Objective To establish TaqMan real-time PCR method for detection and identification of Haemophilus influenzae and Streptococcus pneumonia. Methods Two sets of primers and FAM-labeled probes targeting different genes of Haemophilus influenzae and Streptococcus pneumoniae were designed and synthesized. The bexA gene was used for identification of Haemophilus influenzae and lytA for Streptococcus pneumoniae. The sensitivity and specificity of real-time PCR were assessed for different primers and probes. Cut-off values of cycle threshold (Ct) were determined. Two hundred and seventy-eight cerebrospinal fluid (CSF) specimens from suspected bacterial meningitis cases were detected by real-time PCR assay, latex agglutination test and bacteria culture simultaneously. Results Haemophilus influenzae isolates of serotype a to d could be detected and identified by bexA primers and probe. All Streptococcus pneumoniae isolates of different serotypes could be detected and identified by lytA primers and probe. The respective sensitivities for Haemophilus influenzae and Streptococcus pneumoniae were 10 and 90 genome DNA copies in each PCR reaction. Of the 278 CSF specimens, four were positive by Haemophilus influenzae and seven positive by Streptococcus pneumoniae when detected by real-time PCR. Of the four Haemophilus influenzae positive specimens, two were positive by culture and one positive hy latex. Of the seven Streptococcus pneumonia positive specimens, two were positive by culture and two positive by latex. Conclusions Real-time PCR could rapidly detect and identify Haemophilus influenzae of serotype a to d and Streptococcus pneumoniae of different serotypes with high sensitivity. TaqMan real-time PCR could be widely used for the diagnosis of invasive meningitis caused by Haemophilus influenzae and Streptococcus pneumoniae. It can improve the rate of positivity for diagnosis of suspicious bacterial meningitis cases.

Objective: To observe the relationship between the dynamic changes of plasma levels of urotensin II (UII) and the stability of coronary atherosclerotic plaque in patients with acute coronary syndrome (ACS).
Methods: Our research included 2 groups: ACS group,n=135 consecutive patients treated in our hospital from 2013-03 to 2013-08 that including unstable angina pectoris (UAP) sub-group,n=7, non-ST segment elevation myocardial infarction (NSTEMI) sub-group,n=22 and STEMI sub-group,n=106. In addition, there was a Control group,n=48 healthy subjects. Plasma levels of UII, hs-CRP and NT-proBNP were examined and compared among different groups at different time points.
Results: Compared with Control group at immediate admission, ACS group had increased plasma level of UII (39.82 ± 22.28) pg/ml vs (26.88 ± 6.09) pg/ml,P<0.001; UII level in STEMI sub-group was lower than NSTEMI sub-group (37.41 ± 22.74) pg/ml vs (48.07 ± 15.82) pg/ml,t=2.092,P <0.05. In ACS patients, UII had no correlation to hs-CRP (r=0.041, P=0.639) and NT-proBNP (r=0.112,P=0.261) at immediate admission. There were 58 ACS patients finished the 3 months follow-up study and their UII level was increased than immediate admission as (56.52 ± 20.70) pg/ml vs (51.58 ± 18.70) pg/ml,t=-2.366,P<0.05.
Conclusion: Plasma levels of UII have been changing in different type of ACS patients at immediate admission, UII presented decreasing trend from UAP to NSTEMI to STEMI, while it had increasing trend upon stabilized condition; the admission level of UII had no correlation to inflammatory marker hs-CRP and ventricular overload marker NT-proBNP. UII is not only related to the extent of atherosclerosis, but also related to the nature of atherosclerosis or the stability of plaques.

Objective To evaluate the antigenicity of two proteins of Mycobacteium tuberculosis (M. tuberculosis), Dnak(Rv0350) and MPT83(Rv2873), in order to provide a scientific basis for immuno-logical diagnosis of tuberculosis and research on vaccines. Methods The two antigen proteins, Dnak (Rv0350) and MPT83(Rv2873), were cloned, expressed and purified using the methods of genetic recom-bination and protein purification technology. Blood samples were collected from subjects including tuberculo-sis patients ( TB) , non-tuberculosis patients with other pulmonary diseases ( non-TB) and healthy volunteers (HV). To analyze the immunological properties of the recombinant Dnak (Rv0350) and MPT83 (Rv2873) proteins, they were used as antigens to detect humoral and cellular immunity in the subjects with enzyme linked immunosorbent assay ( ELISA ) and effector T cell enzyme-linked immunospot assay ( ELISPOT ) . Results The recombinant and purified Dnak (Rv0350) and MPT83 (Rv2873) proteins of M. tuberculosis were successfully obtained and used as antigens in the detection of humoral and cellular immunity in the sub-jects. Specific antibodies ( IgG) in the serum samples of 135 TB, 56 non-TB and 94 HV were tested with ELISA. The results showed that the sensitivity, specificity and accuracy of Dnak ( Rv0350 ) protein were 77. 80％ (105/135), 56. 70％ (85/150) and 66. 67％ (190/285). Similarly, the sensitivity, specificity and accuracy of MPT83 (Rv2873) protein were 76. 30％ (103/135), 43. 30％ (65/150) and 58. 95％(168/285). Cellular immunity was tested with the levels of IFN-γproduced by effector T lymphocytes after stimulating peripheral blood monouclear cells ( PBMC) collected form subjects of 59 TB, 65 non-TB and 64 HV with Dnak (Rv0350) and MPT83 (Rv2873) protein antigens. The results showed that the sensitivity, specificity and accuracy of Dnak (Rv0350) and MPT83 (Rv2873) proteins were 66. 10％ (39/59), 62. 79％ (81/129) and 63. 83％ (120/188), and 47. 46％ (28/59), 79. 84％ (103/129) and 69. 68％(131/188), respectively. Conclusions M. tuberculosis Dnak (Rv0350) and MPT83 (Rv2873) proteins have good antigenicity and could stimulate T cells to produce stronger immune responses. The two proteins used in combination might have promising potential in the research of immunodiagnosis of tuberculosis and the development of new anti-tuberculosis vaccines.

Objective:To explore the influencing factors of dietary behavior change of young and middle-aged patients after percutaneous coronary intervention (PCI) from the perspective of the family system, so as to provide the basis for home dietary intervention of patients.Methods:Using the phenomenological research method, 32 young and middle-aged PCI patients and their family members from Department of Cardiology, Xuanwu Hospital, Capital Medical University were interviewed in a semi-structured in-depth way by purposive sampling method from May to October 2022, and the data were analyzed by Colaizzi 7-step analysis method.Results:Among the 32 surveyed individuals, there were 17 males and 15 females, aged 22-61 years old. Two themes of dietary behavior change facilitators and barriers of young and middle-aged patients after PCI were extracted. The facilitators included six subthemes: behavioral autonomy, adaptive change, small family size, motivation for family responsibility, internal family resources, and external family resources. The barriers included five subthemes: bad learned habits, special physical conditions, lack of nutritional literacy, passive dietary environment, and limited economic level.Conclusions:Dietary behavior changes in young and middle-aged patients after PCI were affected by individual and family factors in the family. Medical staff should establish a family-centered dietary management model and integrate the advantages of family resources to give patients targeted individualized nutrition intervention.

China ; Humans ; Streptococcus pneumoniae

Objective To investigate the cross-reactive immune responses to Mycobacterium vac-cae (M. vaccae), Mycobacterium tuberculosis (M. tuberculosis, H37Rv) and Mycobacterium bovis Bacillus Calmette-Guerin ( BCG) for providing reference for the development of new vaccines with M. vaccae. Meth-ods M. vaccae (ATCC95051), M. tuberculosis (H37Rv) and BCG (China strain) were cultured on L-J solid media and harvested. Total bacterial protein antigens prepared by ultrasonic disruption were used to im-munize BALB/c mice. IgG antibodies in serum samples were detected with enzyme-linked immunosorbent assay ( ELISA) to evaluate humoral immune responses. Cellular immunity was assessed by detecting various cytokines with cytokine release assay ( CRA) . Results The mice that were respectively immunized with the three mycobacterial antigens could produce high titers of antibodies ( IgG) and high levels of IFN-γand IL-2, but low levels of IL-4 and IL-10. Results of the cross reactivity tests showed that ATCC95051, H37Rv and BCG were able to cross-react with the immunized mice, and all of them induced high levels of IFN-γ, IL-2 and IgG antibodies. Conclusions The three Mycobacteria mainly elicited Th1 immune responses. There were cross-reactive immune responses to M. vaccae, M. tuberculosis and BCG, which might provide ref-erence for using M. vaccae in the development of new anti-tuberculous vaccines.

Objective:To evaluate the humoral and cellular immunoreactivity of recombinant Mycobacterium tuberculosis ( M. tuberculosis) PstS1 and HspX protein antigens in order to provide reference for immunodiagnosis of tuberculosis and screening of candidates for vaccine antigens. Methods:Purified recombinant M. tuberculosis PstS1 and HspX proteins were obtained using molecular cloning expression and Ni 2+ affinity chromatography. Blood samples and epidemiological data of healthy individuals and patients with M. tuberculosis infection were collected. Specific IgG antibodies and IFN-γ-producing antigen-specific T cells were respectively detected by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT) with the recombinant proteins used as antigens. The humoral and cellular immunoreactivity of the recombinant PstS1 and HspX proteins were assessed with statistical analysis of data. Results:Both the recombinant PstS1 and HspX proteins could induce the secretion of IFN-γ by more specific effector T cells in patient with M. tuberculosis infection, and the differences between the infection and healthy control groups were statistically significant ( P<0.05). The specificity and sensitivity of the recombinant PstS1 and HspX as the diagnostic antigens of ELISPOT were 92.11% (35/38) and 65.96% (31/47), and 68.42% (26/38) and 91.49% (43/47), respectively. The two proteins also possessed some humoral immunoreactivity, but statistically significant difference was only observed in the HspX-specific antibody level between the two groups ( P<0.05). Conclusions:Both the recombinant PstS1 and HspX proteins had good cellular immunoreactivity and were the immunodominant antigens of cellular immunity. They performed well in cellular immunodiagnosis and were good potential candidate antigens for anti-tuberculosis vaccines.

Objective:To preliminarily evaluate the immunogenicity and efficacy of two novel tuberculosis vaccine candidates (a fusion multicomponent protein EPDPA015f and a mixed multicomponent protein EPDPA015m) and to provide a new antigen combination for the development of tuberculosis vaccines.Methods:Recombinant plasmids for the expression of EPDPA015f and EPDPA015m proteins were constructed. Six-week-old BALB/c mice were immunized with EPDPA015f or EPDPA015m in combination with aluminium adjuvant (50 μg/mouse) for three times with an interval of 10 d. The mice were sacrificed 10 d after the last immunization to collect blood and spleen samples. Serum antibody titers and cytokine levels were measured by ELISA, Luminex technique and enzyme-linked immunospot assay (ELISPOT). Mycobacterial growth inhibition assay (MGIA) was used to detect the ability of mouse splenocytes to inhibit the growth of Mtb in vitro. One-way analysis of variance and t-test were used for statistical analysis. Results:Both EPDPA015f and EPDPA015m could induce the production of various cytokines and IgG antibodies at a high level. The levels of cytokines related to Th1 (IL-2, TNF-α, IFN-γ), Th2 (IL-4, IL-6, IL-10) and Th17 (IL-17) as well as other proinflammatory cytokines (GM-CSF, IL-12) were higher in the EPDPA015f group than in the adjuvant group ( P<0.05). The titer of IgG antibody induced by EPDPA015f was as high as 1∶4×10 6. The results of MGIA showed that the numbers of Mtb (lgCFU) in the PBS, adjuvant, EPDPA015f and EPDPA015m groups were 3.46±0.11, 3.51±0.06, 2.98±0.09 and 3.19±0.08, respectively. The number of colonies in the EPDPA015f group was the least as compared with that in the other three groups ( P<0.001, P<0.001, P<0.01). Conclusions:The vaccine candidate EPDPA015f could elicit more comprehensive and high-level cellular and humoral immune responses, and exhibited superior in vitro inhibitory activity against the growth of Mtb. EPDPA015f had the potential to be used as a preventive vaccine or a booster vaccine

Objective To investigate the single nucleotide polymorphism (SNP) of toxinantitoxin-chaperone (TAC) system of Mycobacterium (M.) tuberculosis with different genotypes and its biological significance.Methods A total of 183 clinical M.tuberculosis isolates were collected for spoligotyping.The sequences of higA,higB and Rv1957 were obtained by using PCR and DNA sequencing.The sequences were compared for possible mutations.Functional consequences of nonsynonymous SNPs were predicted by using I-Mutant 2.0 servers.Results Among the 183 M.tuberculosis isolates,138(75.41％) belonged to the Beijing family,while 45(24.59％) belonged to the non-Beijing family.A total of 149(81.42％) isolates showed polymorphisms in the TAC system.We discovered 6 nonsynonymous SNPs and 2 synonymous SNPs.All the synonymous mutations occurred in higA gene,while nonsynonymous SNPs were found in the higA,higB and Rv1957 genes either.All the synonymous mutations and 4 nonsynonymous SNPs were restricted to the Beijing family strains and only 2 nonsynonymous SNPs were observed in the non-Beijing family strains.Of the 6 nonsynonymous SNPs studied,4 were predicted to have ability to affect the stability of respective protein.Conclusion The SNPs in the coding sequences of TAC system in clinical isolates can be relatively high and the Beijing family strains are with higher polymorphism,which might benefit to adapt to different host environment.

Objective: To establish and evaluate a multiplex PCR method for rapid identification of Mycobacterium species in order to provide an approach for rapid diagnosis of pathogens causing tuberculosis. Methods: Four genes including 16S rRNA, Rv0577, RD9 and mtbk_20680 were selected to establish the multiplex PCR method. Specific primers were designed and the reaction system and conditions were optimized. The sensitivity and specificity of the multiplex PCR method were evaluated through identifying Mycobacterium africanum (M.africanum), Mycobacterium bovis (M.bovis), M. bovis BCG, seven common non-tuberculosis Mycobacterium (NTM), seven reference species of common respiratory bacteria and 93 clinical strains of Mycobacterium tuberculosis (MTB) isolated from patients with tuberculosis in Gansu Province of China. Results: The fragments of 16S rRNA, Rv0577, RD9 and mtbk_20680 genes were 543 bp, 786 bp, 369 bp and 231 bp in length, respectively. MTB strains of the Beijing genotype were positive for all of the four genes, while the non-Beijing genotype strains were negative for mtbk_20680. M. africanum, M. bovis and M. bovis BCG strains were negative for 16S rRNA and Rv0577. NTM strains only carried 16S rRNA and none of four genes were detected in the seven species of respiratory bacteria. Among the 93 clinical MTB strains, 80 (86.02%) belonged to the Beijing genotype and the other 13 (13.98%) were non-Beijing genotype strains. The specificity of the multiplex PCR method was 100%. Conclusions The established multiplex PCR method could accurately distinguish Mycobacterium, Mycobacterium tuberculosis complex (MTBC), NTM, MTB, and Beijing and non-Beijing genotype MTB with high sensitivity and specificity.