Rapid antimicrobial susceptibility testing by sensitive detection of precursor rRNA using a novel electrochemical biosensing platform.

Precursor rRNA (pre-rRNA) is an intermediate stage in the formation of mature rRNA and is a useful marker for cellular metabolism and growth rate. We developed an electrochemical sensor assay for Escherichia coli pre-rRNA involving hybridization of capture and detector probes with tail sections that are spliced away during rRNA maturation. A ternary self-assembled monolayer (SAM) prepared on gold electrode surfaces by coassembly of thiolated capture probes with hexanedithiol and posttreatment with 6-mercapto-1-hexanol minimized the background signal and maximized the signal-to-noise ratio. Inclusion of internal calibration controls allowed accurate estimation of the pre-rRNA copy number per cell. As expected, the ratio of pre-rRNA to mature rRNA was low during stationary phase and high during log phase. Pre-rRNA levels were highly dynamic, ranging from 2 copies per cell during stationary phase to ~1,200 copies per cell within 60 min of inoculation into fresh growth medium. Specificity of the assay for pre-rRNA was validated using rifampin and chloramphenicol, which are known inhibitors of pre-rRNA synthesis and processing, respectively. The DNA gyrase inhibitor, ciprofloxacin, was found to act similarly to rifampin; a decline in pre-rRNA was detectable within 15 min in ciprofloxacin-susceptible bacteria. Assays for pre-rRNA provide insight into cellular metabolism and are promising predictors of antibiotic susceptibility.

Target-specific capture enhances sensitivity of electrochemical detection of bacterial pathogens.

We report the concentration and purification of bacterial 16S rRNA by the use of a biotinylated DNA target-specific capture (TSC) probe. For both cultivated bacterial and urine specimens from urinary tract infection patients, TSC resulted in a 5- to 8-fold improvement in the sensitivity of bacterial detection in a 16S rRNA electrochemical sensor assay.

Improved continence in patients with neurogenic sphincteric incompetence with combination tubularized posterior urethroplasty and fascial wrap: the lengthening, narrowing and tightening procedure.

PURPOSE: We evaluated the outcome in 19 patients who underwent bladder neck reconstruction by lengthening, narrowing and tightening the bladder neck with a combined tubularized posterior urethroplasty and circumferential fascial wrap. MATERIALS AND METHODS: We reviewed the records of all patients who underwent bladder neck lengthening, narrowing and tightening between April 1996 and November 2002. Preoperative urodynamic and radiographic data were available on all patients. The surgical technique involved retroperitoneal exposure of the bladder neck with a tubularized posterior urethroplasty over a urethral catheter. The reconstructed urethra was then circumferentially wrapped with a fitted piece of cadaveric fascia. RESULTS: Of the 19 patients 15 remain completely continent at a mean ± SD followup of 35.5 ± 29.1 months. Three patients underwent secondary reconstruction, including bladder neck ligation in all 3 and secondary enterocystoplasty in 2. No patient experienced difficult intermittent catheterization via the urethra postoperatively. CONCLUSIONS: Bladder neck lengthening, narrowing and tightening is effective for managing neurogenic sphincteric incontinence. Outcomes are comparable with those of other reconstructive procedures.

Selective use of voiding cystourethrography in children undergoing renal transplant evaluation.

PURPOSE: Voiding cystourethrography is a routine component in evaluating children awaiting renal transplantation. We examined whether this assessment is necessary in children with renal failure due to dysplasia/aplasia/hypoplasia syndrome and unknown etiology, which account for up to 25% of those with renal failure requiring renal replacement therapies. MATERIALS AND METHODS: We performed an institutional review board approved, retrospective review of 191 children undergoing transplantation between 2002 and 2007. We reviewed clinical factors associated with positive findings on voiding cystourethrogram. We also reviewed cystography results in children with chronic kidney disease due to renal dysplasia and unknown etiology. RESULTS: We identified 113 boys and 78 girls who underwent renal transplantation during the study period. Pre-transplant voiding cystourethrography was documented in 108 children (57%). Predictors of positive pre-transplant results included history of hydronephrosis, urinary tract infections and renal failure due to urological causes. No pre-transplant cystogram was positive in children with renal failure due to dysplasia or unknown etiology. CONCLUSIONS: We recommend selective use of voiding cystourethrography to evaluate children awaiting renal transplantation. We continue to support performing this test in children with renal failure due to urological causes and those with a history of urinary tract infection, hydronephrosis or voiding dysfunction. In the absence of these findings children with renal failure due to renal dysplasia/aplasia/hypoplasia syndrome or unknown etiology need not undergo pre-transplant voiding cystourethrography.

Optimal probe length and target location for electrochemical detection of selected uropathogens at ambient temperature.

We have previously demonstrated the clinical validity of the rapid detection of uropathogens by use of a DNA biosensor. This assay involves the hybridization of capture and detector probe pairs with bacterial 16S rRNA target molecules to form a DNA-RNA sandwich on the sensor surface. Horseradish peroxidase-conjugated antibody binds to the detector probe to enzymatically amplify the hybridization signal. These previous studies involved the hybridization of bacterial 16S rRNA target sequences with 35-mer oligonucleotide probe pairs at 65 degrees C. Achievement of point-of-care technology will be greatly facilitated by ambient-temperature detection. The purpose of this study was to examine the effects of probe length and target location on signal intensity using hybridization temperatures of 20 to 25 degrees C. Signal intensity was found to vary dramatically with hybridization location in the species-specific bulge region of 16S rRNA helix 18. Probe pairs of as short as 10 nucleotides in length were able to produce a significant electrochemical signal, and signal intensity was correlated with probe length for probes of 10 to 20 nucleotides in length. The sensitivity of the Escherichia coli-specific 15-mer probe pairs was approximately 330 cells. These shorter probes allowed differentiation of Klebsiella pneumoniae from Proteus mirabilis 16S rRNA target sequences differing by a single nucleotide. A panel of oligonucleotide probe pairs ranging from 11 to 23 nucleotides in length was able to distinguish among seven groups of urinary tract pathogens. In conclusion, we have developed short oligonucleotide probe pairs for the species-specific identification of uropathogens at ambient temperature by use of an electrochemical sensor.

Uropathogens and host characteristics.

Uropathogens other than Escherichia coli occur with greater frequency in patients with risk factors for urinary tract infection (UTI). However, little is known about associations between uropathogen species and host characteristics. Three hundred nineteen urine specimens containing a balanced distribution of uropathogen species were selected from inpatients and outpatients at a university hospital clinical microbiology laboratory. Information on host characteristics was retrospectively collected by chart review. Differences in the frequencies of host characteristics in UTI groups, as defined by the causative uropathogen, were compared by chi-square/Fisher analysis. Multivariate classification and regression tree analysis were used to identify host characteristic subsets that distinguish among uropathogen groups. In this exploratory study, several uropathogen species were found to be strongly linked to host characteristics relevant to UTI. Patients with Pseudomonas aeruginosa UTIs were more likely to have undergone urinary tract procedures (43% versus 15% overall), to have a neurogenic bladder (29% versus 12% overall), to have received recent antibiotic therapy (52% versus 24% overall), and to be male (76% versus 28% overall). Patients with Proteus mirabilis UTIs were more likely to have a foreign body in the lower urinary tract (48% versus 30% overall). The classification tree identified males over the age of 27 years who had undergone a prior urinary tract procedure as belonging to a host characteristic profile associated with P. aeruginosa UTI: 38% of patients with P. aeruginosa UTIs fit this profile. These data may be useful for planning future targeted prophylaxis studies.

Rapid antimicrobial susceptibility determination of uropathogens in clinical urine specimens by use of ATP bioluminescence.

We describe the first direct testing of the antimicrobial susceptibilities of bacterial pathogens in human clinical fluid samples by the use of ATP bioluminescence. We developed an ATP bioluminescence assay that eliminates somatic sources of ATP to selectively quantify the bacterial load in clinical urine specimens with a sensitivity of <1,000 CFU per milliliter. There was a log-log relationship between light emission and the numbers of CFU in clinical urine specimens. A clinical study was performed to evaluate the accuracy of the ATP bioluminescence assay for determination of the antimicrobial susceptibilities of uropathogens in clinical urine specimens tested in a blinded manner. ATP bioluminescent bacterial density quantitation was used to determine the inoculation volume in growth medium with and without antibiotics. After incubation at 37 degrees C for 120 min, the ATP bioluminescence assay was repeated to evaluate the uropathogen response to antibiotics. The ability of the ATP bioluminescence assay to discriminate between antimicrobial susceptibility and resistance was determined by comparison of the results obtained by the ATP bioluminescence assay with the results obtained by standard clinical microbiology methods. Receiver operator characteristic curves were used to determine the optimal threshold for discriminating between susceptibility and resistance. Susceptibility and resistance were correctly predicted in 87% and 95% of cases, respectively, for an overall unweighted accuracy of 91%, when the results were stratified by antibiotic. For samples in which the pathogen was susceptible, the accuracy improved to 95% when the results for samples with less than a 25-fold increase in the amount of bacterial ATP in the medium without antibiotics were excluded. These data indicate that a rapid bioluminescent antimicrobial susceptibility assay may be useful for the management of urinary tract infections.

An unsteady microfluidic T-form mixer perturbed by hydrodynamic pressure.

An unsteady microfluidic T-form mixer driven by pressure disturbances was designed and investigated. The performance of the mixer was examined both through numerical simulation and experimentation. Linear Stokes equations were used for these low Reynolds number flows. Unsteady mixing in a micro-channel of two aqueous solutions differing in concentrations of chemical species was described using a convection-dominated diffusion equation. The task was greatly simplified by employing linear superimposition of a velocity field for solving a scalar species concentration equation. Low-order-based numerical codes were found not to be suitable for simulation of a convection-dominated mixing process due to erroneous computational dissipation. The convection-dominated diffusion problem was addressed by designing a numerical algorithm with high numerical accuracy and computational-cost effectiveness. This numerical scheme was validated by examining a test case prior to being applied to the mixing simulation. Parametric analysis was performed using this newly developed numerical algorithm to determine the best mixing conditions. Numerical simulation identified the best mixing condition to have a Strouhal number (St)of 0.42. For a T-junction mixer (with channel width = 196 µm), about 75% mixing can be finished within a mixing distance of less than 3 mm (i.e. 15 channel width) at St = 0.42 for flow with a Reynolds number less than 0.24. Numerical results were validated experimentally by mixing two aqueous solutions containing yellow and blue dyes. Visualization of the flow field under the microscope revealed a high level of agreement between numerical simulation and experimental results.

Development of an advanced electrochemical DNA biosensor for bacterial pathogen detection.

Electrochemical sensors have the capacity for rapid and accurate detection of a wide variety of target molecules in biological fluids. We have developed an electrochemical sensor assay involving hybridization of bacterial 16S rRNA to fluorescein-modified detector probes and to biotin-modified capture probes anchored to the sensor surface. Signal is generated by an oxidation-reduction current produced by the action of horseradish peroxidase conjugated to an anti-fluorescein monoclonal Fab. A previous study found that this electrochemical sensor strategy could identify uropathogens in clinical urine specimens. To improve assay sensitivity, we examined the key steps that affect the current amplitude of the electrochemical signal. Efficient lysis and release of 16S rRNA from both gram-negative and -positive bacteria was achieved with an initial treatment with Triton X-100 and lysozyme followed by alkaline lysis, resulting in a 12-fold increase in electrochemical signal compared with alkaline lysis alone. The distance in nucleotides between the target hybridization sites of the detector and capture probes and the location of fluorescein modification on the detector probe contributed to a 23-fold change in signal intensity. These results demonstrate the importance of target-probe and probe-probe interactions in the detection of bacterial 16S rRNA using an electrochemical DNA sensor approach.

Automatic generation of repeated patient information for tailoring clinical notes.

Generating clear, readable, and accurate reports can be a time-consuming task for physicians. Clinical notes, which document patient encounters, often contain a certain set of patient information including demographics, medical history, surgical history, examination results or the current medical condition that is propagated from one clinical note to all subsequent clinical notes for the same patient. To this end, we present a system, which automatically generates this patient information for the creation of a new clinical note. We use semantic patterns and an approximate sequence matching algorithm for capturing the discourse role of sentences, which we show to be a useful feature for determining whether the sentence should be repeated. Our system is shown to perform better than a simple baseline metric using precision/recall results. We believe such a system would allow clinical notes to be more complete, timely, and accurate.

Intracellular Bacterial Communities: A Potential Etiology for Chronic Lower Urinary Tract Symptoms.

Patients with persistent lower urinary tract symptoms and negative urine cultures are often difficult to treat. Infection may go undetected in these patients because the concentrations of bacteria in their urine are beneath the threshold of standard urine culture techniques. Empiric treatment may result in temporary relief, followed by recurrent symptoms. Occult and recurrent urinary tract infection may be due to both invasion of the bladder wall by uropathogenic Escherichia coli and the formation of biofilm-like intracellular bacterial communities. This review examines emerging evidence for a role of intracellular bacterial communities in human infection.

Automatic generation of repeated patient information for tailoring clinical notes.

Dictating clear, readable, and accurate clinical notes can be a time-consuming task for physicians. Clinical notes often contain information concerning the patient's medical history and current medical condition which is propagated from one clinical note to all follow-up clinical notes for the same patient. In this paper, we present a system which, given a clinical note, automatically determines what information should be repeated, and then generates this information for the physician for a new clinical note. We use semantic patterns for capturing the rhetorical category of sentences, which we show to be useful for determining whether the sentence should be repeated. Our system is shown to perform better than a baseline metric based on precision/recall results. Such a system would allow clinical notes to be more complete, timely, and accurate.

Bacterial detection & identification using electrochemical sensors.

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe. Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Timing of orchiopexy in the United States: a quality-of-care indicator.

OBJECTIVE: To investigate whether orchiopexies are occurring later than recommended by American Academy of Pediatrics 1996 guidelines (around age 1). Adherence to guidelines is poorly studied. METHODS: The main cohort consisted of 4103 boys insured from birth (Innovus i3, insurance claims database). The complementary cohort consisted of 17 010 insured and noninsured boys (Pediatric Health Information System, PHIS). The inclusion criteria was age ≤ 5 years at time of International Classification of Disease, 9th revision-defined cryptorchidism diagnosis. The primary outcome was timely surgery (orchiopexy by age 18 months). RESULTS: In Innovus, 87% of boys who underwent an orchiopexy had a timely orchiopexy. Of those who did not undergo surgery (n = 2738), 90% had at least 1 subsequent well-care visit. Those who underwent timely surgery were referred to a surgeon at a younger age compared with those who underwent late surgery (4.1 vs 16.1 months, P < .00005). Predictors of timely surgery were number of well-care visits (odds ratio 1.5, 95% confidence interval 1.3-1.7), continuity of primary care (odds ratio 1.9, 95% confidence interval 1.3-2.7), and use of laparoscopy (odds ratio 4.5, 95% confidence interval 1.4-14.9). Family/internal medicine as referring provider was predictive of delayed surgery (odds ratio 0.5, 95% confidence interval 0.3-0.8). In the Pediatric Health Information System, 61% of those with private insurance had timely surgery compared with 54% of those without private insurance (P < .0001). CONCLUSION: We found an unexpectedly high adherence to guidelines in our continuously insured since birth Innovus population. Primary care continuity and well-care visits were associated with timely surgery. Further studies can confirm these findings in nonprivately insured patients with the ultimate goal of instituting quality improvement initiatives.

Virilization of a female infant by a maternal adrenocortical carcinoma.

OBJECTIVE: To describe a possible mechanism underlying the partial virilization of a 46, XX infant by a functional maternal adrenocortical carcinoma (ACC). METHODS: We performed immunocytochemical staining of tumor sections for luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptors. In addition, related reports in the literature are discussed. RESULTS: A previously healthy mother developed a large cortisol- and androgen-producing stage III adrenal tumor that did not interfere with conception or early morphogenesis. The tumor eluded detection until after delivery of a partially virilized 46, XX female infant with ambiguous genitalia. Immunohistochemical staining of tumor sections revealed overexpression of the LH/hCG receptor. Virilization of the genetically female fetus may have resulted from hCG-stimulated steroid secretion by the ACC. CONCLUSION: Because hypercortisolism and hyperandrogenism are associated with menstrual disturbances and spontaneous abortion, pregnancy in patients with functional adrenal tumors is uncommon. Rarely, maternal steroid excess from a functional adrenal tumor has caused 46, XX disordered sex differentiation. This unusual case demonstrates the influence of hCG on the functionality of an ACC and demonstrates the rare phenomenon of virilization of a female infant by a functional maternal adrenal tumor.

Outcomes of bladder neck closure for intractable urinary incontinence in patients with neurogenic bladders.

OBJECTIVE: Recent reports of outcomes of bladder neck closure for neurogenic urinary incontinence reveal poor initial continence and high vesicourethral fistula rates. We evaluated a large series of patients who underwent complete transection and closure of the bladder neck with modified abdominal stoma creation. MATERIALS AND METHODS: The medical records of 52 consecutive patients (23 males and 29 females) undergoing bladder neck closure by a single surgeon between July 1996 and January 2003 were reviewed. Mean follow up was 20 months (range 2-68 months) and mean age was 13.9 years (range 1.5-58 years). Forty-two patients (81%) underwent concomitant bladder augmentation. Catheterizable stomas included 46 appendicovesicostomies (88%) and six Monti tubes (12%). Of the 52 patients, 22 were confined to wheelchairs (42%), and the majority of patients had spinal cord pathology (40/52, 77%). Mean preoperative vesical leak point pressure was 25 cm/H(2)O (range 4-69 cm/H(2)O). RESULTS: Complete postoperative urinary continence was achieved in 44 patients (88%) after one procedure. Of the six patients who were incontinent (12%), one had a vesicourethral fistula (2%) and five had incontinence at the urinary diversion stoma (10%). Twelve of 50 patients had urinary stomal stenosis (24%), with six requiring urgent evaluation (12%) and six requiring surgical revision (12%). Postoperative urinary continence was unknown in two patients due to inadequate follow-up data. CONCLUSIONS: Our findings suggest that bladder neck closure is a safe and effective method of achieving urinary continence in children with neurogenic voiding dysfunction. While there are risks of stomal stenosis and fistula formation, modifications in surgical technique may reduce them to acceptable levels.

Use of electrochemical DNA biosensors for rapid molecular identification of uropathogens in clinical urine specimens.

We describe the first species-specific detection of bacterial pathogens in human clinical fluid samples using a microfabricated electrochemical sensor array. Each of the 16 sensors in the array consisted of three single-layer gold electrodes-working, reference, and auxiliary. Each of the working electrodes contained one representative from a library of capture probes, each specific for a clinically relevant bacterial urinary pathogen. The library included probes for Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterocococcus spp., and the Klebsiella-Enterobacter group. A bacterial 16S rRNA target derived from single-step bacterial lysis was hybridized both to the biotin-modified capture probe on the sensor surface and to a second, fluorescein-modified detector probe. Detection of the target-probe hybrids was achieved through binding of a horseradish peroxidase (HRP)-conjugated anti-fluorescein antibody to the detector probe. Amperometric measurement of the catalyzed HRP reaction was obtained at a fixed potential of -200 mV between the working and reference electrodes. Species-specific detection of as few as 2,600 uropathogenic bacteria in culture, inoculated urine, and clinical urine samples was achieved within 45 min from the beginning of sample processing. In a feasibility study of this amperometric detection system using blinded clinical urine specimens, the sensor array had 100% sensitivity for direct detection of gram-negative bacteria without nucleic acid purification or amplification. Identification was demonstrated for 98% of gram-negative bacteria for which species-specific probes were available. When combined with a microfluidics-based sample preparation module, the integrated system could serve as a point-of-care device for rapid diagnosis of urinary tract infections.

Rapid, species-specific detection of uropathogen 16S rDNA and rRNA at ambient temperature by dot-blot hybridization and an electrochemical sensor array.

Development of rapid molecular approaches for pathogen detection is key to improving treatment of infectious diseases. For this study, the kinetics and temperature-dependence of DNA probe hybridization to uropathogen species-specific sequences were examined. A set of oligonucleotide probes were designed based on variable regions of the 16S gene of the Escherichia coli, Proteus mirabilis, Klebsiella oxytoca, and Pseudomonas aeruginosa. A universal bacterial probe and probes-specific for gram-positive and gram-negative organisms were also included. The oligonucleotide probes discriminated among 16S genes derived from 11 different species of uropathogenic bacteria applied to nylon membranes in a dot-blot format. Significant binding of oligonucleotide probes to target DNA and removal of nonspecific binding by membrane washing could both be achieved rapidly, requiring as little as 10 min. An oligonucleotide probe from the same species-specific region of the E. coli 16S gene was used as a capture probe in a novel electrochemical 16-sensor array based on microfabrication technology. Sequence-specific hybridization of target uropathogen 16S rDNA was detected through horseradish peroxidase acting as an electrochemical transducer via a second, detector probe. The sensor array demonstrated rapid, species-specific hybridization in a time course consistent with the rapid kinetics of the dot-blot hybridization studies. As in the dot-blot hybridization studies, species-specific detection of bacterial nucleic acids using the sensor array approach was demonstrated both at 65 degrees C and at room temperature. These results demonstrate that molecular hybridization approaches can be adapted to rapid, room temperature conditions ideal for an electrochemical sensor array platform.