Direct-from-specimen microbial growth inhibition spectrums under antibiotic exposure and comparison to conventional antimicrobial susceptibility testing.

Increasing global travel and changes in the environment may escalate the frequency of contact with a natural host carrying an infection and, therefore, increase our chances of encountering microorganisms previously unknown to humans. During an emergency, the etiology of infection may be unknown at the time of patient treatment. The existing local or global Antimicrobial Stewardship Programs may not be fully prepared for emerging/re-emerging infectious disease outbreaks, especially if they are caused by an unknown organism, engineered bioterrorist attack, or rapidly evolving superbug. We demonstrate an antimicrobial efficacy profiling method that can be performed in hours directly from clinical urine specimens. The antimicrobial potency was determined by the level of microbial growth inhibition and compared to conventional antimicrobial susceptibility testing results. The oligonucleotide probe pairs on the sensors were designed to target Gram-negative bacteria, specifically Enterobacterales and Pseudomonas aeruginosa. A pilot study of 10 remnant clinical specimens from the Clinical Laboratory Improvement Amendments-certified labs of New York-Presbyterian Queens was conducted, and only one sample was not detected by the probes. The remaining nine samples agreed with reference AST methods (Vitek and broth microdilution), resulting in 100% categorical agreement. In a separate feasibility study, we evaluated a dual-kinetic response approach, in which we inoculated two antibiotic stripwells containing the same antimicrobial concentrations with clinical specimens at the original concentration (1x) and at a 10-fold dilution (0.1x) to cover a broader range of microbiological responses. The combined categorical susceptibility reporting of 12 contrived urine specimens was 100% for ciprofloxacin, gentamicin, and meropenem over a range of microbial loads from 105 to 108 CFU/mL.

Improved Reversible Cross-Linking-Based Solid-Phase RNA Extraction for Pathogen Diagnostics.

In this study, we developed an amine-functionalized, diatomaceous earth-based, dimethyl suberimidate assisted (ADD) system as a novel binding strategy to improve the solid-phase extraction method for rapid and simple purification of RNA from biological samples including human cells and pathogenic bacteria. This ADD system is based on reversible cross-linking reactions between RNA and the silica matrix. The formation of robust covalent bonds protects RNA from both the sufferance of washing steps and isolation with ribonuclease (RNase)-rich samples, leading to the extraction of higher quality RNA. This improved RNA extraction system integrated with quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is evaluated for pathogen diagnostics. Compared to standard solid-phase extraction based commercial kits, this improved method shows highly enhanced sensitivity with 1000-fold higher sensitivity for human cells and 100-fold higher sensitivity for Brucella bacteria, according to the cycle threshold value of RT-qPCR. We envision that the ADD system can be tailored for commercial applications for RNA expression analysis in forensics studies, as well as for disease diagnostics in clinical applications.

Time to clearance of Chlamydia trachomatis RNA and DNA after treatment in patients coinfected with Neisseria gonorrhoeae - a prospective cohort study.

BACKGROUND: Performing a test of cure (TOC) could demonstrate success or failure of antimicrobial treatment of Chlamydia trachomatis infection, but recommendations for the timing of a TOC using nucleic acid amplification tests (NAATs) are inconsistent. We assessed time to clearance of C. trachomatis after treatment, using modern RNA- and DNA-based NAATs. METHODS: We analysed data from patients with a C. trachomatis and Neisseria gonorrhoeae coinfection who visited the STI Clinic Amsterdam, The Netherlands, from March through October 2014. After treatment with ceftriaxone plus either azithromycin or doxycycline, patients self-collected anal, vaginal or urine samples during 28 consecutive days. Samples were analysed using an RNA-based NAAT (Aptima Combo 2) and a DNA-based NAAT (Cobas 4800 CT/NG). We defined clearance as three consecutive negative results, and defined "blips" as isolated positive results following clearance. RESULTS: We included 23 patients with C. trachomatis and N. gonorrhoeae coinfection. All patients cleared C. trachomatis during follow-up, and we observed no reinfections. The median time to clearance (range) was 7 days (1-13) for RNA, and 6 days (1-15) for DNA. Ninety-five per cent of patients cleared RNA at day 13, and DNA at day 14. The risk of a blip after clearance was 4.4 % (RNA) and 1.7 % (DNA). CONCLUSIONS: If a TOC for anogenital chlamydia is indicated, we recommend performing it at least 14 days after initiation of treatment, when using modern RNA- and DNA-based assays. A positive result shortly after 14 days probably indicates a blip, rather than a treatment failure or a reinfection.

Extra-pulmonary tuberculosis: differential aspects and role of 16S-rRNA in urine.

BACKGROUND: Early diagnosis of extra-pulmonary tuberculosis (EPTB) is important for successful treatment. METHODS: All cases of EPTB diagnosed at Ramon y Cajal Hospital, Madrid, Spain, from 1997 to 2008 were analysed and compared with pulmonary tuberculosis (PTB) patients to identify differential parameters that could serve to predict the presence of EPTB at initial presentation. Different microbiological techniques were analysed, including amplification of 16S-rRNA in urine. RESULTS: During the study period, 814 cases of TB were diagnosed at our centre; 330 (40.5%) were EPTB. Concomitant PTB was detected in 45% of EPTB cases. The main clinical forms of EPTB were lymphadenitis (86, 26%), miliary TB (60, 18%), and multifocal TB (43, 13%). Variables independently associated with EPTB were human immunodeficiency virus (HIV) infection (OR 3.6, 95%CI 2.4-5.4), older age (>60 years) (OR 3.7, 95%CI 2.5-5.6) and mortality (OR 2.9, 95%CI 1.3-6.3). 16S-rRNA in urine was performed in 82 EPTB patients (25%), among whom a positive result was obtained in 70%; in the PTB group, a positive result was found in 5 of 28 patients (18%) (P <0.001). CONCLUSIONS: HIV infection and older age appear to be the main risk factors associated with EPTB. In this study, mortality was significantly higher in patients with EPTB. A positive 16S-rRNA test result in urine is a useful marker of EPTB.

Optimized templates for bottom-up growth of high-performance integrated biomolecular detectors.

Electrochemical deposition of metals represents an important approach in the bottom-up fabrication of nanostructures and microstructures. We have used this approach to generate high-performance chip-based biosensors using silicon as a platform for the generation of sensor arrays. Here, we explore the applicability of different materials to support the electrodeposition and identify the parameters that are essential for robust sensor growth. We show that inexpensive materials can be used as templates for electrodeposition, and demonstrate that these low-cost sensors exhibit clinically-relevant levels of sensitivity and specificity. In particular, we prove herein that the glass-based sensors successfully detect E. coli in urine, when present at the 100 cfu µL(-1) levels found typically in samples of patients with urinary tract infections.

In situ electrokinetic enhancement for self-assembled-monolayer-based electrochemical biosensing.

This study reports a multifunctional electrode approach which directly implements electrokinetic enhancement on a self-assembled-monolayer-based electrochemical sensor for point-of-care diagnostics. Using urinary tract infections as a model system, we demonstrate that electrokinetic enhancement, which involves in situ stirring and heating, can enhance the sensitivity of the strain specific 16S rRNA hybridization assay for 1 order of magnitude and accelerate the time-limiting incubation step with a 6-fold reduction in the incubation time. Since the same electrode platform is used for both electrochemical signal enhancement and electrochemical sensing, the multifunctional electrode approach provides a highly effective strategy toward fully integrated lab-on-a-chip systems for various biomedical applications.

Efficacy of an RNA detection test kit in the diagnosis of genital chlamydial infection.

A nucleic acid amplification method based on DNA detection, the current standard method for the diagnosis of genital infection by Chlamydia trachomatis, has been shown to potentially yield false-positive results after treatment in the clinical setting. RNA detection methods are more appropriate because viable organisms have multiple RNA copies that are surely detected by the method. In this study, we evaluated the efficacy of a new RNA detection test kit, the VIDAS PROBE CT test, in the diagnosis of genital chlamydial infection. For comparison, the standard DNA detection method, Amplicor STD-I, was also used in the study. First voided-urine samples and urethral smears from male patients with urethritis, and first voided-urine samples and cervical smears from female patients with cervicitis served as samples for the detection of C. trachomatis. Of the 60 first voided-urine samples from male patients, 21 were positive and 39 negative with the VIDAS PROBE CT test. Amplicor STD-I achieved exactly the same result. In female patients with cervicitis, the two test kits produced the same result, with 2 positive cervical smears and 38 negative. These results suggest that the VIDAS PROBE CT test is as efficient as Amplicor STD-I in the detection of C. trachomatis. While studies including a greater number of patients will be needed for revealing the unique advantages of the new RNA detection test kit, VIDAS PROBE CT, we concluded from the current study that the test may be clinically useful in the diagnosis of genital chlamydial infection.

Detection of Chlamydia trachomatis in male and female urine specimens by using the amplified Chlamydia trachomatis test.

The amplified Chlamydia trachomatis test (AMP-CT; Gen-Probe), a new diagnostic test for the detection of Chlamydia trachomatis, was evaluated with urine specimens from 1,000 patients visiting the outpatient department for sexually transmitted diseases at the University Hospital Rotterdam, Rotterdam, The Netherlands, by comparing the results to those of cell culture. From February 1996 to July 1996, urine samples for the AMP-CT test and urethral swabs for cell culture were collected from 544 men, while cervical swabs from 456 women were also taken for cell culture. Positive test results were obtained for 130 (13%) of the patients. AMP-CT test and cell culture results were discordant for 70 (7%) specimens. Analysis of the samples with discordant results was performed by an in-house PCR. After resolution of the discordant results, the sensitivity, specificity, and positive and negative predictive values of the AMP-CT test were 84.3, 98.8, 89.6, and 98%, respectively, for samples from females and 100, 99.2, 93.1, and 100%, respectively, for samples from males, while for cell culture these values were 72.5, 99.2, 92.5, and 98%, respectively, for samples from females and 57.4, 99.0, 86.1, and 95.4%, respectively, for samples from males. We conclude that the AMP-CT test is a fast and reliable test for the detection of C. trachomatis in urine specimens from females and, in particular, males.

Evaluation of the Gen-Probe Chlamydia trachomatis transcription-mediated amplification assay with urine specimens from women.

We evaluated the Gen-Probe Chlamydia trachomatis transcription-mediated amplification (TMA) assay with urine specimens for the detection of C. trachomatis infections in women. The novel test, based on the isothermal amplification of chlamydial RNA, was compared with the Roche Amplicor PCR with urine and cell culture with endocervical specimens. First-catch urine and endocervical swab specimens were collected from a total of 561 patients, of whom 70 (12.3%) were confirmed to have chlamydial infection. The diagnostic sensitivity and specificity of TMA with urine were 91.4 and 99.6%, respectively, and those of Amplicor PCR were 97.1 and 99.8%, respectively. By repeated analysis of the specimens with discrepant results, the sensitivity of TMA could be increased to 99%, indicating that some methodological improvements in the assay are still to be expected. The sensitivity of PCR could be increased to 100% by the elimination of DNA polymerase inhibitors in a repeated analysis. The sensitivity and specificity of cell culture with cervical specimens were 85.7 and 100%, respectively. The results indicate that TMA with urine specimens from women is a sensitive and specific assay for the detection of C. trachomatis, providing a new noninvasive technique for the screening of chlamydial infections in women.

A solution hybridization assay for ribosomal RNA from bacteria using biotinylated DNA probes and enzyme-labeled antibody to DNA:RNA.

Rapid, convenient and non-isotopic nucleic-acid hybridization methods are needed for this technology to have practical use in clinical diagnostic tests. A method for hybridization of RNA with a DNA probe in solution followed by capture and measurement of the hybrid is described. DNA probes complementary to 23S rRNAs from Escherichia coli and Bacillus subtilis were labeled with a photoactivable biotin reagent. Hybridization of the biotinylated probes with rRNA was complete in less than 5 min. The resultant hybrids were allowed to bind simultaneously to succinylated avidin immobilized on latex and to beta-galactosidase-labeled Fab' fragments of a monoclonal antibody-specific for DNA:RNA. Finally, beta-galactosidase associated with the captured hybrids was measured colorimetrically. The hybridization method can detect less than 1000 bacteria per assay and has broad specificity to permit detection of the various genera of bacteria that infect the urinary tract.