Akito Terai

Cloning and nucleotide sequencing of Vero toxin 2 variant genes from Escherichia coli O91 : H21 isolated from a patient with the hemolytic uremic syndrome

Cellular DNA extracted from Escherichia coli strain B2F1 (O91:H21) was found to contain two separate DNA sequences that hybridized with a Vero toxin 2 (VT2)-specific gene probe under stringent conditions. These two sequences were cloned and both were shown to encode a variant of Vero toxin 2 (VT2vh). The nucleotide sequences of the operons encoding VT2vh, designated as vtx2ha and vtx2hb, were determined. The two operons were nearly identical (99% overall DNA homology) and both encoded A subunits of 319 amino acid residues and B subunits of 89 amino acid residues, the A and B subunit genes being separated by a stretch of 14 bp. The A and B subunit genes of the vtx2ha operon exhibited 98.6% and 95.5% DNA homology, respectively, with those of the slt-II operon encoding Shiga-like toxin II (or VT2) cloned from a strain from a patient with hemorrhagic colitis, while the A and B subunit genes of the vtx2ha operon showed 94.5% and 82.8% DNA homology, respectively, with those of the slt-IIv operon encoding a SLT-II variant cloned from a strain isolated from a pig with edema disease. The nucleotide sequences of the presumed promoters and presumptive ribosome binding sites in the vtx2ha, vtx2hb, and slt-II, and slt-IIv operons were identical. These results indicate that nucleotide sequences encoding a family of VT2-related toxins are present in various strains of E. coli and that the sequences of the genes for A subunits are better conserved than those of the B subunit genes.

Genetic Evidence Supporting the Fecal-Perineal-Urethral Hypothesis in Cystitis Caused by Escherichia Coli

PURPOSE The fecal-perineal-urethral hypothesis to explain the cause of urinary tract infections (UTI) by enteric bacteria has been supported by longitudinal studies using methods of serotyping and detecting urovirulence factors such as P fimbriae. However, genetic techniques to more accurately characterize Escherichia coli strains have not been exploited. MATERIALS AND METHODS A total of 2,700 E. coli colonies isolated from the urine and rectal swabs of 9 female subjects with acute uncomplicated cystitis and from the rectal swabs of 30 healthy women were serotyped and examined for genes encoding various urovirulence factors by colony hybridization test. The clonality of the urine and fecal isolates of E. coli from the cystitis subjects was further evaluated by pulsed-field gel electrophoresis (PFGE). RESULTS E. coli strains causing cystitis dominated the rectal flora of 7 of 9 patients. In the remaining 2 patients, similar clones comprised at least 20% of the fecal flora. Carriage of E. coli strains with a variety of urovirulence factors was quite common among healthy women. PFGE demonstrated that most of the isolates sharing the same serotypic characteristics and virulence factors in the urine and rectal swab samples from each subject were identical. CONCLUSIONS Based upon precise genetic techniques, our results clearly support the fecal-perineal-urethral hypothesis, indicating that E. coli strains residing in the rectal flora serve as a reservoir for urinary tract infections, e.g., cystitis.

Prevalence of and risk factors for nocturia: Analysis of a health screening program

Background: We examined the prevalence of and risk factors for nocturia in Kurashiki city and the surrounding area, a rural area in Japan.

Diagnostic potential in bladder cancer of a panel of tumor markers (calreticulin, γ‐synuclein, and catechol‐o‐methyltransferase) identified by proteomic analysis

Using proteomic analysis, we previously identified calreticulin (CRT) as a potentially useful urinary marker for bladder cancer. Now, we have also identified γ‐synuclein (SNCG) and a soluble isoform of catechol‐o‐methyltransferase (s‐COMT) as novel candidates for tumor markers in bladder cancer, by means of proteomic analysis. In the process of establishing a superior tumor marker system, we investigated the diagnostic value of a combination assay of these three proteins. Voided urine samples were obtained from 112 bladder cancer and 230 control patients. Urinary CRT, SNCG, and s‐COMT were measured as a combined marker by quantitative western blot analysis. Relative concentration of each protein was calculated and the diagnostic value of a concomitant examination of these markers was evaluated by receiver operator characteristic analysis. With the best diagnostic cutoff, the overall sensitivity of the combined markers was 76.8% (95% confidence interval, 69–81%) with a specificity of 77.4% (72–80%), while those of a single use of CRT were 71.4% and 77.8%, respectively. When evaluated in relation to tumor characteristics, such as grade, stage, size, and outcome of urinary cytology, the diagnostic capacity of the combined markers was equal to or better than that of CRT in all categories. Concomitant use of CRT, SNCG, and s‐COMT had higher sensitivity for detection of bladder cancer than did single use of CRT. Our study suggests that use of this panel of markers will improve the diagnosis of bladder cancer and may allow the development of a protein microarray assay or multi‐channel enzyme‐linked immunosorbent assay.

Urinary Calculi as a Late Complication of the Indiana Continent Urinary Diversion: Comparison with the Kock Pouch Procedure

PURPOSE Although urinary calculi have been frequent late complications of the Kock continent urinary diversion, they have not been regarded as significant problems in patients with the Indiana pouch because of the lack of foreign material present. However, stones developed in a significant percentage of our patients with an Indiana pouch. We investigated the characteristics of stone formation in patients with the Indiana pouch and compared them to those with a Kock pouch. MATERIALS AND METHODS Detailed clinical courses regarding stone formation were reviewed in 72 patients with a Kock pouch and 54 with an Indiana pouch who had a minimum followup of 12 months. RESULTS Stones developed in 7 of 54 patients (12.9%) with an Indiana pouch compared to 31 of 72 (43.1%) with a Kock pouch. The incidence gradually increased with longer followup but it was lower in the Indiana than in the Kock pouch group (5-year stone-free rate 84% versus 66%, respectively). Although the stones consisted principally of a mixture of struvite, carbonate apatite and ammonium hydrogen urate, variable amounts of calcium oxalate were identified in 50% of the Indiana pouch calculi. CONCLUSIONS Not only urinary infections but also metabolic factors were considered to be involved in stone formation within the Indiana pouch. However, the substantially higher rate of stone formation in our Kock and Indiana pouch groups than has been reported in the United States suggested that no or infrequent pouch irrigations in our patients were important risk factors for urinary calculi.

Androgen receptor CAG repeat length polymorphism in benign prostatic hyperplasia (BPH): Correlation with adenoma growth†

The androgen receptor (AR) gene has a polymorphic CAG microsatellite encoding variable‐length glutamine repeats in the AR protein. The purpose of this study was to evaluate the association between the growth of benign prostatic hyperplasia (BPH) and the AR gene CAG repeat length.

Effect of Urinary Intestinal Diversion on Urinary Risk Factors for Urolithiasis

We investigated the effect of urinary intestinal diversion on risk factors for calcium urolithiasis, such as calcium, phosphate, magnesium, uric acid, oxalate and citrate, in 3 groups of patients (Kock pouch, Indiana pouch and ileal conduit). Mean urinary volume was not significantly different among the 3 groups. Mean serum creatinine and 24-hour creatinine clearance in the continent reservoir group were better than in the ileal conduit group. Mean urinary excretion of calcium, phosphate and magnesium was significantly greater in the continent reservoir group than in the ileal conduit group. Although calcium excretion had a positive correlation with 24-hour creatinine clearance, calcium excretion per ml. per minute creatinine clearance still showed a significant difference. Increased calcium excretion is considered to reflect metabolic disturbances resulting from reabsorption of urinary solutes through the intestinal segments. Overall, there was no significant difference in the urinary parameters between the Kock and Indiana pouch groups. While mean urinary oxalate and citrate were within the normal range in all 3 groups, more than a third of the patients in each group were hypocitraturic (less than 100 mg. per day). In none of the 3 groups did the levels of urinary calcium, phosphate and magnesium, as well as other urinary risk parameters show any correlation with the duration of diversion. In summary, our study indicated that the continent urinary reservoir causes a long-term increase in urinary excretion of calcium, phosphate and magnesium. These urinary metabolic alterations might promote the formation of calcium urolithiasis as well as infectious stones. The degree of metabolic alterations may be greater with a continent reservoir than with an ileal conduit.

Typing of verotoxins by DNA colony hybridization with poly- and oligonucleotide probes, a bead-enzyme-linked immunosorbent assay, and polymerase chain reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin‐producing Escherichia coli (VTEC), a sensitive bead‐enzyme‐linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo‐ and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.

The 10-Year Natural History of Simple Renal Cysts

OBJECTIVE We previously reported the natural history of renal cysts, with a mean follow-up of 6 years. Here, we extended the follow-up period to 10 years. METHODS From January 1993 to August 2006, 61 patients diagnosed with renal cysts were followed for up to 14 years (mean 9.9 years). The sequential changes in renal cyst size were plotted against patient age, and the rate of increase in cyst size per year was calculated for each individual. Those cyst characteristics known to predict aggressiveness were analyzed. RESULTS The majority of the cysts increased in size and number. The average size increase and the average rate of enlargement in all cysts were 1.6 mm and 3.9% per year, respectively. Several cysts, especially multiloculated cysts, increased rapidly during the first 2 to 3 years, but then the rate of growth tended to decelerate with time. By using univariate analyses, age, laterality and cyst shape were revealed to be significant predictors of aggressiveness. The multivariate analysis revealed that age was the most significant predictor. Renal neoplasms originating from the renal cysts appeared in 2 patients during the follow-up period. The rate of size increase of the neoplasm-bearing cysts was similar to that of the other benign renal cysts in the same age category. CONCLUSIONS The simple renal cysts continued to increase in size over 10 years, and sometimes increased rapidly, especially in younger patients. However, their growth rates appeared to decrease with age. There seems to be no specific pattern observed in the neoplasm-bearing renal cysts.

ESCHERICHIA COLI VIRULENCE FACTORS AND SEROTYPES IN ACUTE BACTERIAL PROSTATITIS

Background Escherichia coli is the most frequent pathogen in both acute bacterial prostatitis and acute uncomplicated urinary infections. To assess the virulence profiles of E. coli in acute prostatitis, the serotypes and virulence factor (VF) genotypes were determined.