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522Fast detection of bacteria causing urinary tract infection by PCR
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522
D E V E L O P M E N T OF O L I G O N U C L E O T I D E MICRO ARRAY F O R GENOTYPING OF SEXUALLY TRANSMITTED DISEASES
FAST D E T E C T I O N OF B A C T E R I A C A U S I N G U R I N A R Y TRACT INFECTION BY PCR
Moon W.C. ~, Oh M.R. 1, Ko J.E. 1, Park M.S. z, Cho J.H. 3
Hauser S. 1, Lehmann L. a, Malenka T. 2, Stueber F. 2
1Chung-Ang University Hospital, Dept. of Urology, Seoul, South Korea, 2Sunmng Top Urology Clinic, Dept. ofUrology, Seoul, South Korea, 3Goldman Urology Clinic, Dept. of Urology, Seoul, South Korea
INTRODUCTION & OBJECTIVES: Common causative organisms of sexually transmitted diseases (STD) include N. gonorrboea (NG), C trachomatis (CT), U urealyticum (UU), M. genitalium (MG) and human papillomavirus (HPV), all of which are difficult to detect by conventional culture or immunologic assay. STD commonly show mixed infection. Therefore, we need a new molecular test which can reliably detect all the major causative organisms of STD. We herein have developed a prototype oligonucleotide micro array to detect genotype ofNG, CT, UU, MG and HPV and evaluated its diagnostic value. MATERIAL & METHODS: Genomic DNAs were isolated from samples of voided urine or cervical swab from 542 adults attending STD clinics in Seoul, Korea and genntypic information of NG CT, UU, MG, coliform bacteria and HPV were analyzed by PCR, cloning and sequencing. In addition, plasmid DNA of wild and variant-type genomic DNA of each organism was established. Multiple oligonucleotide probes were designed for gene of 16S rRNA, 16-23S intergenic region, cryptic plasmid, tetracycline resistance ofNG, CT, UU, MG and coliform bacteria, E6/E7 and L1 region of HPV, and human beta-globulin gene, and were spotted onto microscopic glass slide to produce DNA chip of STD. The grip of this chip was designed to be able to analyze eight different samples on a single chip. PCR products from plasmid clones and clinical samples containing DNA ofNG, CT, UU, MG, coliform bacteria and HPV, and urine samples from adult men without evidence of STD(N = 50 per each group) were applied onto the DNA chip and hybridization reaction were carried out, which were then analyzed by fluorescence scanner. RESULTS: The STD chip could detect 10 to 100 plasmid clone of each organism with 100% sensitivity in a variety of mixed samples. On analysis of 200 clinical samples with STD and 50 negative control samples, sensitivity, specificity and reproducibility of STD chip to detect genotype of all of NG, CT, UU, MG were 99%, 100% and 99%, respectively. The diagnostic accuracy of STD chip to detect mixed infection was 98%. CONCLUSIONS: The STD genotyping oligonucleotide micro array in the present study can detect major causative organisms of STD including NG, CT, UU and MG in an accurate, high-throughput and cost effective manner. It can also detect HPV and coliform bacteria and provide information on resistance of bacteria to antibacterial drag. This novel STD chip may become a valuable tool for basic research and clinical study of STD.
~University Hospital, Department of Urology, Bonn, Germany, 2University Hospital, Department of Anesthesiology and Intensive Care Medicine, Bonn, Germany
INTRODUCTION & OBJECTIVES: Severe urinary tract infection and its sequel urosepsis are still an important cause of in hospital morbidity and mortality (1). Early diagnoses of the infecting pathogens are important for adequate antibiotic therapy. This study compared a recently developed PCR based method (2) with conventional results obtained by microbiology testing. MATERIAL & METHODS: Urine samples of 76 patients with severe urinary tract infection were collected. Parallel samples were sent for routine microbiology testing and PCR testing, respectively. For PCR testing bacterial D N A was prepared, followed by PCR analysis as described previously (2). A comparison of the microbiological findings with the PCR results was performed. RESULTS: According to microbiology findings 30 samples showed gram positive bacteria, 38 samples were infected with gram negative bacteria and 8 samples had a mixed infection. In the gram negative group 34 samples were identified correctly by the PCR approach which revealed results within 4 hours testing time. Samples showing gram positive microbiology culture results had a lower rate of positive results using PCR testing. 16 different relevant bacteria species were detectable by D N A amplification. CONCLUSIONS: Especially in gram positive and mixed infections conventional microbiology still seems advantageous to a P C R based method. An explanation can be seen in the well known difficult process preparing DNA from gram positive bacteria as opposed to gram negative bacteria. With improved future preparation methods this problem may be circumvented leading to a higher PCR sensitivity. Fast PCR systems detecting microbial D N A appear to be a promising tool References: (1): Infect Contr Hosp Epidemiol 1993 14:73-80. (2):J Clin Microbiol. 2002 40:4304-7.
523
524
GENOTYPING AND NUCLEOTIDE SEQUENCE ANALYSIS OF THE OMPA GENE OF CHLAMYDIA TRACHOMATIS FROM COMMERCIAL SEX WORKERS IN KOREA
SELECTIVE N U C L E A R FACTOR KAPPA B (NFIcB) INHIBITORS, PYROLIDIUM DITHIOCARBAMATE AND SULFASALAZINE, PREVENTS THE NEPHROTOXICITY INDUCED BY GENTAMICIN
Lee G. 1, Park j.1, Kim B.R?, Kim S.A.2, You C.K.2, Seong W.K.2
Tu~cu V. 1, Ozbek E?, Ta~91A?, Somay A. s, Ba~ M. a, Karaca C, 4, Altu~ T.4
1Dankook University Medical College, Urology, Cheonan, Chtmgnam, South Korea, 2National Institute of Health, Korea, Research Center for Pathogen Control, Seoul, South Korea
1Bakirk6y Dr. Sadi Konuk Research and Training Hospital, Urology, Istanbul, Turkey, 2SSK Vaklf Gureba Research and Training Hospital, Urology, Istanbul, Turkey, SSSK Vaklf Gureba Research and Training Hospital, Pathology, Istanbul, Turkey, 4Istanbul University, cerrahpasa Medical School, Animal Research Laboratory, Istanbul, Turkey
INTRODUCTION & OBJECTIVES: Back~'ound; Chlamydia trachomatis is the most common sexually transmitted disease (STD) in Korea, as in many other parts of the world. While some sequences of chlamydial ompA gene were reported in literatures, the assessments of ganotyping in female commercial sex workers (FCSW) in Asia have not been perfonned yet. They not only frequently transmit STD pathogens to male customers, but also are frequently exposed to new infections themselves. To characterize the chlamydial infection in men, it is very important to understand the patterns of chlamydial infection in FCSW. The goal was to determine genotypes in core group, FCSW, in Korea and to assess the significance in chlamydiaI spread by the comparison of sequences. MATERIAL & METHODS: To accomplish this, 40 endo-eervical samples from FCSW in one city of Korea were collected from April 2004 to August 2004. The genomic DNA was extracted with Wizard genomic DNA purification kit. About 300 clinical samples were received for 4 months. OmpA gene of Chlamydia trachomatis from the gcnomic DNA was amplified by PCR.lml dNTP mix, 10mM each, I U ofpfu DNA polymerase (Promega), and 25 pmol of each primer. Amplification consisted of a 6-minute denaturation at 95°C; each consisting of denaturation at 95 °C for 1 minute, annealing at 45 °C for 3 minutes, and chain elongation at 72 °C for 3 minutes; and final extension for 5 minutes at 72 °C. These conditions were used with primers SERO1A and SERO2A in a primary PCR and in a nested PCR with primers PCTM3 and VD42. Of 300 samples, 40 were ompA positive. All samples that turned positive by PCR method were subjected to ompA genotyping. Primers used for sequencing were OMP6S and OMP6AS. Both strands of the ompA gene were sequenced through whole variable domains and some constant domain sequences. RESULTS: The deduced serovars found, in descending order of prevalence, were E (45%), F (20%), G (15%), D (5%), H (5%), J (2.5%), and mixed type (7.5%). While we found various serovars in limited FCSW, only one genotype was found in each E, F, G, D, H, J, and G type. CONCLUSIONS: The majority of pafients were infected with ganotype E. All subjects of E type showed only one type in our study. It suggests serotype E was well conserved under the evolution pressure even in core group who have high possibility of multiple chlamydial infections. While we found various serotypes in limited patients, only one genotype was found in E, F and G type. We believe that ompA DNA polymorphism is not a typical finding in core group but a result of evolution pressure in individual serotype. Interestingly, the abundance of serovar G, KS_G1, was presented in our study that is a minor subtype in European studies. Our subtype G, KS G1, was not found in Birmingham, Indianapolis and New Orleans but only found in Seattle and San Francisco in American study. It may suggest that the high prevalence of KSG1 in Korea has a relation with the high incidence of serovar G in Pacific cities in the United States.
INTRODUCTION & OBJECTIVES: To investigate the effect of selective nuclear factor kapa b (NFKB) inhibitors, pyrolidium dithiocarbamate (PDTC) and sulfasalazine (SULFA), on renal tubular necrosis as well as inducible nitric oxide synthase (i NOS) and NFKB expression induced by gentamicin in rats. MATERIAL & METHODS: Fourty-two adult male Sprague-Dawley rats were divided into six equal groups. In group 1, the rats were control, in group 2 they were injected with gentamicin for 10 days (100 mg/lcg/day, i.p.), in group 3 injected with gentamicin plus PDTC (100 mg/kg/day, i.p), in group 4 injected with gentamicin plus SULFA (75 mg/kg/day, ip), in group 5 injected with gentamicin plus distilled water (vehicle of PDTC), group 6 injected with gentamicin plus ammonium chloride (vehicle of SULFA) for ten days. At 24 h after the last injection, rats were killed and the renal cortex separated from the medulla. A small sample was fixed in formaldehyde solution for histological and immunohistochemical examination. Blood samples were also taken to assess the senlm levels of urea, creatinine, Na+, K+ and gamma-glutamyl transpeptidase (gamma-GT). hnmunohistochemically i NOS and active subunite of NF~zB, P65 were evaluated using mouse monoclonal antibody. Results were evaluated semiquantitively and compared using the Mann-Whitney U-test. RESULTS: On haematoxylene eosine staining compared with the controls rats, gentamicin caused widespread tubular necrosis (grade 2-4) but in group 3 and 4 there was a marked reduction in the extent of tubular damage, hnmunohistochemically there was marked staining in i NOS and P65 expression in gentamicin treated rats compared with control and group 3 and 4. In group 3 and 4 i NOS and P65 expression were significantly reduced compared only with gentamicin treated rats. There was no significant difference in serum levels ofNa+, K+, blood urea nitrogen and creatinine. CONCLUSIONS: These results indicate that gentamicine induces i NOS expression through activation of NF~;B, P65. It is possible to prevent gentamicin induced nephrotoxicity using selective NFKB inhibitors.
European Urology Supplements 4 (2005) No. 3, pp. 133
522
D E V E L O P M E N T OF O L I G O N U C L E O T I D E MICRO ARRAY F O R GENOTYPING OF SEXUALLY TRANSMITTED DISEASES
FAST D E T E C T I O N OF B A C T E R I A C A U S I N G U R I N A R Y TRACT INFECTION BY PCR
Moon W.C. ~, Oh M.R. 1, Ko J.E. 1, Park M.S. z, Cho J.H. 3
Hauser S. 1, Lehmann L. a, Malenka T. 2, Stueber F. 2
1Chung-Ang University Hospital, Dept. of Urology, Seoul, South Korea, 2Sunmng Top Urology Clinic, Dept. ofUrology, Seoul, South Korea, 3Goldman Urology Clinic, Dept. of Urology, Seoul, South Korea
INTRODUCTION & OBJECTIVES: Common causative organisms of sexually transmitted diseases (STD) include N. gonorrboea (NG), C trachomatis (CT), U urealyticum (UU), M. genitalium (MG) and human papillomavirus (HPV), all of which are difficult to detect by conventional culture or immunologic assay. STD commonly show mixed infection. Therefore, we need a new molecular test which can reliably detect all the major causative organisms of STD. We herein have developed a prototype oligonucleotide micro array to detect genotype ofNG, CT, UU, MG and HPV and evaluated its diagnostic value. MATERIAL & METHODS: Genomic DNAs were isolated from samples of voided urine or cervical swab from 542 adults attending STD clinics in Seoul, Korea and genntypic information of NG CT, UU, MG, coliform bacteria and HPV were analyzed by PCR, cloning and sequencing. In addition, plasmid DNA of wild and variant-type genomic DNA of each organism was established. Multiple oligonucleotide probes were designed for gene of 16S rRNA, 16-23S intergenic region, cryptic plasmid, tetracycline resistance ofNG, CT, UU, MG and coliform bacteria, E6/E7 and L1 region of HPV, and human beta-globulin gene, and were spotted onto microscopic glass slide to produce DNA chip of STD. The grip of this chip was designed to be able to analyze eight different samples on a single chip. PCR products from plasmid clones and clinical samples containing DNA ofNG, CT, UU, MG, coliform bacteria and HPV, and urine samples from adult men without evidence of STD(N = 50 per each group) were applied onto the DNA chip and hybridization reaction were carried out, which were then analyzed by fluorescence scanner. RESULTS: The STD chip could detect 10 to 100 plasmid clone of each organism with 100% sensitivity in a variety of mixed samples. On analysis of 200 clinical samples with STD and 50 negative control samples, sensitivity, specificity and reproducibility of STD chip to detect genotype of all of NG, CT, UU, MG were 99%, 100% and 99%, respectively. The diagnostic accuracy of STD chip to detect mixed infection was 98%. CONCLUSIONS: The STD genotyping oligonucleotide micro array in the present study can detect major causative organisms of STD including NG, CT, UU and MG in an accurate, high-throughput and cost effective manner. It can also detect HPV and coliform bacteria and provide information on resistance of bacteria to antibacterial drag. This novel STD chip may become a valuable tool for basic research and clinical study of STD.
~University Hospital, Department of Urology, Bonn, Germany, 2University Hospital, Department of Anesthesiology and Intensive Care Medicine, Bonn, Germany
INTRODUCTION & OBJECTIVES: Severe urinary tract infection and its sequel urosepsis are still an important cause of in hospital morbidity and mortality (1). Early diagnoses of the infecting pathogens are important for adequate antibiotic therapy. This study compared a recently developed PCR based method (2) with conventional results obtained by microbiology testing. MATERIAL & METHODS: Urine samples of 76 patients with severe urinary tract infection were collected. Parallel samples were sent for routine microbiology testing and PCR testing, respectively. For PCR testing bacterial D N A was prepared, followed by PCR analysis as described previously (2). A comparison of the microbiological findings with the PCR results was performed. RESULTS: According to microbiology findings 30 samples showed gram positive bacteria, 38 samples were infected with gram negative bacteria and 8 samples had a mixed infection. In the gram negative group 34 samples were identified correctly by the PCR approach which revealed results within 4 hours testing time. Samples showing gram positive microbiology culture results had a lower rate of positive results using PCR testing. 16 different relevant bacteria species were detectable by D N A amplification. CONCLUSIONS: Especially in gram positive and mixed infections conventional microbiology still seems advantageous to a P C R based method. An explanation can be seen in the well known difficult process preparing DNA from gram positive bacteria as opposed to gram negative bacteria. With improved future preparation methods this problem may be circumvented leading to a higher PCR sensitivity. Fast PCR systems detecting microbial D N A appear to be a promising tool References: (1): Infect Contr Hosp Epidemiol 1993 14:73-80. (2):J Clin Microbiol. 2002 40:4304-7.
523
524
GENOTYPING AND NUCLEOTIDE SEQUENCE ANALYSIS OF THE OMPA GENE OF CHLAMYDIA TRACHOMATIS FROM COMMERCIAL SEX WORKERS IN KOREA
SELECTIVE N U C L E A R FACTOR KAPPA B (NFIcB) INHIBITORS, PYROLIDIUM DITHIOCARBAMATE AND SULFASALAZINE, PREVENTS THE NEPHROTOXICITY INDUCED BY GENTAMICIN
Lee G. 1, Park j.1, Kim B.R?, Kim S.A.2, You C.K.2, Seong W.K.2
Tu~cu V. 1, Ozbek E?, Ta~91A?, Somay A. s, Ba~ M. a, Karaca C, 4, Altu~ T.4
1Dankook University Medical College, Urology, Cheonan, Chtmgnam, South Korea, 2National Institute of Health, Korea, Research Center for Pathogen Control, Seoul, South Korea
1Bakirk6y Dr. Sadi Konuk Research and Training Hospital, Urology, Istanbul, Turkey, 2SSK Vaklf Gureba Research and Training Hospital, Urology, Istanbul, Turkey, SSSK Vaklf Gureba Research and Training Hospital, Pathology, Istanbul, Turkey, 4Istanbul University, cerrahpasa Medical School, Animal Research Laboratory, Istanbul, Turkey
INTRODUCTION & OBJECTIVES: Back~'ound; Chlamydia trachomatis is the most common sexually transmitted disease (STD) in Korea, as in many other parts of the world. While some sequences of chlamydial ompA gene were reported in literatures, the assessments of ganotyping in female commercial sex workers (FCSW) in Asia have not been perfonned yet. They not only frequently transmit STD pathogens to male customers, but also are frequently exposed to new infections themselves. To characterize the chlamydial infection in men, it is very important to understand the patterns of chlamydial infection in FCSW. The goal was to determine genotypes in core group, FCSW, in Korea and to assess the significance in chlamydiaI spread by the comparison of sequences. MATERIAL & METHODS: To accomplish this, 40 endo-eervical samples from FCSW in one city of Korea were collected from April 2004 to August 2004. The genomic DNA was extracted with Wizard genomic DNA purification kit. About 300 clinical samples were received for 4 months. OmpA gene of Chlamydia trachomatis from the gcnomic DNA was amplified by PCR.lml dNTP mix, 10mM each, I U ofpfu DNA polymerase (Promega), and 25 pmol of each primer. Amplification consisted of a 6-minute denaturation at 95°C; each consisting of denaturation at 95 °C for 1 minute, annealing at 45 °C for 3 minutes, and chain elongation at 72 °C for 3 minutes; and final extension for 5 minutes at 72 °C. These conditions were used with primers SERO1A and SERO2A in a primary PCR and in a nested PCR with primers PCTM3 and VD42. Of 300 samples, 40 were ompA positive. All samples that turned positive by PCR method were subjected to ompA genotyping. Primers used for sequencing were OMP6S and OMP6AS. Both strands of the ompA gene were sequenced through whole variable domains and some constant domain sequences. RESULTS: The deduced serovars found, in descending order of prevalence, were E (45%), F (20%), G (15%), D (5%), H (5%), J (2.5%), and mixed type (7.5%). While we found various serovars in limited FCSW, only one genotype was found in each E, F, G, D, H, J, and G type. CONCLUSIONS: The majority of pafients were infected with ganotype E. All subjects of E type showed only one type in our study. It suggests serotype E was well conserved under the evolution pressure even in core group who have high possibility of multiple chlamydial infections. While we found various serotypes in limited patients, only one genotype was found in E, F and G type. We believe that ompA DNA polymorphism is not a typical finding in core group but a result of evolution pressure in individual serotype. Interestingly, the abundance of serovar G, KS_G1, was presented in our study that is a minor subtype in European studies. Our subtype G, KS G1, was not found in Birmingham, Indianapolis and New Orleans but only found in Seattle and San Francisco in American study. It may suggest that the high prevalence of KSG1 in Korea has a relation with the high incidence of serovar G in Pacific cities in the United States.
INTRODUCTION & OBJECTIVES: To investigate the effect of selective nuclear factor kapa b (NFKB) inhibitors, pyrolidium dithiocarbamate (PDTC) and sulfasalazine (SULFA), on renal tubular necrosis as well as inducible nitric oxide synthase (i NOS) and NFKB expression induced by gentamicin in rats. MATERIAL & METHODS: Fourty-two adult male Sprague-Dawley rats were divided into six equal groups. In group 1, the rats were control, in group 2 they were injected with gentamicin for 10 days (100 mg/lcg/day, i.p.), in group 3 injected with gentamicin plus PDTC (100 mg/kg/day, i.p), in group 4 injected with gentamicin plus SULFA (75 mg/kg/day, ip), in group 5 injected with gentamicin plus distilled water (vehicle of PDTC), group 6 injected with gentamicin plus ammonium chloride (vehicle of SULFA) for ten days. At 24 h after the last injection, rats were killed and the renal cortex separated from the medulla. A small sample was fixed in formaldehyde solution for histological and immunohistochemical examination. Blood samples were also taken to assess the senlm levels of urea, creatinine, Na+, K+ and gamma-glutamyl transpeptidase (gamma-GT). hnmunohistochemically i NOS and active subunite of NF~zB, P65 were evaluated using mouse monoclonal antibody. Results were evaluated semiquantitively and compared using the Mann-Whitney U-test. RESULTS: On haematoxylene eosine staining compared with the controls rats, gentamicin caused widespread tubular necrosis (grade 2-4) but in group 3 and 4 there was a marked reduction in the extent of tubular damage, hnmunohistochemically there was marked staining in i NOS and P65 expression in gentamicin treated rats compared with control and group 3 and 4. In group 3 and 4 i NOS and P65 expression were significantly reduced compared only with gentamicin treated rats. There was no significant difference in serum levels ofNa+, K+, blood urea nitrogen and creatinine. CONCLUSIONS: These results indicate that gentamicine induces i NOS expression through activation of NF~;B, P65. It is possible to prevent gentamicin induced nephrotoxicity using selective NFKB inhibitors.
European Urology Supplements 4 (2005) No. 3, pp. 133