Evaluation of a new amplified enzyme immunoassay (EIA) for the detection of Chlamydia trachomatis in male urine, female endocervical swab, and patient obtained vaginal swab specimens.

AIMS: To compare the performance of a new generation dual amplified enzyme immunoassay (EIA) with a molecular method for the diagnosis of Chlamydia trachomatis, using a range of urogenital samples, and to assess the reliability of testing self collected vaginal specimens compared with clinician collected vaginal specimens. METHODS: Two population groups were tested. For the first population group, first void urine samples were collected from 193 male patients with urethritis, and endocervical swabs were collected from 187 high risk commercial sex workers. All urine and endocervical specimens were tested by a conventional assay (IDEIA chlamydia), a new generation amplified immunoassay (IDEIA PCE chlamydia), and the Amplicor polymerase chain reaction (PCR). Discrepant results obtained among the three sample types were confirmed using a nested PCR test with a different plasmid target region. For the second population group, four swab specimens, including one patient obtained vaginal swab, two clinician obtained endocervical swabs, and one clinician obtained vaginal swab, were collected from 91 high risk sex workers. Self collected and clinician collected vaginal swabs were tested by IDEIA PCE chlamydia. Clinician obtained endocervical swabs were assayed by IDEIA PCE chlamydia and Amplicor PCR. RESULTS: The performance of the IDEIA PCE chlamydia test was comparable to that of the Amplicor PCR test when male urine and female endocervical swab specimens were analysed. The relative sensitivities of IDEIA, IDEIA PCE, and Amplicor PCR on male first void urine specimens were 79.3%, 91.4%, and 100%, respectively. The relative sensitivities of the three tests on female endocervical specimens were 85.0%, 95.0%, and 100%, respectively. The positivity rates for patient collected vaginal specimens and clinician collected vaginal specimens by IDEIA PCE were 25.2% and 23.1%, respectively, whereas those for clinician collected endocervical swabs by PCR and IDEIA PCE were both 27.5%. CONCLUSIONS: IDEIA PCE chlamydia is a lower cost but sensitive alternative test to PCR for testing male urine samples and female endocervical swabs. In addition, self collected or clinician collected vaginal specimens tested by IDEIA PCE chlamydia are a reliable alternative to analysing endocervical specimens.

Combination chemotherapy for infections due to methicillin-resistant Staphylococcus aureus with combination therapy by cefuzonam and fosfomycin or minocycline in the urologic field.

To determine the bacterial and clinical effects of methicillin-resistant Staphylococcus aureus (MRSA) on urinary tract infection (UTI), postoperative wound infection, and bacteremia, 22 strains of MRSA from infected patients were examined; minimal inhibitory concentration (MIC), fractional inhibitory concentration (FIC) index, coagulase typing, and change in MIC in the combination of cefuzonam (CZON) and fosfomycin (FOM) or minocycline (MINO), and the clinical effects of the combination therapy of CZON+FOM or CZON+MINO were investigated in 23 patients. MIC distribution was assessed for 11 drugs: methicillin (DMPPC), cefazolin (CEZ), cefotiam (CTM), cefuzonam (CZON), minocycline (MINO), vancomycin (VCM), arbekacin (ABK), imipenem (IPM), fosfomycin (FOM), ofloxacin (OFLX), and clarithromycin (CAM). For VCM and ABK, MICs ranged from 0.2 to 12.5 micrograms/ml. MINO showed a wide range of MIC, from 0.05 to 25 micrograms/ml. All strains were less sensitive to other antimicrobials. This MIC distribution was assessed in categories by coagulase typing. For CAM, type II strains revealed > or = 100 micrograms/ml of MIC50 and MIC90 compared with 0.2 and 3.13 micrograms/ml in type VII strains. For mixed combinations of CZON and MINO at ratios of 10 to 1 and 40 to 1, the rates of blood concentration for drugs 10 and 120 min after the intravenous injection, MIC distribution was observed between MINO and CZON. For mixed combinations of CZON and FOM at ratios of 1 to 1 and 1 to 4, MIC distribution was more sensitive than for FOM or CZON alone. With respect to the clinical effects of combination therapy by CZON+MINO and CZON+FOM, of seven cases of UTI with CZON+MINO, MRSA was eliminated completely in 4 patients (57.1%). In all patients who received CZON+FOM, MRSA was completely eliminated by this treatment regimen. In 4 of 7 (57.1%) patients with would infection and bacteremia, MRSA was eliminated by the combination of CZON+FOM or CZON+MINO. These results suggest that CZON+FOM is an effective combination in treating UTI and other MRSA infections in urology.

[The value of an aseptic intermittent catheterization program in the early management of spinal cord injury patients].

From 1979 to 1989, 269 patients with spinal cord injury were managed by an aseptic intermittent catheterization program during the acute phase of their injuries at the Spinal Injuries Center. One hundred fifty one patients with incomplete cord lesion and 36 males with complete tetraplegia were managed by program I, which protects the shocked bladder from overdistention. In contrast, 82 patients with complete cord lesion excluding male tetraplegia were managed by program II, which allows overdistention of the bladder. Of the 187 patients managed by program I, 137 (73.3%) achieved trigger voiding function, 61.3% of whom were completely dry. Of the 82 patients managed by program II, 62 (75.6%) were put to self- or assisted catheterization, 67.7% of whom were dry. The incidence of a grade I, which means normal bladder configuration, was 88.0% for program I and 87.3% for program II during the follow up course. Upper urinary tract deterioration occurred in only one case. Surgical treatment for urinary tract complications was performed in 14 cases (5.2%). These results suggest that the patients with incomplete cord lesion managed by non-distension regimen of the bladder (program I) and those, especially female, with complete cord lesion managed by overdistention regimen of the bladder (program II) achieve urinary continence with excellent urinary prognosis.

Clitoral involvement by neurofibromatosis: a case report and review of the literature.

We report a case of clitoral involvement by neurofibromatosis in which the clitoris resembled a phallus. The patient was treated successfully by clitoroplasty. This is the seventh such case reported in the English literature.

Antimicrobial resistance of Neisseria gonorrhoeae and high prevalence of ciprofloxacin-resistant isolates in Japan, 1993 to 1998.

To assess the antimicrobial resistance of Neisseria gonorrhoeae isolated from 1993 through 1998 in Japan, susceptibility testing was conducted on 502 isolates. Selected isolates were characterized by auxotype and analysis for mutations within the quinolone resistance-determining region (QRDR) in the gyrA and parC genes, which confer fluoroquinolone resistance on the organism. Plasmid-mediated penicillin resistance (penicillinase-producing N. gonorrhoeae) decreased significantly from 1993-1994 (7.9%) to 1997-1998 (2.0%). Chromosomally mediated penicillin resistance decreased from 1993-1994 (12.6%) to 1995-1996 (1.9%) and then increased in 1997-1998 (10.7%). Chromosomally mediated tetracycline resistance decreased from 1993-1994 (3.3%) to 1997-1998 (2.0%), and no plasmid-mediated high-level tetracycline resistance was found. Isolates with ciprofloxacin resistance (MIC >/= 1 microg/ml) increased significantly from 1993-1994 (6.6%) to 1997-1998 (24.4%). The proline-requiring isolates were less susceptible to ciprofloxacin than the prototrophic or arginine-requiring isolates. Ciprofloxacin-resistant isolates contained three or four amino acid substitutions within the QRDR in the GyrA and ParC proteins.

Beta-D-glucan concentrations detected by Toxicolor and Endospecy tests in the urine of patients with urinary fungal infections.

Beta-D-glucan is an essential component of the cell wall of fungi. We measured its concentration in the urine of patients with funguria using the chromogenic endotoxin assay kits, Toxicolor and Endospecy. These assay systems use the same Limulus coagulation enzymes. Since the Endospecy test detects endotoxin but not factor G, which is activated by beta-D-glucan, the beta-D-glucan concentration can be calculated by subtracting the Endospecy value from the Toxicolor value. Concentrations of beta-D-glucan were found to be significantly higher in urine samples from patients with funguria (> or = 10(3) colony-forming units/ml) than in non-infected samples.

Elevated interleukin-8 levels in the urine of children with renal scarring and/or vesicoureteral reflux.

PURPOSE: Elevation of urinary levels of interleukin-6 and 8 has been observed in patients with acute urinary tract infections. However, to our knowledge there have been no studies concerning the secretion of interleukin-6 and 8 into the urine after acute inflammation has resolved and renal scarring has occurred. On the other hand, it is well known that cytokines are variously related to glomerular diseases and, thus, it is possible that the progression of reflux nephropathy depends on interleukin-6 or 8. Therefore, we assessed urinary levels of interleukin-6 and 8 in children with vesicoureteral reflux and/or renal scarring. MATERIALS AND METHODS: We evaluated interleukin-6 and interleukin-8 levels in the urine of 32 children without a urinary tract infection who presented or were admitted to our hospital because of vesicoureteral reflux between April and December 1994. Interleukin-6 and 8 were determined using a commercially available human enzyme-linked immunosorbent assay kit and the 2-step sandwich method. RESULTS: Urinary interleukin-6 levels were below the lower detection limit (less than 10 pg./ml.) in all samples. There were statistically significant differences between urinary interleukin-8 levels in children with and without renal scarring (p = 0.001), and with and without vesicoureteral reflux (p = 0.0246). CONCLUSIONS: Urinary interleukin-8 is an effective marker for renal scarring and vesicoureteral reflux.

Testicular injury induces cell-mediated autoimmune response to testis.

Studies on testis autoimmunity are needed for a better understanding of immunological male infertility. Evidence has accumulated that the delayed type hypersensitivity (DTH) response plays a key role in the induction and/or maintenance of experimental autoimmune orchitis (EAO), an animal model for human immunological male infertility or aspermatogenesis. We report here that an antigen-specific DTH response to autologous testicular cells (TC) could be induced by bilateral testicular injury (trauma) in mice. Pretreatment of traumatized mice with a high dose of cyclophosphamide (CY) enhances the DTH response in a dose-dependent manner. The DTH response induced by testicular injury reaches its peak on the ninth day. We have shown that the local passive transfer of the footpad reaction to normal recipients by T cells further defines the DTH reaction. These characteristics resemble those of the previously reported DTH response to syngeneic TC induced by subcutaneous immunization with viable syngeneic crude TC. Our present injury model mimics clinical testicular trauma; therefore, this testicular injury model can be very useful in studying the immunological mechanism of EAO and of human immunological male infertility.

Renal scarring is enhanced by phorbol myristate acetate following infection with bacteria with mannose-sensitive pili.

Renal scarring is considered to develop in the later stages of chronic pyelonephritis. In our experimental model of pyelonephritis, bacteria with mannose-sensitive (MS) pili on their surface promoted renal scarring following inoculation into the renal parenchyma. The administration of cyclophosphamide to induce leukopenia and of superoxide dismutase to inactivate superoxide released from polymorphonuclear leukocytes (PMN) at the infection site suppressed any renal scarring following the infection. Conversely, the administration of phorbol myristate acetate at an early stage of infection significantly enhanced the renal scarring. These findings suggest that the PMNs which infiltrate the infection site and the superoxide they release play an important role in any renal scarring following infection with MS-piliated bacteria.

Influence of hyperosmotic environment comparable to the renal medulla upon membrane NADPH oxidase of human polymorphonuclear leukocytes.

Hyperosmotic environment in the renal medulla seems important for bacterial pyelonephritis because it exerts inhibitory influences upon the function of polymorphonuclear leukocytes (PMN). Urea and NaCl primarily contribute to high osmolarity in the renal medulla. We previously reported that PMN function was actually suppressed in phagocytosis, intracellular bacterial killing and superoxide generation in the hyperosmotic solution of urea and NaCl. In the present report, to verify the mechanism of this inhibitory effect, a kinetic study for NADPH oxidase in the cell membrane, the key enzyme complex of superoxide generation, was carried out in the cell membrane-solubilizing system under the hyperosmotic condition caused by urea or NaCl. Urea directly denaturated NADPH oxidase, and its inhibitory mechanism was reversible and uncompetitive with a decrease in Vmax and Km, while NaCl had no effect upon it, maintaining Lineweaver-Burk plots in the same position as those of the control. This result suggests that urea at least produces an inhibitory effect upon PMN through the direct inactivation of NADPH oxidase, although NaCl was unable to do so.

Effect of pili of Serratia marcescens on superoxide production and phagocytosis of human polymorphonuclear leukocytes.

PURPOSE: To determine the role played by superoxide in renal scar formation following renal infection. MATERIALS AND METHODS: The piliation of bacteria was assessed for its capacity to interact with human polymorphonuclear leukocytes (PMNs). Two recombinant strains having either MS or MR pili of Serratia marcescens were constructed. RESULTS: The MS-piliated strain stimulated superoxide production of PMNs twice as much as the MR- or nonpiliated strains did. The MS-piliated strain was more susceptible to phagocytosis than was the MR- or nonpiliated strain. CONCLUSION: These data suggest that the MS-piliated strain stimulates superoxide production of PMNs associated with phagocytosis, which leads to tissue damage in infected organs.

Effect of prednisolone on ascending renal infection due to biofilm disease and lower urinary tract obstruction in rats.

A model of renal infection due to lower urinary tract obstruction and biofilm disease was constructed for the study of renal scarring by inserting glass beads coated with bacterial biofilm into the bladder of rats and then clamping the urethra. We previously reported the effect of antimicrobial therapy used in combination with the anti-inflammatory agent prednisolone to prevent renal scarring. In this study we investigated the effect of prednisolone on renal scar formation using our new model. Renal scarring could not be prevented in the group in which prednisolone was administered in the period during which the urethra was regularly being clamped. In contrast, scarring was prevented in the group that began to receive prednisolone after the period of clamping had ended. Therefore, in cases of lower urinary tract obstruction prednisolone should only be administered for the prevention of renal scarring after the obstruction has been resolved.

Susceptibilities of Neisseria gonorrhoeae isolates containing amino acid substitutions in GyrA, with or without substitutions in ParC, to newer fluoroquinolones and other antibiotics.

We examined the antimicrobial susceptibilities of 85 Neisseria gonorrhoeae isolates, classified according to the presence of amino acid substitutions in the GyrA and ParC proteins, to 12 fluoroquinolones and 7 other antibiotics. Sitafloxacin and HSR-903 showed excellent activity against N. gonorrhoeae, including strains with both GyrA and ParC substitutions. Among the strains with various GyrA substitutions, strains with a serine-91-to-phenylalanine mutation required the highest MICs of all of the fluoroquinolones tested and were cross-resistant to structurally unrelated beta-lactams.

Genotypic evolution in a quinolone-resistant Neisseria gonorrhoeae isolate from a patient with clinical failure of levofloxacin treatment.

Recently, a reduction in the antimicrobial susceptibility of clinical isolates of Neisseria gonorrhoeae to newer fluoroquinolones including levofloxacin in vitro has been recognized in Japan. We examined the quinolone resistance mechanisms in N. gonorrhoeae isolates from a patient with clinical failure of levofloxacin treatment. Man with gonococcal urethritis was treated with oral 100 mg levofloxacin 3 times daily for 7 days. However, clinical failure of the treatment was observed. The minimum inhibitory concentration of levofloxacin for the posttreatment isolate (4.0 microg/ml) was 4-fold higher than that for the pretreatment isolate (1.0 microg/ml). To analyze quinolone resistance mechanisms in the set of isolates, we performed DNA sequencing of the quinolone resistance-determining regions within the gyrA and parC genes. Moreover, we assayed the intracellular levofloxacin and norfloxacin accumulation level in these isolates. The pretreatment isolate contained three substitutions compared to susceptible wild-type isolate, including serine to phenylalanine at position 91 and aspartic acid to asparagine at position 95 in the GyrA protein, and serine to proline at position 88 in the ParC protein. The posttreatment isolate had four substitutions, including the same three substitutions and an additional glutamic acid to glutamine substitution at position 91 in ParC. There was no significant difference in the level of accumulation of levofloxacin and norfloxacin between the pretreatment and posttreatment isolates. Our results indicate that levofloxacin selects a mutant having an additional alteration within the gene cording for the ParC protein during treatment, which may have enhanced quinolone resistance in the organism.

Analysis of quinolone resistance mechanisms in a sparfloxacin-resistant clinical isolate of Neisseria gonorrhoeae.

BACKGROUND AND OBJECTIVES: Recently, a reduction in the susceptibility of clinical isolates of Neisseria gonorrhoeae to newer fluoroquinolones including sparfloxacin in vitro has been recognized in Japan. The quinolone resistance mechanisms in gonococcal isolates from a patient with clinical failure of sparfloxacin treatment was investigated. GOAL: To report a man with gonococcal urethritis in whom clinical failure of sparfloxacin treatment occurred and to examine the quinolone resistance mechanisms in gonococcal isolates from the patient. STUDY DESIGN: A man with gonococcal urethritis was treated with oral 100 mg sparfloxacin three times daily for 5 days. However, clinical failure of the sparfloxacin treatment was observed. The antimicrobial susceptibilities of pretreatment and posttreatment isolates to sparfloxacin and other agents were measured. To analyze quinolone resistance mechanisms in the set of isolates, DNA sequencing of the genes corresponding to the quinolone resistance-determining regions within the GyrA and ParC proteins was performed. We also assayed the intracellular sparfloxacin accumulation level in these gonococcal cells. Moreover, we performed pulsed-field gel electrophoresis analysis to determine whether the pretreatment and posttreatment isolates were isogenic. RESULTS: The minimum inhibitory concentration of sparfloxacin for the posttreatment isolate (4 micrograms/ml) was 16 times higher than that for the pretreatment isolate (0.25 microgram/ml). The pretreatment isolate contained three mutations, including a Ser-91 to Phe mutation and an Asp-95 to Asn mutation in GyrA and a Ser-88 to Pro mutation in ParC. The posttreatment isolate had four mutations, including the same three mutations and an additional Glu-91 to Gly mutation in ParC. The sparfloxacin accumulation level within 30 minutes in the posttreatment isolate was four times less than that in the pretreatment isolate. There were no differences in the pulsed-field gel electrophoresis patterns between the pretreatment and posttreatment isolates from the patient. CONCLUSIONS: The emergence of a fluoroquinolone-resistant N. gonorrhoeae isolate with multiple mutations involving GyrA and ParC reduced the response to sparfloxacin treatment. Multiple dosing and long-term treatment with sparfloxacin seems to induce a mutation in ParC and an alteration leading to reduced drug accumulation that contribute to increasing the fluoroquinolone resistance level.

Neutrophil recruitment and resistance to urinary tract infection.

This study examined the role of neutrophil leukocytes for the antibacterial defense at mucosal infection sites. Urinary tract infection (UTI) was established by injection into the bladder lumen of Escherichia coli 1177, a fully virulent clinical isolate. Infection of C3H/HeN (lpsn, lpsn) mice recruited neutrophils into the urinary tract, and bacteria were cleared from kidneys and bladders. The neutrophil response was absent in C3H/HeJ (lpsd, lpsd) mice, and bacteria persisted in the tissues. Peripheral neutrophil depletion of C3H/HeN mice was subsequently achieved by pretreatment with the granulocyte-specific antibody RB6-8C5. The E. coli-induced neutrophil recruitment was inhibited, as shown by immunohistochemistry and tissue myeloperoxidase quantitation. As a consequence, bacterial clearance from kidneys and bladders was drastically impaired. Antibody treatment of C3H/HeJ mice had only a marginal effect. The results show that neutrophils are essential for bacterial clearance from the urinary tract and that the neutrophil recruitment deficiency in C3H/HeJ mice explains their susceptibility to gram-negative UTI.

Suppression of renal scarring by prednisolone combined with ciprofloxacin in ascending pyelonephritis in rats.

To prevent renal scarring, which occurs at the end stage of chronic pyelonephritis due to vesicoureteral reflux of infected urine, immediate antimicrobial treatment is reported to be essential. When treatment is delayed, the antimicrobial agent is believed to be effective only in eliminating bacteria, not in preventing scar formation. Using the ascending pyelonephritis model in rats, we investigated the effect of immediate or delayed treatment with ciprofloxacin and that of delayed treatment with a combination of ciprofloxacin and prednisolone in preventing renal scarring following infection. An inoculum of 1 x 10(9) colony forming units (cfu)/0.1 ml. of the HM32 strain of Escherichia coli, which was isolated from a patient with a urinary tract infection, was injected directly into the rat bladder, and the urethra was clamped for 4 hours in each rat. Treatment by ciprofloxacin (15 mg./kg., twice a day for 5 days) alone or in combination with prednisolone (2 mg./kg., once a day for 4 days) was initiated 6 or 72 hours after bacterial inoculation. The kidneys of each rat were examined 6 weeks later. Immediate treatment by ciprofloxacin significantly inhibited renal scarring (no scarring was seen in any of the 8 rats), but delayed treatment had no effect on scarring (4 of 8 rats showed scarring) when compared with the untreated controls (7 of 8 rats showed scarring). However, the addition of prednisolone to the delayed treatment with ciprofloxacin significantly inhibited renal scarring (only 1 of 10 rats showed scarring) when compared with the untreated controls (7 of 8 rats showed scarring). These data suggest that prednisolone is effective in preventing renal scarring which occurs due to vesicoureteral reflux when the initiation of antimicrobial treatment is delayed.

Preventive effect of ulinastatin on renal scarring in rat model of pyelonephritis induced by direct or ascending infection with Serratia marcescens or Escherichia coli.

Renal scarring is considered to be a characteristic of reflux nephropathy. The effects of ulinastatin, a strong inhibitor of polymorphonuclear leukocyte elastase, on renal scarring following direct parenchymal or intravesical ascending infection by Serratia marcescens or Escherichia coli were determined. Four days of treatment with ulinastatin initiated 2 or 5 days after infection prevented renal scarring. Doses of 1,000-4,000 units/kg inhibited renal scar formation, but 8,000 units/kg did not. These results suggest that it may be possible to limit renal scar formation in pyelonephritis by the use of an appropriate pharmacologic agent.

Effect of prednisolone on renal scarring in rats following infection with Serratia marcescens.

Renal scarring is considered a criterion of reflux nephropathy and the end stage of pyelonephritis. Prednisolone, a strong anti-inflammatory drug, at doses of 1 or 2 mg/kg prevented renal scarring in rats following infection with Serratia marcescens. Four or 8 mg/kg of prednisolone, however, did not inhibit renal scar formation. In a time course experiment, renal scarring was prevented when 4-day treatment with prednisolone was initiated 2, 5, or 13 days after infection. These results show that prednisolone is effective in preventing such scarring and suggest the clinical use of this drug for preventing renal scar formation after pyelonephritis and reflux nephropathy.

Enhancement of superoxide production of polymorphonuclear neutrophils by ofloxacin and the effects of the inhibitors of protein kinase C.

Polymorphonuclear neutrophils (PMN) represent an important defense mechanism against bacterial infection. Superoxide is one of the most important factors released by PMN following various stimulations including bacteria. Augmentation of chemiluminescence response of PMN stimulated by phorbol myristate acetate was observed following the addition of 25-200 micrograms/ml of ofloxacin, a new quinolone antimicrobial agent. In addition, the effects of two inhibitors of protein kinase C, staurosporine and H-7, were examined. The augmented superoxide production was inhibited by 1 or 2 microM of staurosporine or 50 or 100 microM of H-7. These results suggest that ofloxacin augments superoxide production of PMN and that this augmentation is probably due to the enhancement of leukocyte protein kinase C.