 108 strains (75.0%) of Gram-negative bacteria and 36 strains (25.0%) of Gram-positive cocci. The susceptibility of Gram-negative bacteria to the carbapenems was 98.00%, and the adjustment treatment rate of carbapenems was 3.0%. The susceptibility of Gram-negative bacteria to the other antibiotics was 60.30%, and the adjustment treatment rate was 90.5%. The susceptibility of Grampositive cocci to the carbapenems was 49.3%, and to glycopeptides and linezolid was 100.0%. Comparing all patients'empirical use of antimicrobial agents with the drugs susceptibility results of blood cultures, 80.1% of the patients'initial drug selection was sensitive. Conclusion: The lymphoid neoplasms patients experienced bacteria bloodstream infections most often after receiving the chemotherapy regimens of treating acute lymphoblastic leukemia. The majority type of bacteria was Gram-negative bacteria. Drug susceptibility test showed that susceptibility of Gram-negative bacteria to the carbapenems was the highest, and the treatment adjustment rate was obviously lower. The susceptibility of Gram-positive cocci to glycopeptides and linezolid was high, and which could be applied to the patients with Gram-positive cocci sepsis on basis of susceptibility results in general.

[Determination of cholesterol in lipoprotein fractions by agarose gel electrophoresis].

OBJECTIVE: To evaluate a single step electrophoresis for quantitative determination of cholesterol of high-, low-, very-low-density lipoprotein(HDL, LDL, VLDL) and fast pre-beta lipoprotein [lipoprotein (a), Lp(a)]. METHODS: Quantification of lipoprotein cholesterol was performed by enzymatic staining of cholesterol in a new agarose gel electrophoresis method that allows the separation of LDL, VLDL, HDL, and Lp(a) by Helena REP system. The results of electrophoresis method were compared with those by traditional method like PTA-Mg2+ precipitation method for HDL-C, PVS precipitation method for LDL-C, and Immunoturbidimetric assay(ITA) method for Lp(a). RESULTS: Within-runs CV were 5.16%-7.46%, 1.26%-3.28% and 3.78%-5.86% for VLDL-C, LDL-C and HDL-C, respectively. Between-runs CV were 8.35%-11.25%, 2.78%-4.08% and 4.23%-6.36%, respectively. The linearity of this method was up to 10.35 mmol/L total cholesterol. The recoveries were 90.3%, 94.3% and 89.6%, respectively. No interference were observed when bilirubin(< 342 mumol/L), hemoglobin(< 20 g/L) or triglyceride(< 11.0 mmol/L) were added to pooled serum, respectively. There was good agreement between methods, with r = 0.9557 for HDL-C(electrophoresis method vs PTA-Mg2+ precipitation method), r = 0.9609 for LDL-C(electrophoresis method vs PVS precipitation method) and r = 0.9235 for Lp(a)-C (electrophoresis method) vs Lp(a) (ITA method). CONCLUSIONS: The electrophoresis method offers a simple and inexpensive means of simultaneously measuring HDL-C, VLDL-C, Lp(a)-C and LDL-C.

Uncoupling protein mRNA, mitochondrial GTP-binding, and T4 5'-deiodinase activity of brown adipose tissue in Daurian ground squirrel during hibernation and arousal.

The mRNA level of uncoupling protein (UCP) specific for brown adipose tissue (BAT) in Daurian ground squirrel, was detected by using a [32P]-labeled oligonucleotide probe. The UCP concentration in mitochondria was indirectly determined by titration with its specific ligand [H3]-labeled GTP. Type II T4 5'-deiodinase of BAT was assayed concomitantly. We found two species of mRNA for UCP with lengths of about 1.9 and 1.5 kb, respectively, both occurring in almost the same concentration. UCP mRNA content was elevated significantly during hibernation, but the UCP concentration did not change compared with that of nonhibernating controls kept at room temperature. When hibernating squirrels were aroused, the UCP mRNA remained at the elevated level as during hibernation, but the UCP concentration increased in comparison with that of nonhibernating controls or during hibernating. Changes in T4 5'-deiodinase activity in BAT were similar to the variations of the UCP mRNA level. These results suggest that the activation of T4 5'-deiodinase in BAT may be an important factor for the up-regulation and maintenance of UCP mRNA content needed for the synthesis of sufficient UCP to acquire the thermogenic capacity for arousal from hibernation.

Slow-phase kinetics of nucleotide binding to the uncoupling protein from brown adipose tissue mitochondria.

The kinetics of nucleotide binding to the uncoupling protein (UCP) from brown adipose tissue mitochondria were studied with a filter binding method. Fast and slow phases of binding were observed, corresponding to the two-stage binding model based on equilibrium binding studies (Huang, S. G., and Klingenberg, M. (1996) Biochemistry 35, 7846-7854) (Reaction 1). [reaction: see text] Although this method determines total binding, only the slow phase can be resolved. The fast unresolved phase represents the formation of the initial loose UCP-nucleotide complex (UN; Kd approximately 2 microM), whereas the slow phase reflects the tight binding (U*N) associated with a conformational change induced by the bound nucleotide. Best fits of the binding data yielded, for the slow phase, k+1 values of 3.0 x 10(-3) s-1 for GTP, 4.8 x 10(-3) s-1 for ATP, 0.13 s-1 for GDP, and >0.7 s-1 for ADP and dissociation rate constants (k-1) of 0.10 x 10(-3) s-1 for GTP, 0.58 x 10(-3) s-1 for ATP, 8.8 x 10(-3) s-1 for GDP, and >0.3 s-1 for ADP at pH 6.7 and 4 degrees C. The rates were fairly pH- and temperature-dependent. The distribution constant Kc' (=k+1/k-1) between the tight and loose complexes ranged between 2 and 30, suggesting formation of 71-97% of the tight complex at equilibrium. The Kc' decreases with increasing pH, indicating a progressively less tight complex population. Anions (SO42-) form a loose complex with UCP, thus affecting the initial association step, but not the subsequent transition step. While the kinetic constants were verified by dilution and chase experiments as well as in mass action plots, they were further corroborated with data obtained by fluorescence competition measurements. Taken together, our results show that nucleotide binding to UCP occurs via a two-stage mechanism in which the initial loose complex rearranges slowly into a tight complex.

[The sub-cellular distribution of beta-catenin in the neural differentiation of RA induced P19 EC cells].

Wnt signals have been shown to play an important role in the development of the central nervous system (CNS) of mouse. In our previous work, it was demonstrated that Wnt signal could initiate differentiation of P19 EC cells. In the present investigation, it was examined with RT-PCR whether expression of beta-catenin, a downstream gene of Wnt in its signal transduction pathway, is regulated. It was found that the level of protein or transcript beta-catenin during P19 neuronal differentiation was not changed. However, immunostaining data showed that beta-catenin was translocalized into nuclei after retinoic acid induced P19 cell aggregates were trypsinized and cultured in serum free N2 medium for 2 and 4 d. In this period, transcription of En-2, a downstream target gene of Wnt signal, increased evidently. The above data suggest that Wnt signals are involved in the early stage of neuronal differentiation process of P19 cell. Meanwhile, the distribution of beta-catenin on the neurites indicates that this protein may also be involved in neuritis outgrowth process.

Stearylamine liposome as a new efficient reagent for DNA transfection of eukaryotic cells.

The efficiency of stearylamine (SA) liposome in transfecting DNA into eukarytic cells was evaluated and compared with that of the Lipofectin reagent and the calcium phosphate transfection method. Results with the 21 cell lines tested varied, depending on the cell type. The overall indication was that SA liposomes had certain advantages over the Lipofectin reagent and both of them were superior to the calcium phosphate method, showing the greatest effect in 8 different cell lines, while the calcium phosphate method was the best in only 4 cell lines. There were 2 cell lines which could only be transfected by SA liposomes, another 2 only by the calcium phosphate method, but none depended solely on the Lipofectin reagent. The number of cell lines failed to be transfected was 2 for SA liposomes, 3 for calcium phosphate method, and 4 for the Lipofectin reagent. The advantage of the SA liposome method is discussed.

[Qualitative and quantitative studies of 1000 cases of isolated ventricular septal defect and follow-up using two-dimensional color Doppler echocardiography].

Color Doppler echocardiography (CDE) was used to evaluate 1,000 cases of isolated ventricular septal defect (IVSD). The types of IVSD included perimembrane (854), outflow tract (131) and muscular (15). Compared to surgical findings, CDE had 100% of sensitivity in the diagnosis of IVSD. The accuracy was 98.0% in determining the types of IVSD. CDE was significantly correlated with surgery in assessing sizes of defects (r = 0.893, P < 0.001). Continuous wave Doppler was significantly correlated with catheterization in measuring pulmonary systolic pressure (r = 0.838, P < 0.001). MPA/Ao value > 1.27 and enlargement of right ventricle were both considered good indices for reflecting pulmonary hypertension. A follow-up datum showed that 21 out of 80 cases of IVSD were closed spontaneously in their age of 7 months to 8 years, and the spontaneous closure was related to the types of defects. The complications of IVSD were also discussed.

Spectra properties of rat liver mitochondrial choline dehydrogenase.

Choline dehydrogenase contains the prosthetic group FAD, non-haem iron and acid labile sulfur. However, the absorption spectra of the purified enzyme do not change after adding substrate. The reduced absorption spectra of choline dehydrogenase can only be determined after the addition of dithionite. Those choline dehydrogenases situated in the mitochondrial inner membrane can be reduced by substrate and exist in the reduced state. When cholate was used to solubilize the substrate-reduced choline dehydrogenase, the reduced spectra will gradually disappear. However, if solubilization is carried out under anaerobic conditions, the reduced spectra can be retained, suggesting that the solubilized choline dehydrogenase can use oxygen as an acceptor.

Purification and some properties of glycerol-3-phosphate dehydrogenase from rabbit skeletal muscle mitochondria.

Glycerol-3-phosphate dehydrogenase (E. C. 1. 1. 99. 5) was solubilized from rabbit skeletal muscle mitochondria by Triton X-100 and purified through hydroxyapatite column chromatography, DEAE-Sepharose CL-6B column chromatography and sucrose density gradient ultracentrifugation. The preparation was electrophoretically pure, the total recovery was 10% and the specific activity had been increased 200-fold. The apparent molecular weight of the enzyme polypeptide was 69,000, it existed in the form of enzyme-Triton X-100 complex with a Stokes' radius of 59 A and a sedimentation coefficient of 10.7 S. There were 1.7 mg Triton X-100 and 26 micrograms phospholipid per mg protein of the preparation. The enzyme absorbed at 410 and 460 nm which could be attributed to non-haem iron and FAD respectively. Both of the absorption would be largely diminished by adding the substrate glycerol-3-phosphate.