OBJECTIVE: To investigate the value of clinical application of simultaneous amplification and testing of RNA (SAT-RNA) for detecting Chlamydia trachomatis (CT) and Ureaplasma urealyticum (UU) by comparing with the polymerase chain reaction testing of DNA (PCR-DNA) method. METHODS: Specimens from both urethra swab and the first avoid urine which should be at least one hour after the previous urination were collected from 163 men who were scheduled for in vitro fertilization and embryo transfer (IVF-ET) treatment due to female factors at Center for Reproductive Medicine, Shengjing Hospital of China Medical University during the period of April 2016 to April 2017. Among the 163 men, 109 simultaneously provided semen that was collected after 3-7 days of sexual abstinence for the testing. Urine and semen specimens were detected for CT and UU with SAT-RNA, while urethra swab specimens were detected for CT and UU with standard PCR-DNA. Detection results of the SAT-RNA were compared with those of the PCR-DNA method. RESULTS: The positive rate of UU in the urethra swab detected with PCR-DNA and that of UU in the urine with SAT-RNA were 47.24% and 47.85%, respectively, and the coincidence rate was 93.25%. In addition, the positive and negative coincidence rates were 93.51% and 93.02%, respectively, and the concordance between the two methods was very good (Kappa=0.865). On the other hand, the positive rate of CT in the swab specimen tested with PCR-DNA was 3.07% and that of CT in urine with SAT-RNA was 4.29%, and the coincidence rate was 97.55%. Moreover, the positive and negative coincidence rates were 80.00% and 98.10%, respectively, and the concordance between the two methods was good (Kappa=0.654). Regarding SAT-RNA detection of UU in the urine and semen specimen of the 109 patients, the positive rates of UU in the urine and semen specimens were 50.46% and 44.95%, respectively; and the coincidence rate between the two specimens was 88.99%. In addition, the positive coincidence rate and the negative coincidence rate was 93.88% and 85.00%, respectively, and the concordance between the two specimens was good (Kappa=0.780). Similarly, SAT-RNA detection of CT in the urine and semen specimens showed the positive rate was 5.50% and 3.67%, respectively; and the two specimens showed 98.17% coincidence rate. The positive and negative coincidence rates were 100.00% and 98.10%, respectively, and the concordance was also good (Kappa=0.791). CONCLUSION SAT-RNA detection of CT and UU in the urine specimen showed good concordance with the PCR-DNA detection of CT and UU in the urethra swab specimen. In addition, the concordance was also good between the urine and semen specimens detected with SAT-RNA. These results indicate that, as a less invasive and equally accurate procedure, SAT-RNA may be more suitable for clinical application.
Chlamydia Infections/epidemiology* ; Chlamydia trachomatis/genetics* ; Female ; Humans ; Infertility, Male ; Male ; Neisseria gonorrhoeae/genetics* ; Polymerase Chain Reaction ; Ureaplasma urealyticum/genetics*

Lipooligosacharide (LOS) of Neisseria gonorrhoeae (gonococci, GC) is involved in the interaction of GC with host cells. Deletion of the alpha-oligosaccharide (alpha-OS) moiety of LOS (lgtF mutant) significantly impairs invasion of GC into epithelial cell lines. GC opacity (Opa) proteins, such as OpaI, mediate phagocytosis and stimulate chemiluminescence responses in neutrophils in part through interaction with members of the carcinoembryonic antigen (CEA) family, which includes CEACAM3 (CD66d), a human neutrophil specific receptor for phagocytosis of bacteria. In the present work, we examined the effects of OpaI-expressing lgtF mutant on phagocytosis by HeLa-CEACAM3 cells and chemiluminescence responses in neutrophils. The results showed that lgtF mutant even expressing OpaI completely lost the ability to promote either phagocytosis mediated by CEACAM3 interaction in HeLa cells or chemiluminescence responses in neutrophils. These data indicated that Opa proteins in the lgtF mutant, which might result from the conformational change, cannot be functional.
Antigens, Bacterial ; chemistry ; genetics ; immunology ; metabolism ; Carbohydrate Sequence ; Carcinoembryonic Antigen ; genetics ; immunology ; Gene Expression Regulation ; HeLa Cells ; Host-Pathogen Interactions ; Humans ; Lipopolysaccharides ; chemistry ; immunology ; Luminescent Measurements ; Mutation ; Neisseria gonorrhoeae ; genetics ; metabolism ; pathogenicity ; Neutrophils ; immunology ; microbiology ; Phagocytosis

PURPOSE: The detection of high-level tetracycline-resistant strains of Neisseria gonorrhoeae (TRNG) can make important epidemiological contributions that are relevant to controlling infections from this pathogen. In this study, we aimed to determine the incidence of TRNG isolates over time and also to investigate the characteristics and genetic epidemiology of these TRNG isolates in Korea. MATERIALS AND METHODS: The antimicrobial susceptibilities of 601 isolates of N. gonorrhoeae from 2004 to 2011 were tested by standard Clinical and Laboratory Standards Institute methods. To determine the molecular epidemiological relatedness, N. gonorrhoeae multi-antigen sequence typing was performed. RESULTS: The incidence of TRNG increased from 2% in 2004 to 21% in 2011. The minimum inhibitory concentration distributions of ceftriaxone and susceptibility of ciprofloxacin in TRNG were different from non-TRNG and varied according to the year of isolation. Most of the TRNG isolates collected from 2004 to 2007 exhibited genetic relatedness, with sequence type (ST) 1798 being the most common. From 2008 to 2011, the STs of the isolates became more variable and introduction of genetically unrelated TRNG were noted. CONCLUSION: The increased incidence of TRNG strains until 2007 appears to be due, at least in part, to clonal spread. However, we propose that the emergence of various STs since 2008 could be associated with foreign import.
Anti-Bacterial Agents/*pharmacology ; Ceftriaxone/pharmacology ; Ciprofloxacin/pharmacology ; DNA, Bacterial/analysis ; Drug Resistance, Multiple, Bacterial/*genetics ; Gonorrhea/drug therapy/epidemiology/microbiology ; Humans ; Incidence ; Microbial Sensitivity Tests ; Molecular Epidemiology ; Neisseria gonorrhoeae/*drug effects/*genetics/isolation & purification ; Republic of Korea/epidemiology ; Sequence Analysis, DNA ; Tetracycline/pharmacology ; Tetracyclines/*pharmacology

OBJECTIVETo investigate the genotype and characteristics of Neisseria gonorrhoeae (N.gonorrhoeae) isolates with reduced susceptibility to ceftriaxone in Shenzhen from 2009 to 2011.

METHODSA total of 296 N.gonorrhoeae isolates were collected in Shenzhen from 2009 to 2011.ceftriaxone strains (minimum inhibitory concentration between 0.06 and 0.50 µg/ml) were determined by agar dilution method.Logistic regression was used to analyze the associated factors of ceftriaxone N.gonorrhoeae infection.Neighbor-joining (NJ) phylogenetic tree analysis and N.gonorrhoeae multi antigen sequence typing (NG-MAST) were performed on all ceftriaxone isolates and susceptible control isolates randomly selected in accordance with the principle of 1: 1 sampling.

RESULTSNo isolates displayed resistance to ceftriaxone, whereas 53(17.9%) showed reduced susceptibility to ceftriaxone among 296 isolates.Only antibiotic use in recent two months was associated with ceftriaxone isolates infection (OR = 3.080, 95%CI: 1.376-6.894) . Among the ceftriaxone isolates, 48 different ST were identified including 5 STs (ST1768, ST3927, ST641, ST7076 and ST7078) containing 2 isolates and 43 single STs. There were 26 STs previously reported from HongKong in China.Low sensitive strains clustering was not observed by NJ phylogenetic tree.

CONCLUSIONThe proportion of ceftriaxone strains among the 296 N.gonorrhoeae isolates collected from 2009 to 2011 in Shenzhen is high. The STs of ceftriaxone strains may have unique epidemic features in Shenzhen.

Adult ; Anti-Bacterial Agents ; pharmacology ; Ceftriaxone ; pharmacology ; China ; epidemiology ; Drug Resistance, Bacterial ; genetics ; Female ; Gonorrhea ; epidemiology ; microbiology ; Humans ; Male ; Middle Aged ; Molecular Epidemiology ; Neisseria gonorrhoeae ; classification ; drug effects ; genetics ; isolation & purification ; Young Adult

Chlamydia trachomatis ; genetics ; Chlamydiaceae Infections ; diagnosis ; genetics ; Genes, Bacterial ; genetics ; Genes, Viral ; genetics ; Gonorrhea ; diagnosis ; genetics ; Herpes Simplex ; diagnosis ; genetics ; Herpesvirus 2, Human ; genetics ; Humans ; Multiplex Polymerase Chain Reaction ; Neisseria gonorrhoeae ; genetics ; Ureaplasma Infections ; diagnosis ; genetics ; Ureaplasma urealyticum ; genetics

OBJECTIVETo detect the genes of Neisseria spp. isolated from patients with male genitourinary tract infections, and to study the pathogenicity of non-gonococcal strains of Neisseria and the laboratory diagnosis for the infections caused by Neisseria spp.

METHODSUsing polymerase chain reaction and nucleotide sequencing, we amplified and sequenced 4 genes of Neisseria spp. isolated from patients with male genitourinary tract infections, including 16S rRNA, orfl, cppB and nspA.

RESULTSFourteen Neisseria strains were identified through analysis of the 16S rRNA gene, including 3 N. mucosa strains, 3 N. cinerea strains, 2 N. gonorrhoea strains, 2 N. sicca strains, 2 N. subflava strains, 1 N. lactamica strain, and 1 N. polysaccharea strain. Among them, 9 showed positive results in gonococcal fluorescence-labeled multiplex-PCR detection, 1 in cppB gene reaction, 5 in orfl gene reaction, and 3 in nspA gene reaction. The consistency rate was 85.7% between the above results from our gene detection and those from the routine bacteriological methods.

CONCLUSIONThe cppB gene is absent in the non-gonococcal strains of Neisseria spp. that can cause male genitourinary tract infection. Most of the strains not only lack virulence-associated orfl and nspA genes, but also show positive results in gonococcal fluorescence-labeled multiplex-PCR detection, which is one of the important reasons for the misdiagnosis and missed diagnosis of gonorrhea infection. The combination of routine bacteriological methods and gene detection in laboratory examinations may help improve the accuracy rates of Neisseria species identification and clinical diagnosis of the infections caused by Neisseria spp.

Genes, Bacterial ; Gonorrhea ; diagnosis ; microbiology ; Humans ; Male ; Neisseria gonorrhoeae ; classification ; genetics ; Polymerase Chain Reaction ; Urinary Tract Infections ; microbiology

A 42-yr-old man with hepatitis B virus associated liver cirrhosis was admitted to the emergency room because of multiple seizures, a history of chills and myalgia over the previous 2 weeks, and 3 days of melena. He was febrile with a temperature of 38.0degrees C. There were no symptoms and signs related to the genitourinary system, skin, or joints. Three sets of blood cultures were obtained and oxidase-positive, gram-negative diplococci were detected after 25.9-26.9 hr of incubation in all aerobic vials. The organism was positive for catalase and oxidase, and was identified as Neisseria gonorrhoeae, using a Vitek Neisseria-Haemophilus Identification card (bioMerieux Vitek, Inc., USA). Further, 16S rRNA sequencing of this isolate revealed a 99.9% homology with the published sequence of N. gonorrhoeae strain NCTC 83785 (GenBank Accession No. NR_026079.1). Acute bleeding by variceal rupture seems to be a likely route of introduction of N. gonorrhoeae from the mucosa into the blood. To the best of our knowledge, this is the first case of gonococcal bacteremia in Korea.
Adult ; Bacteremia/complications/*diagnosis/microbiology ; Catalase/metabolism ; Esophageal and Gastric Varices/complications/*diagnosis ; Gastrointestinal Hemorrhage/*etiology ; Gonorrhea/complications/*diagnosis/microbiology ; Humans ; Ligation ; Liver Cirrhosis/diagnosis ; Male ; Neisseria gonorrhoeae/genetics/*isolation & purification ; Oxidoreductases/metabolism ; RNA, Ribosomal, 16S/chemistry/genetics ; Sequence Analysis, DNA

OBJECTIVETo construct a fused expression vector of the outer membrane protein gene PorB of Neisseria gonorrhoeae, express the fusion protein in the prokaryotic system, and obtain a gene recombination protein, for the purpose of preparing the ground for further research on the pathopoiesis and immune protective response of PorB.

METHODSA pair of primers were designed according to the known sequence of the PorB gene, and the PorB gene was amplified by PCR from the genome of Neisseria gonorrhoeae 29403 and cloned into the prokaryotic expression plasmid pGEX-4T-1 to generate pGEX-4T-PorB recombinants. The recombinant plasmid pGEX4T-PorB was transferred into competent cells E. coli BL21. After confirmed by restriction endonuclease digestion, PCR and DNA sequencing analysis, the recombinant protein was induced to express by isopropyl-beta-D-thiogalactoside (IPTG), and examined by SDS-PAGE and Western blotting.

RESULTSRestriction endonuclease digestion, PCR amplification and DNA sequencing analysis showed that the PorB gene of 1 047 bp was amplified from Neisseria gonorrhoeae DNA, and the recombinant plasmid pGEX-4T-PorB was successfully constructed and highly expressed in E. coli.

CONCLUSIONThe prokaryotic expression vector of pGEX-4T-PorB was successfully constructed and efficiently expressed in the prokaryotic system, which has provided a basis for further study on the biological activity of the PorB protein, as well as animal immune experiment and detection of Neisseria gonorrhoeae, and its application as a mucosal immune vaccine.

Bacterial Outer Membrane Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; metabolism ; Gene Expression ; Genetic Vectors ; Molecular Sequence Data ; Neisseria gonorrhoeae ; genetics ; metabolism ; Plasmids ; Porins ; genetics ; metabolism

The detection method of gene chip based on SPR principle is a potential high-throughput microanalysis method without labelling. With the use of Self-assembled monolayer (SAM) technology, the gene chip of Neisseria gonorrhoeae probe lattice has been prepared, detected and analyzed using the Surface plasmon resonance (SPR) and SPR imaging (SPRI) gene chip detection system here-in provided for research in the hybridizatin reaction on the probe lattice of gene chip. The result indicates that there is an obvious resonance assimilate peak on the SPR resonance curve. And after hybridization, the refractive index and resonance as well as molecular weight of the probe have increased. So whether a hybridization takes place or whether the wanted ingredient is in the sample under examination can be determined by using SPR to watch the detecting interface or the resonance curve. The SPRI detection system is available for observing the happening of a hybridization on the probe of gene chip in real-time and straighforwardly. The SPR and SPRI system can do analysis qualitatively and quantitatively.
DNA Probes ; genetics ; Neisseria gonorrhoeae ; genetics ; Oligonucleotide Array Sequence Analysis ; instrumentation ; methods ; Surface Plasmon Resonance ; methods ; Surface Properties

The surface plasmon resonance (SPR)-based gene chip was prepared according to the following processes: First, a film of nanogold, which was synthesized by using Frens' method, was plated on chip by Chlorauric acid/hydroxylamine method. Then probes were fixed on nanogold film by Self-assembled monolayer (SAM) technology. Subsequently, the fixing time and concentration of probes, the sensitivity and the specificity of the chip were optimized. Our results suggested that the chip plated with 2.5 nm nanogold film has a better SPR reflection, and when fixed by probes for 4.5 h at the concentration of 1 500 nmol/L, the gene chip also shows a fine performance of detection and can identify accurately the mismatch between bases in SPR detection system. The gene chip constructed in the research can be used for SPR sensor detection.
Gold ; chemistry ; Nanoparticles ; chemistry ; Neisseria gonorrhoeae ; genetics ; Oligonucleotide Array Sequence Analysis ; Sensitivity and Specificity ; Surface Plasmon Resonance ; methods