Standardization and regularization for antimicrobial susceptibility testing in clinical microbiology laboratory is obvious and vital to ensure reliable and reproducible results to guide early and rapid therapy for clinical physicians.Clinical microbiology laboratory should play an important role in antimicrobial therapy.

Objective To develop a selective isolation media which has a high isolation ratio for Legionella.Methods We compared the growth of Legionella pneumonia on BCYE? plate (with DGVP), BCYE?(with GVPC) and BCYE? (with CCCV) by colony count and compared with BCYE? plate. The comparison of isolation ratio of Legionella pneumonia from air-conditions cooling water with 3 kinds of BCYE? plate containing different anti-reagents was performed.Results The results of colony count on BCYE? plate (with DGVP) and BCYE?(with GVPC) was identical with that on BCYE? plate at same concentration. But the result of colony count on BCYE? plate (with CCCV) was half of the former 2 kinds of plate. 5 strains of Legionella pneumonia isolated from 9 central air-condition cooling water were used by BCYE? plate (with DGVP) and BCYE? (with GVPC), while only 2 strains of Legionella pneumonia were isolated by BCYE? plate (with CCCV). Legionella pneumonia wasn′t detected by BCYE? plate.Conclusion BCYE? plate with antibiotics system of CCCV showed obvious suppression to Legionella pneumonia, and so that there was a low isolation ratio to Legionella pneumonia. But BCYE? plate with antibiotics system of DGVP and GVPC had a few suppression and showed a high isolatio ratio for Legionella pneumonia. Moreover, BCYE? plate with antibiotics system of DGVP and GVPC can effectively inhibit non-Legionella and increase isolation ratio of Legionella. They should be the first selection media for Legionella from environment and clinical samples.

Objective To discuss the techniques and efficacy of Ho:YAG laser incision by using uteroscopy for ureterostenosis.Methods From July 2004 to April 2007,52 patients with ureterostenosis received ureteral incision by using Ho:YAG laser under a endoscope.Two double pigtail stents(F5 or F6) were placed in the ureters after the operation and left indwelling for 8 to 12 weeks.Ultrasonography and excretion urography were performed 3 to 6 months after extubation.Results Follow-up was available for 3 to 24 months(mean,17 months) in 46 patients,of which 40(87%) were cured after the treatment.In the cured patients,hydronephrosis,ureteral dilation,and ureterostenosis were improved,and the pain in the kidney region was relieved;none of them showed signs of infection.In the other 6 patients,4 were improved after the treatment(no deterioration of the symptoms of hydronephrosis,ureteral dilation,and pain in the kidney region,and no infection);and 2 failed(the symptoms of hydronephrosis and ureteral dilation deteriorated,and pain in the kidney region and infection were developed).Conclusion Endoscopic ureteral excision using holmium laser combined with indwelling of two double pigtail stents is effective and safe for ureterostenosis.

Objective To establish a real‐time fluorescent polymerase chain reaction and detect 16S rRNA gene of Legionella strains isolated from sputum specimens of patients with pulmonary infection by using this method .Methods 16s rRNA gene of Le‐gionella was used to design primers and probes .The reaction system and reaction conditions were optimized and the specificity ,sen‐sitivity and repeatability of this method were verified by detecting Legionella pneumophila ,non‐Legionella pneumophila and other bacteria .A total of 557 sputum specimens of patients with pulmonary infection were detected ,and PCR‐digestion identification method was carried out as control .Otherwise ,sequences of 16S rRNA were verified in patients with positive detection results .Re‐sults The results showed that all reference strains of Legionella were positive ,while all of other bacteria were negative ,and the sensitivity was 102 CFU/mL .Among sputum specimens collected from 577 cases of patients with pulmonary infection ,the positive rate of Legionella detected by using real‐time fluorescent PCR and PCR‐digestion identification method was 23 .1% and 19 .9% re‐spectively ,while the positive rate was 17 .2% by verifying the sequences of 16s rRNA .There were no statistically significant differ‐ences of positive rate among the three methods(P>0 .05) .Conclusion The real‐time fluorescent PCR is fast and convenient in de‐tection of Legionella strains isolated from sputum specimens of patients ,which could be an assisted method for clinically diagnosing Legionella infection .

[ ABSTRACT] AIM:To study the effect of idazoxan ( IDA) on the permeability of blood-brain barrier ( BBB) and the expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in mouse ex-perimental autoimmune encephalomyelitis (EAE).METHODS: Female C57BL/6 mice (n=36) were randomly divided into control group, EAE group and IDA group, with 12 mice in each group.EAE was induced by myelin oligodendrocyte glycoprotein 35-55 ( MOG35-55 ) .IDA (2 mg/kg, ip, bid) was administered for 15 d after immunization.The neurological defects of the mice were observed daily and scored.The pathological changes were observed under microscope with HE stai-ning and LFB myelin staining.The BBB permeability was detected by Evans blue extravasation.The expression of MMP-9 and TIMP-1 in the brain of EAE mice was determined by Western blotting.RESULTS: Compared with EAE group, the score of neurological defects in IDA group was decreased, the inflammation was relieved, the BBB permeability was re-duced, and the expression MMP-9 and the ratio of MMP-9/TIMP-1 were decreased ( P<0.05 ) .CONCLUSION: The neuroprotective effect of IDA on mouse EAE might be related to the down-regulation of MMP-9 and the ratio of MMP-9/TIMP-1, thus reducing the degradation of BBB and the permeability of BBB, and ameliorating the pathologic process of EAE.

Objectives To identify and characterize 10 strains of Francisella philomiragia-like organisms isolated from blood samples and environmental water.Methods The 10 clinical and environmental isolates were identified by traditional morphological examination and biochemical characterization,matrix-assisted laser desorption/ionization time of flight(MALDI-TOF) mass spectrometry(MS) systems and sequencing based on 16S rRNA gene.The minimum inhibitory concentrations were tested by E-test methods.Results All the 10 isolates were gram-negative coccobacilli appearing tiny and faint counterstain of safranin,negative for urease,nitrate reduction and X and/or V factor requirement,but positive for oxidase and catalase.The isolates grew rapidly in sheep blood agar,chocolate agar and BCYE plate forming white opaque,colorless transparent or gray smooth colonies with about 2-mm diameters,but did not grow in M-H agar and MacConkey agar.The sequencing for 16S rRNA gene indicated that the 10 isolates shared more than 99.6％ similarity to Francisella philomiragia,and fell into the same clusters of Francisella philomiragia on phylogenetic tree.The MALDI-TOF MS analysis also showed the typical peaks with 6 153 m/z,5 180 m/z,7 757 m/z and 9 392 m/z which were similar to Francisella philomiragia ATCC 25015.However,they may be misidentified to be Sphingomonas paucimobilis by using Vitek 2 GN cards,Neisseria cinerea by using Vitek 2 NH cards,Myroides odoratimimus by using API 20NE strips and Haemophilus by using API NH cards.The results of antimicrobial susceptibility showed that they were all sensitive to chloramphenicol,doxycycline,tetracycline,gentamicin,ofloxacin and ciprofloxacin.Conclusion The 10 isolates could be identified as Francisella philomiragia,so we should pay more attention to the infrequent pathogen for its inactive biochemical reaction and the misidentification by commercial detection systems.

Objective To identify one runny mucoid-like Gram-negative bacteria with pink pigment isolated from clinical pus sample. Methods The pus sample was aseptically extracted from a deep lesions of one patient, then stored in Amies medium at room temperature for transportation. One sheep blood plate and one chocolate plate were used to detect the possible pathogens from the specimens. After inoculation, the plates were placed in a humidified incubator with 5% CO2 at 35 ℃. To identify the obtained isolates, we used the commercial Vitek2 and API systems, combining some traditional morphological examination and classical biochemical and physiological characteristics. For pure cultures, the cellular fatty acids were extracted, methylated, and determined by gas chromatography method. The 16S rRNA gene was amplified and sequenced by a commercial broad-spectrum PCR primers. The phylogenetic tree based on 16S rRNA gene was constructed by Mega 4.1 software using the neighbour-joining methods with 1 000 bootstrap replications. Results One runny mucoid-like Gram-negative bacterium, named K8756, was isolated both on sheep blood and chocolate plates after 72 h incubation. The API 20NE profile was 1245045 after a 3-day culture, which would be identified as Ochrobactrum anthropi with a good confidence of 98% probability. It was identified as Ralstonia pickettii and Bordetella bronchiseptica by VITEK 2 GN kits. However, further comparative 16S rRNA gene sequences showed that strain K8756 was closely related to the valid published Roseomonas mucosa MDA 5527 with 100% identity. Colonial morphologic features, phenotypic characteristics and major cellular fatty acid composition were also with high similarity to Roseomonas mucosa. Conclusions Strain K8756( = GIMCC 1.0030 ) is identified as Roseomonas mucosa by the polyphasic phenotypic and genotypic characteristics. The comparative analysis based on 16S rRNA gene sequences is a useful method for identifying the problematic and newly named bacteria.

Objective To investigate the clinical features and disease-causing mutations of familial hypomagnesaemia with hypercalciuria and nephrocalcinosis.Methods In February 2016,a 24 year old female patient with left kidney stone and nephrocalcinosis in bilateral kidneys was admitted to our hospital.One month prior to this admission,she had been treated by PCNL to remove the most part of left kidney stone in otherhospital.Mter admission,She was found hypomagnesaemia (serum magnesium 0.65 mmol/ L) and hypercalciuria (24h urine calcium 364.0 mg) but with normal renal function (serum creatinine 101.5μmol/L).And the remained part of left kidney stone was removed by flexible ureteroscope.As she was considered probably with an autosomal recessive FHHNC,an analysis of CLDN16 and CLDN19 gene mutations was performed using her and her parents'peripheral white blood cells.Results Mutation analysis revealed this patient had two heterozygous mutations in the CLDN16.One is an one-base deletion mutation in the 123th codon in exon 2:368delA.The other is a missense mutation in the 139th codon in exon 2:416C →T which resulted in an amino acid change Ala139Val.Her parents respectively had one of each heterozygous mutation.In the six months follow-up,an oral administration with hvdrochlorothiazide,potassium citrate,and calcium magesium supplements significantly reduced her hypomagnesaemia (serum magnesiun 1.0 mmol/L) and hypercalciuria (24-h urine calcium 156.0 mg),and no stone recurrence and aggravation of nephrocalcinosis and renal dysfunction occurred.Conclusions We diagnosed a patient with FHHNC who had a novel compound heterozygous mutation of CLDN16.This rare disease should be suspected if there are three constant clinical features of hypomagnesaemia,hypercalciuria and nephrocalcinosis,and verified with CLDN16 and CLDN19 gene test.Currently the option for treatment of FHHNC is symptomatic treatment until severe deterioration of renal function.The hydrochlorothiazide,potassium citrate,and calcium magesium supplements may have considerable effects on hypomagnesaemia and hypercalciuria.

Objective To introduce a improved method for the production of hemoglobin liquid by use of the hemoglobin electro‐phoresis alkaline .Methods First ,we used the pipette to absorb the settlement of red blood cells from a batch of EDTA anticoagula‐ted whole blood specimens ,then dropped them into the 0 .9% saline washed human erythrocytes .With the help of the pipette nozzle we pipet from the liquid surface to bottom repeatedly ,made the red cells suspended in the liquid evenly .The samples should be cen‐trifuged and the red blood cells fully deposited at the bottom ,then poured the supernatant after centrifugation to remove the impuri‐ties in plasma and leave the allowance of red blood cells .We add distilled water or hemolysin to lysis RBC .Hands up test tubes rack using wrist gently back and forth several times until the hemolysis was clear and transparent .Results Compared the results of the modified method and the traditional method ,then the two results compared with the results of HPLC recommended by international association of thalassemia method .Compared the results of three screening methods with that of the thalassemia gene identification method .So we could objectively evaluate the reliability of the test .Conclusion The result of the improved method are same to the electrophoretogram by SOP operation ,it will be more efficient and reduce the cost of reagent .The whole process of preparing hemo‐globin solution doesn′t contact with any chemical reagent .So there will be no pollution to the environment ,and it also reduces the harmful of toxic reagent to the human body .

Objective To verify result consistency of the improved examination method and the standard operation method with the erythrocyte osmotic fragility test (OFT).Methods The samples and reagents were reduced half volume and used different testing equipment to evaluate methods for OFT.Selected 100 samples that the brittleness were increased and de-creased (50 samples positive and negative respectively),used a kind of improved examination method and traditional test method to evaluate the consistency.Results The detection result of improved examination method and the detection results of the manufacturer standard method were consistent (t=1.660 8,P >0.05).So,there was no significant difference of con-trast between two groups of data.Conclusion Improved mothed OFT alternatives to traditional OFTscreening method could be faster,more objectively the results of clinical service,and could effectively reduce reagent and manpower cost,improve the efficiency of work.Therefore,this method could be used to groups detection and lack of equipment for the primary care of hospital screening thalassemia desease method is preferred.