Objective: To establish a simple and rapid loop-mediated isothermal amplification (LAMP) to detect Candida albicans (CA) and explore its clinical value. Methods: The Primer Explorer 5.0 software was used to design 4 primers for amplification of CA by LAMP. The system and conditions of LAMP reaction were optimized to evaluate its specificity and the minimum limit in the detection. The vaginal swabs were collected from 123 vulvovaginal candidiasis (VVC) patients and 42 healthy individuals. Fungal culture, LAMP test, PCR test and 1.79 mol/L KOH microscopy were conducted in parallel. Fungal culture was used as the reference method for VVC diagnosis. The positive rates between two groups were compared by Pearson χ² test. The consistency of the results from LAMP, PCR, microscopy and culture were analyzed by Kappa test and P＜0.05 was considered as statistically significant difference. ROC curve analysis was used to evaluate LAMP and PCR test for diagnosis of VVC. Results: The optimum reaction temperature of LAMP was 61 ℃ with high specificity. No cross reaction with other strains was found. The minimum detection limit was 10³ copies/ul. The positive rates of LAMP, PCR and microscopy between VVC and healthy group showed statistically significant difference（LAMP: χ²=68.576；PCR: χ²=64.918；microscopy: χ²=50.076，P＜0.01）. LAMP detection and PCR showed good consistency (κ=0.744, 0.720), but microscopy examination showed poor consistency (κ=0.533). LAMP showed diagnostic sensitivity of 87.62%, specificity of 88.33%, positive predictive value of 92.93% and negative predictive value of 80.30%. The area under the curves of LAMP and PCR were 0.873 and 0.888, respectively. No difference in efficacies between LAMP and PCR was found (Z=0.849, P=0.395 6), but the lowest detection time of LAMP was shorter than 1 hour. Conclusion A rapid, reliable, sensitive and specific LAMP technique for detecting CA was established. The comprehensive screening performance should be superior to the routine method in laboratories, so LAMP could be used for the supplementary diagnosis and monitoring therapeutic effects in CA infection.

Objective: To study the effects of serum and its components on biofilm formation of Pseudomonas aeruginosa. Methods: 96 well microplates combined with crystal violet staining was used to detect the effects of serum, albumin and transferrin on biofilm formation of Pseudomonas aeruginosa. And confocal laser scanning microscope was used to observe the morphology of the biofilm. Results: The biofilm of PAO1 was significantly enhanced from 2.26±0.42 to 3.42±0.08（t=4.71, p<0.01）with horse serum and but reduced to 0.807±0.10（t=4.71,p<0.01） by human serum; And the total biofilm biomass was significantly increased and clump-changed with horse serum, but decreased and scattered in distribution by human serum. Besides, horse serum could also enhance the biofilm formation of part of the clinical isolates of Pseudomonas aeruginosa, however, human serum could inhibit the biofilm formation of all of the clinical isolates. And 2.5g/L albumin could significantly enhance the biofilm of PAO1 from 1.96±0.22 to 2.54±0.18（t=3.55，p<0.05）, but 5 g/L could reduce the biofilm of PAO1 from 1.85±0.36 to 0.84±0.24（t=4.03，p<0.05）. Conclusion Horse serum and albumin could significantly promote the biofilm formation of Pseudomonas aeruginosa, but human serum and transferrin could decrease its biofilm formation.

Objective: To investigate the distribution and expression of T3SS virulence genes in clinically isolated Pseudomonas aeruginosa (P. aeruginosa) strains and its correlation with drug resistance. Methods: A total of 68 P. aeruginosa isolates were collected from the First Affiliated Hospital of Wenzhou Medical University in 2015. The antimicrobial susceptibility was detected by the agar dilution method. The distribution of virulence genes such as exoU, exoS, exoT and exoY from different isolates was detected by PCR. The expression levels of transcriptional regulator genes (ptrA and exsA) and effector-related genes (exoT and exoS) in some isolates were determined by real-time fluorescence quantitative PCR, and the Pearson correlation analysis was used to analyze the results. Results: The detection rates of exoT and exoY in 68 P. aeruginosa isolates were higher, accounting for 79.4% and 75.0%, respectively. The detection rate of exoT in wound-sourced isolates was significantly higher than that in sputum (97.0% vs 61.8%, P＜0.01). In addition, the genotype of exoU - /exoS + was the most common, accounting for 51.5% (35/68). The resistance rates of sputum-sourced isolates to imipenem and meropenem were significantly higher than that of wound-sourced isolates (47.1% vs 8.8%, 47.1% vs 14.7%, P＜0.01). The resistance rates of isolates carrying exoU gene to carbapenems, aminoglycosides and fluoroquinolones were higher than those of isolates carrying exoS, exoT or exoY genes. Pearson correlation analysis showed that the expression level of ptrA gene was negatively correlated with those of exoT, exoS and exsA genes (P＜0.05). Conclusion The P. aeruginosa isolates from our hospital carrying T3SS virulence genes exoT and exoY are common, and the virulence genes are related to the drug resistance of P. aeruginosa. In addition, ptrA may be a potential negative regulatory gene for the expression of T3SS virulence genes.

Objective: To analyze the susceptibility and distribution of β-lactamase encoding and efflux pump genes as well as the inhibitory effect of efflux pump inhibitors on the carbapenem resistance of the carbapenem resistant Klebsiella pneumoniae without producing carbapenemase (CRKPPC). Methods: One hundred and eight strains of carbapenem non-susceptible Klebsiella pneumoniae were collected from our hospital during 2012-2014. The strains producing carbapenemase were screened by Mastdiscs combi Carba plus disc system. For CRKPPC strains, the susceptibilities to antimicrobial agents were determined by micro-dilution broth methods. PCR and DNA sequencing technology were used to analyze the prevalence of β-lactamase encoding extended-spectrum β-lactamase (ESBL) and plasmid mediated AmpC genes as well as efflux genes including acrAB-tolC, kexD, kdeA, kpnEF and kpnGH. The inhibitory experiments were implemented by using carbonylcyanide-m-chlorophenylhydrazone (CCCP) and Reserpine to observe the role of efflux pumps on the carbapenem resistance. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to analyze the expression of outer membrane proteins OmpK35 and OmpK36. Results: Twenty-six strains out of the 108 carbapenem non-susceptible Klebsiella pneumoniae were identified to be CRKPPC strains which displayed quite high resistance to β-lactam and high resistance to amikacin, gentamicin, levofloxacin and ciprofloxacin. Very good sensitivities were observed to tigecycline, polymyxin, ceftazidime/avibatan and aztreonam/avibatan. The high prevalence of bla CTX-M and bla SHV were also displayed with the prevalent rates being 76.9% and 57.7%, respectively. The loss of outer membrane proteins OmpK35 and OmpK36 with varying degrees was observed, among which 57.7% strains lost ompK35 and 19.2% strains lost OmpK36. More than 80% strains contained efflux genes including acrAB-tolC, kexD, kdeA, kpnEF and kpnGH. The results of inhibitory experiments showed that CCCP displayed quite obviously inhibitory effects on the carbepenem resistance whereas no inhibitory effect was observed for Reserpine. Conclusion Tigecycline and polymyxin could be used as the basis of combined drug for CRKPPC. The prevalence of AmpC, ESBLs and loss of OmpK35 and OmpK36 with varying degrees among these strains were observed, but the over-expression of efflux pumps should be the main mechanism of the carbapenem resistance in the Klebsiella pneumoniae strains, and efflux inhibitors may have potential value in treating the infections caused by these bacteria.

Objective: To investigate the clinical significance of combined examinations for neutrophil-lymphocyte ratio (NLR), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and fecal occult blood (OB) in the differential diagnosis of Crohn′s disease and irritable bowel syndrome. Methods: A total of 129 patients with Crohn′s disease and 120 patients with irritable bowel syndrome from October 2014 to October 2017 in Changhai Hospital were enrolled in this study. The results of NLR, CRP, ESR and OB were recorded. Logistic regression was used to study the association of the four indicators. The combined impact of the four indicators was explored with multivariable regression. ROC curve was used to compare the diagnostic value of the combined examinations with the four indicators for Crohn′s disease. The diagnosis was performed by substituting the data of individual patient into regression model. Results: The levels of NLR, CRP, ESR and OB in Crohn′s disease group were higher than those in irritable bowel syndrome group (Z=-7.067--4.148, P＜0.01). The area under the curve of combined diagnostic indicator was 0.881, which was higher than that of single NLR, CRP, ESR or OB (0.759, 0.695, 0.652, 0.643) respectively (Z=3.19-5.60, P＜0.01). When the cutoff value was 0.498, the sensitivity was 79.1%, the specificity was 83.3% and the diagnostic accuracy was 81.1%. A patient who was not included within the statistical range of this experimental study was randomly assigned to the model and 0.831 of P value was obtained, which was higher than the cutoff value of 0.498, indicating that the patient suffered from Crohn′s disease with accuracy of 81.1%. Conclusion The logistic regression model established with the combined diagnostic indicators, which was formulated by examinations of NLR, CRP, ESR and OB, exhibited higher diagnostic value than any single indicator in the differential diagnosis of Crohn′s disease and irritable bowel syndrome.

Objective: To analyze the molecular epidemiological characteristics of Escherichia coli producing NDM-5 carbapenemase in the neonatal department of our hospital. Methods: Three carbapenem-resistant Escherichia coli strains（E1, E2 and E3） isolated from neonatal ward of our hospital from August to September of 2017 were collected. Vitek 2 Compact system combined with K-B disk method was used for drug sensitivity test. The resistance genes were detected by PCR amplification. Plasmid replicon typing was detected by PCR. Plasmid conjugation tests were performed to explore the conjugating transfer of plasmids in the three strains. The homology of the three strains was analyzed by multiple locus sequence typing (MLST) and pulse field gel electrophoresis (PFGE). Results: Drug susceptibility test showed that the three bacteria were resistant to most β-lactam antibiotics except Aztreonam, and resistant to quinolones and SMZ-TMP, but sensitive to aminoglycosides drugs. PCR and sequencing results indicated that the three strains carried bla SHV gene and extended-spectrum beta-lactamase gene (bla SHV , bla TEM and bla CTX-M ). The plasmid replicon type was IncX3. Transfer test of E1 strain was successful. MLST results indicated that all the three strains were ST1642 type. MLST and PFGE results indicated that the bands of the three bacteria were identical. Conclusion Both NDM-5 carbapenemase and extended-spectrum beta-lactamase were detectable in the three strains of carbapenem-resistant bacteria from neonatal department. MLST and PFGE results suggested that the three strains were from the same clonal source.

Objective: To investigate the values of T lymphocyte-bound complement activation products such as T-C3d and T-C4d, B lymphocyte-bound complement activation products such as B-C3d and B-C4d and erythrocyte-bound complement activation products such as E-C3d and E-C4d in the diagnosis of systemic lupus erythematosus (SLE). Methods: Peripheral blood samples from 68 SLE patients, 70 patients with non-SLE autoimmune diseases and 68 healthy controls were collected randomly, and the expression levels of T-C4d, B-C4d, E-C4d, T-C3d, B-C3d and E-C3d in these samples were detected by flow cytometry. Meanwhile, antinuclear antibodies (ANA), anti-double stranded DNA antibodies (anti-dsDNA), peripheral blood cell count and other markers were also detected. The differences of cell-bound complement activation products in three groups were analyzed with the area under the receiver operating characteristic curve (AUC), nonparametric test, sensitivity and specificity. Results: The specific median fluorescence intensity (SMFI) of T-C4d, B-C4d, E-C4d, T-C3d, B-C3d and E-C3d in SLE patients were significantly higher than those in the patients with non-SLE autoimmune diseases and healthy controls (all P＜0.05). The SMFI (median \[P 25, P 75\]) of T-C4d, B-C4d and E-C4d in SLE patients were 3.8(1.2, 13.1), 22.1(6.2, 67.9) and 19.6(1.8, 52.4), respectively. The SMFI of T-C4d, B-C4d and E-C4d in SLE patients with reduced red blood cells and/or lymphocytes were significantly higher than that with normal red blood cell and lymphocyte count (all P＜0.05). The AUCs of T-C4d, B-C4d, E-C4d, T-C3d, B-C3d and E-C3d were 0.711, 0.763, 0.663, 0.631, 0.611 and 0.615, respectively (all P＜0.05). The sensitivity of the combination of T-C4d with B-C4d (73.5%) in the diagnosis of SLE was superior to that of anti-dsDNA (36.8%). Conclusion The cell-bound complement activation products (CB-CAPs) are specifically expressed in SLE patients, and their combination detection is helpful for the diagnosis of SLE.

Objective: To compare the consistency of thyroid stimulating hormone (TSH) results from four chemiluminescence assays. Methods: A total of 102 fresh serum samples from Peking Union Medical College Hospital during March 2018 and April 2018 were collected for precision evaluation and methodological comparison referring to CLSI EP15-A2 and EP9-A2 protocols. The levels of serum TSH were detected by Abbott i2000 (system A), Beckman DXI800 (system B), Siemens ADVIA Centaur XP (system C) and Roche e601 (system D) automatic chemiluminescence analyzers and their matching reagents, respectively. The obtained results were compared with the passing-bablok and Bland Altman methods. Taking 0.27 μIU/mL and 5.33 μIU/mL as the medical decision level, the expected bias of each detection system was compared. Results: The precisions of systems A,B,C and D were 1.7%-3.3%， 2.3%- 3.9%，0.7%-2.3% and 0.6%-1.5%,respectively. The median (P 25,P 75) of TSH concentrations detected by systems A,B,C and D were 1.898 (0.518,4.809)μIU/mL, 2.819 (0.719,7.020)μIU/mL,2.502 (0.692,6.888)μIU/mL and 3.105 (0.886, 7.905)μIU/mL, respectively. The coefficients of determination (R 2 ) of regression equation were above 0.975 for 4 detection systems. The correlation coefficients (r), intercepts and slopes of 4 detection systems were 0.993 5-0.997 1, 0-0.06 and 0.59-1.15, respectively, and systems B and C had the best correlations with 1.02 of slope and 0 of intercept. The deviation plot showed that the bias% of 4 detection systems was between -48.1% and 17.3%. Among them, systems A and D had the largest bias, while systems B and C had the lowest bias. The expected bias of 4 detection systems at the medical decision level was -40.7%-37.2%. Conclusion The consistency between Beckman and Siemens TSH detection systems is good, while those of Roche and Abbott TSH detection systems are different from the other two.

Objective: To establish the information solution for the classification and assessment of specimen quality based on the assembly line. Methods: Before the samples entered into the assembly line, they were took pictures and screened by the image results. For the suspected samples, serum index was detected. Then, the classification criteria of specimen quality were set, and the alarm thresholds of serum indices for each item suitable for our laboratory were established. The results of serum indices were compiled into the corresponding text descriptions and automatically written into the notes of the result reports. The pictures of blood collection tubes were stored in the laboratory information management system (LIMS) and could be accessed at any time for verification. The samples intercepted by the automatic review were further reviewed by manual. Results: The intra-assay coefficients of variation (CV) of serum indices for haemolysis (H), lipaemia (L) and icterus (I) were 0.6%, 0.7% and 1.3%, respectively, indicating that the precision was good. Among 657 770 samples detected by the assembly line, 11.9% of samples were screened out before they entered the assembly line. The detection of serum indices of these samples demonstrated that the samples with haemolysis, lipaemia and icterus accounted for 1.6%, 1.2% and 0.3% of the total samples, respectively. According to the results of the interference experiment, the alarm threshold of hemolytic serum index was set in 11 items, and those of lipaemia and icterus were set in 1 item. Conclusion By establishing the information solution of specimen quality based on the assembly line, the real-time classification prompting of specimen quality is realized, and the missed detection is avoided, which is helpful to reduce the pre-analysis errors caused by serum quality and simplify the laboratory workflow.

Objective: To investigate the consistency of plasma prothrombin time (PT) results detected by the STAGO STA-R Evolution and Mindray Precil C3510 automatic coagulation analyzers. Methods: The PTs from 69 plasma samples were detected by the STA-R Evolution and Precil C3510 coagulation analyzers, respectively, and the obtained results were compared. Based on the CLSI EP9-A3 protocol, the ESD test was used to detect outliers, the scatter plot, difference plot, and frequency distribution plot were drawn, and the method comparison and bias evaluation were performed using the Passing-Bablok regression and Bland-Altman plot. Results: The PTs (median \[P 25, P 75\]) detected by the STA-R Evolution and Precil C3510 analyzers were 19.00 (13.85, 25.65) s and 20.50 (13.83, 26.30) s, respectively, and there was no significant difference between them (P＞0.05). No outliers were detected by the ESD test, and the variation of PTs (CV) was constant. There were no systematic, random and proportional differences in PT results from two coagulation analyzers. The bias between two coagulation analyzers was within the acceptable range (1/2 CLIA′88 TEa). The predicted bias of PT at each medical decision point was also within the acceptable range. Conclusion The results of PT detected by the Precil C3510 and STA-R Evolution coagulation analyzers are comparable, and the bias is within the acceptable range, which can meet the needs of clinical diagnosis and treatment.