ABSTRACT
Objective:To investigate the application effect of case-based learning (CBL) combined with flipped classroom in the teaching of experimental diagnostics in the integrated course of Diagnosis and Treatment Fundamentals. Methods:The cluster random sampling method was used to select the class of 2019 in the eight-year program and the class of 2020 in the five-year program, with the major of stomatology in Air Force Medical University. The 24 students in the observation group received CBL combined flipped classroom, and the 37 students in the control group received traditional teaching. The two groups were compared in terms of theoretical assessment score, classroom assessment score, comprehensive ability, self-learning ability, and degree of satisfaction with teaching. SPSS 22.0 was used for the t-test. Results:The observation group had a significantly higher theoretical assessment score than the control group [(74.88±3.46) vs. (71.89±4.45), P<0.05]. The observation group had significantly better scores of practical skill assessment than the control group ( P<0.05). Compared with the control group, the observation group had significantly better scores of comprehensive ability and self-learning ability ( P<0.05). The observation group had significantly better scores of satisfaction with teaching than the control group ( P<0.05). Conclusion:The application of CBL combined with flipped classroom in the teaching of experimental diagnostics in the integrated course of Diagnosis and Treatment Fundamentals can improve theoretical knowledge, practical skills, comprehensive quality, and satisfaction with teaching among students, and therefore, it holds promise for application in teaching.
ABSTRACT
Objective:To evaluate the performance of the automated digital cell morphology instrument in detecting platelet (PLT) clumps.Methods:A total of 4271 blood samples whose PLT reached the reviewing rules of thrombocytopenia were selected from inpatients having blood analysis in Xijing Hospital from January 1 st to June 30 th, 2019, including 2 200 males and 2 071 females,with a median age of (35±7.03) years old. The smears for these cases were made, stained by Wright-Giemsa, and examined to capture PLT clumps by digital cell morphology system and manual microscope separately. The digital cell analysis system (hereinafter referred to as the instrument method) as an evaluation method and the microscope method as a reference method were used to calculate the positive rate of platelet clump detection and evaluate the comparison of two methods and bias assessments. The chi-square test was used to compare counting data rates. Results:Among 4, 271 samples reaching the reviewing rule of thrombocytopenia, 128 cases with platelet clumps were detected by manual microscope(initial) with a positive detection rate of 96.24%, and a total 133 of cases with PLT clumps were detected by microscope (initial+reconfirmation) with a positive detection rate of 100 %. Meanwhile, 129 cases with platelet clumps were detected by instrument method with a positive detection rate of 96.9%. There was no significant difference in terms of positive rate of PLT clumps detection between the instrumental method and the microscope method (initial) ( χ2 =0.115, P=0.73); the positive rate of clumps detection by the instrumental method was lower than microscope method (initial+reconfirmation), and the difference was statistically significant (χ 2 =4.061, P=0.04). For instrument method, the positive rate of PLT clumps detection by simultaneous observation of RBC analysis interface+PLT aggregation interface+WBC analysis interface was higher than only observation of PLT aggregation interface, and the difference was statistically significant (χ 2 =5.090, P=0.02). The average error of the deviation of PLT counting results before and after correction of the cases with PLT plumps missed by instrument method was significantly higher than microscope method (initial), and the difference was statistically significant (χ 2 =56.26, P<0.001). Conclusion:The automated digital cell morphology system has a good consistency with manual microscope(initial) in terms of the sensitivity of platelet clumps detection and can be used as a supplementary method for detecting platelet aggregation.
ABSTRACT
Objective:To investigate the genetic etiology of a Marfan syndrome pedigree, and the impact of c.4336G>A variant on the splicing process of FBN1 gene.Methods:The proband was admitted to the Department of Cardiovascular Surgery of Xijing Hospital due to thoracic aortic aneurysm and dissection in August 2019. Multiplex PCR and next generation sequencing technology were used to detect 15 genes associated with hereditary aortic diseases in the proband. Then the pathogenic sites were further verified by Sanger sequencing, and above examinations were also performed among the family members of the proband. The effect of the mutation on mRNA splicing was predicted by splicing prediction software. RNAs from peripheral blood cells of the proband and the healthy person were extracted, and the effect of the mutation on mRNA splicing was verified by reverse transcription PCR and Sanger sequencing. The pathogenicity was analyzed by the recommendations from the American College of Medical Genetics (ACMG).Results:The gene panel detected a missense mutation of FBN1 gene (c.4336G>A) in the proband. Sanger sequencing results were consistent with that of panel. Sanger sequencing results showed that 4 family members were carriers of the same variant, and 3 out of the 4 family members presented signs of thoracic aortic aneurysm and dissection. The dbscSNV_ada_score and dbscSNV_rf_score software predicted that this mutation would lead to the occurrence of abnormal splicing of mRNA. The skipping of exon 35 was verified in the subsequent examinations by reverse transcription PCR and Sanger sequencing. The variant was classified as"pathogenic"according to ACMG guideline.Conclusion:FBN1 c.4336G>A mutation can cause the skipping of exon 35, and this might be the genetic mechanistic of severe cardiovascular abnormalities observed in this Marfan syndrome pedigree.
ABSTRACT
As a new type of intercellular signaling rector, extracellular vesicles (EV) are involved in almost the whole process of tumorigenesis, progression, metastasis, and drug resistance. Therefore, EV have become the ideal biomarker candidates and research hotspots for cancer diagnosis and treatment. However, EV tumor biomarker research mainly focused on RNA and protein, and a small part of the research focused on lipids at the early stage. EV DNA has received little attention and its diagnostic value has gradually been recognized in recent years. Study on the biological characteristics and function of EV DNA may highlight its potential in tumor diagnosis.
ABSTRACT
Objective:A multi-center and large sample volume study was conducted on the verification and improvement of the early established criteria for intelligent routine urinalysis validation (including the microscopic review rules and manual validation rules, referred to as intelligent criteria for short), in order to improve the clinical application of this intelligent criteria.Methods:A total of 31 456 urine specimens were collected from the inpatients and outpatients in six hospitals in China, from March to September 2019. Firstly, 3105 specimens were analyzed for preliminary verification and improvement of the intelligent criteria based on the results of the microscopic examination and manual validation. Secondly, 28 351 specimens were used to verify the clinical application of the improved intelligent criteria. All samples were manually validated as reference.Results:The approval inconsistency rate of the manual validation rules in the original intelligent criteria was 8.59% (202/2 352), and the interception inconsistency rate was 8.84% (208/2 352). The false negative rate and the microscopic review rate of the microscopic review rules were similar to the previous results. Based on an in-depth analysis of big data and the discussions by senior technicians from eight hospitals, one microscopic review rules and four manual validation rules were added, meanwhile two manual validation rule was deleted. The manual validation standards were unified. Finally, the intelligent criteria was improved. Based on the improved intelligent criteria, for microscopic review rules, the false positive rate, false negative rate (misdiagnosis rate), and microscopic review rate did not change significantly, which were 14.72% (457/3 105), 4.06% (126/3 105), and 24.73% (768/3 105), respectively. The approval inconsistency rate and the interception inconsistency rate of manual validation rules were both reduced to 0; the total manual validation rate of the intelligent criteria was 50.89% (1 580/3 105), and the auto-validation rate was 49.11% (1 525/3 105). The large sample volume verification results were consistent with the preliminary verification results of the improved intelligent criteria.Conclusion:This multi-center and large sample volume study had shown that the improved intelligent criteria had better clinical performance.
ABSTRACT
Objective@#To compare the efficacy of femoral triangle versus adductor canal approach to saphenous nerve block for postoperative analgesia in the patients undergoing knee arthroplasty.@*Methods@#Sixty American Society of Anesthesiologists physical status Ⅰ or Ⅱ patients of both sexes, aged 53-68 yr, scheduled for elective total knee arthroplasty under general anesthesia, were assigned into 2 groups (n=30 each) using a random number table method: femoral triangle approach to saphenous nerve block group (group F) and adductor canal approach to saphenous nerve block group (group A). Femoral triangle and adductor canal approach to saphenous nerve block was performed by injecting 0.5% ropivacaine 20 ml in group F and group A, respectively.Patient-controlled saphenous nerve block analgesia was used in two groups, and the analgesic pump solution contained 1% ropivacaine 400 mg diluted to 160 ml in 0.9% sodium chloride injection.The analgesic pump was set up with a 5 ml bolus dose, a 30-min lockout interval and background infusion at a rate of 5 ml/h, and analgesia lasted until 72 h after operation.When visual analog scale score > 4 and pain was not relived after 30-min pressing by patients, pethidine hydrochloride 100 mg was intramuscularly injected as rescue analgesic.The muscle strength of quadriceps femoris was assessed by manual muscle test at 4, 8, 24, 48 and 72 h after operation.The patient′s satisfaction score was assessed and recorded at 72 h after operation.Rescue analgesia and development of adverse reactions (local anesthetic intoxication, itching, dizziness, urinary retention, nausea and vomiting) were recorded within 72 h after operation.@*Results@#Compared with group F, the muscle strength of quadriceps femoris was significantly increased at 4, 8 and 24 h after operation, the rate of postoperative rescue analgesia was decreased (P<0.05), and no significant change was found in patient′s satisfaction score or incidence of adverse reactions in group A (P>0.05).@*Conclusion@#Adductor canal approach to saphenous nerve block provides better efficacy for postoperative analgesia than femoral triangle approach to saphenous nerve block in the patients undergoing knee arthroplasty.
ABSTRACT
Objective To compare the efficacy of femoral triangle versus adductor canal approach to saphenous nerve block for postoperative analgesia in the patients undergoing knee arthroplasty.Methods Sixty American Society of Anesthesiologists physical status Ⅰ or Ⅱ patients of both sexes,aged 53-68yr,scheduled for elective total knee arthroplasty under general anesthesia,were assigned into 2 groups (n=30 each) using a random number table method:femoral triangle approach to saphenous nerve block group (group F) and adductor canal approach to saphenous nerve block group (group A).Femoral triangle and adductor canal approach to saphenous nerve block was performed by injecting 0.5％ ropivacaine 20 ml in group F and group A,respectively.Patient-controlled saphenous nerve block analgesia was used in two groups,and the analgesic pump solution contained 1％ ropivacaine 400 mg diluted to 160 ml in 0.9％ sodium chloride injection.The analgesic pump was set up with a 5 ml bolus dose,a 30-main lockout interval and background infusion at a rate of 5 ml/h,and analgesia lasted until 72 h after operation.When visual analog scale score ＞ 4 and pain was not relived after 30-min pressing by patients,pethidine hydrochloride 100 mg was intramuscularly injected as rescue analgesic.The muscle strength of quadriceps femoris was assessed by manual muscle test at 4,8,24,48 and 72 h after operation.The patient's satisfaction score was assessed and recorded at 72 h after operation.Rescue analgesia and development of adverse reactions (local anesthetic intoxication,itching,dizziness,urinary retention,nausea and vomiting) were recorded within 72 h after operation.Results Compared with group F,the muscle strength of quadriceps femoris was significantly increased at 4,8 and 24 h after operation,the rate of postoperative rescue analgesia was decreased (P＜0.05),and no significant change was found in patient's satisfaction score or incidence of adverse reactions in group A (P＞0.05).Conclusion Adductor canal approach to saphenous nerve block provides better efficacy for postoperative analgesia than femoral triangle approach to saphenous nerve block in the patients undergoing knee arthroplasty.
ABSTRACT
Objective To compare the sensitivity of four kinds of drug susceptibility test method in detecting sensitivity of tigecycline against Acinetobacter baumannii.Methods The susceptibility of 72 clinically isolated strains of carbapenemase-resistant Acinetobacter baumannii(CRAB) to tigecycline in vitro was detected with disk diffusion method,VITEK 2 Compact system,E-test and MIC test strip(MTS) test strip respectively,according to FDA standards,and the differences of four kinds of drug susceptibility test methods were compared.Results The susceptibility rates of 72 strains of CRAB to tigecycline by disk diffusion method,VITEK 2 Compact system,E-test and MIC test strip were 50.00%,69.44%,36.11% and 98.61% respectively,the intermediate rates were 48.61%,29.17%,26.39% and 1.39% respectively,the resistant rates were 1.39%,1.39%,37.50% and 0.00% respectively.Compared with MTS,the classification consistency rates of E-test,disk diffusion method and VITEK 2 Compact system were 36.11%,51.39% and 70.83% respectively.Conclusion There is difference among four kinds of method for conducting the drug susceptibility testing of tigecycline against CRAB,the consistency of disk diffusion method,VITEK 2 Compact system and E-test is lower.Detecting mediation or drug resistant strains of CRAB by disk diffusion method,VITEK 2 Compact system and E-test needs to be verified by MTS or Broth dilution method.
ABSTRACT
Objective To evaluate the diagnostic performance of Xpert MTB/RIF for the diagnosis of tuberculous meningitis (TBM).Methods This was a prospective, single center clinical trial.A total of 116 consecutive patients with suspected meningitis who were admitted to Xijing Hospital from October 2013 to June 2015 were recruited.Mycobacterium tuberculosis ( MTB) and rifampicin ( RIF) resistance mutations in 1 ml cerebrospinal fluid ( CSF) were detected with Xpert MTB/RIF and the remaining sample was tested by Ziehl-Neelsen staining , MGIT960 liquid culture and other laboratory tests .And the enrolled patients were grouped according to the 2010 South African expert consensus .The diagnostic performance of Xpert MTB/RIF was evaluated by comparing against clinical score >5 points and MGIT960 liquid culture as reference standards respectively .The comparison was performed using a χ2 test or Fisher′s exact test for categorical variables and a nonparametric rank sum test for continuous variables .Results Among the enrolled 116 subjects, 23 subjects were diagnosed as definite-TBM by MGIT960 liquid culture, 16 subjects were classified as probable TBM , 27 subjects were classified as possible TBM , and 50 subjects were classified as non-TBM.When clinical score >5 points was used as a reference standard , the sensitivity of Xpert MTB/RIF (39.4%) was comparable with that of MGIT960 liquid culture (34.8%) (χ2 =0.292, P=0.589), and significantly better than that of Ziehl-Neelsen staining (9.9%) (χ2 =16.500, 12.771, P<0.001). No significant differences were found among the specificities of Xpert MTB /RIF, MGIT960 liquid culture and Ziehl-Neelsen staining ( 98.0%, 100.0% vs 98.0%, χ2 =1.014, P=0.602 ) .When tested against MGIT960 liquid culture as a reference standard , the sensitivity of Xpert MTB/RIF was 91.3%. Conclusions Xpert MTB/RIF is a rapid and specific method to detect MTB and RIF resistance in CSF .It exhibits a good rule in value for the diagnosis of TBM and a comparable sensitivity with MGIT 960 liquid culture, thus it can be used as the initial method for the diagnosis of tuberculous meningitis .
ABSTRACT
Objective To study the characteristics of antimicrobial resistance and molecular epidemiology of Staphylococcus aureus (S .aureus)in the intensive care units(ICUs)of a hospital.Methods Clinical isolates of S .aureus collected from ICUs between January and December 2014 were identified and performed antimicrobial susceptibility testing,then typed by staphylococcal protein A (spa)typing and multilocus sequence typing (MLST) methods.Results Of 160 isolates of S .aureus ,120 (75.00%)were methicillin-resistant S .aureus (MRSA). Resistance rates of MRSA to erythromycin,clindamycin,and levofloxacin were all > 80%;methicillin-sensitive S .aureus (MSSA)were sensitive to cefazolin,resistance rates to erythromycin,clindamycin,and levofloxacin were 62.50%,35.00%,and 10.00% respectively.spa typing and MLST results showed that the main types of 120 isolates of MRSA were ST239-t030,ST239-t037,and ST5-t2460,the major epidemic strains were ST239-t030 (n=105,87.50%),and were isolated from 8 ICUs;MSSA had more types,ST59-t437 were detected only from depart-ment of neurology(n =8)and department of digestive diseases(n =2),ST6-t701 ,ST398-t3625,ST398-t1793,and ST121-t2092 were isolated from departments of neurology(n=7),anesthesiology(n=5),neurosurgery(n=4),and cardiac surgery(n=4)respectively.Conclusion Isolation rate of MRSA in ICUs in this hospital is high,ST239-t030 is the main type,which prevailed in hospital;different types of MSSA have epidemic trends in various departments.
ABSTRACT
Objective To understand the clindamycin resistance gene and molecular epidemic of Staphylococcus aureus in the pa‐tients with bloodstream infection in our hospital during 2014 .Methods The clinical isolates of Staphylococcus aureus in clinical bloodstream infection were collected during 2014 .The phenotype of erythromycin to clindamycin induced resistance was assessed by D test .The erm gene was detected by PCR .The different erm types of the molecular epidemiology of Staphylococcus aureus were studied by spa and MLST typing .Results In 33 strains of Staphylococcus aureus ,the isolation rate of MRSA strains were 78 .79% ,moreover all of MRSA strains carried ermA gene .In 7 strains of methicillin sensitive Staphylococcus aureus(MSSA) ,4 strains respectively carried ermB or ermC gene .The results of MLST and spa results showed that the main type of MRSA in our hospital was ST239‐t030 .But MSSA had more types ,such as ST59‐t437 ,ST398‐t3625 and so on .Conclusion MRSA has higher i‐solation rate in our hospital ,which is dominated by ST239‐t030 type .For the detection of gene erm ,ermA (86 .67% ) is the main type .The strains are obviously resistant to antibacterial drugs .The laboratory should strengthen the detection of clindamycin in‐duced resistance for guiding the clinical rational use of antibacterial drugs .
ABSTRACT
Objective To investigate the distribution and antimicrobial resistance of multidrug‐resistant organisms(MDROs) . Methods The distribution and antimicrobial resistance of MDROs ,isolated from 2010 to 2014 ,were retrospectively analyzed . MDROs were identified according to international consensus .The WHONET5 .6 software was used to analyze data .Results A to‐tal of 5 709 strains of MDROs were isolated in five years ,in which 2 441 strains were Staphylococcus(42 .76% ) ,2 091 strains were non‐fermentive bacterial(36 .63% ) ,737 strains were Enterococcus(12 .90% ) ,440 strains were Enterobacter(7 .71% ) .Of the 5 709 MDROs isolates ,55 .04% were isolated from respiratory tract specimens .The resistant rate of multidrug‐resistant E .coli and K . pneumoniae against cefoperazone/sulbactam ,imipenem and meropenem was less than 30% .The resistance of multidrug‐resistant A . baumanii was higher than 90% ,except to minocycline and cefoperazone/sulbactam ,20 .2% and 50 .6% respectively .The resistant rate of multidrug‐resistant P .aeruginosa was 71 .4% -97 .0% against other antimicrobial agents ,except to polymyxin B .The resist‐ance of multidrug‐resistant E .faecium against the antimicrobials was higher than 90% ,except 13 .8% to minocycline and less than 3% to linezolid ,teicoplanin and vancomycin .Meanwhile ,1 linezolid resistant strain was identified in 1 914 methicillin resistant S .au‐reus(MRSA) strains and all MRSA strains were susceptible to vancomycin and teicoplanin .Conclusion MDROs could be predomi‐nated by A .bauman and MRSA in this hospital .Monitoring and control measures to healthcare‐associated infections should be in‐tensified to prevent the spread of MDROs .
ABSTRACT
GeneXpert is an advanced molecular biological detection system developed in recent years, it integrates and automates the sample preparation , nucleic acid purification, gene amplification, results report of the fluorescent quantitative PCR process.As, an accurate, rapid, biosafe technology , GeneXpert has become one of the novel molecular POCT detection technology platforms for diagnosis of infectious pathogens and especially declared it a major milestone for global TB diagnosis .This paper introduces the detection principle , clinical application in the detection of infectious pathogens , limitation and prospect of GeneXpert.
ABSTRACT
Objective To express recombinant HBHAΔC and HBHAΔN protein,and compare the HBHA series protein activity with each other.It will be provide a experimental basis for the research on clinical diagnostic of HBHA.Methods The HBHAΔC and HBHAΔN gene fragments were cloned and expressed by transforming E.coli BL-21.Test the protein heparin binding ability by CL-6B column.And then added protein to the BCG 7H9 culture medium,to observe the induced BCG aggregation.Results nHB-HA,rHBHA and HBHAΔN protein have heparin binding ability.Meanwhile nHBHA,rHBHA and HBHA Δ C protein have in-duced BCG aggregation effect.Conclusion The HBHAΔC and HBHAΔN protein were successfully obtained.It was proved that the HBHA C-terminal could be combined with heparin and the N-terminal involved could induce the aggregation of BCG.This results provide a basis for further study on molecular mechanism of TB infection and clinical application.
ABSTRACT
Objective To evaluate the value of T-cell spot of tuberculosis test (T-spot.TB) in differential diagnosis of Crohn's disease and intestinal tuberculosis.Methods From May 2010 to October 2010,in Xijing hospital,Fourth Military Medical University,the peripheral blood samples of 126 patients were collected and peripheral blood mononuclear cells were isolated with density gradient centrifugation.T-spot.TB was conducted according to the kit instructions.The clinical diagnosis of Crohn's disease and intestinal tuberculosis was according to clinical manifestations, imaging,endoscopy,pathology,laboratory tests and on empirical anti-TB treatment response.The sensitivity and specificity of T-spot.TB in diagnosis of Crohn's disease and intestinal tuberculosis was analyzed.Results Fifteen patients were diagnosed as Crohn's disease (11.9％,15/126),14 patients were intestinal tuberculosis (11.1％,14/126) and 40 patients were extraintestinal tuberculosis (31.7％,40/126).The positive rate of T- spot.TB in Crohn's disease,intestinal tuberculosis,extra-intestinal tuberculosis and other diseases was 1/15,12/14,70％ (28/40) and 0％ (0/57),respectively.Thedifference between the groups was statistically significant (P =0.00).There was statistically significant difference of T-spot.TB positive rate between Crohn's disease and intestinal tuberculosis (x2 =70.58,P=0.00).The sensitivity and specificity of T- spot.TB in Crohn's disease detection was 93.3％(14/15) and 87.5％(14/16),in intestinal tuberculosis was 85.7％(12/14) and 93.3％ (14/15).The negatively predictive value of Crohn's disease was higher [87.5％ (14/16)] than that of intestinal tuberculosis [12.5％ (2/16)].Conclusion T-spot.TB is helpful for differential diagnosis of Crohn's disease and intestinal tuberculosis.
ABSTRACT
ObjectiveTo screen out the two-component system associated with drug resistance of Mycobacterium tuberculosis by detecting the differential expression of two-component system regulator genes between multidrug resistant Mycobacterium tuberculosis strains and drug sensitive strains. MethodsTotal RNA of MTB was extracted from cultured MTB during the logarithmic phase in the 7H9 brook medium, and then its purity was identified. Reverse transcription was further completed. The expressing levels of TCS response regulators were quantified using SYBR Green I qRT-PCR, which aimed at finding the differential expressions between multidrug resistant strains and sensitive strains. Finally, all of differentially expressed TCS were screened out under the stress of INH, SM and LFA. Results Compared with sensitive strains,multidrug resistant strains of Rv0491, Rv3133c, Rv3143 and Rv3246c were up-regulated 1. 03, 7.11,3.48and 1.37 folds, respectively (t/t' =5. 623, -4. 196, -3. 559 and -3. 016, respectively, P ＜0. 01 ). The expressing level of other regulators had no statistical significance between muhidrug resistant strains and drug sensitive strains. Under the antibiotic pressure, the expression of Rv1027c, Rv3246c and Rv3143 showed significant changes compared with no antibiotic group. ConclusionRv3246c and Rv3143 may be associated with MTB drug resistance and the differentially expressed genes in multi-drug resistant strains may be used as potential drug targets against drug resistant tuberculosis.
ABSTRACT
Objective To purify native and recombinant heparin-binding hemagglutinin(HBHA)protein,and investigate the activity of HBHA polyclonal antibody against aggregation of Bacillus CalmetteGuerin(BCG)induced by HBHA.Methods After growing BCG to the stationary phase in the 7H9 liquid medium,the native HBHA protein(nHBHA)was obtained by CL-6B column chromatography.At the same time,the HBHA gene fragment was cloned and expressed by transforming Escherichia coli BL-21.Then the polyclonal antibody against rHBHA was prepared by immunizing rabbit.Different comcentration of the HBHA protein was added to the BCG liquid medium,and the aggregation of the BCG was observed.Then,add the HBHA protein that incubated with anti-HBHA antibodies to the BCG culture medium and observe the aggregation of BCG.Results The purity of native HBHA was 99% and the concentration was 1.016 mg/ml.The expressed product contained 36% of total somtic protein.After purified,the purity of the recombinant HBHA protein was 97.1% and the concentration was 10.98 mg/ml.Both the rHBHA and nHBHA could induce the aggregation of BCG.When then concentration of nHBHA is 0.2μg/ml,BCG could be induced to aggregate,while the rHBHA concentration is 2μg/ml could induce the aggregation.Both aggregations could be suppressed by the polyclonal antibody against rHBHA.Conclusions The native and recombinant HBHA are successfully obtained.It is proved that the rHBHA could induce the aggregation of BCG similar as nHBHA,and polyclonal antibody against rHBHA could also suppress the activity of nHBHA.It suggested that rHBHA could be further used in clinical diagnosis and vaccination.
ABSTRACT
Objective To investigate the relationship between the phenotypes and the patterns of genetic mutations in the corresponding resistance genes (rpoB, katG, inhA, ahpC, rrs, rpsL, embB and gyrA) in resistant Mycobacterium tuberculosis (MTB) isolates. Methods Rifampicin-resistant gene (rpoB), isoniazid-resistant genes (katG, inhA, ahpC), streptomycin-resistant genes (rrs, rpsL), ethambutol-resistant gene (embB) and quinolinone-resistant gene (gyrA) were amplified by PCR with sequence-specific primers, then mutants screened by single-stranded conformation polymorphism (SSCP) were sequenced. Results rpoB mutation with predominant Ser450Trp pattern was 94. 9% (56/59) in 59 rifampicin-resistant isolates;katG mutation rate was 38. 9% (35/90) and the main pattern was Ser315Thr, but only 3 inhA mutants and no ahpC mutation were determined in 90 isoniazid-resistant isolates;gyrA mutation with main Asp94Gly then Ala90Val pattern was 82.4% (28/34) in 34 quinolinone-resistant isolates;the total mutation rate was 77.4% in 31 streptomycin-resistant isolates, of which 15 isolates mutated in rrs with main pattern A514C or A1041G, 10 isolates mutated in rpsL Lys88Arg;and embB mutation with main Met306Val accounted for 19.4% (6/31) in 31 ethambutol-resistant isolates. Conclusions The results showed that resistance of resistant MTB may be complicated, and DNA sequencing-based mutation analysis could efficiently detect the molecular makers such as rpoB, katG, gyrA, rrs, rpsL and embB in resistant MTB isolates. Meanwhile, it is notable that the rpoB mutation pattern in our isolates is different from previous report, further effort are needed to confirm the characteristics. The spectrum of potential resistance-related mutations in MTB clinical isolates may lay substantial foundation for the rapid molecular diagnosis and rational use of drug to MTB patients.
ABSTRACT
Objective To investigate antifungal activities of AMB, ICZ, VRC, CBF against 72 strains of filamentous fungi in vitro. Methods Based on CLSI M38-P and M38-A scheme, MIC of antifungal drugs were determined. The growing inhibitory concentration of 100%, 100%,≥80%, for AMB, VRC ,ICZ act as respective MIC. For caspofungin, the minimal effective concentration (MEC) was determined as the lowest drug concentration showing morphology change of filaments. The fractional inhibitory concentration (FIC) was used to evaluate the effect of combination therapy. FIC was calculated by the following equation: FIC = MICcombination/MICA drug alone+ MICcombination/MICB drug alone. Results MIC90 of AMB, ICZ, CBF, VRC against 72 isolates of filamentous fungi were 8 μg/ml, 4 μg/ml, 2 μg/ml, 8 μg/ml, respectively. MICs range of combined AMB + ICZ, AMB + VRC, ICZ + VRC were 0. 125-16. 97, 0. 2452-1.25, and 0.0625-8. 25 μg/ml respectively. The percent of synergistic interaction of AMB + VRC against filamentous fungi (20.0%-88.9% ) was higher than those of AMB + ICZ ( 10.0% -62.5% ) and ICZ + VRC ( 20.0% - 44.4% ) ( P=0.007 <0.05 ). Conclusions The antifungal activities of four kinds antifungal drugs against 72 strains of filamentous fungi vary in vitro. The therapy of AMB combined with VRC is maybe better than AMB + ICZ and ICZ + VRC for severe fungi infection.
ABSTRACT
Objective To detect the isoniazid (INH) and rifampin (RIF) resistance of Mycohaeterium tuberculosis isolates in the single tube with multiplex-polymerase chain reaction-single strand conformation polymorphism(muhi-PCR-SSCP) system. Methods According to the sequences of inhA, katG and rpoB genes of the Mycohacterium tuberculosis, three pairs of oligonucleotide primes were designed to examine the INH and RIF resistance with the multi-PCR-SSCP. The validity of the newly developed method was evaluated with 116 clinical isolates of Mycohacterium tuberculosis( 70 isolates that were INH-resistant and 66 isolates that were RIF-resistant). Results The validity of the method was assessed with multiplex PCR-SSCP with the bacteria culture with susceptibility test as golden standard. The three genes, katG, inhA and rpoB, in the 116 clinical isolates and H37Rv strain were amplified successfully in single PCR reactions,except 4 isolates with katG deletion mutants. Compared with strain H37Rv, forty-six isolates had katG gene mutations, thirteen had inhA mutations and fifty-eight had rpoB mutations. Thirty-eight isolates had simultaneous katG and rpoB mutations and 4 isolates had both inhA and rpoB mutations. Four isolates had inhA and katG mutations and 2 isolates had mutations in all three genes simultaneously. The sensitivity of the newly developed multiplex-PCR-SSCP assay was 80% and 82% for INH and RIF, respectively. The specificity of the assay was 100% and 92% for INH and RIF, respectively. Conclusion Muhiplex-PCRSSCP provides a rapid, specific and cost-effective method of detecting multidrug-resistant TB. It laid a solid foundation for the further study of drug resistant gene.